Phenolic acid lipid-based cationic lipids

Abstract

This invention provides a cationic lipid that is important for the delivery of liposome-encapsulated nucleic acids, and that can be synthesized efficiently and inexpensively without forming potentially toxic byproducts. [Solution] The present invention provides phenolic lipid compounds of formula (I) and its subformulas, or pharmaceutically acceptable salts thereof. The provided compounds may be useful for the delivery and expression of mRNA and encoded proteins, for example, as components of liposome delivery vehicles, and therefore may be useful in the treatment of various diseases, disorders, and conditions, such as those associated with the deficiency of one or more proteins. TIFF2026048786000258.tif43105

Description

Background Art

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 003,698, filed Apr. 1, 2020, which is hereby incorporated by reference in its entirety.

[0002] The delivery of nucleic acids has been extensively studied as a potential therapeutic option for certain medical conditions. Specifically, messenger RNA (mRNA) therapies are becoming increasingly important options for the treatment of a variety of diseases, including those associated with a deficiency of one or more proteins.

[0003] The efficient delivery of nucleic acids encapsulated in liposomes remains an active area of research. Cationic lipid components play an important role in facilitating the efficient encapsulation of nucleic acids during liposome loading. Furthermore, cationic lipids can play an important role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. A variety of cationic lipids suitable for in vivo use have been found. However, there remains a need to identify lipids that can be synthesized efficiently and at low cost without forming potentially toxic by - products.\

[0004] Phenolic acids have many advantageous features that make them a good starting point for the synthesis of cationic lipids for use in in vivo settings. For example, phenolic acids are non - toxic, are readily available in large quantities, and can be easily derivatized. Generally, phenolic acids can be divided into two groups, benzoic acids and cinnamic acids, and their derivatives.

[0005] Examples of benzoic acids that can be used to synthesize the cationic lipids of the present invention include the following.

Chemical Formula

[0006] Examples of cinnamic acid that can be used to synthesize the cationic lipids of the present invention include the following: . [ka]

[0007] In some embodiments, examples of cinnamic acid that can be used to synthesize the cationic lipids of the present invention include the following: [ka]

[0008] In some embodiments, examples of cinnamic acid that can be used to synthesize the cationic lipids of the present invention include the following: [ka]

[0009] In some embodiments, examples of cinnamic acid that can be used to synthesize the cationic lipids of the present invention include the following: [ka]

[0010] In some embodiments, examples of cinnamic acid that can be used to synthesize the cationic lipids of the present invention include the following: [ka] [Overview of the project]

[0011] The present invention particularly provides novel cationic lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids. These compounds are intended to enable highly effective in vivo delivery while maintaining a favorable toxicity profile.

[0012] The cationic lipids of the present invention can be synthesized from readily available starting reagents such as phenolic acids, benzoic acids, and cinnamic acids. The cationic lipids of the present invention also have unexpectedly high encapsulation efficiencies. The cationic lipids of the present invention also include cleavable groups (e.g., esters and disulfides) that are intended to improve biodegradability and thus contribute to their favorable toxicity profiles.

[0013] In one aspect, as used herein, a cationic lipid having a structure according to the following formula (I):

Chemical formula

[0014] In one embodiment, a cationic lipid that is a pharmaceutically acceptable salt of formula (I) is provided herein.

[0015] In one embodiment, a composition comprising a cationic lipid, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids is provided herein. In one embodiment, the composition is lipid nanoparticles, optionally liposomes.

[0016] In one embodiment, a composition containing the cationic lipid of the present invention may be used in therapy. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows in vivo protein expression after intratracheal administration of lipid nanoparticles containing one of the cationic lipid compounds 1-12. The cationic lipid-containing lipid nanoparticles described herein are effective for in vivo delivery of FFL mRNA based on positive luciferase activity. [Modes for carrying out the invention]

[0018] definition To make the present invention more easily understood, certain terms are first defined below. Further definitions of the following terms and other terms are provided throughout this specification. Publications and other reference materials referenced herein to provide background to the present invention and to offer further details relating to its implementation are incorporated herein by reference.

[0019] Amino Acids: As used herein, the term “amino acid” means, in its broadest sense, any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, amino acids have the general structure H2N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids, in some embodiments, amino acids are D-amino acids, and in some embodiments, amino acids are L-amino acids. “Standard amino acid” means any of the 20 standard L-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” means any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. As used herein, “synthetic amino acid” includes, but is not limited to, salts, amino acid derivatives (such as amides), and / or substitutions of chemically modified amino acids. Amino acids, including carboxy-terminal and / or amino-terminal amino acids in peptides, may be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the cyclic half-life of the peptide without adversely affecting their activity. Amino acids may be involved in disulfide bonds. An amino acid may include one or more post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, acetate group, acetyl group, phosphate group, formyl moiety, isoprenoid group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.). The term “amino acid” is used interchangeably with “amino acid residue” and may refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to free amino acids or peptide residues will be clear from the context in which it is used.

[0020] Animals: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human at any developmental stage. In some embodiments, “animal” refers to a non-human animal at any developmental stage. In certain embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0021] Approximately or about: As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​similar to the specified reference values. In certain embodiments, unless otherwise indicated or evident from the context (unless such number exceeds 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than any of the specified reference values ​​(greater than or less than them).

[0022] Biologically active: As used herein, the term “biologically active” refers to the characteristic of any drug that is active in a biological system, in particular in a living organism. For example, a drug that, when administered to an organism, has a biological effect on that organism is considered biologically active.

[0023] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery encompasses situations in which mRNA is delivered to a target tissue, the encoded protein is expressed, and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which mRNA is delivered to a target tissue, the encoded protein is expressed, secreted into the patient’s circulatory system (e.g., serum), distributed throughout the body, and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery”).

[0024] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into an intact protein (e.g., an enzyme), and / or post-translational modification of polypeptides or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production,” as well as their grammatical synonyms, are used interchangeably.

[0025] Functionality: As used herein, “functional” biomolecule is a biomolecule in which it exhibits the properties and / or activity that characterize it.

[0026] Half-life: As used herein, the term “half-life” refers to the time required for an amount of nucleic acid or protein, such as concentration or activity, to decrease to half of its initial measured value over a given period.

[0027] Helper Lipids: As used herein, the term “helper lipids” refers to any natural lipid material, including cholesterol, or any amphoteric lipid material. While we do not wish to be bound by any particular theory, helper lipids may impart stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles.

[0028] To improve, increase, or decrease: As used herein, “improve,” “increase,” or “decrease,” or grammatical synonyms, refer to a value compared to a baseline measurement, e.g., a measurement in the same individual before the initiation of the treatment described herein, or a measurement in a control subject (or control subjects) in the absence of the treatment described herein. A “control subject” is a subject suffering from the same disease form as the subject being treated and being approximately the same age as the subject being treated.

[0029] In vitro: As used herein, the term “in vitro” refers to events occurring in an artificial environment, such as in a test tube or reaction vessel, or under cell culture conditions, rather than within a multicellular organism.

[0030] In vivo: As used herein, the term “in vivo” refers to events occurring within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0031] Isolated: As used herein, the term “isolated” means (1) a substance and / or entity that has been separated from at least some of the components to which it was originally associated (whether in a natural or experimental environment) and / or (2) has been artificially produced, prepared, and / or manufactured. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components to which they were originally associated. In some embodiments, the isolated agent is ultrapure of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the purity percentage of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0032] Liposome: As used herein, the term "liposome" refers to any layered, Liposomes refer to multilayered or solid nanoparticle vesicles. Typically, liposomes as used herein can be formed by mixing one or more lipids, or by mixing one or more lipids with a polymer. In some embodiments, liposomes suitable for the present invention comprise cationic lipids and optionally non-cationic lipids, optionally cholesterol-based lipids, and / or optionally PEG-modified lipids.

[0033] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The term “modified mRNA” is associated with mRNA containing at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. If necessary, for example, in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs such as chemically modified bases or sugars, or analogs with skeletal modifications, etc. mRNA sequences are presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, mRNA is a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine), a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine) The bases are or include C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite bonds).

[0034] Nucleic Acids: As used herein, the term “nucleic acid” means, in its broadest sense, any compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into a polynucleotide chain via phosphodiester bonds. In some embodiments, “nucleic acid” means individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” means a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” includes RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, “nucleic acid” encompasses ribonucleic acid (RNA), including but not limited to one or more of the following: interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), multimer-coding nucleic acid (MCNA), polymer-coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA). In some embodiments, “nucleic acid” encompasses deoxyribonucleic acid (DNA), including but not limited to one or more of the following: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, “nucleic acid” encompasses both RNA and DNA. In several embodiments, DNA includes antisense DNA, plasmid DNA, portions of plasmid DNA, pre-condensed DNA, polymerase chain reaction (PCR) products, and vectors. This can take the form of (e.g., P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In multiple embodiments, RNA includes messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), transfer RNA (tRNA), transfer-messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splice reader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (lncRNA), microRNA (miRNA), Piwi-binding RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), and 73K This can take the form of RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is mRNA that encodes a protein such as an enzyme.

[0035] Patient: As used herein, the terms “patient” or “subject” mean any organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventive, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. Humans include prenatal and postnatal forms.

[0036] Pharmacologically acceptable: As used herein, the term "pharmaceutically acceptable" means a substance that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and that is commensurate with a reasonable benefit / risk ratio.

[0037] pharmaceutically acceptable salts: pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Suitable pharmaceutically acceptable salts of the compounds of the present invention include those derived from inorganic and organic acids, as well as inorganic and organic bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfonate. Examples include lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, and valersates. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4 Examples include alkyl(4) salts. Typical alkali metal salts or alkaline earth metal salts. These include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates, where appropriate. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines, using a suitable electrophile, such as an alkyl halide, to form a quaternary alkylated amino salt.

[0038] Whole-body distribution or delivery: As used herein, the terms “whole-body distribution” or “whole-body delivery,” or their grammatical synonyms, refer to a delivery or distribution mechanism or approach that affects the whole body or organism. Typically, whole-body distribution or delivery is achieved through the body’s circulatory system, e.g., blood flow. Compare this to the definition of “local distribution or delivery.”

[0039] Subject: As used herein, the term “Subject” means a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient and refers to a human being who visits a healthcare provider for the diagnosis or treatment of a disease. The term “Subject” is used herein interchangeably with “individual” or “patient.” The subject may be susceptible to or prone to a disease or disorder, and may or may not exhibit symptoms of the disease or disorder.

[0040] Substantially: As used herein, the term “substantially” refers to a qualitative state that exhibits all or nearly all range or degree of the desired characteristics or properties. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, complete and / or reach completion, or achieve or avoid absolute results. Therefore, the term “substantially” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0041] Target tissue: As used herein, the term “target tissue” refers to any tissue affected by the disease being treated. In some embodiments, target tissue includes tissue exhibiting a disease-related condition, symptom, or feature.

[0042] Therapeutic dose: As used herein, the term “therapeutic dose” of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to such disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutic dose is typically administered in a dosing regimen containing at least one unit dose.

[0043] Treatment: As used herein, the terms “treatment,” “medication,” or “to treat” mean any method used to partially or completely alleviate, improve, reduce, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be applied to subjects who are not showing signs of the disease, and / or who are showing only early signs of the disease, with the aim of reducing the risk of developing a condition associated with that disease.

[0044] chemical definition Acyl: Where used herein, the term "acyl" means R Z -(C=O)- refers to the equation, and in the formula, R Z For example, any alkyl, alkenyl, alkynyl, heteroalkyl, These are heteroalkylenes.

[0045] Aliphatic: As used herein, the term aliphatic means C 1- C 40 This term refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be linear, branched, or cyclic. For example, C1-C 20 Aliphatic species include C1-C 20 Alkyl (e.g., linear or branched C1-C) 20 Saturated alkyl), C2-C 20 Alkenyls (e.g., linear or branched C4-C) 20 Dienyl, linear or branched C6-C 20 Trienyl, etc., and C2-C 20 Alkynyl (e.g., linear or branched C2-C) 20 (Alkinyl) may be included. C1-C 20 Aliphatic species include C3-C 20 Cyclic aliphatic (e.g., C3-C) 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20It may include (cycloalkynyl). In certain embodiments, the aliphatic may include one or more cycloaliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and optionally may be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. The aliphatic group is unsubstituted or substituted with one or more of the substituents described herein. For example, the aliphatic may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'', wherein each example of R'' is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl, or C1-C3 alkyl). In multiple embodiments, R'' is independently unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 30 Alkyl, or C1-C3 alkyl). In multiple embodiments, R'' is independently unsubstituted C1-C3 alkyl. In multiple embodiments, the aliphatic is unsubstituted. In multiple embodiments, the aliphatic contains no heteroatoms. Alkyl: As used herein, the term "alkyl" means acyclic straight-chain and branched hydrocarbon groups, e.g., "C1-C 30"Alkyl" refers to an alkyl group having 1 to 30 carbons. The alkyl group can be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentylhexyl, isohexyl, etc. The term "lower alkyl" means a linear or branched alkyl of an alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be readily apparent to those skilled in the art in view of the benefits of the present disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents described herein. For example, the alkyl group can be substituted with one or more of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), where each example of R'' is independently C1-C 20 aliphatic (e.g., C1-C 20 alkyl, C1-C 15 alkyl, C1-C 10 alkyl, or C1-C3 alkyl). In multiple embodiments, R'' is independently an unsubstituted alkyl (e.g., unsubstituted C1-C 20 alkyl, C1-C 15 alkyl, C1-C 10 alkyl, or C1-C3 alkyl). In multiple embodiments, R'' is independently an unsubstituted C1-C3 alkyl. In multiple embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein). In multiple embodiments, the alkyl group is substituted with an -OH group and can also be referred to herein as a "hydroxyalkyl" group, where the prefix indicates the -OH group and "alkyl" is as described herein.

[0046] As used herein, "alkyl" also refers to a radical of a linear or branched saturated hydrocarbon group having 1 to 50 carbon atoms ("C1-C 50 alkyl") In some embodiments, the alkyl group has 1 to 40 carbon atoms ("C1-C 40 Alkyl). In some embodiments, the alkyl group has 1 to 30 carbon atoms ("C1-C"). 30 Alkyl). In some embodiments, the alkyl group has 1 to 20 carbon atoms ("C1-C"). 20 Alkyl). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C1-C"). 10 1-C2 alkyl) In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C1-C6 alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C1-C5 alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), and isopropyl (C2). 3)Examples of alkyl groups include n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7) and n-octyl (C8). Unless otherwise specified, each example of alkyl group is independently either unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, alkyl groups are unsubstituted C1-C 50 It is alkyl. In certain embodiments, the alkyl group is a substituted C1-C 50 It is alkyl.

[0047] When the suffix "-ene" is added to a base compound, it becomes the divalent part. For example, arylene is the divalent part of aryl, and heteroarylene is the divalent part of heteroaryl.

[0048] Alkylene: As used herein, the term “alkylene” refers to a saturated divalent linear or branched hydrocarbon group, exemplified by methylene, ethylene, isopropylene, and the like. Similarly, as used herein, the term “alkenylene” refers to an unsaturated divalent linear or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, and the term “alkynylene” as used herein refers to an unsaturated divalent linear or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur at any stable point along the chain. In certain embodiments, the alkylene group, alkenylene group, or alkynylene group may contain one or more cyclic aliphatic and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. For example, alkylene, alkenylene, or alkynylene may be substituted with one or more of the following (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents): halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'', where each example of R'' is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl, or C1-C3 alkyl). In several embodiments, R'' is independently an unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl (or C1-C3 alkyl) In some embodiments, R'' is independently an unsubstituted C1-C3 alkyl group. In certain embodiments, alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, alkylene, alkenylene, or alkynylene does not contain any heteroatoms. Alkenyl: As used herein, “alkenyl” means any linear or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds that can occur at any stable point along the chain, e.g., “C2-C 30 An "alkenyl" refers to an alkenyl group having 2 to 30 carbon atoms. For example, alkenyl groups include prop-2-enyl, buta-2-enyl, buta-3-enyl, 2-methylprop-2-enyl, hexa-2-enyl, hexa-5-enyl, and 2,3-dimethylbuta-2-enyl. In some embodiments, the alkenyl contains one, two, or three carbon-carbon double bonds. In some embodiments, the alkenyl contains a single carbon-carbon double bond. In some embodiments, multiple double bonds (e.g., two or three) are conjugated. The alkenyl group may be unsubstituted or may be substituted with one or more substituents as described herein. For example, the alkenyl group may be substituted with one or more of the following (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents): halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'', where each example of R'' is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl, or C1-C3 alkyl). In several embodiments, R'' is independently an unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10The group is alkyl, or C1-C3 alkyl. In some embodiments, R'' is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkenyl is unsubstituted. In some embodiments, the alkenyl is substituted (for example, with one, two, three, four, five, or six substituents as described herein). In some embodiments, the alkenyl group is substituted with an -OH group, which may also be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates an -OH group and "alkenyl" is as described herein.

[0049] As used herein, “alkenyl” also refers to a radical of a linear or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). 50 This refers to an alkenyl group. In some embodiments, the alkenyl group has 2 to 40 carbon atoms ("C2-C2"). 40 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 30 carbon atoms ("C2-C2"). 30 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 20 carbon atoms ("C2-C2"). 20 ("Alkenyl"). In some embodiments, the alkenyl group has 2 to 10 carbon atoms ("C2-C"). 10In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C2-C9 alkenyl"). In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C2-C7 alkenyl"). In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C2-C5 alkenyl"). In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C2-C4 alkenyl"). In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C2-C3 alkenyl"). In some embodiments, the alkenyl group has 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds may be internal (e.g., 2-butenyl) or terminal (e.g., 1-butenyl). Examples of C2-C4 alkenyl groups include, but are not limited to, ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), and butadienyl (C4). Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups, as well as pentenyl (C5) and pentadienyl. Examples include nyl (C5) and hexenyl (C6). Additional examples of alkenyls include heptenyl (C7), octenyl (C8), and octatrienyl (C8). Unless otherwise specified, each example of an alkenyl group is either unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, the alkenyl group is an unsubstituted C2-C 50 It is an alkenyl. In certain embodiments, the alkenyl group is a substituted C2-C 50 It is Alkenil.

