Sterol-based cationic lipids having aromatic head groups

Abstract

The present invention is, in part, based on formula (I): [Case 1] This invention provides sterol-based cationic lipids having the aromatic head group of TIFF2026524780000208.tif29170 and its partial formula, or pharmaceutically acceptable salts thereof. The compounds provided herein 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 treating a variety of diseases, disorders and conditions, including those associated with the deficiency of one or more proteins.

Description

[Technical Field]

[0001] Related applications This application claims priority to European Patent Application No. 23305928.6 and European Patent Application No. 23305934.4, both filed on June 12, 2023, the full disclosures of which are incorporated herein by reference. [Background technology]

[0002] Nucleic acid delivery is being widely studied as a potential treatment option for certain disease conditions. In particular, messenger RNA (mRNA) therapy is becoming an increasingly important option for the prevention and treatment of various diseases (e.g., the use of vaccines).

[0003] The efficient delivery of liposomal-encapsulated nucleic acids remains an active area of ​​research. Liposomal-encapsulated nucleic acids can be administered intramuscularly (IM). Currently approved intramuscular (IM) vaccines exhibit robust systemic immunity but suffer from the drawback of low IgA levels in the respiratory mucosa and insufficient airway immunity. This invention addresses the problem of airway immunodeficiency by providing liposomes (and cationic lipids for said liposomes) that can be administered intranasally (IN). Indeed, a mouse model of influenza virus infection has demonstrated that intranasal (IN) immunity induces local, rather than systemic, IgA secretion (Oh, JE, et al. (2021), Intranasal Priming Induces Local Lung-Resident B cell Populations that Secrete Protective Mucosal Antiviral IgA. Science Immunology, 6(66)).

[0004] The cationic lipid components of liposomes play a crucial role in promoting the effective encapsulation of nucleic acids during liposome loading. In addition, cationic lipids can play a vital role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. Various cationic lipids suitable for in vivo use have been discovered. However, there remains a need to identify cationic lipids effective for transnasal / pulmonary delivery of mRNA. Furthermore, there is a need to identify cationic lipids that can be synthesized efficiently and inexpensively without producing toxic byproducts.

[0005] The inventors have surprisingly found that lipid nanoparticles composed of cationic lipids having an aromatic head group and a sterol tail are highly effective for nasal / pulmonary delivery of mRNA encapsulated within the lipid nanoparticles. In fact, the lipid nanoparticles composed of the cationic lipid of the present invention showed high levels of peptide or protein expression when delivering the mRNA encoding the peptide or protein by pulmonary delivery (e.g., intratracheal delivery). For example, lipid nanoparticles containing the cationic lipid of the present invention and encapsulating firefly luciferase (FFL) mRNA, including ICE, showed improved FFL mRNA expression when administered to mice via intratracheal catheterization compared to lipid nanoparticles encapsulating FFL mRNA (see Figure 5). [Overview of the Initiative] [Means for solving the problem]

[0006] The present invention provides, in particular, a novel class of cationic lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids. These compounds are intended to enable highly effective in vivo intranasal / intrapulmonary delivery of therapeutic agents and vaccines. These compounds are also intended to enable in vivo intranasal / intrapulmonary delivery of therapeutic agents and vaccines while maintaining a favorable safety profile.

[0007] The cationic lipids of the present invention also contain cleavable groups (e.g., esters) that are intended to contribute to their favorable safety profile by improving their biodegradability.

[0008] In one embodiment, the formula (I): [ka] A cationic lipid having a structure according to, or a pharmaceutically acceptable salt thereof, During the ceremony, [ka] This is selected from optionally substituted arylenes or optionally substituted heteroarylenes. In the formula, L2 is selected from a bond, an optionally substituted (C1-C6) alkylene, or an optionally substituted (C2-C6) alkenylene. In the formula, X 1 It is selected from O, In the formula, R 1 teeth, [ka] And, In the formula, a is selected from 0, 1, 2, 3, 4, or 5. In the formula, R 2 and R 3 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, R 4 and R 5 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, L1 is selected from D or E-L3-C(=O)O-, and the right side of the cited structure is, [ka] It is connected, In the formula, D is selected from -(C1~C3)alkyl-O-, -OC(=O)O-, -SC(=O)O-, -OC(=O)S-, -(C1~C3)alkyl-OC(=O)O-, or -C(=O)O-, where the right side of each specified structure is: [ka] It is connected, In the formula, E is selected from -O- or -OC(=O)-, and the right side of each structure is bonded to L3. In the formula, L3 is selected from (C1-C6) alkylenes that are optionally substituted, or from (C2-C6) alkenylenes that are optionally substituted, and [ka] These are naturally occurring sterols or non-naturally occurring sterols.

[0009] In one embodiment, a cationic lipid that is a pharmaceutically acceptable salt of formula (I) is provided herein.

[0010] In one embodiment, the present invention comprises a cationic lipid or a pharmaceutically acceptable salt thereof, and further includes: (i) One or more noncationic lipids, (ii) One or more cholesterol-based lipids, (iii) One or more PEG-modified lipids and Compositions comprising the above are provided herein.

[0011] In one embodiment, the herein provides a cationic lipid of the present invention (e.g., compound III or compound IV) or a pharmaceutically acceptable salt thereof, and further: (i) One or more noncationic lipids, (ii) One or more PEG-modified lipids and It is a composition containing [the specified ingredient].

[0012] In one embodiment, the composition is lipid nanoparticles, optionally liposomes.

[0013] In one embodiment, a composition containing the cationic lipid of the present invention may be used for therapeutic purposes. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows Scheme 1, which is the reaction scheme for Example 1. [Figure 2] This figure shows Scheme 2, which is the reaction scheme for Example 2. [Figure 3] This figure shows Scheme 3, which is the reaction scheme for Example 3. [Figure 4] This figure shows Scheme 4, which is the reaction scheme for Example 4. [Figure 5] This figure demonstrates in vivo firefly luciferase (FFL) protein production resulting from catheter-mediated intratracheal delivery of FFL mRNA using lipid nanoparticles containing compounds III and IV described herein. As shown in this figure, the use of these compounds as part of lipid nanoparticles can result in high levels of in vivo FFL protein production after administration. [Modes for carrying out the invention]

[0015] definition To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are provided throughout this specification. Publications and other reference materials mentioned herein to provide background to the present invention and to offer additional details regarding its implementation are incorporated herein by reference.

[0016] Amino Acids: As used herein, the term “amino acid” in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. “Standard amino acid” refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. “Non-standard amino acid” refers to any amino acid that is not 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, chemically modified amino acids, including salts, amino acid derivatives (amides, etc.), and / or substitutions. Amino acids, including carboxy-terminal and / or amino-terminal amino acids in peptides, can 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 its activity. Amino acids can be involved in disulfide bonds. Amino acids may have one or more post-translational modifications, such as association with one or more chemical entities (e.g., 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 it refers to free amino acids or peptide residues should be clear from the context in which the term is used.

[0017] 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 worms. In some embodiments, animals may be transgenic animals, genetically modified animals, and / or clones.

[0018] Approximately or about: As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​that are similar to the stated reference values. In certain embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “about” refer to values ​​that fall within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the stated reference value (except where such numbers exceed 100% of the possible values).