[0050] Alkynyl: As used herein, “alkynyl” means any hydrocarbon chain in either a linear or branched configuration having one or more carbon-carbon triple bonds occurring at any stable point along the chain, for example, “C2-C30 "Alkynyl" refers to an alkynyl group having 2 to 30 carbon atoms. Examples of alkynyl groups include prop-2-inyl, buta-2-inyl, buta-3-inyl, penta-2-inyl, 3-methylpenta-4-inyl, hexa-2-inyl, hexa-5-inyl, etc. In some embodiments, the alkynyl contains one carbon-carbon triple bond. The alkynyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group may be substituted with one or more of halogens, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), where each example of R'' is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl, or C1-C3 alkyl). In several embodiments, R'' is independently an unsubstituted alkyl (e.g., unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 The alkyl (or C1-C3 alkyl) is used. In some embodiments, R'' is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkynyl is unsubstituted. In some embodiments, the alkynyl is substituted (for example, with one, two, three, four, five, or six substituents as described herein).

[0051] As used herein, "alkynyl" also refers to a radical of a linear or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds). 50This refers to an "alkynyl" group. An alkynyl group having one or more triple bonds and one or more double bonds is also called an "en-yne" group. In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("C2-C2"). 40 In some embodiments, the alkynyl group has 2 to 30 carbon atoms ("C2-C"). 30 In some embodiments, the alkynyl group has 2 to 20 carbon atoms ("C2-C"). 20 In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("C2-C"). 10 In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C2-C9 alkynyl"). In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C2-C8 alkynyl"). In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C2-C7 alkynyl"). In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C2-C6 alkynyl"). In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C2-C5 alkynyl"). In some embodiments, the alkynyl group has 2 to 4 carbon atoms ("C2-C4 alkynyl"). In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C2-C3 alkynyl"). In some embodiments, the alkynyl group has 2 carbon atoms ("C2 alkynyl"). One or more carbon-triple bonds may be internal (e.g., 2-butynyl) or terminal (e.g., 1-butynyl). Examples of C2-C4 alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), and 2-butynyl (C4). C2-C6 alkenyl groups Examples include the aforementioned C2-C4 alkynyl groups, as well as pentynyl (C5) and hexynyl (C6). Further examples of alkynyls include heptynyl (C7) and octinyl (C8). Unless otherwise specified, each example of an alkynyl group is either unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, the alkynyl group is an unsubstituted C2-C 50 It is an alkynyl group. In certain embodiments, the alkynyl group is a substituted C2-C 50 It is alkinyl.

[0052] Aryl: As with "aralkyl," the term "aryl," used alone or as part of a larger term, refers to a monocyclic, bicyclic, or tricyclic carbocyclic structure having a total of 6 to 14 ring members, wherein the aforementioned ring structure has a single bond with the rest of the molecule, at least one ring in the system is aromatic, and each ring in the system contains 4 to 7 ring members. In several embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl"; for example, anthracyl. "Aryl" also includes a ring system in which an aryl ring is condensed with one or more carbocyrillic or heterocyclyl groups, as defined above, where the bonding radical or bond site is on the aryl ring, and in such examples the number of carbon atoms still specifies the number of carbon atoms in the aryl ring system. Examples of aryls include phenyl, naphthyl, and anthracene.

[0053] As used herein, “aryl” also refers to a ring of 6 to 14 carbon atoms that provide an aromatic ring system ("C6- 14The term refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having an aryl group and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl"; for example, anthracyl. "Aryl" also includes a ring system in which an aryl ring is condensed with one or more carbocyrillic or heterocyclyl groups, as defined above, where the bonding radical or bond site is on the aryl ring, and in such examples the number of carbon atoms still specifies the number of carbon atoms in the aryl ring system. Unless otherwise specified, each example of an aryl group is independently either unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is an unsubstituted C6-C 14 It is aryl. In certain embodiments, the aryl group is substituted C6-C 14 It is Ariel.

[0054] Arylene: As used herein, the term "arylene" refers to a divalent aryl group (i.e., having two bonding sites with respect to the molecule). Examples of arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene).

[0055] Carbocyclyl: As used herein, "carbocyclyl" or "carbocyclic" refers to a ring of 3 to 10 carbon atoms in a non-aromatic ring system ("C3- 10This refers to a radical of a non-aromatic cyclic hydrocarbon group having 0 heteroatoms ("carbocyclyl"). In some embodiments, the carbocyclyl group has 3 to 8 ring carbon atoms ("C3-C8 carbocyclyl"). In some embodiments, the carbocyclyl group has 3 to 7 ring carbon atoms ("C3-C7 carbocyclyl"). In some embodiments, the carbocyclyl group has 3 to 6 It has a ring carbon atom ("C3-C6 carbocyrill"). In some embodiments, the carbocyrill group has 4 to 6 ring carbon atoms ("C4-C6 carbocyrill"). In some embodiments, the carbocyrill group has 5 to 6 ring carbon atoms ("C5-C6 carbocyrill"). In some embodiments, the carbocyrill group has 5 to 10 ring carbon atoms ("C5-C6 carbocyrill"). 10 The C3-C6 carbocyclyl group has a carbocyclyl group. Examples of C3-C6 carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), and cyclohexadienyl (C6). Examples of C3-C8 carbocyclyl groups include, but are not limited to, the aforementioned C3-C6 carbocyclyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), and bicyclo[2.2.2]octanyl (C8). Simplistic C3-C 10 The carbocyclyl group is not limited to the aforementioned C3-C8 carbocyclyl group, as well as cyclononyl (C9), cyclononenyl (C9), and cyclodecyl (C9). 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C9) 10 ), spiro[4.5]decanil(C 10Examples include the following. As shown in the examples above, in certain embodiments, the carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., including fusions, bridges, or spirocyclic systems such as bicyclic ("bicyclic carbocyclyl") or tricyclic ("tricyclic carbocyclyl")), and may be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes a cyclic system in which the carbocyclyl ring is fused with one or more aryl or heteroaryl groups, as defined above, with the bonding site on the carbocyclyl ring, in which case the carbon number still specifies the carbon number of the carbocyclic system. Unless otherwise specified, each example of a carbocyclyl group is independently either unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is an unsubstituted C 3- C 10 It is a carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C3- C It is 10 carbocyclyl.

[0056] In some embodiments, "carbocyrill" or "carbocyclic" refers to "cycloalkyl," i.e., a monocyclic saturated carbocyclyl group having 3 to 10 ring carbon atoms ("C3- 10 It is called a "cycloalkyl" group. In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, the cycloalkyl group has 4 to 6 ring carbon atoms ("C4-C6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms ("C5-C6 cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). 10The cycloalkyl group has a cycloalkyl element. Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the aforementioned C5-C6 cycloalkyl groups, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each example of a cycloalkyl group is either unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl group is unsubstituted C3-C 10 It is a cycloalkyl group. In certain embodiments, the cycloalkyl group is a substituted C3-C 10 It is a cycloalkyl group.

[0057] Halogen: As used herein, the term "halogen" refers to fluorine, chlorine, odor It means element, or iodine.

[0058] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P, in addition to 1 to 14 carbon atoms. Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyls may optionally contain monocyclic, bicyclic, or tricyclic rings, each ring preferably having 3 to 6 members. Examples of heteroalkyls include polyethers such as methoxymethyl and ethoxyethyl.

[0059] Heteroalkylene: As used herein, the term "heteroalkylene" refers to the divalent form of the heteroalkyl group described herein.

[0060] Heteroaryl: As used herein, the term “heteroaryl” means a fully unsaturated heteroatom-containing ring in which at least one ring atom is a heteroatom such as, but not limited to, nitrogen and oxygen.

[0061] As used herein, “heteroaryl” also refers to a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) radical having a ring carbon atom and one or more ring heteroatoms (e.g., 1, 2, 3, or 4 ring heteroatoms) provided to the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the bond site can be a carbon atom or a nitrogen atom, as the valence allows. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems in which a heteroaryl ring is fused with one or more carbocyryl or heterocyclyl groups, as defined above, where the bond site is on the heteroaryl ring, and in such examples, the number of ring members still specifies the number of ring members in the heteroaryl ring system. "Hyperaryl" also includes a ring system in which a heteroaryl ring is fused with one or more aryl groups, as defined above, and the bond site is on either the aryl ring or the heteroaryl ring, in which case the number of ring members specifies the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. Polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) may have the bond site on either ring, i.e., the ring containing a heteroatom (e.g., 2-indolyl) or the ring not containing a heteroatom (e.g., 5-indolyl).

[0062] In some embodiments, the heteroaryl group is a 5- to 10-membered aromatic ring system having a ring carbon atom provided to the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 10-membered heteroaryl"). In some embodiments, the heteroaryl group is a 5- to 8-membered aromatic ring system having a ring carbon atom provided to the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 8-membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having a ring carbon atom provided to the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl is oxygen, sulfur, nitrogen, boron, silicon, and They have one or more (e.g., 1, 2, or 3) ring heteroatoms selected from phosphorus. In some embodiments, 5-6 membered heteroaryls have one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, 5-6 membered heteroaryls have one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each example of a heteroaryl group is independently either unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

[0063] Exemplary five-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary five-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary five-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary six-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetradinyl, respectively. Exemplary seven-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranil, benzoisofuranil, benzimidazolyl, benzoxazolyl, benzoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolidinyl, and prinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthylidinyl, pteridinyl, quinolinyl, isoquinolinyl, sinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, phenanthridine, dibenzofuranil, carbazolyl, acridinil, phenothiazinil, phenoxadinil, and phenazinil.

[0064] As used herein, “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having a ring carbon atom and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each heteroatom independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“3- to 14-membered heterocyclyl”). In heterocyclyl groups containing one or more nitrogen atoms, the bond sites can be carbon atoms or nitrogen atoms, as long as the valence allows. Heterocyclyl groups can be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., condensed, bridging, or spirocyclic systems, e.g., bicyclic systems (“bicyclic heterocyclyl”) or tricyclic systems (“tricyclic heterocyclyl”)), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes a ring system in which a heterocyclyl ring as defined above is fused with one or more carbocyclyl groups (where the bond site is located on either the carbocyclyl ring or the heterocyclyl ring), or a ring system in which a heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups (where the bond site is located on the heterocyclyl ring), in which case the number of ring members specifies the number of ring members in the heterocyclyl ring system. Continued. Unless otherwise specified, each example of a heterocycline is independently either unsubstituted ("unsubstituted heterocycline") or substituted with one or more substituents ("substituted heterocycline"). In certain embodiments, the heterocycline group is an unsubstituted 3- to 14-membered heterocycline. In certain embodiments, the heterocycline group is a substituted 3- to 14-membered heterocycline.

[0065] In some embodiments, the heterocyclyl group is a 5- to 10-membered non-aromatic ring system having a ring carbon atom and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 10-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5- to 8-membered non-aromatic ring system having a ring carbon atom and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 8-membered heterocyclyl"). In some embodiments, the heterocyclyl group is a 5- to 6-membered non-aromatic ring system having a ring carbon atom and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 6-membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclil has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclil has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclil has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0066] Exemplary three-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azildinyl, oxyranil, and thiorenyl. Exemplary four-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanil, and thietanil. Exemplary five-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranil, dihydrofuranil, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary five-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanil, oxathiolanil, and dithiolanil. Exemplary five-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary six-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranil, dihydropyridinyl, and thianyl. Exemplary six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinil, dithianyl, and dioxanil. Exemplary six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinyl. Exemplary seven-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary eight-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azokanyl, oxekanyl, and thiokanyl.Examples of bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydroclomenyl, octahydroisoclomenyl, decahydronaphthilidinyl, decahydro-1,8-naphthilidinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthaliumidyl, naphthaliumidyl, chromanyl, clomenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, and 5,6-dihy. This includes, but is not limited to, dro-4H-fl[3,2-b]pyrrolyl, 6,7-dihydro-5H-fl[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofl[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofl[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthilidinyl, etc.

[0067] Heterocycloalkyl: As used herein, the term “heterocycloalkyl” means a non-aromatic ring in which at least one atom is a heteroatom, such as but not limited to nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocycloalkyl groups may be substituted or unsubstituted.

[0068] As can be understood from the above, alkyl groups, alkenyl groups, alkynyl groups, acyl groups, carbosicryl groups, heterocyclyl groups, aryl groups, and heteroaryl groups are optionally substituted in certain embodiments. Optionally substituted means a group that may be substituted or unsubstituted (for example, a "substituted" or "unsubstituted" alkyl group, a "substituted" or "unsubstituted" alkenyl group, a "substituted" or "unsubstituted" alkynyl group, a "substituted" or "unsubstituted" heteroalkyl group, a "substituted" or "unsubstituted" heteroalkenyl group, a "substituted" or "unsubstituted" heteroalkynyl group, a "substituted" or "unsubstituted" carbosicryl group, a "substituted" or "unsubstituted" heterocyclyl group, a "substituted" or "unsubstituted" aryl group, or a "substituted" or "unsubstituted" heteroaryl group). Generally, the term “substituted” means that at least one hydrogen atom present on a group is substituted with an acceptable substituent, for example, a substituent that, upon substitution, results in a stable compound, such as a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reactions. Unless otherwise indicated, a “substituted” group has substituents at one or more substituteable positions of the group, and if multiple positions in any given structure are substituted, the substituents are either identical or different at each position. The term “substituted” is intended to include substitution with all acceptable substituents of an organic compound, any substituent described herein that results in the formation of a stable compound. The present invention intends for all such combinations to arrive at a stable compound. For the purposes of the present invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0069] Exemplary carbon atom substituents include, but are not limited to, halogens, -CN, -NO2, -N3, -SO2, -SO3H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3+X-, -N(ORcc)Rbb, -SeH, -SeRaa, -SH, -SRaa, -SSRcc, -C(=O)Raa, -CO2H, -CHO, -C(ORcc)2, -CO2Raa, -OC(=O)Raa, -OCO2Raa, -C(=O)N(Rbb)2, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NRbbCO2Raa, -NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)ORaa, -OC(=NRbb)Raa, -OC(=NRb b)ORaa, -C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb)2, -NRbbC(=NRbb)N(Rbb)2, -C(=O)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -SO2ORaa, -OSO2Raa, -S(=O)Raa, -OS(=O)Raa, -Si(Raa)3 -OSi(Raa)3-C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, -SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRa a, -SC(=O)ORaa, -SC(=O)Raa, -P(=O)2Raa, -OP(=O)2Raa, -P(=O)(Raa)2, -OP(=O)(Raa)2, -OP(= O)(ORcc)2, -P(=O)2N(Rbb)2, -OP(=O)2N(Rbb)2, -P(=O)(NRbb)2, -OP(=O)(NRbb)2, -NRbbP(=O)(ORcc)2 , -NRbbP(=O)(NRbb)2, -P(Rcc)2, -P(Rcc)3, -OP(Rcc)2, -OP(Rcc)3, -B(Raa)2, -B(ORcc)2, -BRaa(ORcc) Examples include C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C14 carbocyrill, 3-14 member heterocyclyl, C6-C14 aryl, and 5-14 member heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5Rdd groups;

[0070] Alternatively, two geminal hydrogens on a carbon atom may be substituted with =O, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb, or =NORcc groups;

[0071] Each example of Raa is independently selected from C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, 3-14 member heterocyclyl, C6-C14 aryl, and 5-14 member heteroaryl, or two Raa groups together form a 3-14 member heterocyclyl or 5-14 member heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5Rdd groups;

[0072] Each example of Rbb is independently hydrogen, -OH, -ORaa, -N(Rcc)2, -CN, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRcc)ORaa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc )2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, -C(=S)SRcc, -P(=O)2Raa, -P(=O)(Raa)2, -P(=O)2N(Rcc)2, -P(=O)(NRcc)2, C1- Selected from C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyryl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rbb groups together with the heteroatom to which they are bonded to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5Rdd groups;

[0073] Each example of Rcc is independently selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, 3-14 member heterocyclyl, C6-C14 aryl, and 5-14 member heteroaryl, or two Rcc groups together with the heteroatom they are bonded to form a 3-14 member heterocyclyl or 5-14 member heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5Rdd groups;

[0074] Each example of Rdd is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORee, -ON(Rff)2, -N(Rff)2, -N(Rff)3+X-, -N(ORee)Rff, -SH, -SRee, - SSRee, -C(=O)Ree, -CO2H, -CO2Ree, -OC(=O)Ree, -OCO2Ree, -C(=O)N(Rff)2, -OC(=O)N(Rff)2, -NRffC(=O)Ree, -NRffCO2Ree, -NRffC(=O)N(Rff)2, -C(=NRff)ORee, -OC(=NRff)Ree, -OC(=NRff)ORee, -C(=NRff)N(Rff)2, -OC(=NRff)N(Rff)2, -NRffC(=NR ff)N(Rff)2, -NRffSO2Ree, -SO2N(Rff)2, -SO2Ree, -SO2ORee, -OSO2Ree, -S(=O)Ree, -Si(Ree)3, -OSi(Re e)3, -C(=S)N(Rff)2, -C(=O)SRee, -C(=S)SRee, -SC(=S)SRee, -P(=O)2Ree, -P(=O)(Ree)2, -OP(=O)(Ree)2 Selected from -OP(=O)(ORee)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, 3-10 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl, each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl can independently be substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or two geminal Rdd substituents can combine to form =O or =S;

[0075] Each example of Ree is independently selected from C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, C6-C10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, and each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;

[0076] Each example of Rff is independently selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, 3-10 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl, or two Rff groups together with the heteroatom they are bonded to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyrill, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and

[0077] Each example of Rgg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C50alkyl, -ON(C1-C50alkyl)2, -N(C1-C50alkyl)2, -N(C1-C50alkyl)3+X-, -NH(C1-C50alkyl)2+X-, -NH2(C1-C50alkyl)+X-, -NH3+X-, -N(OC1-C50alkyl)(C1-C50alkyl), -N(OH)(C1-C50alkyl), -NH(OH), -SH, -SC1-C50alkyl, -SS(C1- C50alkyl), -C(=O)(C1-C50alkyl), -CO2H, -CO2(C1-C50alkyl), -OC(=O)(C1-C50alkyl), -OCO2(C1-C50alkyl), -C(=O)NH2, -C(=O)N(C1-C50alkyl)2, -OC(=O)NH(C1-C50alkyl), -NHC(=O)(C1-C50alkyl), -N(C1-C50alkyl)C(=O)(C1-C50alkyl), -NHCO2(C1-C50alkyl), -NHC(=O)N(C1-C50alkyl)2, -NH C(=O)NH(C1-C50alkyl), -NHC(=O)NH2, -C(=NH)O(C1-C50alkyl), -OC(=NH)(C1-C50alkyl), -OC(=NH)OC1-C50alkyl, -C(=NH)N(C1-C50alkyl)2, -C(=NH)NH(C1-C50alkyl), -C(=NH)NH2, -OC(=NH)N(C1-C50alkyl)2, -OC(NH)NH(C1-C50alkyl), -OC(NH)NH2, -NHC(NH)N(C1-C50alkyl)2, -NHC(=NH)NH2 -NHSO2(C1-C50alkyl), -SO2N(C1-C50alkyl)2, -SO2NH(C1-C50alkyl), -SO2NH2, -SO2(C1-C50alkyl), -SO2O(C1-C50alkyl), -OSO2(C1-C6alkyl), -SO(C1-C6alkyl), -Si(C1-C50alkyl)3, -OSi(C1-C6alkyl)3, -C(=S)N(C1-C50alkyl)2, C(=S)NH(C1-C50alkyl), C(=S)NH2, -C(=O)S(C1-C6alkyl), -C(= Selected from S)S(C1-C6 alkyl), -SC(=S)S(C1-C6 alkyl), -P(=O)2(C1-C50 alkyl), -P(=O)(C1-C50 alkyl)2, -OP(=O)(C1-C50 alkyl)2, -OP(=O)(OC1-C50 alkyl)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyrill, C6-C10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents can come together to form =O or =S; X- is the counterion.