[0019] Biologically active: As used herein, the term “biologically active” refers to the characteristic of any drug that is active in a biological system and, in particular, in a living organism. For example, a drug that has a biological effect on an organism when administered to that organism is considered biologically active.

[0020] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery includes situations in which mRNA is delivered to a target tissue, the encoded protein is expressed, and it is 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, it is secreted into the patient’s circulatory system (e.g., serum), systematically distributed, and absorbed by other tissues (also referred to as “systemic distribution” or “systemic delivery”).

[0021] 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 fully assembled proteins (e.g., enzymes). Herein, the terms “expression” and “production,” as well as their grammatical synonyms, are used interchangeably.

[0022] Functional: As used herein, a “functional” biomolecule is a biomolecule in which it exhibits the properties and / or activity that characterize it.

[0023] Half-life: As used herein, the term “half-life” refers to the time required for an amount of a nucleic acid or protein, such as its concentration or activity, to fall to half of its initial measured value over a given period.

[0024] Helper Lipids: As used herein, the term “helper lipids” refers to any neutral or zwitterionic lipid material, including cholesterol. Without being constrained by any particular theory, helper lipids can impart stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles.

[0025] To improve, increase, or decrease: As used herein, the terms “improve,” “increase,” or “decrease,” or grammatical synonyms, refer to relative values ​​to baseline measurements, such as measurements in the same individual prior to the initiation of the treatment described herein, or measurements in a control subject (or control subject) that has not received the treatment described herein. A “control subject” is a subject suffering from the same form of disease as the subject receiving treatment and being of approximately the same age as the subject receiving treatment.

[0026] In vitro: As used herein, the term “in vitro” refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, or in a cell culture, rather than within a multicellular organism.

[0027] 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).

[0028] Liposome: As used herein, the term “liposome” refers to any layer, multilayer, or solid nanoparticle vesicle. Typically, liposomes as used herein may 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 a cationic lipid and optionally further: (i) Noncationic lipids, (ii) Cholesterol-based lipids, and / or (iii) Contains PEG-modified lipids

[0029] In some embodiments, liposomes suitable for the present invention comprise a cationic lipid (e.g., compound III or compound IV) and optionally further: (i) noncationic lipids, and / or (ii) Contains PEG-modified lipids

[0030] 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 used in relation to 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 selectively purified, chemically synthesized, etc. Where appropriate, for example, in 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); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynylcytidine, C5-propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-amino Adenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 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) or comprising them.

[0031] Naturally occurring sterols: As used herein, the term “naturally occurring sterols” has the ordinary meaning in the art. These are sterols found in living systems such as plants, animals, bacteria, and fungi. A non-limiting example of naturally occurring sterols is cholesterol.

[0032] Non-natural sterols: As used herein, the term “non-natural sterols” has the ordinary meaning in the art. For example, synthetic sterols that do not exist naturally in biological systems such as plants, animals, bacteria, and fungi. For example, non-natural sterols have the following ring structure: [ka] The formula may include one or more C atoms which may be substituted and / or unsaturated. Non-exclusive examples of non-natural sterols are synthetic analogs or derivatives of cholesterol, for example, fluorinated analogs or derivatives of cholesterol.

[0033] Nucleic Acids: As used herein, the term “nucleic acid” in its broadest sense refers to 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 by phosphate diester bonds. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” includes RNA and 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-coated 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 the embodiments, the DNA may be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, polymerase chain reaction (PCR) products, vectors (e.g., P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.In the embodiment, RNA includes messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), transcription RNA (tRNA), transcription messenger RNA (tmRNA), micronuclear RNA (snRNA), micronucleolar RNA (snoRNA), SmY RNA, small Cajal-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), spliced ​​leader 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-interacting RNA (piRNA), small interfering RNA (siRNA), transaction siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), and 73K It may be in the form of RNA, retrotransposons, viral genomes, viloids, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is mRNA that encodes a protein such as an enzyme.

[0034] Patient: As used herein, the terms “patient” or “subject” refer to 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.

[0035] Pharmacologically acceptable: As used herein, the term "pharmaceutically acceptable" means a substance that, within the bounds of reliable medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable risk-benefit ratio.

[0036] 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. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed by inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or by organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using 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, glucoheptone, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxyethanesulfone. Examples of salts include acid salts, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, p-toluenesulfons, undecanoates, and valersates. Salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium and N + (C 1~4Examples include alkyl(4) salts. Typical alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, 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 electrophiles suitable for the formation of quaternized alkylated amino salts, such as alkyl halides.

[0037] Whole-body distribution or delivery: As used herein, the terms “whole-body distribution” or “whole-body delivery,” or their grammatical synonyms, refer to a mechanism or method of delivery or distribution that affects the entire body or organs. Typically, whole-body distribution or delivery is achieved through the body’s circulatory system, e.g., blood flow. Compare with the definition of “local distribution or delivery.”

[0038] 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). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human being. The subject may be a patient, referring 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 a person suffering from or susceptible to a disease or disorder, the symptoms of which may or may not be present.

[0039] Substantially: As used herein, the term "substantially" refers to a qualitative state indicating an overall or nearly overall degree or extent of a targeted feature or characteristic. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0040] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, target tissue includes tissue that exhibits a pathological condition, symptom, or characteristic associated with the disease.

[0041] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition, when administered to a subject afflicted with or at risk of such disease, disorder, and / or condition. One of ordinary skill will understand that a therapeutically effective amount is typically administered in a dosing regimen that includes at least one unit dose.

[0042] Treating: As used herein, the terms "treating," "treatment," or "treat" refer to any method used to partially or completely alleviate, relieve, mitigate, inhibit, 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 can be administered to a subject who does not exhibit symptoms of a disease and / or who exhibits only early symptoms of a disease for the purpose of reducing the risk that a disease-related pathological condition will develop.

[0043] Chemical Definitions Acyl: When used herein, the term "acyl" means R Z-(C=O)-(wherein, R Z This refers to, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene.

[0044] Aliphatic: As used herein, the term aliphatic means (C1-C 50 ) refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be linear, branched, or cyclic. For example, (C1~C 20 )Aliphatic is (C1~C 20 )alkyl (for example, linear or branched (C1~C) 20 (Saturated alkyl), (C2~C 20 ) Alkenyl (for example, linear or branched (C4~C) 20 ) Dienyl, straight chain or branched (C6~C 20 (e.g., trienyl) and (C2~C 20 )Alkinyl (for example, linear or branched (C2~C) 20 (C1~C 20 )Aliphatic is (C3~C 20 )Cyclic aliphatic (for example, (C3~C 20 )Cycloalkyl, (C4~C 20 )Cycloalkenyl or (C8~C 20 It may include cycloalkynyls. In certain embodiments, the aliphatic group may comprise one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. The aliphatic group may be unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more 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'' may independently be (C1~C 20 )Aliphatic (for example, (C1~C 20 ) alkyl, (C1~C 15) alkyl, (C1~C 10 )alkyl or (C1~C3)alkyl). In 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 embodiments, R'' is independently an unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not contain any heteroatoms. Alkyl: As used herein, the term "alkyl" means acyclic straight-chain hydrocarbon groups and branched hydrocarbon groups, for example "(C1-C 30 "Alkyl" refers to an alkyl group having 1 to 30 carbon atoms. Alkyl groups 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 group having 1 to 6 carbon atoms. Other alkyl groups will be readily apparent to those skilled in the art, given the advantages of this disclosure. Alkyl groups can be unsubstituted or substituted with one or more substituents, as described herein. For example, an alkyl 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 (for example, (C1~C 20 ) alkyl, (C1~C 15 ) Alkyl, C1~C 10 It is an alkyl or C1-C3 alkyl. In the embodiment, R'' is independently an unsubstituted alkyl (e.g., unsubstituted (C1-C 20 ) alkyl, (C1~C 15 ) alkyl, (C1~C 10The alkyl group is either (C1-C3) alkyl or (C1-C3) alkyl. In embodiments, R'' is independently an unsubstituted (C1-C3) alkyl group. In embodiments, the alkyl group is substituted (for example, with the 1, 2, 3, 4, 5, or 6 substituents described herein). In embodiments, the alkyl group is substituted with an -OH group, which may also be referred to herein as a "hydroxyalkyl" group, where the prefix represents the -OH group and "alkyl" is as described herein.

[0045] As used herein, "alkyl" also refers to a linear or branched saturated hydrocarbon group having 1 to 50 carbon atoms ("(C1-C 50 This refers to an alkyl radical having 1 to 40 carbon atoms. In some embodiments, an 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)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, alkyl groups have 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), isopropyl (C3), 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). Further examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise specified, each example of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted (C1-C 50 ) is alkyl. In certain embodiments, the alkyl group is substituted (C1~C 50 It is alkyl.

[0046] Adding the suffix "-en" to a base indicates that the base is a divalent part; for example, arylene is the divalent part of aryl, and heteroarylene is the divalent part of heteroaryl.

[0047] 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 as used herein, the term "alkynylene" 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, alkenylene, or alkynylene group may contain one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. For example, alkylene, alkenylene, or alkynylene may be substituted with one or more of the following substituents (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 instance of R'' is independently (C1~C 20 )Aliphatic (for example, (C1~C 20 ) alkyl, (C1~C 15 ) alkyl, (C1~C 10 )alkyl or (C1~C3)alkyl). In 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 embodiments, R'' is independently an unsubstituted (C1-C3)alkyl. 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 straight 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 "Alkenyl" refers to an alkenyl group having 2 to 30 carbon atoms. For example, alkenyl groups include propa-2-enyl, buta-2-enyl, buta-3-enyl, 2-methylpropa-2-enyl, hexa-2-enyl, hexa-5-enyl, 2,3-dimethylbuta-2-enyl, etc. In embodiments, the alkenyl contains 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl contains a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated. The alkenyl group may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkenyl group may be substituted with one or more of the following substituents (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 (for example, (C1~C 20 ) alkyl, (C1~C 15 ) alkyl, (C1~C 10 )alkyl or (C1~C3)alkyl). In embodiments, R'' is independently an unsubstituted alkyl (e.g., unsubstituted (C1~C 20 ) alkyl, (C1~C 15 ) alkyl, (C1~C 10The group is an alkyl or (C1-C3) alkyl group. In embodiments, R'' is independently an unsubstituted (C1-C3) alkyl group. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (for example, with the 1, 2, 3, 4, 5, or 6 substituents described herein). In embodiments, the alkenyl group is substituted with an -OH group, which may also be referred to herein as a "hydroxyalkenyl" group, where the prefix represents the -OH group and "alkenyl" is as described herein.

[0048] As used herein, “alkenyl” also refers to 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 radical. 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~C 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 10)Alkenyl). In 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., in 2-butenyl) or terminal (e.g., in 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), pentadienyl (C5), and hexenyl (C6). Further 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 unsubstituted (C2-C2). 50 ) is an alkenyl. In certain embodiments, the alkenyl group is substituted (C2~C 50 ) It is Alkenil.

[0049] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain of straight-chain or branched configuration having one or more carbon-carbon triple bonds present at any stable point along the chain, e.g., "(C2-C 30 ) alkynyl" refers to an alkynyl group having 2 to 30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. In an embodiment, 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 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), and each instance 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 an embodiment, R'' is independently unsubstituted alkyl (e.g., unsubstituted (C1-C 20 ) alkyl, (C1-C 15 ) alkyl, (C1-C 10 [[ID=1?]]) alkyl or (C1-C3) alkyl). In an embodiment, R'' is independently unsubstituted (C1-C3) alkyl. In an embodiment, alkynyl is unsubstituted. In an embodiment, alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0050] As used herein, "alkynyl" also means a straight-chain 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) "(C2-C 50This refers to the radical of an alkynyl group. An alkynyl group having one or more triple bonds and one or more double bonds is also called an "en-yne". In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("(C2-C 40 )alkynyl). In some embodiments, the alkynyl group has 2 to 30 carbon atoms ("(C2~C 30 )alkynyl). In some embodiments, the alkynyl group has 2 to 20 carbon atoms ("(C2~C 20 )alkynyl). In some embodiments, the alkynyl group has 2 to 10 carbon atoms ("(C2~C 10)alkynyl). 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-carbon triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 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). Examples of (C2-C6) alkynyl groups 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 independently either unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, the alkynyl group is unsubstituted (C2-C 50 ) is an alkynyl group. In certain embodiments, the alkynyl group is substituted (C2~C 50 ) This is Alkinnil.

[0051] Aryl: Used alone or as part of a larger term such as “aralkyl”, the term “aryl” refers to a monocyclic, bicyclic, or tricyclic carbocyclic system having a total of 6 to 14 ring members, wherein the system has a single bond to 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 some 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 ring systems in which the aryl ring defined above is condensed with one or more carbocyrillic or heterocyclyl groups, and the radical or bond site is on the aryl ring, in which case the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Exemplary aryls include phenyl, naphthyl, and anthracene.

[0052] As used herein, “aryl” also refers to 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 6 to 14 ring carbon atoms and zero heteroatoms provided to the aromatic ring system (“(C6~C 14 )aryl). 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" means that the aryl ring defined above is condensed with one or more carbocyclic or heterocyclic groups, and the radical or bonding point is on the aryl ring. In such cases, the number of carbon atoms also includes ring systems that continue to specify the number of carbon atoms in the aryl ring system. Unless otherwise specified, each example of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl group is unsubstituted (C6-C 14 ) aryl. In certain embodiments, the aryl group is substituted (C6-C 14 ) aryl.

[0053] Arylene: As used herein, the term "arylene" refers to an aryl group that is divalent (i.e., has two bonding points in the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted phenylene or substituted phenylene).