[0078] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iod, -I).

[0079] As used herein, “counterion” is a negatively charged group bonded to a positively charged quaternary amine in order to maintain electronic neutrality. Exemplary counterions include halides (e.g., F-, Cl-, Br-, I-), NO3-, ClO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.) and carboxylate ions (e.g., acetate, ethaneate, propaneate, benzoate, glycerolate, lactate, tartrate, glycolate, etc.).

[0080] Nitrogen atoms can be substituted or unsubstituted, to the extent their valence allows, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -ORaa, -N(Rcc)2, -CN, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)ORaa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, -C(=S)SRcc, -P(=O)2Raa, -P(=O)(Raa)2, and -P(=O)2N(Rcc) Examples include )2, -P(=O)(NRcc)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyryl, 3-14 member heterocyclyl, C6-C14 aryl, and 5-14 member heteroaryl, or two Rcc groups together with the N atom to which they are bonded to form a 3-14 member heterocyclyl or 5-14 member heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups, where Raa, Rbb, Rcc, and Rdd are as defined above.

[0081] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also called an amino protecting group). Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGMWuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0082] For example, nitrogen protecting groups such as amide groups (e.g., -C(=O)Raa) are not limited to, but include formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetamide Examples include cetoamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazofenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0083] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)ORaa) are not limited to these, but include methyl carbamate, ethyl carbamate (carbamante), 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluorenyl methyl carbamate, and 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl]]methyl carbamate (D BD-Tmoc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (hZ), 1-(1-adamantyl (adamanty1))-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl -2,2,2-Trichloroethylcarbamate (TCBOC), 1-Methyl-1-(4-biphenylyl)ethylcarbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'-and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamide)ethylcarbamate, t-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), Allylcarbamate (Alloc), 1-Isopropylallylcarbamate (Ipaoc), Cinnamylcarbamate (Coc), 4-Nitrocinnamylcarbamate (Noc), 8-Quinolylcarbamate, N-Hydroxypiperidinylcarbamate, Alkyldithiocarbamate, Benzylcarbamate (Cbz), p-Methoxybenzylcarbamate (Moz), p-Nitobenzylcarbamate, p-Bromobenzylcarbamate, p-Chlorobenzylcarbamate, 2,4-Dichlorobenzylcarbamate, 4-Methylsulfinylbenzylcarbamate (Msz), 9-Anthrylmethylcarbamate, Diphenylmethylcarbamate, 2-Methylthioethylcarbamate, 2-Methylsulfonylethylcarbamate, 2-(p-Toluenesulfonyl)ethylcarbamate, [2-(1,3-Dithianyl)]methylcarbamate (Dmoc), 4-Methylthiophenylcarbamate (Mtpc), 2,4-Dimethylthiophenyl Rubamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenyl Carbamates, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate Rubamate, 2,2-dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamide)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornyl carbamate, isobutylcarbamate, isonicotin, Examples include ylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-l-cyclopropylmethylcarbamate, 1-methyl-1(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-l-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, and 2,4,6-trimethylbenzylcarbamate.

[0084] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)2Raa) are not limited to p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6 Examples include trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0085] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, and N-1,1,4,4-tetramethyldi Silyl azacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexa-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline (pyrooli n)-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimeth Luthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivatives, N-diphenylboric acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphine amide (Dpp, Examples include dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

[0086] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also called a hydroxyl protecting group). Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGMWuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0087] Examples of oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, and 2-methoxyethoxymethyl. 2-(2-chloroethoxy)methyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-oxide, 1-[(2-chloro-4-methyl)phenyl [L]-4-methoxypiperidine-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloro Roethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-Dibenzosberyl, Triphenylmethyl, α-Naphthyldiphenylmethyl, p-Methoxyphenyldiphenylmethyl, Di(p-Methoxyphenyl)phenylmethyl, Tri(p-Methoxyphenyl)methyl, 4-(4'-Bromophenacyloxyphenyl)diphenylmethyl, 4,4',4”-Tris(4,5-Dichlorophthalimidophenyl)methyl, 4,4',4”-Tris(Lebrinoyloxyphenyl)methyl, 4,4',4”-Tris(Benzoyloxyphenyl)methyl, 3-(Imidazole-1-yl)bis(4',4”-Dimethoxyphenyl)methyl, 1,1-Bis(4- Methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, Diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), piva Roate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkylmethyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkylethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenyl ethyl) Sulfonioethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthotyl - Bonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,Examples include 1-dimethylpropyl)phenoxyacetate, chlorodiphenyl acetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamide, alkyl N-phenylcarbamate, borate, dimethylphosphinoticoyl, alkyl 2,4-dinitrophenyl sulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0088] In certain embodiments, substituents present on the sulfur atom are sulfur protecting groups (also known as thiol protecting groups). Sulfur protecting groups are well known in the art and include those described in detail in *Protecting Groups in Organic Synthesis*, TW Greene and PGMWuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0089] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxide, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosberyl, triphenylmethyl, diphenyl Nyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidinomethyl, acetamidemethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbonylaminomethyl, phenylacetamidemethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-thia Examples include noethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carboethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylprop-2-yl, acetyl, benzoyl, trifluoroacetyl, N-[[(p-biphenylyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonates, sulfenylthiocarbonates, 3-nitro-2-pyridinesulfenylsulfide, and oxathiolone.

[0090] Compound of the present invention Liposome-based vehicles are considered attractive carriers for therapeutics and remain the subject of ongoing development efforts. While liposome-based vehicles containing cationic components have shown promising results in terms of encapsulation, stability, and site localization, there remains a strong need for improvements in liposome-based delivery systems. For example, significant shortcomings of liposome delivery systems relate to the construction of liposomes with sufficient cell culture or in vivo stability to reach desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release their encapsulated materials into such target cells.

[0091] Specifically, there remains a need for improved lipid compounds that exhibit enhanced pharmacokinetic properties and can deliver macromolecules such as nucleic acids with high efficiency to a wide variety of cell types and tissues. Importantly, there also remains a specific need for novel lipid compounds characterized by reduced toxicity and capable of efficiently delivering encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0092] This specification describes novel cationic lipid compounds aimed at improving the in vivo delivery of therapeutic agents, such as nucleic acids. In particular, the cationic lipids described herein may be used in combination with other lipids to prepare lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents, such as nucleic acids for therapeutic use (e.g., DNA, siRNA, mRNA, microRNA).

[0093] In some embodiments, the compounds of the present invention described herein may provide one or more desired features or properties. That is, in certain embodiments, the compounds of the present invention described herein may be characterized as having one or more properties that provide advantages of such compounds compared to other similarly classified lipids. For example, the compounds disclosed herein may enable control and modification of the properties of liposome compositions (e.g., lipid nanoparticles) in which they are components. Specifically, the compounds disclosed herein may be characterized by enhanced transfection efficiency and their ability to produce specific biological outcomes. Such outcomes may include, for example, enhanced cellular uptake, endosomal / lysosome disruption ability, and / or enhanced release of encapsulated material (e.g., polynucleotides) within cells. Furthermore, the compounds described herein may have advantageous pharmacokinetic performance, biodistribution, and efficacy (e.g., due to the various dissociation rates of the polymer groups used).

[0094] This application demonstrates that the cationic lipids of the present invention are not only synthetically transportable from readily available starting materials, but also possess unexpectedly high encapsulation efficiency.

[0095] Furthermore, the cationic lipids of the present invention have cleavable groups such as ester groups and disulfides. These cleavable groups (e.g., esters and disulfides) are intended to improve biodegradability and thus contribute to their favorable toxicity profile. .

[0096] The compound of the present invention This specification provides for compounds that are cationic lipids. For example, the cationic lipid of the present invention is a compound having a structure according to the following formula (I), [ka] In the formula, L1 is a bond, (C1-C6) alkyl or (C2-C6) alkenyl, In the formula, X is either O or S. In the formula, R 1 , R 2 , R 3 , R 4 and R 5 Each is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) alkoxy, and -OC(O)R', In the formula, R 1 , R 2 , R 3 , R 4 or R 5 At least one of them is -OC(O)R', In the formula, R' is as follows: [ka] In the formula, R 6 The following is: [ka] In the formula, m and p are independently 0, 1, 2, 3, 4, or 5. In the formula, R 7 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A Selected from, In the formula, R 8 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B Selected from, In the formula, R 9is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C Selected from, In the formula, R 10 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D Selected from, In the formula, k, n, q, and r are each independently 1, 2, 3, 4, or 5. Or in the formula, (i)R 7 and R 8 , or (ii)R 9 and R 10 Together, they form optionally substituted 5-membered or 6-membered heterocycloalkyl or heteroaryl groups, each containing 1 to 3 heteroatoms selected from N, O, and S. In the formula, R 11 , R 12 , R 13 , and R 14 Each is independently selected from H, methyl, ethyl, or propyl. In the formula, R A , R B , R C and R D Each of these can be substituted independently and arbitrarily (C6-C 20 )alkyl, optionally substituted (C6-C 20 ) Alkenyl, optionally substituted (C6-C 20 )Alkinyl, optionally substituted (C6-C 20Selected from acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, and -NH2. In the formula, R 7 , R 8 , R 9 , R 10 At least one of them is R A , R B , R C or R D Including the portion, R A , R B , R C or R D These are independently and optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 ) Alkenyl, optionally substituted (C6-C 20 )Alkinyl, optionally substituted (C6-C 20 ) Acyl, optionally substituted -OC(O)(C6-C 20 )alkyl or optionally substituted -OC(O)(C6-C 20 ) Compounds selected from alkenyls, or a pharmaceutically acceptable salt thereof.

[0097] In several embodiments, any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)acyl, (C1-C6)alkoxy, halogen, -COR, -CO2H, -CO2R, -CN, -OH, -OR, -OCOR, -OCO2R, -NH2, -NHR, -N(R)2, -SR, or -SO2R, or two geminal hydrogens on a carbon atom are substituted with the group =NH, and each example of R is independently a C1-C10 aliphatic alkyl.

[0098] In some embodiments, L1 is a coupling.

[0099] In several embodiments, L1 is a (C1-C6) alkyl group.

[0100] In several embodiments, L1 is a (C2-C6) alkenyl.

[0101] In several embodiments, L1 is a C2 alkenyl.

[0102] In some embodiments, RA and RB are the same. In some embodiments, RC and RD are the same. In some embodiments, RA and RB are the same, and RC and RD are the same.

[0103] In multiple embodiments, RA and RB are different. In multiple embodiments, RC and RD are different. In multiple embodiments, RA and RB are different, and RC and RD are different.

[0104] In some embodiments, RA, RB, RC, and RD are identical.

[0105] In multiple embodiments, RA, RB, RC, and RD are different.

[0106] In some embodiments, RA, RB, RC, or RD are each independently selected from optionally substituted (C6-C20) alkyl, optionally substituted (C6-C20) alkenyl, optionally substituted (C6-C20) alkynyl, optionally substituted (C6-C20) acyl, optionally substituted -OC(O)(C6-C20) alkyl, or optionally substituted -OC(O)(C6-C20) alkenyl.

[0107] In some embodiments, RA, RB, RC, or RD are identical and selected from optionally substituted (C6-C20) alkyl, optionally substituted (C6-C20) alkenyl, optionally substituted (C6-C20) alkynyl, optionally substituted (C6-C20) acyl, optionally substituted -OC(O)(C6-C20) alkyl, or optionally substituted -OC(O)(C6-C20) alkenyl.

[0108] In some embodiments, RA and RB are independently selected from optionally substituted (C6-C20) alkyl, optionally substituted (C6-C20) alkenyl, and optionally substituted (C6-C20) alkynyl.

[0109] In some embodiments, RA and RB are identical and are selected from optionally substituted (C6-C20) alkyl, optionally substituted (C6-C20) alkenyl, or optionally substituted (C6-C20) alkynyl.

[0110] In some embodiments, RA and RB are each independently an optionally substituted (C6-C20) alkyl group.

[0111] In several embodiments, RA and RB are identical and optionally substituted (C6-C20) alkyl groups.

[0112] In some embodiments, RA and RB are each independently an optionally substituted (C6-C20) alkenyl.

[0113] In several embodiments, RA and RB are identical and optionally substituted (C6-C20) alkenyls.

[0114] In some embodiments, RA and RB are each independently an optionally substituted (C6-C20) alkynyl.

[0115] In several embodiments, RA and RB are identical and optionally substituted (C6-C20) alkynyls.

[0116] In some embodiments, RA and RB are each independently an optionally substituted (C6-C20) acyl.

[0117] In several embodiments, RA and RB are identical and optionally substituted (C6-C20) acyls.

[0118] In several embodiments, RA and RB are each independently an optionally substituted -OC(O)(C6-C20) alkyl group.

[0119] In several embodiments, RA and RB are identical and optionally substituted -OC(O)(C6-C20)alkyl groups.

[0120] In several embodiments, RA and RB are each independently an optionally substituted -OC(O)(C6-C20) alkenyl.

[0121] In several embodiments, RA and RB are identical and optionally substituted -OC(O)(C6-C20) alkenyls.

[0122] In several embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are independently selected from the following: [ka]

[0123] In several embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, RA and RB are the same and selected from the following: [ka]

[0124] In several embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both RA and RB are C8H17.

[0125] In several embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both RA and RB are C10H21.

[0126] In several embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both RA and RB are C12H25.

[0127] In multiple embodiments, R7 = -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both RA and RB are [ka] That is the case.

[0128] In some embodiments, X is O.

[0129] In some embodiments, X is S.

[0130] In multiple embodiments, only one of R1, R2, R3, R4, and R5 is -OC(O)R'. In multiple embodiments, only one of R1, R2, R3, R4, and R5 is -OC(O)R', and none of R1, R2, R3, R4, or R5 are OH.

[0131] In multiple embodiments, two of R1, R2, R3, R4, and R5 are -OC(O)R'. In multiple embodiments, two of R1, R2, R3, R4, and R5 are -OC(O)R', and none of R1, R2, R3, R4, or R5 are OH.

[0132] In some embodiments, three of R1, R2, R3, R4, and R5 are -OC(O)R'.

[0133] In some embodiments, R1 is -OC(O)R'. In some embodiments, R5 is -OC(O)R'. In some embodiments, both R1 and R5 are -OC(O)R'.

[0134] In some embodiments, R2 is -OC(O)R'. In some embodiments, R4 is -OC(O)R'. In some embodiments, both R2 and R4 are -OC(O)R'.

[0135] In several embodiments, R3 is -OC(O)R'.

[0136] In several embodiments, R3 is -OC(O)R' and R2 is OMe.

[0137] In several embodiments, L1 is a bond, R3 is -OC(O)R', and R2 is OMe.

[0138] In some embodiments, R3 is -OC(O)R', and R2 and R4 are OMe. .

[0139] In some embodiments, L1 is a bond, R3 is -OC(O)R', and R2 and R4 are OMe.

[0140] In several embodiments, L1 is a (C2-C6) alkenyl, R3 is -OC(O)R', and R2 and R4 are OMe.

[0141] In several embodiments, L1 is a C2 alkenyl, R3 is -OC(O)R', and R2 and R4 are OMe.

[0142] In several embodiments, R7 is -(CH2)kCH(OR11)RA.

[0143] In several embodiments, R7 is -(CH2)1CH(OR11)RA.

[0144] In several embodiments, R7 is -(CH2)1CH(OH)RA.

[0145] In several embodiments, R8 is -(CH2)nCH(OR12)RB.

[0146] In several embodiments, R8 is -(CH2)1CH(OR12)RB.

[0147] In several embodiments, R8 is -(CH2)1CH(OH)RB.

[0148] In several embodiments, R7 is -(CH2)kCH(OR11)RA and R8 is -(CH2)nCH(OR12)RB.

[0149] In several embodiments, R7 is -(CH2)1CH(OR11)RA and R8 is -(CH2)1CH(OR12)RB.

[0150] In some embodiments, R7 is -(CH2)1CH(OH)RA and R8 is -(CH2)1CH(OH)RB.

[0151] In several embodiments, R7 and R8 are each optionally substituted (C1-C6) alkyl groups, for example, -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 40 It is alkyl. In some embodiments, R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula.aa is C1-C 30 It is alkyl. In some embodiments, R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 20 It is alkyl.

[0152] In several embodiments, R7 and R8 are identical, each being an optionally substituted (C1-C6) alkyl, for example, a (C1-C6) alkyl substituted with -CO2Raa, where Raa is a C1-C50 alkyl. In several embodiments, R7 and R8 are identical, each being a (C1-C6) alkyl substituted with -CO2Raa, where Raa is a C1-C40 alkyl. In several embodiments, R7 and R8 are identical, each being a (C1-C6) alkyl substituted with -CO2Raa, where Raa is a C1-C30 alkyl. In several embodiments, R7 and R8 are each a (C1-C6) alkyl substituted with -CO2Raa, where Raa is a C1-C20 alkyl.