[0054] Carbocyclic: As used herein, "carbocyclic" or "carbocyclic ring" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("(C3-C 10 ) carbocyclic") and zero heteroatoms in the non-aromatic ring system. In some embodiments, the carbocyclic group has 3 to 8 ring carbon atoms ("(C3-C8) carbocyclic"). In some embodiments, the carbocyclic group has 3 to 7 carbon atoms ("(C3-C7) carbocyclic"). In some embodiments, the carbocyclic group has 3 to 6 carbon atoms ("(C3-C6) carbocyclic"). In some embodiments, the carbocyclic group has 4 to 6 carbon atoms ("(C4-C6) carbocyclic"). In some embodiments, the carbocyclic group has 5 to 6 carbon atoms ("(C5-C6) carbocyclic"). In some embodiments, the carbocyclic group has 5 to 10 carbon atoms ("(C5-C 10("carbocyclic"). Exemplary (C3-C6) carbocyclic groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Exemplary (C3-C8) carbocyclic groups include, but are not limited to, the aforementioned (C3-C6) carbocyclic groups, and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrieneyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), etc. Exemplary (C3-C 10 ) carbocyclic groups include, but are not limited to, the aforementioned (C3-C8) carbocyclic groups, and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), etc. As shown in the above examples, in certain embodiments, the carbocyclic group is either monocyclic ("monocyclic carbocyclic") or polycyclic (e.g., bicyclic system ("bicyclic carbocyclic") or tricyclic system (including fused, bridged or spiro ring systems such as "tricyclic carbocyclic", etc.), and can be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclic" means that the carbocyclic ring defined above is fused with one or more aryl groups or heteroaryl groups, and the radical or bonding point is on the carbocyclic ring. In such cases, the number of carbon atoms also includes ring systems that continue to specify the number of carbon atoms in the carbocyclic ring system. Unless otherwise specified, each instance of the carbocyclic group is independently unsubstituted ("unsubstituted carbocyclic") or substituted with one or more substituents ("substituted carbocyclic"). In certain embodiments, the carbocyclic group is unsubstituted (C3-C 10) is a carbocyclyl. In certain embodiments, the carbocyclyl group is substituted (C3~C 10 It is carbocyclyl.

[0055] In some embodiments, "carbocyrill" or "carbocyclic" is referred to as "cycloalkyl," that is, a monocyclic saturated carbocyclyl group having 3 to 10 ring carbon atoms ("(C3~C 10 )cycloalkyl). In some embodiments, the cycloalkyl group has 3 to 8 carbon atoms ("(C3-C8)cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 carbon atoms ("(C3-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 4 to 6 carbon atoms ("(C4-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 carbon atoms ("(C5-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 10 carbon atoms ("(C5-C 10 (C5-C6) cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). (C3-C6) cycloalkyl groups include the aforementioned (C5-C6) cycloalkyl groups, as well as cyclopropyl (C3) and cyclobutyl (C4). (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 ) is a cycloalkyl. In certain embodiments, the cycloalkyl is substituted (C3~C 10 It is a cycloalkyl compound.

[0056] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0057] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyls may optionally include monocyclic, bicyclic, or tricyclic rings, each ring preferably having 3 to 6 ring members. Examples of heteroalkyls include polyethers such as methoxymethyl and ethoxyethyl.

[0058] Heteroalkylene: When used herein, the term "heteroalkylene" refers to the divalent form of the heteroalkyl group described herein.

[0059] Heteroaryl: As used herein, the term "heteroaryl" refers to a fully unsaturated heteroatom-containing ring in which at least one ring atom is a heteroatom such as nitrogen and oxygen, but is not limited to these.

[0060] 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) in an 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 long 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 the heteroaryl ring as defined above is condensed with one or more carbocyryl or heterocyclyl groups, and the bond site is on the heteroaryl ring, in which case the number of ring members continues to specify the number of ring members in the heteroaryl ring system. "Hyperaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, and the bond site is on an aryl or heteroaryl ring, in which case the number of ring members specifies the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. A polycyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) may have a bond site on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

[0061] 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 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 heteroaryl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each example of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl group.

[0062] 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. Examples of seven-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Examples of 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranil, benzoisofuranil, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolidinyl, and purinyl. Examples of 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 phenadinil.

[0063] As used herein, “heterocyclyl” or “heterocyclic” refers to a 3- to 14-membered non-aromatic cyclic radical 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 (“3- to 14-membered heterocyclyl”). In heterocyclyl groups containing one or more nitrogen atoms, the bond sites may be carbon atoms or nitrogen atoms, as long as their valence allows. A heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a bicyclic system ("bicyclic heterocyclyl") or a tricyclic system (condensed, bridged, or spirocyclic systems such as "tricyclic heterocyclyl"), and may be saturated or contain one or more carbon-carbon double or triple bonds. A heterocyclyl polycyclic ring system may contain one or more heteroatoms in one or both rings. A "heterocyclyl" is a ring system in which the heterocyclyl ring as defined above is condensed with one or more carbocykyl groups, and the bond site is on the carbocykyl ring or the heterocyclyl ring, or as described above. The defined heterocyclyl ring is condensed with one or more aryl or heteroaryl groups, and the bonding site includes ring systems on the heterocyclyl ring, in which case the number of ring members continues to specify the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of a heterocyclyl group is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 14-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 14-membered heterocyclyl.

[0064] In some embodiments, the heterocyclyl group is a 5-10 member 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 ("5-10 member heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-8 member 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 ("5-8 member heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-6 member 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 ("5-6 member 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.

[0065] Examples of three-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxyranyl, and thiorenyl. Examples of four-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Examples of five-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Examples of five-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Examples of five-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Examples of six-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranil, dihydropyridinyl, and thianil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinil, dithianil, and dioxanil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinyl. Examples of seven-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanil, oxepanil, and thiepanil. Examples of eight-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azokanil, oxecanil, and thiokanil.Examples of bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydroclomenyl, octahydroisoclomenyl, decahydronaphthyldinyl, decahydro-1,8-naphthyldinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, clomenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7- Examples include, but are not limited to, tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-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, and 1,2,3,4-tetrahydro-1,6-naphthilidinyl.

[0066] Heterocycloalkyl: As used herein, the term "heterocycloalkyl" refers to a non-aromatic ring in which at least one atom is a heteroatom such as nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocycloalkyl groups may be substituted or unsubstituted.