[0153] In some embodiments, R7 and R8 are respectively [ka] That is the case.

[0154] In some embodiments, R7 and R8 are respectively [ka] That is the case.

[0155] In some embodiments, R9 and R10 are independently selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, and optionally substituted (C2-C6) alkynyl.

[0156] In some embodiments, R9 and R10 are independently an optionally substituted (C1-C6) alkyl or an optionally substituted (C2-C6) alkenyl.

[0157] In several embodiments, both R9 and R10 are optionally substituted (C1-C6) alkyl or optionally substituted (C2-C6) alkenyl.

[0158] In several embodiments, both R9 and R10 are optionally substituted (C1-C6) alkyl groups.

[0159] In some embodiments, both R9 and R10 are -CH3.

[0160] In several embodiments, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both R9 and R10 are -CH3.

[0161] In some embodiments, R7 is -(CH2)1CH(OR11)RA, R8 is -(CH2)1CH(OR12)RB, and both R9 and R10 are -CH3.

[0162] In some embodiments, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, and both R9 and R10 are -CH3.

[0163] In several embodiments, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, both RA and RB are C8H17, and both R9 and R10 are -CH3.

[0164] In some embodiments, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, both RA and RB are C8H17, and both R9 and R10 are -CH3.

[0165] In multiple embodiments, R7 is -(CH2)kCH(OR11)RA, and R8 is -(CH2 )nCH(OR12)RB, where both RA and RB are C10H21, and both R9 and R10 are -CH3.

[0166] In some embodiments, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, both RA and RB are C10H21, and both R9 and R10 are -CH3.

[0167] In several embodiments, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, both RA and RB are C12H25, and both R9 and R10 are -CH3.

[0168] In some embodiments, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, both RA and RB are C12H25, and both R9 and R10 are -CH3.

[0169] In several embodiments, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, both RA and RB are C16H29, and both R9 and R10 are -CH3.

[0170] In some embodiments, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, both RA and RB are C16H29, and both R9 and R10 are -CH3.

[0171] In multiple embodiments, p, q, and r are the same. In multiple embodiments, one or more of p, q, and r are different. In multiple embodiments, q and r are the same, and p is different. In multiple embodiments, p and q are the same, and r is different. In multiple embodiments, p and r are the same, and q is different. In multiple embodiments, p, q, and r are different.

[0172] In multiple embodiments, k, m, and n are the same. In multiple embodiments, one or more of k, m, and n are different. In multiple embodiments, k and m are the same, and n is different. In multiple embodiments, m and n are the same, and k is different. In multiple embodiments, k and n are the same, and m is different. In multiple embodiments, k, m, and n are different.

[0173] In multiple embodiments, m is 1, 2, 3, 4, or 5. In multiple embodiments, m is 0. In multiple embodiments, m is 1. In multiple embodiments, m is 2. In multiple embodiments, m is 3. In multiple embodiments, m is 4. In multiple embodiments, m is 5. In multiple embodiments, m is 0, 1, 2, 3, or 4.

[0174] In multiple embodiments, p is 1, 2, 3, 4, or 5. In multiple embodiments, p is 0. In multiple embodiments, p is 1. In multiple embodiments, p is 2. In multiple embodiments, p is 3. In multiple embodiments, p is 4. In multiple embodiments, p is 5. In multiple embodiments, p is 0, 1, 2, 3, or 4.

[0175] In some embodiments, m is 2 and p is 2.

[0176] In several embodiments, m is 3 and p is 2.

[0177] In some embodiments, k and n are 1, and m is 2.

[0178] In some embodiments, k and n are 1, and m is 3.

[0179] In several embodiments, q and r = 1, and p = 2.

[0180] In some embodiments, k, n, q, and r are = 1, m = 2 or 3, and p = 2, respectively.

[0181] In some embodiments, R' is as follows: [ka]

[0182] In some embodiments, R' is [ka] k and n=1, and m=2 or 3.

[0183] In some embodiments, R' is [ka] k and n=1, m=2.

[0184] In some embodiments, R' is [ka] k and n=1, m=3.

[0185] In some embodiments, R' is [ka] And R 11 and R 12 H is H.

[0186] In some embodiments, R' is [ka] And k and n=1, m=2 or 3, R 11 and R 12 H is H.

[0187] In some embodiments, R' is [ka] And k and n=1, m=2, R11 and R 12 H is H.

[0188]

[0189] In some embodiments, R' is [ka] And k and n=1, m=3, R 11 and R 12 H is H.

[0190] In some embodiments, R' is as follows: [ka]

[0191] In any of the above embodiments, if R' has the following structure [ka] R A and R B This may be defined as in any of paragraphs

[0104] to

[0129] .

[0192] In multiple embodiments, R 6 The following applies: [ka]

[0193] In multiple embodiments, R 6 teeth [ka] Therefore, q and r = 1, and p = 2.

[0194] In multiple embodiments, R 6 teeth [ka] And R 13 and R 14 H is H.

[0195] In multiple embodiments, R 6 teeth [ka] And q and r = 1, p = 2, R 13 and R 14 H is H.

[0196] In multiple embodiments, R 6 The group is selected from the following: [ka]

[0197] In multiple embodiments, R 6 The group is selected from the following: [ka]

[0198] In multiple embodiments, R 6 The group is selected from the following: [ka]

[0199] In multiple embodiments, R 6 The following applies: [ka]

[0200] In multiple embodiments, R 6 The following applies: [ka]

[0201] In multiple embodiments, R 6 The following applies: [ka]

[0202] In multiple embodiments, R 6 The group is selected from the following: [ka]

[0203] In multiple embodiments, R 6 The following applies: [ka]

[0204] In multiple embodiments, R 6 The following applies: [ka]

[0205] In multiple embodiments, R 6 The following applies: [ka]

[0206] In multiple embodiments, R 6 The following applies: [ka]

[0207] In multiple embodiments, R 6 The following applies: [ka]

[0208] In multiple embodiments, R 6and R ’ They are the same.

[0209] In multiple embodiments, R 6 teeth [ka] And R ’ teeth [ka] Therefore, m is 2 and p is 2.

[0210] In multiple embodiments, R 6 teeth [ka] And R ’ teeth [ka] Therefore, m is 3 and p is 2.

[0211] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0212] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] That is the case.

[0213] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0214] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0215] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0216] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0217] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0218] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0219] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 1, R 4 , and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0220] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0221] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0222] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 1 , R 4 , and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0223] In some embodiments, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0224] In some embodiments, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0225] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0226] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0227] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0228] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] That is the case.

[0229] In multiple embodiments, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl.

[0230] In multiple embodiments, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa C1-C 50 It is alkyl, m is 2, and p is 2. In some embodiments, R 7 and R 8 They are the same. In some embodiments, L1 is coupled And R 3 is -OC(O)R', and R 2 and R 4 It is OMe.

[0231] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 and R4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same.

[0232] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0233] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same. In some embodiments, m is 2.

[0234] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same. In some embodiments, m is 2.

[0235] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0236] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0237] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 Oh biR 8 They are the same. In some embodiments, m is 2.

[0238] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same. In some embodiments, m is 2.

[0239] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 1 , R 4 , and R 5 H is R 6 teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same. In some embodiments, m is 2.

[0240] In some embodiments, L1 is a coupling, and R 3 is -OC(O)R', and R 2 is OMe, R 6 teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0241] In multiple embodiments, X=O, L1 is a bond, and R 3 is -OC(O)R', and R 2 is OMe, R 1 , R 4 and R 5 H is R 6 teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R7 and R 8 They are the same.

[0242] In some embodiments, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa (C1-C6) alky replaced by It is R, and in the formula, aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same.

[0243] In some embodiments, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0244] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same.

[0245] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl. In some embodiments, R 7 and R 8 They are the same.

[0246] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0247] In some embodiments, X=O, L1 is a C2 alkenyl, and R 3 is -OC(O)R', and R 2 and R 4 is OMe, R 1 and R 5 H is R 6 teeth [ka] And R ’ teeth [ka] And R 7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, and m is 2. In some embodiments, R 7 and R 8 They are the same.

[0248] In any of the above embodiments, R 7 and R 8 -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, R aa Instead, C1-C 40 Alkyl is also acceptable.

[0249] In any of the above embodiments, R 7 and R 8 -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, R aa Instead, C1-C 30 Alkyl is also acceptable.

[0250] In any of the above embodiments, R 7 and R 8 -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, R aa Instead, C1-C 20 Alkyl is also acceptable.

[0251] In any of the above embodiments, R 7 and R 8 -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, R 7 and R 8 Each [ka] That's fine.

[0252] In any of the above embodiments, R7 and R 8 These are respectively -CO2R aa A (C1-C6) alkyl group substituted with R in the formula. aa is C1-C 50 It is alkyl, R 7 and R 8 Each [ka] That's fine.

[0253] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (II), [ka] or comprising a pharmaceutically acceptable salt thereof, where R 1 ~R 6 And X are as already defined herein.

[0254] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIA), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’ , R 6 And X are as already defined herein.

[0255] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formulas (IIB), (IIC), (IID), (IIE), (IIJ), or (IIK). [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0256] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0257] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0258] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0259] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0260] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIF), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’ , R 6 And X are as already defined herein.

[0261] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIG), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’, R 6 And X are as already defined herein.

[0262] In several embodiments, the cationic lipid of the present invention includes a compound having a structure according to the following formula (IIH): [ka] In the formula, one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are independently -OC(O)R', and in the formula, R ’ , R 6 And X is as already defined herein.

[0263] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (III), [ka] or comprising a pharmaceutically acceptable salt thereof, where R 1 ~R 6 And X are as already defined herein.

[0264] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIIA), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’ , R 6 And X are as already defined herein.

[0265] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIIB), [ka] or comprising a pharmaceutically acceptable salt thereof, where R A , R Band p are as already defined herein.

[0266] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or having a pharmaceutically acceptable salt thereof, in the formula R A and R B This is as already defined herein.

[0267] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or having a pharmaceutically acceptable salt thereof, in the formula R A and R B This is as already defined herein.

[0268] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0269] In several embodiments, the cationic lipid of the present invention has the following structure: [ka] or a pharmaceutically acceptable salt thereof.

[0270] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIID), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’ , R 6 And X are as already defined herein.

[0271] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formulas (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ), or (IIIK). [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0272] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0273] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0274] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0275] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0276] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0277] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0278] In several embodiments, the cationic lipid of the present invention has the following structure. [ka]

[0279] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IIIL), [ka] or comprising a pharmaceutically acceptable salt thereof, where R ’ , R 6 And X are as already defined herein.

[0280] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formula (IV): [ka] In the formula, M is a compound selected from H, OH, OMe, or Me. or comprising a pharmaceutically acceptable salt thereof, where R A , R B m and p are as already defined herein.

[0281] In several embodiments, the cationic lipid of the present invention is a compound having a structure according to the following formulas (VI), (VII), (VIII), (IX), or (X), [ka] or a pharmaceutically acceptable salt thereof, In the formula, one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are independently -OC(O)R', and in the formula, R ’ , R 6 And X is as already defined herein.

[0282] In some embodiments, one of Y and Z is OH and the other is -OC(O)R'.

[0283] In several embodiments, Y is OH and Z is -OC(O)R'.

[0284] In several embodiments, Y is -OC(O)R' and Z is OH.

[0285] In some embodiments, both Y and Z are -OC(O)R'.

[0286] In several embodiments, compositions comprising one cationic lipid, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids are provided herein. In several embodiments, the composition is lipid nanoparticles. In several embodiments, one or more cationic lipids constitute about 30 mol% to 60 mol% of the lipid nanoparticles. In several embodiments, one or more non-cationic lipids constitute about 10 mol% to 50 mol% of the lipid nanoparticles. In several embodiments, one or more PEG-modified lipids constitute about 1 mol% to 10 mol% of the lipid nanoparticles. In several embodiments, cholesterol-based lipids constitute 10 mol% to 50 mol% of the lipid nanoparticles. In several embodiments, the lipid nanoparticles encapsulate mRNA encoding nucleic acids, optionally peptides or proteins. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 70% of mRNA. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 75% of mRNA. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 80% of mRNA. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 85% of mRNA. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 90% of mRNA. In several embodiments, the lipid nanoparticles have an encapsulation rate of at least 95% of mRNA.

[0287] In some embodiments, one of the compositions described above is for use in therapy.

[0288] In some embodiments, any one of the compositions of the embodiments described above is intended for use in a method of treating or preventing a disease suitable for treatment or prevention with a peptide or protein encoded by mRNA, wherein the disease is optionally (a) a protein deficiency, optionally a protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infection, or (d) cancer.

[0289] In several embodiments, the composition is administered via spray, optionally by intravenous, subarachnoid, intramuscular, or pulmonary delivery.

[0290] Exemplary Compounds Examples of compounds include those listed in Tables 1-8. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0291] [Table 2-1] [Table 2-2] [Table 2-3]

[0292] [Table 3-1] [Table 3-2] [Table 3-3]

[0293] [Table 4-1] [Table 4-2] [Table 4-3]

[0294] [Table 5-1] [Table 5-2] [Table 5-3]

[0295] [Table 6-1] [Table 6-2] [Table 6-3]

[0296] [Table 7]

[0297] [Table 8]

[0298] Any of the compounds specified in Tables 1-8 above may be provided in the form of pharmaceutically acceptable salts, and such salts are intended to be included by the present invention.

[0299] Unless otherwise specified, R=C 16 H 29 It has the following structure: [ka]

[0300] Unless otherwise specified, R=C 16 H 31It has the following structure: [ka]

[0301] The compounds of the present invention described herein can be prepared according to methods known in the art, including exemplary synthesis of the examples provided herein.

[0302] nucleic acid Compositions useful for nucleic acid delivery can be prepared using the compounds of the present invention described herein.

[0303] Nucleic acid synthesis Nucleic acids according to the present invention can be synthesized according to any known method. For example, mRNA according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system which may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, mutant T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitors. The exact conditions vary depending on the specific application.

[0304] In some embodiments, the DNA template is transcribed in vitro for the preparation of mRNA according to the present invention. A suitable DNA template typically has a promoter for in vitro transcription, e.g., a T3, T7, mutant T7, or SP6 promoter, followed by a desired nucleotide sequence of the desired mRNA and a termination signal.

[0305] A desired mRNA sequence according to the present invention can be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), a virtual backtranslation is performed based on the degeneracy of the genetic code. Subsequently, an optimization algorithm can be used for the selection of suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content on the one hand, and to give maximum consideration to the frequency of tRNA according to codon usage on the other hand. The optimized RNA sequence can be established and displayed, for example, using a suitable display device and compared to the original (wild-type) sequence. Secondary structures can also be analyzed to calculate the stabilizing and destabilizing properties or regions of the RNA, respectively.

[0306] modified mRNA In some embodiments, the mRNA according to the present invention may be synthesized as unmodified mRNA or modified mRNA. Modified mRNA includes nucleotide modifications to the RNA. Therefore, the modified mRNA according to the present invention may include nucleotide modifications such as skeletal modifications, sugar modifications, or base modifications. In some embodiments, mRNA may be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -Methyl-inosine, pseudouracil (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetate methyl ester, 5-methylaminomethyluracil, 5-methyl These can be synthesized as toxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate(v), 1-methyl-psoidouracil, quosin, beta-D-mannosylquosin, weybutoxosin, and phosphoramidites, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine, etc. The preparation of such analogues is known to those skilled in the art, for example, from U.S. Patents No. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated in their entirety by reference.

[0307] Pharmaceutical preparations of cationic lipids and nucleic acids In certain embodiments, the compounds of the present invention described herein, as well as pharmaceutical compositions and liposome compositions containing such lipids, can be used in formulations to facilitate the delivery of encapsulated material (e.g., one or more polynucleotides such as mRNA) to one or more target cells and subsequent transfection. For example, in certain embodiments, cationic lipids described herein (and compositions such as liposome compositions containing such lipids) may be characterized as providing one or more releaseable properties that offer advantages of such compounds compared to receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and macropinocytosis, fusion, endosomal or lysosome disruption and / or other similarly classified lipids.

[0308] According to the present invention, nucleic acids encoding proteins (e.g., full-length, fragment, or partial protein) as described herein, such as mRNA, can be delivered via a delivery vehicle containing the compounds of the present invention as described herein.

[0309] As used herein, the terms “delivery vehicle,” “import vehicle,” and “nanoparticles,” or their grammatical synonyms, are to be used interchangeably.

[0310] For example, the present invention provides a composition (e.g., a pharmaceutical composition) comprising the compounds described herein and one or more polynucleotides. The composition (e.g., a pharmaceutical composition) may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and / or one or more PEG-modified lipids.

[0311] In certain embodiments, the composition exhibits an enhanced (e.g., improved) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally involve the step of contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation containing one or more polynucleotides, comprising the compounds described herein), thereby transfecting one or more target cells within it. The encapsulated material (e.g., one or more polynucleotides) is to be transfected. As used herein, the terms “transfect” or “transfection” refer to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and / or polynucleotides) into a cell, or preferably a target cell. The introduced polynucleotides may be stable or transiently maintained in the target cell. The term “transfection efficiency” refers to the relative amount of such encapsulated material (e.g., polynucleotides) taken up by, introduced into, and / or expressed by the target cell being transfected. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cell after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby improving the likelihood that an appropriate dose of the encapsulated material (e.g., one or more polynucleotides) will be delivered to the disease site and subsequently expressed, while minimizing potential systemic side effects or toxicity associated with the compound or its encapsulated contents.

[0312] For example, after transfection of one or more target cells with polynucleotides encapsulated in one or more lipid nanoparticles comprising a pharmaceutical composition or liposome composition disclosed herein, the production of the product encoded by such polynucleotides (e.g., polypeptides or proteins) may be preferably stimulated, enhancing the ability of such target cells to express the polynucleotides and, for example, produce the target polypeptide or protein. For example, transfection of target cells with one or more compounds or pharmaceutical compositions that encapsulate mRNA enhances (i.e., increases) the production of proteins or enzymes encoded by such mRNA.

[0313] Furthermore, the delivery vehicles described herein (e.g., liposome delivery vehicles) may be prepared to preferentially distribute to other target tissues, cells, or organs, such as the heart, lungs, kidneys, or spleen. In several embodiments, the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical compositions and liposome compositions described herein may be delivered to and / or transfected to target cells or tissues. In some embodiments, the encapsulated polynucleotides (e.g., mRNA) are expressed, and a functional polypeptide product is produced by the target cells (and, in some examples, excreted), thereby conferring beneficial properties to, for example, the target cells or tissues. Such encapsulated polynucleotides (e.g., mRNA) may encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest.

[0314] Liposome delivery vehicle In some embodiments, the composition is a suitable delivery vehicle. In some embodiments, the composition is a liposome delivery vehicle, such as lipid nanoparticles.

[0315] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0316] The enrichment of a liposome composition with one or more of the cationic lipids disclosed herein may be used as a means to improve (e.g., reduce) toxicity, or otherwise to impart one or more desired properties to such enriched liposome composition (e.g., improved delivery of encapsulated polynucleotides to one or more target cells and / or reduced in vivo toxicity of the liposome composition). Therefore, a pharmaceutical composition comprising one or more of the cationic lipids disclosed herein, specifically liposome A compositing solution is also being considered.

[0317] Accordingly, in certain embodiments, the compounds of the present invention described herein may be used as components of a liposome composition to facilitate or enhance the delivery and release of encapsulated material (e.g., one or more therapeutic agents) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells).

[0318] As used herein, liposome delivery vehicles, such as lipid nanoparticles, are typically characterized as microvesicles having an internal aqueous space isolated from an external medium by one or more bilayer membranes. The liposome bilayer membrane is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer membrane may also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In connection with the present invention, liposome delivery vehicles typically play a role in transporting desired mRNA to target cells or tissues.

[0319] In certain embodiments, such a composition (e.g., a liposome composition) is loaded with or encapsulates, for example, one or more biologically active polynucleotides (e.g., mRNA).

[0320] In several embodiments, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a protein encapsulated within a liposome. In several embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, the at least one cationic lipid being one of the compounds of the present invention as described herein. In several embodiments, the composition comprises mRNA encoding a protein (e.g., any of the proteins described herein). In several embodiments, the composition comprises mRNA encoding a cystic fibrosis membrane conductance regulator (CFTR) protein. In several embodiments, the composition comprises mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0321] In several embodiments, the composition (e.g., a pharmaceutical composition) comprises nucleic acids encapsulated within liposomes, the liposomes comprising compounds described herein.

[0322] In several embodiments, the nucleic acid is mRNA encoding a peptide or protein. In several embodiments, the mRNA encodes a peptide or protein for use in delivery to or treatment of the lung or lung cells of a target (for example, the mRNA encodes a cystic fibrosis membrane conductance regulator (CFTR) protein). In several embodiments, the mRNA encodes a peptide or protein for use in delivery to or treatment of the liver or liver cells of a target (for example, the mRNA encodes an ornithine transcarbamylase (OTC) protein). Other exemplary mRNAs are described herein.

[0323] In several embodiments, the liposome delivery vehicle (e.g., lipid nanoparticles) may have a net positive charge.

[0324] In several embodiments, the liposome delivery vehicle (e.g., lipid nanoparticles) may have a net negative charge.

[0325] In several embodiments, the liposome delivery vehicle (e.g., lipid nanoparticles) is net It may possess a sex charge.

[0326] In several embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein.

[0327] For example, the amount of the compound of the present invention described herein in a composition may be expressed as a percentage ("weight %) of the total dry weight of all lipids in the composition (e.g., the total dry weight of all lipids present in the liposome composition).

[0328] In some embodiments of the pharmaceutical compositions described herein, the compounds of the present invention described herein are present in an amount of about 0.5% to about 30% by weight (e.g., about 0.5% to about 20% by weight) of the total dry weight of all lipids present in the composition (e.g., liposome composition).

[0329] In some embodiments, the compounds of the present invention described herein are present in amounts of about 1% to about 30% by weight, about 1% to about 20% by weight, about 1% to about 15% by weight, about 1% to about 10% by weight, or about 5% to about 25% by weight of the total dry weight of all lipids present in the composition (e.g., liposome composition). In some embodiments, the compounds of the present invention described herein are present in amounts of about 0.5% to about 5% by weight, about 1% to about 10% by weight, about 5% to about 20% by weight, or about 10% to about 20% by weight of the total dry weight of all lipids present in the composition such as a liposome delivery vehicle.

[0330] In some embodiments, the amount of the compounds of the present invention described herein is present in an amount of at least about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight, about 30% by weight, about 35% by weight, about 40% by weight, about 45% by weight, about 50% by weight, about 55% by weight, about 60% by weight, about 65% by weight, about 70% by weight, about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, about 95% by weight, about 96% by weight, about 97% by weight, about 98% by weight, or about 99% by weight of the total dry weight of the total lipids in the composition (e.g., liposome composition).

[0331] In some embodiments, the amount of the compounds of the present invention described herein is present in an amount of about 5% by weight or less, about 10% by weight or less, about 15% by weight or less, about 20% by weight or less, about 25% by weight or less, about 30% by weight or less, about 35% by weight or less, about 40% by weight or less, about 45% by weight or less, about 50% by weight or less, about 55% by weight or less, about 60% by weight or less, about 65% by weight or less, about 70% by weight or less, about 75% by weight or less, about 80% by weight or less, about 85% by weight or less, about 90% by weight or less, about 95% by weight or less, about 96% by weight or less, about 97% by weight or less, about 98% by weight or less, or about 99% by weight or less, based on the total dry weight of the total lipids in the composition (e.g., liposome composition).

[0332] In several embodiments, the composition (e.g., a liposome delivery vehicle such as lipid nanoparticles) contains about 0.1% to about 20% by weight (e.g., about 0.1% to about 15% by weight) of the compound described herein. In several embodiments, the delivery vehicle (e.g., a liposome delivery vehicle such as lipid nanoparticles) contains about 0.5% by weight, about 1% by weight, about 3% by weight, about 5% by weight, or about 10% by weight of the compound described herein. In several embodiments, the delivery vehicle (e.g., a liposome delivery vehicle such as lipid nanoparticles) contains up to about 0.5% by weight, up to about 1% by weight, up to about 3% by weight, up to about 5% by weight, up to about 10% by weight, up to about 15% by weight, or up to about 20% by weight of the compound described herein. In several embodiments, this percentage results in an improved beneficial effect (e.g., improved delivery to target tissues such as the liver or lungs).

[0333] The amount of the compound of the present invention described herein in the composition is also the total molar amount of the total lipids in the composition ( For example, it can be expressed as a percentage ("mol%") of the total molar amount of all lipids present in the liposome delivery vehicle.

[0334] In some embodiments of the pharmaceutical compositions described herein, the compounds of the present invention described herein are present in an amount of about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%) of the total molar amount of all lipids present in the composition, such as a liposome delivery vehicle.

[0335] In some embodiments, the compounds of the present invention described herein are present in amounts of about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol%, or about 45 mol% to about 60 mol% of the total molar amount of all lipids present in a composition such as a liposome delivery vehicle. In some embodiments, the compounds of the present invention described herein are present in amounts of about 1 mol% to about 60 mol%, about 1 mol% to about 50 mol%, about 1 mol% to about 40 mol%, about 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol%, or about 5 mol% to about 25 mol% of the total molar amount of all lipids present in a composition such as a liposome delivery vehicle.

[0336] In certain embodiments, the compounds of the present invention described herein may comprise about 0.1 mol% to about 50 mol%, or 0.5 mol% to about 50 mol%, or about 1 mol% to about 25 mol%, or about 1 mol% to about 10 mol%, of the total amount of lipids in the composition (e.g., liposome delivery vehicle).

[0337] In certain embodiments, the compounds of the present invention described herein may contain more than about 0.1 mol%, or more than about 0.5 mol%, or more than about 1 mol%, more than about 5 mol%, more than about 10 mol%, more than about 20 mol%, more than about 30 mol%, or more than about 40 mol% of the total amount of lipids in the lipid nanoparticles.

[0338] In certain embodiments, the compounds described may comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, or less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipids in the composition (e.g., liposome delivery vehicle).

[0339] In some embodiments, the amount of the compounds of the present invention described herein is present in an amount of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the total molar amount of total lipids in the composition (e.g., liposome composition).

[0340] In several embodiments, the amount of the compound of the present invention described herein is present in an amount of about 5 mol% or less, about 10 mol% or less, about 15 mol% or less, about 20 mol% or less, about 25 mol% or less, about 30 mol% or less, about 35 mol% or less, about 40 mol% or less, about 45 mol% or less, about 50 mol% or less, about 55 mol% or less, about 60 mol% or less, about 65 mol% or less, about 70 mol% or less, about 75 mol% or less, about 80 mol% or less, about 85 mol% or less, about 90 mol% or less, about 95 mol% or less, about 96 mol% or less, about 97 mol% or less, about 98 mol% or less, or about 99 mol% or less of the total molar amount of the total lipids in the composition (e.g., liposome composition).

[0341] In some embodiments, this percentage results in improved beneficial effects (e.g., liver This results in improved delivery to target tissues such as organs or lungs.

[0342] In a typical embodiment, the composition of the present invention (e.g., a liposome composition) comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is one of the compounds of the present invention described herein. For example, a composition suitable for carrying out the present invention has four lipid components, comprising one of the compounds of the present invention described herein as a cationic lipid component, a non-cationic lipid, a cholesterol-based lipid, and a PEG-modified lipid. The non-cationic lipid may be DOPE or DEPE. The cholesterol-based lipid may be cholesterol. The PEG-modified lipid may be DMG-PEG2K.

[0343] In several embodiments, the composition of the present invention comprises the cationic lipid of the present invention, DMG-PEG2000, cholesterol, and DOPE, wherein the molar ratio of cationic lipid:DMG-PEG2000:cholesterol:DOPE is 40:5:25:30.

[0344] In further embodiments, the pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids, non-cationic lipids, and cholesterol lipids. In other embodiments, such pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol lipids. In even further embodiments, such pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids and one or more cholesterol lipids.

[0345] In several embodiments, the composition for encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) (e.g., lipid nanoparticles) comprises one or more compounds of the present invention as described herein, and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids.

[0346] In several embodiments, a composition (e.g., lipid nanoparticles) for encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprises one or more compounds of the present invention as described herein; one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids; and further comprises cholesterol-based lipids. Typically, such a composition has four lipid components, comprising a cationic lipid component of the compounds of the present invention as described herein, a non-cationic lipid (e.g., DOPE), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K).

[0347] In several embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) include one or more compounds of the present invention as described herein, as well as one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, PEGylated lipids, and cholesterol-based lipids.

[0348] According to various embodiments, the selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids, including lipid nanoparticles, and the selection of the relative molar ratios of such lipids to each other, are based on the characteristics of the selected lipids, the properties of the target cells, and the characteristics of the delivered mRNA. Further considerations include, for example, the saturation of the alkyl chains of the selected lipids, as well as their size, charge, pH, pKa, fusionability, and toxicity. Therefore, the molar ratios can be adjusted as appropriate.

[0349] In some embodiments, the ratio of cationic lipids: non-cationic lipids: cholesterol-based lipids: PEG-modified lipids is approximately 30-60:20-40:20-30:1-10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol lipids to PEG-modified lipids is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol lipids to PEG-modified lipids is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol lipids to PEG-modified lipids is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipids:non-cationic lipids:cholesterol lipids:PEG-modified lipids is approximately 50:25:20:5.

[0350] Cationic lipids In addition to any of the compounds of the present invention described herein, the composition may also contain one or more additional cationic lipids.

[0351] In some embodiments, liposomes may contain one or more additional cationic lipids. As used herein, the term “cationic lipid” refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH. Several cationic lipids are documented in the literature, and many of them are commercially available.

[0352] Additional cationic lipids suitable for use in this composition include the cationic lipids described in this document.

[0353] Helper lipids A composition (e.g., a liposome composition) may also contain one or more helper lipids. Such helper lipids include noncationic lipids. As used herein, the term “noncationic lipid” refers to any neutral lipid, amphoteric lipid, or anionic lipid. As used herein, the term “anionic lipid” refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-diylcoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (P This includes, but is not limited to, OPE, dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), or mixtures thereof. A suitable noncationic or helper lipid for carrying out the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-dielcyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as a noncationic or helper lipid.

[0354] In some embodiments, the noncationic lipid is a neutral lipid, i.e., a lipid that has no net charge under the conditions in which the composition is formulated and / or administered.

[0355] In some embodiments, noncationic lipids constitute about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, and about the total lipids present in the composition. They may be present in a molar ratio (mol%) of 10% to about 70%, about 10% to about 50%, or about 10% to about 40%. In some embodiments, total noncationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of noncationic lipids in liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of total noncationic lipids in liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of noncationic lipids in liposomes may be less than or equal to about 5 mol%, less than or equal to about 10 mol%, less than or equal to about 20 mol%, less than or equal to about 30 mol%, or less than or equal to about 40 mol%. In some embodiments, the percentage of total noncationic lipids in liposomes may be less than or equal to about 5 mol%, less than or equal to about 10 mol%, less than or equal to about 20 mol%, less than or equal to about 30 mol%, or less than or equal to about 40 mol%.

[0356] In some embodiments, noncationic lipids may be present in the composition at a weight ratio (weight %) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, total noncationic lipids may be present in the composition at a weight ratio (weight %) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of non-cationic lipids in liposomes may be less than or equal to about 5% by weight, less than or equal to about 10% by weight, less than or equal to about 20% by weight, less than or equal to about 30% by weight, or less than or equal to about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in liposomes may be less than or equal to about 5% by weight, less than or equal to about 10% by weight, less than or equal to about 20% by weight, less than or equal to about 30% by weight, or less than or equal to about 40% by weight.

[0357] Cholesterol-based lipids In some embodiments, the composition (e.g., a liposome composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for carrying out the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleylaminopropyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179,280 (1991), Wolf et al. BioTechniques 23,139 (1997), U.S. Patent No. 5,744,335), or imidazole cholesterol esters (ICE) having the following structures. [ka]

[0358] In some embodiments, cholesterol-based lipids may be present in the liposome at a molar ratio (mol%) of about 1% to about 30% or about 5% to about 20% of the total lipids present. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles may be less than or equal to about 5 mol%, less than or equal to about 10 mol%, less than or equal to about 20 mol%, less than or equal to about 30 mol%, or less than or equal to about 40 mol%.

[0359] In some embodiments, cholesterol lipids may be present in the liposome at a weight ratio (wt%) of about 1% to about 30% or about 5% to about 20% of the total lipids present. In some embodiments, the percentage of cholesterol lipids in lipid nanoparticles may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of cholesterol lipids in lipid nanoparticles may be less than or equal to about 5% by weight, less than or equal to about 10% by weight, less than or equal to about 20% by weight, less than or equal to about 30% by weight, or less than or equal to about 40% by weight.

[0360] PEGylated lipids In some embodiments, the composition (e.g., a liposome composition) comprises one or more further PEGylated lipids. A suitable PEGylated lipid for carrying out the present invention is 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0361] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER) including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide), in combination with one or more compounds of the present invention described herein, and in some embodiments together with other lipids including liposomes, is also intended by the present invention. In some embodiments, particularly useful interchangeable lipids are those with shorter acyl chains (e.g., C8 PEG-2000 ceramide). 14 or C 18 It is a PEG-ceramide that has )

[0362] Further PEG-modified lipids intended (also referred to herein as PEGylated lipids, this term is interchangeable with PEG-modified lipids) include C6-C 20 The PEG-modified or PEGylated lipid may include, but is not limited to, polyethylene glycol chains up to 5 kDa in length covalently bonded to a lipid having a long alkyl chain. In some embodiments, the PEG-modified or PEGylated lipid may be PEGylated cholesterol or PEG-2K. The addition of such components may prevent aggregation of the complex, increase its circulating lifetime, and provide a means for increasing the delivery of the lipid-nucleic acid composition to target cells (Klibanov et al. (1990) FEBS). Letters, 268(1):235-237) or these ingredients are invincible. It may be selected to rapidly replace the formulation in a bottle (see U.S. Patent No. 5,885,613).

[0363] Further PEG-modified phospholipids and derivatized lipids of the present invention may be present in a molar ratio (mol%) of about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, or about 3% to about 5% of the total lipids present in the composition (e.g., liposome composition).

[0364] Pharmaceutical formulations and therapeutic use The compounds of the present invention described herein may be used in the preparation of compositions (e.g., construction of liposome compositions) to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells).

[0365] For example, if a liposome composition (e.g., lipid nanoparticles) contains one or more of the compounds disclosed herein, or is otherwise concentrated therewith, a phase transition in the lipid bilayer of one or more target cells may facilitate the delivery of an encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in lipid nanoparticles) to one or more target cells.

[0366] Similarly, in certain embodiments, the compounds of the present invention described herein may be used to prepare liposome vehicles characterized by reduced in vivo toxicity. In certain embodiments, the reduced toxicity is a function of the high transfection efficiency associated with the compositions disclosed herein, such that a reduced amount of such composition can be administered to a subject to achieve a desired therapeutic response or outcome.

[0367] Accordingly, pharmaceutical formulations containing the compounds described herein and the nucleic acids provided by the present invention can be used for a variety of therapeutic purposes. To facilitate in vivo delivery of nucleic acids, the compounds and nucleic acids described herein may be formulated in combination with one or more additional pharmaceutical carriers, targeted ligands, or stabilizing reagents. In some embodiments, the compounds described herein may be formulated via a pre-mixed lipid solution. In other embodiments, compositions containing the compounds described herein may be formulated using a post-insertion technique of nanoparticles into a lipid membrane. Formulation techniques and drug administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. (latest edition).

[0368] Preferred routes of administration include, for example, oral administration, rectal administration, vaginal administration, transmucosal administration, pulmonary administration including intratracheal or inhalation administration, or intestinal administration, parenteral delivery including intradermal injection, transdermal (local) injection, intramuscular injection, subcutaneous injection, and intrathecal injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or nasal administration. In certain embodiments, intramuscular administration is performed in muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, this administration results in the delivery of nucleic acids to muscle cells. In some embodiments, this administration results in the delivery of nucleic acids to hepatocytes (i.e., liver cells).