[0067] As can be understood from the foregoing, the alkyl, alkenyl, alkynyl, acyl, carbocyryl, heterocyclyl, aryl, and heteroaryl groups as defined herein are optionally substituted in certain embodiments. Optionally substituted means a group that may be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl, a "substituted" or "unsubstituted" alkenyl, a "substituted" or "unsubstituted" alkynyl, a "substituted" or "unsubstituted" heteroalkyl, a "substituted" or "unsubstituted" heteroalkenyl, a "substituted" or "unsubstituted" heteroalkynyl, a "substituted" or "unsubstituted" carbocyryl, a "substituted" or "unsubstituted" heterocyclyl, a "substituted" or "unsubstituted" aryl, or a "substituted" or "unsubstituted" heteroaryl group). Generally, the term "substituted" means that at least one hydrogen atom present on the group is spontaneously subject to conversion by an acceptable substituent, such as a compound that is stable when substituted, such as by reconstitution, cyclization, removal, or other reactions. This means that the group is substituted with substituents that result in a compound that does not exist. Unless otherwise indicated, a “substituted” group has substituents at one or more of its substituted positions, and if two or more positions in any given structure are substituted, the substituents are either the same or different at each position. The term “substituted” is intended to include substitution with any of the substituents described herein that result in the formation of a stable compound, among all acceptable substituents of an organic compound. The present invention intends any and 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 preferred substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0068] Examples of carbon atom substituents include halogens, -CN, -NO2, -N3, -SO2, -SO3H, -OH, and -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3+X - , -N(OR cc )R bb -SeH, -SeR aa、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa-SC(=O)R aa -P(=O)2R aa -OP(=O)2R aa -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 14 )Carbocyclyl, 3-14 membered ring heterocyclyl, (C6-C 14 Examples include, but are not limited to, aryls and 5-14 membered heteroaryl rings. Each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl ring independently contains 0, 1, 2, 3, 4, or 5 R groups. dd It is substituted with the base, Alternatively, the two geminal hydrogens on a carbon atom form the group =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa ,=NNR bb C(=O)OR aa ,=NNR bb S(=O)2R aa ,=NR bb , or =NOR cc Replaced by, R aaEach example is independent of (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 ) Carbocyclyl, 3-14 member heterocyclyl, (C6-C 14 ) Selected from aryls and 5-14 member heteroaryls, or two R aa The groups are linked together to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R groups. dd It is substituted with the base, R bb Each example is independently of hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa -C(=O)N(R cc )2, -CO2R aa , -SO2R aa -C(=NR cc )OR aa -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc Selected from -SO2OR cc -SOR aa -C(=S)N(R cc )2, -C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3-C 10 ) Carbocyclyl, 3-14 member heterocyclyl, (C6-C 14 )aryl, and 5-14 member heteroaryl, or two R bbThe groups, together with the heteroatoms to which they are bonded, form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocykryl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R groups. dd It is substituted with the base, R cc Each example is independent of hydrogen, (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 ) Carbocyclyl, 3-14 member heterocyclyl, (C6-C 14 ) Selected from aryls and 5-14 member heteroaryls, or two R cc The groups, together with the heteroatoms to which they are bonded, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocykryl, heterocyclyl, aryl, and heteroaryl has 0, 1, 2, 3, 4, or 5 R groups. dd It is independently substituted at the base, R dd Each example is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3+X - , -N(OR ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee , -OCO2R ee -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(Rff )2, -C(=NR ff )OR ee -OC(=NR ff )R ee -OC(=NR ff )OR ee -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 )Carbocyclyl, 3-10 member heterocyclyl, (C6-C 10 ) Selected from aryls and 5-10 membered heteroaryls, each alkyl, alkenyl, alkynyl, carbocykrill, heterocyclyl, aryl, and heteroaryl has 0, 1, 2, 3, 4, or 5 R gg Either independently substituted at the base, or two geminal R dd Substituents can link together to form =O or =S. R ee Each example is independent of (C1~C 50 ) Alkyl, (C2~C50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 )Carbocyclyl, (C6~C 10 ) Selected from aryls, 3-10 membered heterocyclines, and 3-10 membered heteroaryls, where each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl has 0, 1, 2, 3, 4, or 5 R gg It is independently substituted at the base; R ff Each example is independent of hydrogen, (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 ) Carbocyclyl, 3-10 member heterocyclyl, (C6-C 10 ) Selected from aryls and 5-10 member heteroaryls, or two R ff These groups, together with the heteroatoms to which they are bonded, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocykryl, heterocyclyl, aryl, and heteroaryl has 0, 1, 2, 3, 4, or 5 R groups. gg It is independently substituted at the base, R gg Each example is independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -O(C1~C 50 )alkyl, -ON((C1~C 50 )alkyl)2,-N((C1~C 50 )alkyl)2,-N((C1~C 50 )alkyl)3+X - -NH((C1~C 50 )alkyl)2+X - -NH2((C1~C 50 )alkyl)+X - -NH3+X - -N(O(C1~C 50 )alkyl)(((C1~C 50 )alkyl), -N(OH)(((C1~C 50)alkyl), -NH(OH), -SH, -S(C1~C 50 )Alkyl, -SS((C1~C 50 )alkyl), -C(=O)(((C1~C 50 )alkyl), -CO2H, -CO2(((C1~C 50 )alkyl), -OC(=O)((C1~C 50 )alkyl), -OCO2((C1~C 50 )alkyl), -C(=O)NH2, -C(=O)N((C1~C 50 )alkyl)2,-OC(=O)NH((C1~C 50 )alkyl), -NHC(=O)((C1~C 50 )alkyl), -N((C1~C 50 )alkyl)C(=O)((C1~C 50 )alkyl), -NHCO2((C1~C 50 )alkyl), -NHC(=O)N((C1~C 50 )alkyl)2,-NHC(=O)NH((C1~C 50 )alkyl), -NHC(=O)NH2, -C(=NH)O((C1~C 50 )alkyl), -OC(=NH)O((C1~C 50 )alkyl), -OC(=NH)O((C1~C 50 )alkyl, -C(=NH)N((C1~C 50 )alkyl)2,-C(=NH)NH((C1~C 50 )alkyl), -C(=NH)NH2, -OC(=NH)N((C1~C 50 )alkyl)2,-OC(NH)NH((C1~C 50 )alkyl), -OC(NH)NH2, -NHC(NH)N((C1~C 50 )alkyl)2,-NHC(=NH)NH2,-NHSO2((C1~C 50 )alkyl), -SO2N((C1~C 50 )alkyl)2,-SO2NH((C1~C 50 )alkyl), -SO2NH2, -SO2((C1~C 50 )alkyl), -SO2O((C1~C 50)alkyl), -OSO2((C1~C6)alkyl), -SO((C1~C6)alkyl), -Si((C1~C 50 )alkyl)3,-OSi((C1~C6)alkyl)3,-C(=S)N((C1~C 50 )alkyl)2, C(=S)NH((C1~C 50 )alkyl), C(=S)NH2, -C(=O)S((C1~C6)alkyl), -C(=S)S((C1~C6)alkyl), -SC(=S)S((C1~C6)alkyl), -P(=O)2((C1~C 50 )alkyl), -P(=O)((C1~C 50 )alkyl)2,-OP(=O)((C1~C 50 )alkyl)2,-OP(=O)(O(C1~C 50 )alkyl)2, (C1~C 50 ) Alkyl, (C2~C 50 ) Alkenil, (C2~C 50 ) Alkinyl, (C3~C 10 )Carbocyclyl, (C6~C 10 )aryl, 3-10 member heterocyclyl, 5-10 member heteroaryl, or two geminal R gg Substituents can bond to form =O or =S, X - It is a counterion.

[0069] As used herein, the terms "halo" or "halogen" refer to fluorine (fluoro, -F), chlorine (chloro, ~Cl), bromine (bromo, -Br), or iodine (iod, -I).

[0070] As used herein, “counterion” is a negatively charged group that binds to a positively charged quaternary amine to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F - Cl - , Br - , I - ), NO3 - ClO4 - , OH - H2PO4 - HSO4 -Examples include sulfonic acid ions (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, 10-camphorsulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1-sulfonic acid-5-sulfonic acid, ethane-1-sulfonic acid-2-sulfonic acid, etc.) and carboxylic acid ions (e.g., acetic acid, ethanoic acid, propanoic acid, benzoic acid, glyceric acid, lactic acid, tartaric acid, glycolic acid, etc.).

[0071] The nitrogen atom can be substituted or unsubstituted as long as the valence allows, and includes primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include hydrogen, -OH, -OR aa 、-N(R cc )2、-CN、-C(=O)R aa 、-C(=O)N(R cc )2、-CO2R aa 、-SO2R aa 、-C(=NR<着 bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2、-C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R[[ID=H43]] aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2、(C1~C 50 ) alkyl, (C2~C 50 ) alkenyl, (C2~C 50 ) alkynyl, (C3~C 10 ) carbocyclic, 3- to 14-membered heterocyclic, (C6~C 14 ) aryl, and 5- to 14-membered heteroaryl, but are not limited thereto, or two R cc It should be noted that there seems to be an incorrect tag "着0000387" in the original text, which is retained as is during translation. You may want to check and correct this in the original source if necessary.The groups, together with the N atoms to which they are bonded, form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyryl, heterocyclyl, aryl, and heteroaryl has 0, 1, 2, 3, 4, or 5 R atoms. dd It is independently substituted at the base, R aa , R bb , R cc and R dd This is defined as above.

[0072] 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 is incorporated herein by reference.

[0073] For example, an amide group (for example, -C(=O)R aa Examples of nitrogen protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetacetamide (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.

[0074] Carbamate group (e.g., -C(=O)OR) aaExamples of nitrogen protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfo)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluoroenyl methyl carbamate, 2,7-di-t-butyl-[9-(10,10 dioxo-10,10,10,10 tetrahydrothioxantyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), and 2-trimethyl Tylsilyl ethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t- Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamide)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl Dithiocarbamate, benzylcarbamate (Cbz), p-methoxybenzylcarbamate (Moz), p-nitrobenzylcarbamate, 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-dimethylthiophenylcarbamate (Bmpc), 2-phosphonoethylcarbamate (Peoc), 2-triphenylphosphonoisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chloro Monylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 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, 2,2-Dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamide)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamide)propylcarbamate, 1,dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isovorincarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-Methylcyclohexylcarbamate, 1-Methyl-1-cyclopropylmethylcarbamate, 1-Methyl-1(3,5-dimethoxyphenyl)ethylcarbamate, 1-Methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-Methyl-1-phenylethylcarbamate, 1-Methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate and 2,4,Examples include, but are not limited to, 6-trimethylbenzylcarbamate.

[0075] Sulfonamide group (e.g., -S(=O)2R) aa Examples of nitrogen protecting groups include 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), and 2,4,6-trimethylbenzenesulfonamide (Mts). Examples include, but are not limited to, 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.

[0076] Other nitrogen protecting groups include 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-dithiascinimide (DTS), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1, 3,5-Triazacyclohexane-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-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosberylamine, N-tri Phenylmethylamine (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-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)methyl]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(pentaacylchrom- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide,Examples include, but are not limited to, diphenylphosphinamide (DPP), dimethylthiophosphinamide (MPT), diphenylthiophosphinamide (PPT), dialkylphosphoramide, dibenzylphosphoramide, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (NPS), 2-,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

[0077] 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 is incorporated herein by reference.

[0078] Examples of oxygen protecting groups include methyl, methoxylmethyl (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, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, and bis(2-chloro)methyl. Roethoxymethyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidine-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiopyranyl Ophranyl, 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-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 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',4,4''-tris(levrinoyloxyphenyl)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, Benzoisothiazolyl S, S-dioxide, Trimethylsilyl (TMS), Triethylsilyl (TES), Triisopropylsilyl (TIPS), Dimethylisopropylsilyl (IPDMS), Diethylisopropylsilyl (DEIPS), Dimethylhexylsilyl, 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 (rebrinate), 4,4-(ethylenedithio)pentanoate (rebrinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethyl Benzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (PSEC), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate,Alkyl p-methoxybenzyl, alkyl 3,4-dimethoxybenzyl, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyldithiocarbonate, 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 Examples include, but are not limited to, (1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butanoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamide, alkyl N-phenylcarbamate, borate, dimethylphosphintioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (TS).

[0079] 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.

[0080] Examples of sulfur protecting groups include 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-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbonylaminomethyl, phenyl Nylacetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl(2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carboethoxy)ethyl(1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylpropane-2-yl, acetyl, benzoyl, t Examples include, but are not limited to, difluoroacetyl, 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.

[0081] Compound of the present invention Liposome-based vehicles are considered attractive carriers for therapeutic agents and are the subject of ongoing development efforts. While liposome-based vehicles containing specific lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a great need for improvements to 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 encapsulated material into such target cells.

[0082] In particular, there remains a need for cationic lipids effective for transnasal / intrapulmonary delivery of mRNA. Improved lipid compounds exhibiting enhanced pharmacokinetic properties and capable of delivering macromolecules such as nucleic acids to a wide variety of cell types and tissues with enhanced efficiency are still needed. Importantly, there remains a specific need for novel lipid compounds characterized by an improved safety profile and the ability to efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0083] This specification describes a novel class of cationic lipid compounds for improving the in vivo delivery of therapeutic agents such as nucleic acids. In particular, the cationic lipids described herein can be optionally used in combination with other lipids to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic use, such as for disease treatment and prevention (vaccination).

[0084] In embodiments, the compounds of the present invention described herein can provide one or more desired features or properties. That is, in certain embodiments, the compounds of the present invention described herein can be characterized by having one or more properties that give such compounds an advantage compared to other similarly classified lipids. For example, the compounds disclosed herein can enable control and modification of the properties of liposome compositions (e.g., lipid nanoparticles) in which they are components. In particular, the compounds disclosed herein can be characterized by enhanced transfection efficiency and their ability to induce specific biological outcomes. Such outcomes may include, for example, enhanced cellular uptake, endosomal / lysosome disruption ability and / or enhanced intracellular release of encapsulated material (e.g., polynucleotides). The compounds disclosed herein can also be characterized by achieving high levels of peptide or protein expression when mRNA encoding the peptide or protein is delivered by pulmonary delivery (e.g., intratracheal delivery) or intranasal delivery. Furthermore, the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution and efficiency.

[0085] This application demonstrates that the cationic lipids of the present invention are not only easy to synthesize from readily available starting materials, but also possess unexpectedly high encapsulation efficiency.

[0086] In addition, the cationic lipids of the present invention have cleavable groups such as ester groups. These cleavable groups (e.g., esters) are intended to improve biodegradability and therefore contribute to a favorable safety profile of the lipids.

[0087] Compounds that are cationic lipids are provided herein. For example, the cationic lipid of the present invention is a compound having the structure of formula (I): [ka] or containing a pharmaceutically acceptable salt thereof, During the ceremony, [ka] This is selected from optionally substituted arylenes or optionally substituted heteroarylenes. In the formula, L2 is selected from a bond, an optionally substituted (C1-C6) alkylene, or an optionally substituted (C2-C6) alkenylene. In the formula, X 1 It is selected from O, In the formula, R 1 teeth, [ka] And, In the formula, a is selected from 0, 1, 2, 3, 4, or 5. In the formula, R 2 and R 3 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, R 4 and R 5 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, L1 is selected from D or E-L3-C(=O)O-, and the right side of the cited structure is, [ka] It is connected, In the formula, D is selected from -(C1~C3)alkyl-O-, -OC(=O)O-, -SC(=O)O-, -OC(=O)S-, -(C1~C3)alkyl-OC(=O)O-, or -C(=O)O-, where the right side of each specified structure is: [ka] It is connected, In the formula, E is selected from -O- or -OC(=O)-, and the right side of each structure is bonded to L3. In the formula, L3 is selected from (C1-C6) alkylenes that are optionally substituted, or from (C2-C6) alkenylenes that are optionally substituted, and [ka] These are naturally occurring sterols or non-naturally occurring sterols.