[0369] A common route for administering the liposomal composition of the present invention may be intravenous delivery, particularly when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency). Alternatively, depending on the disease or disorder being treated, the liposomal composition may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposomal composition of the present invention is typically administered intramuscularly. Diseases or disorders affecting the eye may be treated by intravitreal administration of the liposomal composition of the present invention.

[0370] Alternatively, the pharmaceutical formulations of the present invention may be administered topically rather than systemically, for example, by direct injection of the pharmaceutical formulation into a targeted tissue, preferably in a sustained-release formulation. Topical delivery can be achieved in a variety of ways depending on the targeted tissue. Exemplary tissues to which the delivered mRNA may be delivered and / or expressed include, but are not limited to, the liver, kidneys, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In some embodiments, the targeted tissue is the liver. For example, an aerosol containing the composition of the present invention may be inhaled (in the case of nasal, tracheal, or bronchial delivery), the composition of the present invention may be injected, for example, into a site of injury, disease sign, or pain, the composition may be provided as a lozenge for oral, tracheal, or esophageal administration, supplied in liquid, tablet, or capsule form for gastric or intestinal administration, supplied in suppository form for rectal or vaginal administration, or delivered to the eye by using a cream, droplet, or even injection.

[0371] The compositions described herein may include mRNA encoding a peptide (e.g., a polypeptide such as a protein) as described herein.

[0372] In several embodiments, mRNA encodes a polypeptide.

[0373] In several embodiments, mRNA encodes a protein.

[0374] Examples of mRNA-encoded peptides (e.g., mRNA-encoded proteins) are described herein.

[0375] The present invention provides a method for delivering a composition having a full-length mRNA molecule encoding a peptide or protein for use in the treatment of a target, such as a human target, or cells of a human target, or cells that are treated and delivered to a human target.

[0376] Accordingly, in certain embodiments, the present invention provides a method for producing a therapeutic composition comprising full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target lung or lung cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a cystic fibrosis membrane conductance regulator (CFTR) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an ATP-binding cassette subfamily A member 3 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a dynein axonemal intermediate chain 1 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a dynein axonemal heavy chain 5 (DNAH5) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an alpha-1-antitrypsin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a forkhead box P3 (FOXP3) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding one or more surfactant proteins, for example, surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.

[0377] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target liver or liver cells. Such peptides and polypeptides are urea-containing This may include disorders related to crustaceans, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery of enriched full-length mRNA to the liver or hepatocytes, or treatment therewith, provides a therapeutic benefit.

[0378] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein associated with urea cycle disorders. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding argininosuccinate synthetase 1 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding carbamoyl phosphate synthetase I protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding argininosuccinate lyase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding arginase protein.

[0379] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein associated with lysosome storage dysfunction. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an alpha-galactosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a glucocerebrosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an iduronate-2-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an iduronidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an N-acetyl-alpha-D-glucosaminidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a heparan-N-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a galactosamine-6-sulfatase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a beta-galactosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a lysosomal lipase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the transcription factor EB (TFEB).

[0380] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein associated with glycogen storage impairment. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an acid alpha-glucosidase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a liver glycogen phosphorylase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a muscle phosphoglycerate mutase protein. The present invention provides a method for producing a therapeutic composition having full-length mRNA encoding glycogen debranching enzyme.

[0381] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein related to amino acid metabolism. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a phenylalanine hydroxylase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a glutaryl-CoA dehydrogenase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a propionyl-CoA carboxylase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an oxalase alanine-glyoxylaminotransferase enzyme.

[0382] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein related to lipid metabolism or fibrous disorders. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an mTOR inhibitor. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the ATPase phospholipid transporter 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding one or more NF-kappa B inhibitors, such as I-kappa B alpha, interferon-associated developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a PPAR-gamma protein or an active variant.

[0383] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding protein-related methylmalonic acidemia. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding methylmalonyl-CoA mutase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding methylmalonyl-CoA epimerase protein.

[0384] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA that can provide therapeutic benefits when delivered to or treated with the liver. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding ATP7B protein, also known as Wilson's disease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the porphobilinogen deaminase enzyme. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding one or more coagulation enzymes such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding human hemochromatosis (HFE) protein.

[0385] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target cardiovascular structure or cardiovascular cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding vascular endothelial growth factor A protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding relaxin protein. In certain embodiments The present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the osteomorphogenetic protein-9 protein. In a specific embodiment, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the osteomorphogenetic protein-2 receptor protein.

[0386] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target muscle or muscle cell. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a dystrophin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a frataxin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target cardiac muscle or cardiomyocyte. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein that modulates one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein that modulates the Kv7.1 channel in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a protein that modulates the Nav1.5 channel in muscle tissue or muscle cells.

[0387] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target nervous system or nervous system cells. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the survival motor neuron 2 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the frataxin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding the CLN3 protein.

[0388] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of target blood or bone marrow, or blood cells or bone marrow cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a beta-globin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding one or more coagulation enzymes, for example, factor VIII, factor IX, factor VII, and factor X.

[0389] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target kidney or kidney cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding type IV collagen alpha 5 chain (COL4A5) protein.

[0390] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in delivery to or treatment of a target eye or eye cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a retinosuxin protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a retinosuxin-specific 65kDa (RPE65) protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a 290kDa centrosome protein (CEP290).

[0391] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a peptide or protein for use in vaccine delivery or vaccine therapy to a target or target cells. For example, in certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from an infectious pathogen such as a virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from influenza virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from respiratory syncytial virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from rabies virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from cytomegalovirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from rotavirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from hepatitis viruses such as hepatitis A virus, hepatitis B virus, or hepatitis C virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from human papillomavirus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from herpes simplex virus, such as herpes simplex virus type 1 or herpes simplex virus type 2. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from human metapneumovirus.In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from a malaria virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from a Zika virus. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen derived from a Chikungunya virus.

[0392] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen associated with a target cancer or an antigen identified from the target cancer cells. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen determined from the target's own cancer cells, i.e., personalized cancer The present invention provides a method for providing kutin. In a particular embodiment, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antigen expressed from a mutant KRAS gene.

[0393] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody against CD3. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an antibody against CD19.

[0394] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an immunomodulatory factor. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding interleukin-12. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding interleukin-23. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding interleukin-36 gamma. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding one or more constitutively active variants of interferon gene-stimulating (STING) proteins.

[0395] In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an endonuclease. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding an RNA-guided DNA endonuclease protein, such as Cas9 protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a meganuclease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the present invention provides a method for producing a therapeutic composition having full-length mRNA encoding a zinc finger nuclease protein.

[0396] Delivery method The delivery routes used in the methods of the present invention enable non-invasive, self-administration of the compounds of the present invention. In some embodiments, the methods involve intratracheal or intrapulmonary administration by aerosolization, spraying, or dropping of a composition containing mRNA encoding a therapeutic protein in a suitable transfection or lipid carrier vehicle as described above. In some embodiments, the protein is encapsulated in liposomes. In some embodiments, the liposomes contain lipids that are the compounds of the present invention. Hereinafter as used herein, administration of the compounds of the present invention includes administration of a composition containing the compounds of the present invention.

[0397] While local cells and tissues of the lung represent potential targets that can function as biological depots or reservoirs for the production and secretion of mRNA-encoded proteins, the applicant has found that administration of the compounds of the present invention to the lungs via aerosolization, spraying, or dropping results in the distribution of even non-secretory proteins to the outside of lung cells. While not intended to be bound by the argument, the nanoparticle compositions of the present invention are intended to cross the blood-pulmonary barrier and result in the translation of intact nanoparticles into non-pulmonary cells and tissues, such as the heart, liver, and spleen, leading to the production of encoded proteins in these non-pulmonary tissues. Thus, the usefulness of the compounds and methods of the present invention extends beyond the production of therapeutic proteins in lung cells and lung tissues and can be used for delivery to non-pulmonary target cells and / or tissues. These are useful in the management and treatment of numerous diseases, particularly peripheral diseases resulting from both secretory and non-secretory protein and / or enzyme deficiencies (e.g., one or more lysosomal storage disorders). In certain embodiments, the compounds of the present invention used in the methods of the present invention result in the distribution of mRNA-encapsulated nanoparticles and the production of encoded proteins in the liver, spleen, heart, and / or other non-pulmonary cells. For example, administration of the compounds of the present invention to the lungs by aerosolization, spraying, or dropping allows the composition itself and its protein products (e.g., functional beta-galactosidase protein) to be detected in both local cells and tissues of the lung, as well as in peripheral target cells, tissues, and organs as a result of translocation of mRNA and delivery vehicle to non-lung cells.

[0398] In certain embodiments, the compounds of the present invention may be used in the methods of the present invention to specifically target peripheral cells or tissues. After pulmonary delivery, the compounds of the present invention are intended to cross the blood-airway barrier and be distributed to cells other than local lung cells. Accordingly, the compounds disclosed herein can be administered to a target by a pulmonary administration route using various approaches known to those skilled in the art (e.g., inhalation) and distributed to both local target cells and tissues of the lung, as well as peripheral non-lung cells and tissues (e.g., cells of the liver, spleen, kidney, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both local cells of the lung and peripheral non-lung cells can function as biological reservoirs or depots capable of producing and / or secreting translation products encoded by one or more polynucleotides. Thus, the present invention is not limited to the treatment of lung diseases or conditions, but rather can be used as a non-invasive means to promote the delivery of polynucleotides to peripheral organs, tissues, and cells (e.g., hepatocytes), or the production of enzymes and proteins encoded therein, which would otherwise only be achieved by systemic administration. Examples of peripheral non-pulmonary cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0399] After administration of the composition to the subject, the mRNA-encoded protein product (e.g., functional protein or enzyme) is detectable in peripheral target tissue for at least approximately 1 to 7 days or longer after administration of the compound to the subject. The amount of protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the encoded protein, and the patient's condition. For example, the protein product may be detected at a concentration of at least 0.025 to 1.5 μg / ml (e.g., at least 0.050 μg / ml, at least 0.075 μg / ml, at least 0.1 μg / ml, at least 0.2 μg / ml, less than) for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer after administration of the compound to the subject. It may be detectable in peripheral target tissue at concentrations (e.g., therapeutic concentrations) of at least 0.3 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.7 μg / ml, at least 0.8 μg / ml, at least 0.9 μg / ml, at least 1.0 μg / ml, at least 1.1 μg / ml, at least 1.2 μg / ml, at least 1.3 μg / ml, at least 1.4 μg / ml, or at least 1.5 μg / ml).

[0400] Nucleic acids are produced by intratracheal administration of a liquid suspension of the compound and by liquid nebulizer. It has been demonstrated that it can be delivered to the lungs by inhalation of the aerosol mist or by use of a dry powder device such as that described in U.S. Patent No. 5,780,014, which is incorporated herein by reference.

[0401] In certain embodiments, the compounds of the present invention may be formulated to be delivered to a subject as a particulate liquid or solid by aerosolization or other means before or during administration. Such compounds may be administered with the assistance of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions) to generate particles that can be easily breathed or inhaled by the subject. In some embodiments, such devices (e.g., metered-dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, nebulizer-type inhalers or injectors) facilitate the administration of a predetermined mass, volume, or dose (e.g., about 0.5 mg / kg of mRNA per dose) of the composition to the subject. For example, in certain embodiments, the compounds of the present invention are administered to a subject using a metered-dose inhaler containing a suspension or solution comprising the compound and a suitable nebulizer. In certain embodiments, the compounds of the present invention may be formulated as particulate powder intended for inhalation (e.g., breathable dry particles). In certain embodiments, the compositions of the present invention, formulated as breathable particles, are appropriately sized so that they may be breathable by the subject or can be delivered using a suitable device (e.g., particle sizes with an average D50 or D90 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, or less than or equal to 2.5 μm). In yet other embodiments, the compounds of the present invention are formulated to contain one or more lung surfactants (e.g., lamellar bodies).In some embodiments, the compound of the present invention is present in amounts of 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 mg / kg, at least 15 mg / kg, at least 20 mg / kg, and at least 25 mg / kg. The subject is administered a concentration of 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 / kg body weight in a single dose. In some embodiments, the compounds of the present invention are administered to a subject in a single or multiple dose so that a total amount of mRNA 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 is administered. [Examples]

[0402] While certain compounds, compositions, and methods of the present invention are specifically described according to certain embodiments, the following examples serve only to illustrate the compounds of the present invention and are not intended to limit them.

[0403] Synthetic scheme of vanillic phosphate lipids [ka] Synthesis of 3-(dimethylamino)propyl 4-hydroxy-3-methoxybenzoate (3) [ka]

[0404] To a suspension of vanillic acid 1 (1.0 g, 5.9 mmol) in 25 mL of dichloromethane, oxalyl chloride (2.0 mL, 23.8 mmol) was added at 0°C, followed by dimethylformamide (1 drop). The resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propan-1-ol 2 (0.7 mL, 5.9 mmol) was slowly added, and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered to obtain 3-(dimethylamino)propyl 4-hydroxy-3-methoxybenzoate 3 as a white solid (1.18 g, 79%).

[0405] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3-methoxybenzoate (4) [ka]

[0406] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate AIM-3-E12 (1.0 g, 1.43 mmol) in 20 mL of dichloromethane, oxalyl chloride (0.15 mL, 1.72 mmol) was added at 0°C, followed by dimethylformamide (1 drop), and the mixture was stirred at 0°C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propyl 4-hydroxy-3-methoxybenzoate 3 (0.18 g, 0.7 mmol), followed by pyridine (0.4 mL, 4.9 mmol), was added, and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with ice, the organic layer was washed with brine, and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to obtain 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3-methoxybenzoate 4 as a pale yellow oil (330 mg, 50%).

[0407] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3-methoxybenzoate (VA-3-E12-DMAPr) [ka]

[0408] To a solution of 3-(dimethylamino)propyl 4-((4-((bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3-methoxybenzoate 4 (330 mg, 0.35 mmol) in 10 mL of tetrahydrofuran, hydrofluoric acid (70%, 2.5 mL) in pyridine was added dropwise at 0°C, and the reaction mixture was stirred overnight at room temperature. Saturated sodium bicarbonate solution was added to adjust the pH to 7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was subjected to reverse-phase column chromatography (C18: 5-95%). The compound was purified using MeCN / water / 0.1% TFA to obtain 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3-methoxybenzoate as the TFA salt (120 mg, 48%). This compound was stored in 2-butanol to prevent degradation.

[0409] All other lipids were prepared in similar yields following typical procedures.

[0410] Synthetic scheme of syringate lipids [ka] Synthesis of 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate (6) [ka]

[0411] To a suspension of syringic acid 5 (7.5 g, 0.04 mol) in 100 mL of dichloromethane, oxalyl chloride (12.8 mL, 0.15 mol) was added at 0°C, followed by dimethylformamide (5 drops). The resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 100 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propan-1-ol 2 (4.5 mL, 40 mmol) was slowly added, and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered to obtain 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate 6 as a white solid (6.2 g, 58%).

[0412] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate (7) [ka]

[0413] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate AIM-3-E12 (0.99 g, 1.41 mmol) in 20 mL of dichloromethane, oxalyl chloride (0.15 mL, 1.72 mmol) was added at 0°C, followed by dimethylformamide (1 drop), and the mixture was stirred at 0°C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate 6 (0.2 g, 0.7 mmol), followed by pyridine (0.35 mL, 4.34 mmol), was added, and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with ice, the organic layer was washed with brine, and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to obtain 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate 7 as a pale yellow oil (380 mg, 50%).

[0414] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate (SA-3-E12-DMAPr) [ka]

[0415] To a solution of 3-(dimethylamino)propyl 4-((4-((bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate 7 (380 mg, 0.39 mmol) in 10 mL of tetrahydrofuran, hydrofluoric acid (70%, 2.5 mL) in pyridine was added dropwise at 0°C, and the reaction mixture was stirred overnight at room temperature. Saturated sodium bicarbonate solution was added to adjust the pH to 7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse-phase column chromatography (C18: 5-95% MeCN / water / 0.1% TFA) to obtain 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate as the TFA salt (94 mg, 32%).

[0416] All other lipids were prepared in similar yields following typical procedures.

[0417] Synthetic scheme of synaptic lipids [ka] Synthesis of 3-(dimethylamino)propyl(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate (9) [ka]

[0418] To a suspension of sinapic acid 8 (5 g, 22 mmol) in 100 mL of dichloromethane, oxalyl chloride (7.5 mL, 90 mmol) was added at 0°C, followed by dimethylformamide (5 drops). The resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 100 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propan-1-ol 2 (2.64 mL, 22 mmol) was slowly added, and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered to obtain 3-(dimethylamino)propyl(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate 9 as a pale yellow solid (2.66 g, 39%).

[0419] Synthesis of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate (10) [ka]

[0420] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoic acid AIM-3-E12 (1.8 g, 2.59 mmol) in 20 mL of dichloromethane, oxalyl chloride (0.3 mL, 3.09 mmol) was added at 0°C, followed by dimethylformamide (1 drop), and the mixture was stirred at 0°C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0°C, 3-(dimethylamino)propyl(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate 9 (0.4 g, 1.29 mmol), followed by pyridine (0.62 mL, 7.7 mmol), was added, and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with ice, the organic layer was washed with brine, and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to obtain (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate 10 as a pale yellow oil (540 mg, 42%).

[0421] Synthesis of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-hydroxydodecyl)amino)butanoate (SI-3-E12-DMAPr) [ka]

[0422] To a solution of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate 10 (540 mg, 0.55 mmol) in 10 mL of tetrahydrofuran, hydrofluoric acid (70%, 2.5 mL) in pyridine was added dropwise at 0°C, and the reaction mixture was stirred overnight at room temperature. Saturated sodium bicarbonate solution was added to adjust the pH to 7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse-phase column chromatography (C18: 5-95% MeCN / water / 0.1% TFA) to obtain (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxoprop-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-hydroxydodecyl)amino)butanoate as the TFA salt (330 mg, 80%).

[0423] All other lipids were prepared in similar yields following typical procedures.