[0088] In the embodiment, the cationic lipid of the present invention is a compound having the structure of formula (Ia): [ka] or a pharmaceutically acceptable salt thereof.

[0089] In the embodiment, the cationic lipid of the present invention is a compound having the structure of formula (Ia1): [ka] or a pharmaceutically acceptable salt thereof.

[0090] In the embodiment, the cationic lipid of the present invention is a compound having the structure of formula (Ib): [ka] or a pharmaceutically acceptable salt thereof.

[0091] In the embodiment, the cationic lipid of the present invention is a compound having the structure of formula (IB1): [ka] or a pharmaceutically acceptable salt thereof.

[0092] In the embodiment, [ka] This is an aryl that is optionally substituted. In the embodiment, [ka] This is a heteroarylene that is optionally substituted.

[0093] In the embodiment, [ka] However, it is selected from optionally substituted phenylene, optionally substituted pyridinylene, optionally substituted pyrrolylene, optionally substituted thiophenylene, optionally substituted furanylene, optionally substituted thiazolylen, optionally substituted imidazolylen, optionally substituted indolylene, optionally substituted tetrazolylen, optionally substituted piperidinylene, or optionally substituted pyrrolidinylene. In embodiments, [ka] This is phenylene that has been optionally substituted.

[0094] In the embodiment, [ka] teeth, [ka] [ka] Selected from the above, the right-hand side of each illustrated structure is connected to L2. In the formula, X 2 is N or -C(R 16 )- and, In the formula, X 3 is N or -C(R 17 )- and, In the formula, X 4 is NH, O, or S, In the formula, X 5 is N, and In the formula, R 13 , R 14 , R 15 , R 16 , R 17 、 R 18 , R 19 , R20 and R 21 If present, each is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy. In the embodiment, [ka] teeth, [ka] Therefore, the right-hand side of the illustrated structure is connected to L2.

[0095] In this embodiment, X 2 is N. In this embodiment, X 2 is -C(R 16 )-. In this embodiment, X 3 is N. In this embodiment, X 3 is -C(R 17 )-. In this embodiment, X 4 is NH. In this embodiment, X 4 is O. In this embodiment, X 4 S is.

[0096] In the embodiment, [ka] teeth, [ka] And in the formula, X 3 is -C(R 17 )- and R 13 and R 17 H is R 14 and R 16 These are (C1-C6) alkoxy molecules that have been selectively substituted, and the right side of the illustrated structure is bonded to L2.

[0097] In the embodiment, [ka] teeth, [ka] And in the formula, R 13 ~R 15 , R 17 or R 18 Two of the substituents are absent, one of the absent substituents is substituted by a bond with L1, and the other absent substituent is substituted by a bond with L2. In the formula, X 2 is N or -C(R 16 )- and In the formula, R 13 , R 14 , R 15 , R 16 , R 17 and R 18 If present, each of these is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy.

[0098] In this embodiment, X 2 is N. In this embodiment, X 2 is -C(R 16 )-is.

[0099] In the embodiment, [ka] teeth, [ka] Therefore, the right-hand side of the illustrated structure is connected to L2.

[0100] In the embodiment, L2 is a bond. In the embodiment, L2 is an optionally substituted (C1-C6) alkylene. In the embodiment, L2 is an optionally substituted (C2-C6) alkenylene.

[0101] In this embodiment, R 1 teeth, [ka] In this embodiment, R 1 teeth, [ka] That is the case.

[0102] In this embodiment, a is 0. In this embodiment, a is 1. In this embodiment, a is 2. In this embodiment, a is 3. In this embodiment, a is 4. In this embodiment, a is 5.

[0103] In this embodiment, R 2 is hydrogen. In this embodiment, R 2 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 3 is hydrogen. In this embodiment, R 3 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 4 is hydrogen. In this embodiment, R 4 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 4 is methyl. In this embodiment, R 5 is hydrogen. In this embodiment, R 5 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 5 is methyl. In this embodiment, R 4 and R 5 is methyl. In embodiments (for example, compounds of formula (I), formula (Ib), or formula (Ib1)), R 2 , R 3 , R 4 and R 5 It is hydrogen.

[0104] In the embodiment (for example, the compound of formula (I)), R 1 teeth, [ka] In the embodiment (for example, the compound of formula (I)), R 1 teeth, [ka] In the embodiment (for example, the compound of formula (I)), R 1 teeth, [ka] In the embodiment (for example, the compound of formula (I)), R 1 teeth, [ka] That is the case.

[0105] In the embodiment, L1 is D. In the embodiment, D is -(C1~C3)alkyl-O-, and the right side of the enumerated structure is [ka] In this embodiment, D is -OC(=O)O-. In this embodiment, D is -SC(=O)O-, and the right side of the enumerated structure is [ka] In this embodiment, D is -OC(=O)S-, and the right side of the enumerated structure is [ka] In this embodiment, D is -(C1~C3)alkyl-OC(=O)O-, and the right side of the listed structures is [ka] In this embodiment, D is -C(=O)O-, and the right side of the enumerated structure is [ka] That is the case.

[0106] In this embodiment, L1 is E-L3-C(=O)O-, and the right side of the enumerated structure is [ka] In this embodiment, E is -O-. In this embodiment, E is -OC(=O)-, and the right side of the enumerated structure is bonded to L3.

[0107] In the embodiment, L3 is an optionally substituted (C1-C6) alkylene. In the embodiment, L3 is an optionally substituted (C2-C6) alkenylene.

[0108] In embodiments (for example, compounds of formula (I), formula (Ia), formula (Ia1), formula (Ib), or formula (Ib1)), L1 is [ka] In the embodiment (for example, formula (Ia) or formula (Ia1)), L1 is [ka] In the embodiments (for example, formula (I), formula (Ia), formula (Ia1), formula (Ib), or formula (Ib1)), L1 is [ka] The right side of the illustrated structure is [ka] In the embodiments (for example, in formula (Ia), formula (Ia1), formula (Ib), or formula (Ib1), L1 is [ka] The right side of the illustrated structure is [ka] That is the case.

[0109] In the embodiment, [ka] These are naturally derived sterols. In the embodiment, [ka] These are non-naturally derived sterols.

[0110] In the embodiment, [ka] Equation (II): [ka] It has a structure that follows the formula, where R 6 and R 7 Each of these is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy; In the formula, R 8 (C1~C 30 ) alkyl, optionally substituted (C2~C 30 ) Alkenyl, optionally substituted (C2~C 30 ) Alkinyl, optionally substituted (C1~C 30 )alkoxy, optionally substituted (C1~C 10 )Alkylene-C(O)O-Optionally substituted (C1~C 20 ) Selected from alkyl groups; and optionally substituted (C1~C 10 ) is alkylene-C(O)OH, In the formula, R 9 H is R 10 Is it OH, or R 9 and R 10 Both are H, or neither is present, R9 and R 10 If R does not exist, 9 and R 10 A C=C double bond exists between the carbon atoms to which it is bonded, and In the formula, R 11 OH is R 12 Is H or R 11 and R 12 Both are H, or neither is present, R 11 and R 12 If it does not exist, the C=C double bond is R 11 and R 12 It exists between the bonded carbon atoms.