[0424] Synthesis scheme A of phenolic lipids [ka] Synthesis of intermediate (3a) in synthesis scheme A: [ka]

[0425] To a solution of (1) (2.00 g, 2.86 mmol) in anhydrous CH2Cl2 (10 mL), oxalyl chloride (0.98 mL, 4.0 equivalents) was added dropwise at 0°C, and the reaction mixture was gradually warmed to room temperature and stirred for 2 hours. After removing excess solvent and oxalyl chloride under reduced pressure, the remaining residue was redissolved in anhydrous CH2Cl2 (30 mL). To the stirred acid chloride solution, (2a) (555 mg, 1.0 equivalent), DMAP (349 mg, 1.0 equivalent), and then triethylamine (3.18 mL, 8.0 equivalents) were added at 0°C. The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature for 16 hours. After 16 hours, the reaction mixture was diluted with CH2Cl2 and saturated NaHCO3 3(aq) Washed with solution. The separated organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to deliver the crude substance. The crude substance was first purified with 50% siRNA in hexane, and then re-purified with 15% siRNA in CH2Cl2 to obtain (3a) (623 mg, 25%) as a viscous oil. MS(ESI+) calculated value C 50 H 93 NO7Si2, [M+H] + =876.65, measured value =876.6.

[0426] Synthesis of intermediate (3b) in synthesis scheme A: [ka]

[0427] Using (2b), (3b) (557 mg, 23%) was obtained as viscous oil by following the procedure of (3a). MS(ESI+) calculated value C 49 H 91 NO6Si2, [M+H] + =846.64, measured value =846.6.

[0428] Synthesis of intermediate (5a) in synthesis scheme A: [ka]

[0429] To solution (3a) (308 mg, 0.351 mmol) in anhydrous CH2Cl2 (3 mL), oxalyl chloride (0.15 mL, 5.0 equivalents) was added at room temperature, and the mixture was stirred at the same temperature for 2 hours. After removing excess solvent and oxalyl chloride under reduced pressure, the remaining residue was redissolved in anhydrous CH2Cl2 (3 mL). To the stirred acid chloride solution, 3-dimethylaminopropanol (4) (109 mg, 3 equivalents) was added at 0°C, followed by triethylamine (0.10 mL, 2.0 equivalents). The reaction mixture was slowly warmed to room temperature, and then stirred at the same temperature for 16 hours. After the completion of the reaction, which was monitored by MS, the reaction mixture was concentrated under reduced pressure and dried, and purified using 0-10% MeOH in CH2Cl2 to obtain (5a) (204 mg, 60%) as a viscous oil. MS (ESI+) calculated value C 55 H 104 N2O7Si2, [M+H] + =961.74, measured value =961.7.

[0430] Synthesis of intermediate (5b) in synthesis scheme A: [ka]

[0431] Using (3b), (5b) (248 mg, 90%) was obtained as viscous oil following the procedure of (5a). MS(ESI+) calculated value C 54 H 102 N2O6Si2, [M+H] + =931.73, measured value =931.7.

[0432] Synthesis of TBL-0731 compound 355(6a) in synthesis scheme A: [ka]

[0433] To a stirred solution of (5a) (204 mg, 0.212 mmol) in anhydrous THF (3 mL), 70% hydrogen fluoride in pyridine (1.09 mL, 197 equivalents) was added dropwise at 0°C, and the mixture was gradually heated to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the completion of the reaction, which was monitored by MS, the reaction mixture was cooled to 0°C and quenched by batch addition of solid NaHCO3. After gas formation was minimized, the resulting mixture was diluted with ethyl acetate and saturated with NaHCO3. 3(aq) The solution was neutralized. The separated organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to deliver the crude substance. The crude substance was purified using 0-20% MeOH in CH2Cl2 to obtain TBL-0731(6a) (132 mg, 85%) as a viscous oil. MS(ESI+) calculated value C 43 H 76 N2O7, [M+H] + =733.57, measured value =733.5. 1 H NMR(500MHz,CDCl3)δ7.64(d,J=15.9Hz,1H),7.14-7.08(m,2H),7.05(d,J=8.1, 3.5Hz,1H),6.37(d,J=16.0Hz,1H),4.27(t,J=6.4Hz,2H),3.86(s,3H),3.72-3.6 3(m,2H),2.78-2.70(m,2H),2.67-2.47(m,6H),2.46-2.40(m,2H),2.36(s,6H), 2.02-1.90(m,4H),1.49-1.35(m,4H),1.34-1.15(m,32H),0.87(t,J=6.9Hz,6H).

[0434] Synthesis of TBL-0750 compound 467(6b) in synthesis scheme A: [ka]

[0435] Using (5b), TBL-0750(6b) (153 mg, 82%) was obtained as a viscous oil following the procedure in (6a). MS(ESI+) calculated value C 42 H 74 N2O6, [M+H] +=703.55, measured value =703.6. 1 H NMR(400MHz,CDCl3)δ7.66(d,J=16.0Hz,1H),7.53(d,J=8.7,1.3Hz,2H),7.12(d ,J=8.6,1.5Hz,2H),6.38(d,J=16.0,0.9Hz,1H),4.26(t,J=6.4Hz,2H),3.70-3.6 3(m,2H),2.73-2.64(m,2H),2.64-2.46(m,6H),2.45-2.40(m,2H),2.34(s,6H), 1.99-1.88(m,4H),1.43-1.34(m,4H),1.31-1.21(m,32H),0.87(t,J=6.8Hz,6H).

[0436] Synthesis scheme B for phenolic lipids [ka] Synthesis of intermediate (3) in synthesis scheme B: [ka]

[0437] To a solution of acid intermediate (1) (4.58 g) in CH2Cl2 (30 mL, anhydrous), oxalyl chloride (1.04 mL, 2 equivalents) was added and the mixture was stirred at room temperature for 2 hours. All volatile substances were removed under reduced pressure, and the remaining residue was redissolved in CH2Cl2 (30 mL, anhydrous). Syringic acid (2) (1.32 g, 1.1 equivalents), followed by pyridine (2.93 mL, 6 equivalents), was added to this solution, and the resulting mixture was stirred overnight at room temperature. After stirring overnight, the solvent was removed under reduced pressure, and the remaining residue was solubilized with a minimum amount of CH2Cl2. The mixture was filtered through a short silica gel plug with 100% siRNA as the eluent. The clear filtrate was concentrated to dryness to obtain the crude product, which was purified using an MPLC over 10 CV with a 10–100% siRNA gradient in hexane to obtain acid product (3) (3.70 g, 78%). MS(ESI+) Calculated value C 53 H 101 NO8Si2, [M+H] + =936.7, measured value =936.7.

[0438] Synthesis of intermediate (5a) in synthesis scheme B: [ka]

[0439] To a solution of benzoic acid (3) (400 mg) in CH2Cl2 (4 mL, anhydrous), oxalyl chloride (0.18 mL, 5 equivalents) was added and the mixture was stirred at room temperature for 2 hours. All volatile substances were removed under reduced pressure, and the remaining residue was redissolved in CH2Cl2 (3 mL, anhydrous). The resulting solution was cooled in an ice bath, and then a solution of 2-aminoethanol (4a) (76 mg) in CH2Cl2 (1 mL) was added. The reaction mixture was heated to room temperature and stirred at the same temperature for 16 hours. After monitoring by LC-MS and complete consumption of the starting materials, the reaction mixture was concentrated to dryness, and the crude product was purified by MPLC over 12 CV using a 0-20% MeOH gradient in CH2Cl2 to obtain product (5a) as viscous oil (225 mg, 52%). MS(ESI+) calculated value C 57 H 110 N2O8Si2, [M+H] + =1007.8, measured value =1007.6.

[0440] Synthesis of intermediate (5b) in synthesis scheme B: [ka]

[0441] Using 4-aminobutanol (4b), product (5b) was obtained as a viscous oil (360 mg, 81%) following the synthesis procedure of (5a). MS(ESI+) calculation value C 59 H 114 N2O8Si2, [M+H] + =1035.8, measured value =1035.6.

[0442] Synthesis of intermediate (5c) in synthesis scheme B: [ka]

[0443] Using intermediate (3) (500 mg) and 3-morpholinopropanol (4c), the synthesis procedure for (5a) was followed to obtain product (5c) (350 mg, 62%). MS(ESI+) calculated value C 60 H 114 N2O9Si2, [M+H] + =1063.8, measured value =1063.6.

[0444] Synthesis of intermediate (5d) in synthesis scheme B: [ka]

[0445] Using intermediate (3) (500 mg) and 2-pyridinylethanol (4d), the synthesis procedure of (5a) was followed to obtain product (5d) (237 mg, 43%). MS(ESI+) calculated value C 60 H 108 N2O8Si2, [M+H] + =1041.8, measured value =1041.6.

[0446] Synthesis of intermediate (5e) in synthesis scheme B: [ka]

[0447] Using intermediate (3) (843 mg) and 4-methylpiperazinylethanol (4e), product (5e) (346 mg, 36%) was obtained by following the synthesis procedure for 5a. MS(ESI+) calculation value C 60 H 115 N3O8Si2, [M+H] + =1062.8, measured value =1062.7.

[0448] Synthesis of TBL-0507 compound 48(6a) in synthesis scheme B: [ka]

[0449] In a non-glass plastic polymer scintillation vial, a stirred solution of TBS-protected intermediate (5a) (225 mg) in THF (3 mL, anhydrous) was mixed with triethylamine (0.16 mL, 5 equivalents), followed by the dropwise addition of triethylamine-3HF (0.36 mL, 10 equivalents). The reaction mixture was stirred overnight at 50°C. The reaction was then completed by adding two drops of ethylamine to the reaction mixture, which consisted of ethylamine and NaHCO₃. 3(aq) The starting material was suspended between the layers and monitored by analysis of the organic layer (TLC or LC-MS). Once the starting material was consumed, excess HF and volatiles were removed by blowing with N2 gas in a fume hood, and the remaining material was diluted with siRNA and neutralized with saturated NaHCO3 aqueous solution (checked with pH paper). The separated organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to obtain the crude material. The crude material was subjected to a 0-40% MeOH gradient in CH2Cl2 over 10 CV using MPLC. The sample was used and purified to obtain TBL-0507(6a) (90 mg, 52%). MS(ESI+) calculated value C 45 H 82 N2O8, [M+H] + =779.6, measured value =779.5.

[0450] Synthesis of TBL-0508 compound 49(6b) in synthesis scheme B: [ka]

[0451] Using the TBS protective intermediate (5b) (360 mg), TBL-0508 (6b) (71 mg, 25%) was obtained according to the procedure in (6a). MS(ESI+) calculated value C 47 H 86 N2O8, [M+H] + =807.6, measured value =807.5.

[0452] Synthesis of TBL-0517 compound 562(6c) in synthesis scheme B: [ka]

[0453] Using the TBS-protected intermediate (5c) (350 mg), purification was performed according to the procedure in (6a) with a 0-10% MeOH gradient in CH2Cl2 to obtain TBL-0517 (6c) (164 mg, 60%). MS(ESI+) calculated value C 48 H 86 N2O9, [M+H] + =835.6, measured value =835.5.

[0454] Synthesis of TBL-0518 compound 563(6d) in synthesis scheme B: [ka]

[0455] Using the TBS-protected intermediate (5d) (237 mg), purification was performed according to the procedure in (6a) with a 0-10% MeOH gradient in CH2Cl2 to obtain TBL-0518 (6d) (111 mg, 60%). MS(ESI+) calculated value C 48 H 80 N2O8, [M+H] + =813.6, measured value =813.5.

[0456] Synthesis of TBL-0535 compound 564(6e) in synthesis scheme B: [ka]

[0457] Using the TBS-protected intermediate (5e) (346 mg), purification was performed according to the procedure in (6a) with a 0-20% MeOH gradient in CH2Cl2 to obtain TBL-0535 (6e) (88 mg, 32%). MS(ESI+) calculated value C 48 H 87 N3O8, [M+H] + =834.6, measured value =834.6.

[0458] Synthesis scheme C of phenolic lipids [ka] Synthesis of intermediate (9a) in synthesis scheme C: [ka]

[0459] To a flask containing amino acid (7) (500 mg) and dodecyl acrylate (8a) (2.91 g, 2.5 equivalents), isopropanol (5 mL) and triethylamine (1.35 mL, 2 equivalents) were added. The resulting mixture was heated at 90°C for 3 hours. After the reaction was completed, as monitored by MS, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The remaining substance was purified using MPLC with 0-12% MeOH in CH2Cl2 to obtain product (9a) (987 mg, 35%). MS(ESI+) calculated value C 34 H 65 NO6, [M+H] + = 584.5, measured value = 584.5.

[0460] Synthesis of intermediate (9b) in synthesis scheme C: [ka]

[0461] Using amino acid (7) (1.00 g), tetradecyl acrylate (8b) (6.51 g, 2.5 equivalents), isopropanol (10 mL), and triethylamine (2.70 mL, 2 equivalents), product (9b) (1.80 g, 29%) was obtained according to the procedure in (9a). MS(ESI+) calculated value C 38 H 73 NO6, [M+H] + =640.5, measured value =640.5.

[0462] Synthesis of TBL-0484 compound 565(11a) in synthesis scheme C: [ka]

[0463] To a solution of intermediate (9a) (300 mg) in anhydrous CH2Cl2 (3 mL), oxalyl chloride (1.0 mL, 23 equivalents) was added at room temperature and the mixture was stirred at the same temperature for 2 hours. After removing excess solvent and oxalyl chloride under reduced pressure, the remaining residue was redissolved in anhydrous CH2Cl2 (3 mL). To the stirred acid chloride solution, phenolic acid (10) (146 mg, 1.0 equivalent) and pyridine (0.21 mL, 5 equivalents) were added at room temperature and the mixture was stirred at the same temperature for 16 hours. After the reaction was completed, as monitored by MS, the reaction mixture was concentrated under reduced pressure. The remaining crude product was purified using MPLC with 0-10% MeOH in CH2Cl2 to obtain TBL-0484 (11a) (90 mg, 21%). MS (ESI+) calculated value C 48 H 84 N2O 10 [M+H] + =849.6, measured value =849.5.

[0464] Synthesis of TBL-0485 compound 566(11b) in synthesis scheme C: [ka]

[0465] Using intermediate (9b) (300 mg), TBL-0485 (11b) (80 mg, 19%) was obtained following the procedure in (11a). MS(ESI+) calculated value C 52 H 92 N2O 10 [M+H] + =905.7, measured value =905.6.

[0466] Example 1: Lipid Nanoparticle Formulation The cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, preferred methods include those described in International Publication No. WO2018 / 089801, which is incorporated in whole by reference herein.

[0467] One exemplary process for the formulation of lipid nanoparticles is Process A of WO2018 / 089801 (see, for example, Example 1 and Figure 1 of WO2018 / 089801). Process A ("A") relates to a conventional method of encapsulating mRNA by mixing mRNA with a lipid mixture without first preforming the lipids into lipid nanoparticles. In the exemplary process, an ethanol lipid solution and an mRNA buffer aqueous solution were prepared separately. A solution of the lipid mixture (cationic lipids, helper lipids, amphoteric lipids, PEG lipids, etc.) was prepared by dissolving the lipids in ethanol. The mRNA solution was prepared by dissolving the mRNA in citrate buffer. The two mixtures were then heated to 65°C and mixed. These two solutions were then mixed using a pump system. In some cases, these two solutions were mixed using a gear pump system. In certain embodiments, these two solutions were mixed using a "T" junction (or "Y" junction). The mixture was then purified by dialysis filtration using a TFF process. The resulting formulation was concentrated and stored at 2-8°C until use.

[0468] A second exemplary process for the formulation of lipid nanoparticles is Process B of WO2018 / 089801 (see, for example, Example 2 and Figure 2 of WO2018 / 089801). Process B ("B") relates to a process for encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with mRNA. Different ranges of conditions, e.g., different temperatures (i.e., heating or not heating the mixture), buffers, and concentrations can be used in Process B. In the exemplary process, lipids dissolved in ethanol and citrate buffer were mixed using a pump system. The instantaneous mixing of these two flows formed hollow lipid nanoparticles, which was a self-assembly process. The resulting formulation mixture consisted of hollow lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process, in which buffer exchange occurred. The resulting preformed empty lipid nanoparticle suspension was then mixed with mRNA using a pump system. In the case of certain cationic lipids, heating the mixed solution increased the percentage of mRNA-containing lipid nanoparticles and resulted in a higher overall mRNA yield.

[0469] The lipid nanoparticle formulations in Table 5 were prepared by either process A or B. It contains mRNA encoding firefly luciferase protein (FFL mRNA) and lipids (cationic lipids: DMG-PEG2000, cholesterol: DOPE), as listed in Table 5 (mol% ratio). [Table 9]

[0470] Delivery of FFL mRNA by intratracheal administration The lipid nanoparticle formulations containing FFL mRNA shown in Table 5 were administered to male CD1 mice (6-8 weeks old) under anesthesia via a single intratracheal aerosol dose using Microsprayer® (50 μl / animal). Approximately 24 hours after administration, the animals were administered luciferin at a dose of 150 mg / kg (60 mg / mL) by intraperitoneal injection at a rate of 2.5 mL / kg. After 5-15 minutes, all animals were imaged using the IVIS imaging system to measure luciferase production in the lungs. Figure 1 shows that the lipid nanoparticles containing cationic lipids described herein are effective for in vivo delivery of FFL mRNA based on positive luciferase activity.

[0471] Numbered Embodiments • Cationic lipids having a structure according to formula (I), [ka] In the formula, L1 is a bond, (C1-C6) alkyl or (C2-C6) alkenyl, In the formula, X is either O or S. In the formula, R 1 , R 2 , R 3 , R 4 and R 5 These are, independently, H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, and optionally substituted Selected from (C2-C6)alkynyl, optionally substituted (C1-C6)alkoxy, and -OC(O)R', In the formula, R 1 , R 2 , R 3 , R 4 or R 5 At least one of them is -OC(O)R', In the formula, R' is as follows: [ka] In the formula, R 6 The following is: [ka] In the formula, m and p are independently 0, 1, 2, 3, 4, or 5. In the formula, R 7 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A Selected from, In the formula, R 8 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B Selected from, In the formula, R 9 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C Selected from, In the formula, R 10 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D Selected from, In the formula, k, n, q, and r are each independently 1, 2, 3, 4, or 5. Or in the formula, (i)R 7and R 8 , or (ii)R 9 and R 10 Together, they form optionally substituted 5-membered or 6-membered heterocycloalkyl or heteroaryl groups, each containing 1 to 3 heteroatoms selected from N, O, and S. In the formula, R 11 , R 12 , R 13 , and R 14 Each is independently selected from H, methyl, ethyl, or propyl. In the formula, R A , R B , R C and R D Each of these can be substituted independently and arbitrarily (C6-C 20 )alkyl, optionally substituted (C6-C 20 ) Alkenyl, optionally substituted (C6-C 20 )Alkinyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O) alkyl, optionally substituted -OC(O) alkenyl, optionally substituted (C1-C6) monoalkylamino, optionally substituted (C1-C6) dialkylamino, optionally Selected from (C1-C6) alkoxy, -OH, and -NH2 substituted by intent, In the formula, R 7 , R 8 , R 9 , R 10 At least one of them is R A , R B , R C or R D Including the portion, R A , R B , R C or R D These are independently and optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 ) Alkenyl, optionally substituted (C6-C 20 )Alkinyl, optionally substituted (C6-C 20 ) Acyl, optionally substituted -OC(O)(C6-C 20)alkyl or optionally substituted -OC(O)(C6-C 20 ) Cationic lipids selected from alkenyls, or a pharmaceutically acceptable salt thereof. 2. Any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)acyl, (C1-C6)alkoxy, halogen, -COR, -CO2H, -CO2R, -CN, -OH, -OR, -OCOR, -OCO2R, -NH2, -NHR, -N(R)2, -SR, or -SO2R, or two geminal hydrogens on carbon atoms are substituted with the group =NH, and each example of R is independently C1-C 10 A cationic lipid according to the numbered embodiment 1, which is an aliphatic alkyl, or a pharmaceutically acceptable salt thereof. 3. i)R A and R B They are identical, and / or ii)R C and R D The cationic lipid or a pharmaceutically acceptable salt thereof described in any of the numbered embodiments 1 to 2 is identical. 4. i)R A and R B They are different, and / or ii)R C and R D A cationic lipid or a pharmaceutically acceptable salt thereof as described in a different, numbered embodiment 1 or 2. 5.R A , R B , R C and R D A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 3, wherein the two are identical. 6.R A , R B , R C and R DA cationic lipid or a pharmaceutically acceptable salt thereof as described in any one of the numbered embodiments 1 to 4, wherein one or more of the embodiments are different. 7. A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 6, wherein X is O. 8. A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 6, wherein X is S. 9.R 1 , R 2 , R 3 , R 4 and R 5 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 8, wherein only one of the is -OC(O)R'. 10.R 1 , R 2 , R 3 , R 4 or R 5 A cationic lipid or a pharmaceutically acceptable salt thereof as described in the numbered embodiment 9, wherein none of the above are OH groups. 11.R 1 , R 2 , R 3 , R 4 and R 5 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 8, wherein two of the molecules are -OC(O)R'. 12.R 1 , R 2 , R 3 , R 4 or R 5 A cationic lipid or a pharmaceutically acceptable salt thereof as described in the numbered embodiment 11, wherein none of the above are OH groups. 13.R 1 , R 2 , R 3 , R 4 and R 5 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 8, wherein three of the molecules are -OC(O)R'. 14.R 1and / or R 5 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 13, wherein is -OC(O)R'. 15.R 2 and / or R 4 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 14, wherein is -OC(O)R'. 16.R 3 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 15, wherein is -OC(O)R'. 17. i) p, q, and r are identical, or ii) One or more of p, q, and r are different, or iii) A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 16, wherein q and r are the same and p is different. 18. i) k, m, and n are identical, or ii) One or more of k, m, and n are different, or iii) A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 17, wherein k and n are the same and m is different. 19. A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 18, wherein m is 1, 2, or 3. 20. A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 19, wherein p is 1, 2, or 3. 21.R ’ The cationic lipid or a pharmaceutically acceptable salt thereof as described in any one of the numbered embodiments 1 to 20, wherein the cationic lipid or a pharmaceutically acceptable salt thereof is as follows: [ka] twenty two. i) k, m and n = 1, or ii) k, m and n=1, and R 11 and R 12 =H, or iii) k and n=1, and m=2, or iv) k and n=1, m=2, and R 11 and R 12 =H, or v) k and n=1, and m=3, or vi) k and n=1, m=3, and R 11 and R 12 A cationic lipid or a pharmaceutically acceptable salt thereof, as described in the numbered embodiment 21, wherein =H. 23.R 6 The cationic lipid or a pharmaceutically acceptable salt thereof as described in any one of the numbered embodiments 1 to 22, wherein the following is the cationic lipid or a pharmaceutically acceptable salt thereof. [ka] twenty four. i) p, q and r = 1, or ii) p, q and r = 1, and R 13 and R 14 is H, or iii) q and r=1 and p=2, or iv) q and r=1, p=2, and R 13 and R 14 A cationic lipid or a pharmaceutically acceptable salt thereof, as described in the numbered embodiment 23, wherein H is present. 25.R 6 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of the numbered embodiments 1 to 22, wherein the following is selected from the group consisting of: [ka] 26.R 6 The cationic lipid or a pharmaceutically acceptable salt thereof as described in the numbered embodiment 25, wherein the following applies: [ka] 27. Cationic lipids according to any one of the numbered embodiments 1 to 26 having a structure according to formula (II): [ka] or a pharmaceutically acceptable salt thereof. 28. Cationic lipids according to numbered embodiment 27 having a structure according to formula (IIA) [ka] or a pharmaceutically acceptable salt thereof. 29. Cationic lipids according to numbered embodiment 28, having a structure according to one of formulas (IIB), (IIC), (IID), or (IIE). [ka] [ka] or a pharmaceutically acceptable salt thereof. 30. Cationic lipids according to numbered embodiment 27 having a structure according to formula (IIF) [ka] or a pharmaceutically acceptable salt thereof. 31. Cationic lipids according to numbered embodiment 27 having a structure according to formula (IIG) [ka] or a pharmaceutically acceptable salt thereof. 32. A cationic lipid according to numbered embodiment 27 having a structure according to formula (IIH), [ka] A cationic lipid or a pharmaceutically acceptable salt thereof, wherein one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are independently -OC(O)R'. 33. Cationic lipids according to any one of the numbered embodiments 1 to 26 having a structure according to formula (III): [ka] or a pharmaceutically acceptable salt thereof. 34. Cationic lipid according to numbered embodiment 33 having a structure according to formula (IIIA) [ka] or a pharmaceutically acceptable salt thereof. 35. Cationic lipids according to numbered embodiment 33 or numbered embodiment 34 having a structure according to formula (IIIB) [ka] or a pharmaceutically acceptable salt thereof. 36. Cationic lipid according to numbered embodiment 35 having a structure according to formula (IIIC) [ka] or a pharmaceutically acceptable salt thereof. 37. Cationic lipid according to numbered embodiment 33 having a structure according to formula (IIID) [ka] or a pharmaceutically acceptable salt thereof. 38. Cationic lipids according to numbered embodiment 37, having a structure selected from formula (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ), or (IIIK). [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof. 39. Cationic lipid according to numbered embodiment 33 having a structure according to formula (IIIL) [ka] or a pharmaceutically acceptable salt thereof. 40. A cationic lipid according to numbered embodiment 33 having a structure according to formula (IV), [ka] In the formula, M is a cationic lipid selected from H, OH, OMe, or Me, or a pharmaceutically acceptable salt thereof. 41. Cationic lipids according to numbered embodiment 33 having a structure according to formula (VI), (VII), (VIII), (IX), or (X), [ka] A cationic lipid or a pharmaceutically acceptable salt thereof, wherein one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are independently -OC(O)R'. 42. A cationic lipid or a pharmaceutically acceptable salt thereof according to the numbered embodiment 41, wherein one of Y and Z is OH and the other is -OC(O)R'. 43. A cationic lipid or a pharmaceutically acceptable salt thereof according to the numbered embodiment 42, wherein Y is OH and Z is -OC(O)R'. 44. A cationic lipid or a pharmaceutically acceptable salt thereof according to the numbered embodiment 42, wherein Y is -OC(O)R' and Z is OH. 45. Described in numbered embodiment 41, where both Y and Z are -OC(O)R'. Cationic lipids or their pharmaceutically acceptable salts. 46. ​​A compound selected from those listed in Tables 1-8, or a pharmaceutically acceptable salt thereof. 47. A composition comprising a cationic lipid, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, as described in any one of the numbered embodiments 1 to 46. 48. The composition according to the numbered embodiment 47, wherein the composition is lipid nanoparticles, optionally liposomes. 49. The composition according to the numbered embodiment 48, wherein one or more cationic lipids constitute about 30 mol% to 60 mol% of the lipid nanoparticles. 50. The composition according to any one of the numbered embodiments 48 or 49, wherein one or more noncationic lipids constitute about 10 mol% to 50 mol% of the lipid nanoparticles. 51. The composition according to any one of the numbered embodiments 48 to 50, wherein one or more PEG-modified lipids constitute about 1 mol% to 10 mol% of the lipid nanoparticles. 52. A composition according to any one of the numbered embodiments 48 to 51, wherein cholesterol-based lipids constitute approximately 10 mol% to 50 mol% of the lipid nanoparticles. 53. A composition according to any one of the numbered embodiments 48 to 52, wherein lipid nanoparticles encapsulate nucleic acids, optionally mRNA encoding peptides or proteins. 54. The composition according to any one of the numbered embodiments 48 to 52, wherein lipid nanoparticles encapsulate mRNA encoding a peptide or protein. 55. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 70% mRNA. 56. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 75% mRNA. 57. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 80% mRNA. 58. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 85% of mRNA. 59. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 90% mRNA. 60. The composition according to the numbered embodiment 54, wherein the lipid nanoparticles have an encapsulation rate of at least 95% of mRNA. 61. A composition according to any one of the numbered embodiments 54 to 60 for use in therapy. 62. A composition according to any one of the numbered embodiments 54 to 60 for use in a method of treating or preventing a disease which is suitable for treatment or prevention by a peptide or protein encoded by mRNA, wherein the disease is optionally (a) a protein deficiency, optionally (b) an autoimmune disease in which the protein deficiency affects the liver, lungs, brain or muscles, (c) an infection, or (d) cancer. 63. Compositions for use according to numbered embodiments 61 or 62, wherein the composition is administered via spray, optionally by intravenous, subarachnoid, intramuscular, or pulmonary delivery. 64. A method for treating or preventing a disease, the method comprising administering a composition described in any one of the numbered embodiments 54 to 60 to a subject in need thereof, wherein the disease is suitable for treatment or prevention by a peptide or protein encoded by mRNA, and optionally the disease is (a) a protein deficiency, optionally the protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infection, or (d) cancer. 65. The method according to the numbered embodiment 64, wherein the composition is administered via spray, optionally by intravenous, subarachnoid, intramuscular, or pulmonary delivery.

Claims

1. A cationic lipid having a structure according to formula (I), 【Chemistry 1】 In the ceremony, L １ is a combination, (C １ -C ６ ) alkyl or (C ２ -C ６ ) Alkenil, In the formula, X is either O or S, wherein, R １ , R ２ , R ３ , R ４ and R ５ are each independently selected from H, OH, optionally substituted (C １ -C ６ ) alkyl, optionally substituted (C ２ -C ６ ) alkenyl, optionally substituted (C ２ -C ６ ) alkynyl, optionally substituted (C １ -C ６ ) alkoxy and -OC(O)R', In the formula, R １ , R ２ , R ３ , R ４ or R ５ At least one of them is -OC(O)R', In the formula, R' is as follows: 【Chemistry 2】 In the formula, R ６ The following is: 【Transformation 3】 In the formula, m and p are independently 0, 1, 2, 3, 4, or 5. In the formula, R ７ is H, optionally substituted (C １ -C ６ ) alkyl, optionally substituted (C ２ -C ６ ) alkenyl, optionally substituted (C ２ -C ６ ) Alkinyl, optionally substituted (C １ -C ６ ) Acyl, - (CH ２ ) ｋ R Ａ or - (CH ２ ) ｋ CH(OR １１ ) R Ａ Selected from, In the formula, R ８ is H, optionally substituted (C １ -C ６ ) alkyl, optionally substituted (C ２ -C ６ ) alkenyl, optionally substituted (C ２ -C ６ ) Alkinyl, optionally substituted (C １ -C ６ ) Acyl, - (CH ２ ) ｎ R Ｂ or - (CH ２ ) ｎ CH(OR １２ ) R Ｂ Selected from, In the formula, R ９ is H, optionally substituted (C １ -C ６ ) alkyl, optionally substituted (C ２ -C ６ ) alkenyl, optionally substituted (C ２ -C ６ ) Alkinyl, optionally substituted (C １ -C ６ ) Acyl, - (CH ２ ) ｑ R Ｃ or - (CH ２ ) ｑ CH(OR １３ ) R Ｃ Selected from, In the formula, R １０ is H, optionally substituted (C １ -C ６ ) alkyl, optionally substituted (C ２ -C ６ ) alkenyl, optionally substituted (C ２ -C ６ ) Alkinyl, optionally substituted (C １ -C ６ ) Acyl, - (CH ２ ) ｒ R Ｄ or - (CH ２ ) ｒ CH(OR １４ ) R Ｄ Selected from, In the formula, k, n, q, and r are each independently 1, 2, 3, 4, or 5. Or in the formula, (i) R ７ and R ８ , or (ii) R ９ and R １０ together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, and the heterocycloalkyl or heteroaryl contains 1 to 3 heteroatoms selected from N, O and S, In the formula, R １１ , R １２ , R １３ , and R １４ Each is independently selected from H, methyl, ethyl, or propyl. wherein R Ａ 、R Ｂ 、R Ｃ and R Ｄ are each independently selected from optionally substituted (C ６ -C ２０ ) alkyl, optionally substituted (C ６ -C ２０ ) alkenyl, optionally substituted (C ６ -C ２０ ) alkynyl, optionally substituted (C ６ -C ２０ ) acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C １ -C ６ ) monoalkylamino, optionally substituted (C １ -C ６ ) dialkylamino, optionally substituted (C １ -C ６ ) alkoxy, -OH, -NH ２ ; In the formula, R ７ , R ８ , R ９ , R １０ At least one of them is R Ａ , R Ｂ , R Ｃ or R Ｄ Including the portion, R Ａ , R Ｂ , R Ｃ or R Ｄ (C) is independently and optionally substituted. ６ -C ２０ ) alkyl, optionally substituted (C ６ -C ２０ ) alkenyl, optionally substituted (C ６ -C ２０ ) Alkinyl, optionally substituted (C ６ -C ２０ ) Acyl, optionally substituted -OC(O)(C ６ -C ２０ ) alkyl or optionally substituted -OC(O)(C ６ -C ２０ ) Cationic lipids selected from alkenyls, or a pharmaceutically acceptable salt thereof.

2. A cationic lipid or a pharmaceutically acceptable salt thereof according to claim 1, wherein X is O.

3. A cationic lipid or a pharmaceutically acceptable salt thereof according to claim 1 or claim 2, wherein m is 1, 2, or 3.

4. A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein p is 1, 2, or 3.

5. R ’ A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the following is also true. 【Chemistry 4】

6. vii) k, m and n = 1, or viiii) k, m and n = 1, and R １１ and R １２ = H, or ix) k and n = 1, and m = 2, or x) k and n=1, m=2, and R １１ and R １２ = H, or xi) k and n = 1, and m = 3, or xi) k and n=1, m=3, and R １１ and R １２ A cationic lipid or a pharmaceutically acceptable salt thereof according to claim 5, wherein = H.

7. R' 【Transformation 5】 And R ７ and R ８ Each of these is arbitrarily substituted, -CO ２ R ａａ Replaced with (C １ -C ６ ) is alkyl, and in the formula, R ａａ is C １ -C ５０ It is alkyl, preferably R ７ and R ８ Each of these is -CO ２ R ａａ Replaced with (C １ -C ６ ) is alkyl, and in the formula, R ａａ is C １ -C ２０ Alkyl, more preferably R ７ and R ８ Each of them, 【Transformation 6】 A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4.

8. R ６ A cationic lipid or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the following is present. 【Transformation 7】

9. A cationic lipid having a structure according to the following, (a) Equation (II): 【Transformation 8】 or a pharmaceutically acceptable salt thereof (b) Formula (IIA): 【Chemistry 9】 or a pharmaceutically acceptable salt thereof (c) Formula (IIF): 【Chemistry 10】 or a pharmaceutically acceptable salt thereof (d) Equation (IIG): 【Chemistry 11】 or a pharmaceutically acceptable salt thereof (e) Formula (IIH): 【Chemistry 12】 In the formula, one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are cationic lipids, or pharmaceutically acceptable salts thereof, each independently of the other being -OC(O)R'. (f) Formula (III): 【Chemistry 13】 or a pharmaceutically acceptable salt thereof (g) Formula (IIIA): 【Chemistry 14】 or a pharmaceutically acceptable salt thereof (h) Formula (IIIB): 【Chemistry 15】 or a pharmaceutically acceptable salt thereof (i) Formula (IIID): 【Chemistry 16】 or a pharmaceutically acceptable salt thereof (j) Formula (IIIL): 【Chemistry 17】 or a pharmaceutically acceptable salt thereof, (k) Formula (IV): [Chemistry 18] The cationic lipid according to any one of claims 1 to 8, wherein M is a cationic lipid selected from H, OH, OMe, or Me, or a pharmaceutically acceptable salt thereof.

10. A cationic lipid having a structure according to formula (VI), (VII), (VIII), (IX), or (X), 【Chemistry 19】 The cationic lipid according to any one of claims 1 to 8, wherein one of Y and Z is OH and the other is -OC(O)R', or both Y and Z are cationic lipids or pharmaceutically acceptable salts thereof, each independently being -OC(O)R'.

11. A compound selected from those listed in Tables 1-8, or a pharmaceutically acceptable salt thereof.

12. A composition comprising a cationic lipid according to any one of claims 1 to 11, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein the composition is optionally lipid nanoparticles.

13. The composition according to claim 12, wherein the composition is lipid nanoparticles, and the lipid nanoparticles encapsulate nucleic acids, optionally peptides, or mRNA encoding proteins.

14. The composition according to claim 13, wherein the lipid nanoparticles have an mRNA encapsulation rate of at least 70%.

15. A composition according to any one of claims 12 to 14 for use in treatment.

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