[0111] In the embodiment, [ka] Equation (IIa): [ka] It has a structure that follows [the specified format].

[0112] In this embodiment, R 6 is H. In this embodiment, R 6 is OH. In the embodiment, R 6 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 6 is an optionally substituted (C2-C6) alkenyl. In the embodiment, R 6 is an optionally substituted (C2-C6) alkynyl. In the embodiment, R 6 This is an alkoxy that may be optionally substituted (C1-C6).

[0113] In this embodiment, R 7 is H. In this embodiment, R 7 is OH. In the embodiment, R 7 is an optionally substituted (C1-C6) alkyl group. In the embodiment, R 7 is methyl. In this embodiment, R 7is an (C2-C6) alkenyl that is optionally substituted. In the embodiment, R 7 is an alkynyl (C2-C6) that is optionally substituted. In the embodiment, R 7 These are (C1-C6) alkoxys that are optionally substituted.

[0114] In this embodiment, R 8 These are replaced by arbitrary substitutions (C1~C 30 ) is alkyl. In this embodiment, R 8 (C2~C 30 ) is rukenil. In the embodiment, R 8 (C2~C 30 ) is an alkynyl. In the embodiment, R 8 These are replaced by arbitrary substitutions (C1~C 30 ) is an alkoxy. In this embodiment, R 8 These are replaced by arbitrary substitutions (C1~C 10 )Alkylene-C(O)O- is optionally substituted (C1~C 20 ) is alkyl. In this embodiment, R 8 These are replaced by arbitrary substitutions (C1~C 10 It is alkylene-C(O)OH.

[0115] In this embodiment, R 9 H is R 10 is OH. In the embodiment, R 9 and R 10 Both are H. In the embodiment, R 9 and R 10 Neither of them exists.

[0116] In this embodiment, R 11 OH is R 12 is H. In this embodiment, R 11 and R 12 Both are either H, or neither exists.

[0117] In the embodiment, [ka] This is selected from animal sterols, or in embodiments thereof, or their reduced forms; phytosterols, or in embodiments thereof, or their reduced forms; synthetic sterols (e.g., non-naturally derived), or in embodiments thereof, or their reduced forms; bile acids or their alkyl esters, or their oxidized forms, or their reduced forms. [ka] (Cholesterol) or its oxidized or reduced form; [ka] In the formula, R 22 (C1~C 20 ) Alkyl (alkyl lithocholate) or its oxidized or reduced form; [ka] (Stigmasterol) or its oxidized or reduced form; [ka] (Stigma malt) or its oxidized or reduced form; [ka] (Campesterol) or its oxidized or reduced form; [ka] (Ergosterol) or its oxidized or reduced form; [ka] (Sitosterol) or its oxidized or reduced form; or [ka] Selected from, in the formula, R23 is replaced by H or optionally (C1~C 20 ) Alkyl (cholic acid) or its oxidized or reduced form.

[0118] In the embodiment, [ka] This is an animal sterol or its oxidized or reduced form. In embodiments, [ka] These are animal sterols. In the embodiment, [ka] This is an oxidized form of animal sterol. In an embodiment, [ka] This is a reduced form of animal sterol.

[0119] In the embodiment, [ka] This is a phytosterol or its oxidized or reduced form. In the embodiment, [ka] This is a phytosterol. In the embodiment, [ka] This is an oxidized form of phytosterol. In the embodiment, [ka] This is the reduced form of phytosterols.

[0120] In the embodiment, [ka] This is a synthetic sterol or its oxidized or reduced form. In the embodiment, [ka] It is a synthetic sterol. In the embodiment, [ka] This is the oxidized form of synthetic sterols. In an embodiment, [ka] This is the reduced form of synthetic sterols.

[0121] In the embodiment, [ka] This is a sterol selected from the group consisting of cholesterol, oxidized forms of cholesterol, reduced forms of cholesterol, alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, and sitosterol.

[0122] In the embodiment, [ka] This is a sterol selected from the group consisting of oxidized forms of cholesterol, reduced forms of cholesterol, alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, and sitosterol.

[0123] In the embodiment, [ka] The sterol is selected from the following: [ka] In the formula, R 22 (C1~C20 ) is alkyl (alkyl lithocholate); [ka] In the formula, R 23 is replaced by H or optionally (C1~C 20 Alkyl (cholic acid).

[0124] In the embodiment, [ka] The sterol is selected from the following: [ka] In the formula, R 22 (C1~C 20 ) is alkyl (alkyl lithocholate); [ka]

[0125] In the embodiment, [ka] The sterol is selected from the following: [ka] In the formula, R 22 (C1~C 20 ) is alkyl (alkyl lithocholate); [ka]

[0126] In the embodiment, [ka] The sterol is selected from the following: [ka] In the formula, R 22 (C1~C 20 ) is alkyl (alkyl lithocholate); [ka] In the formula, R 23 is replaced by H or optionally (C1~C 20 Alkyl (cholic acid).

[0127] In the embodiment, [ka] This is a sterol selected from cholesterol, alkyl lithocholate, stigmasterol, stigmatanol, campesterol, ergosterol, and sitosterol, or a sterol selected from any oxidized or reduced form thereof.

[0128] In the embodiment, [ka] This is a sterol selected from cholesterol, alkyllithocholic acid, stigmasterol, stigmatanol, campesterol, ergosterol, and sitosterol.

[0129] In the embodiment, [ka] These are sterols, which are the following oxidized forms: cholesterol, alkyl lithocholates, stigmasterols, stigmatanols, campesterols, ergosterols, or sitosterols.

[0130] In the embodiment, [ka] These are sterols, which are the following reduced forms: cholesterol, alkyl lithocholate, stigmasterol, stigmatol, campesterol, ergosterol, or sitosterol.

[0131] In the embodiment, [ka] This is a sterol which is cholesterol, oxidized cholesterol, or reduced cholesterol. In the embodiment, [ka] This is cholesterol. In the embodiment, [ka] This is an oxidized form of cholesterol. In the embodiment, [ka] This is a reduced form of cholesterol.

[0132] In the embodiment, [ka] Equation (IIb): [ka] It has a structure that follows [the specified format].

[0133] In the embodiment, [ka] Equation (IIc): [ka] It has a structure that follows [the specified format].

[0134] In the embodiment, [ka] These are sterols that are alkyl lysocholates, oxidized forms of alkyl lysocholates, or reduced forms of alkyl lysocholates.

[0135] In the embodiment, [ka] It is a sterol that is stigmasterol, an oxidized form of stigmasterol, or a reduced form of stigmasterol.

[0136] In the embodiment, [ka] It is a sterol that is stigmathanol, an oxidized form of stigmathanol, or a reduced form of stigmathanol.

[0137] In the embodiment, [ka] These are sterols, which are campesterol, an oxidized form of campesterol, or a reduced form of campesterol.

[0138] In the embodiment, [ka] It is a sterol, which is ergosterol, oxidized ergosterol, or reduced ergosterol.

[0139] In the embodiment, [ka] These are sterols, which are sitosterol, an oxidized form of sitosterol, or a reduced form of sitosterol.

[0140] In the embodiment, [ka] This is cholic acid or its alkyl ester, or a sterol which is its oxidized form or reduced form.

[0141] In the embodiment, [ka] These are sterols, which are bile acids or their alkyl esters, or their oxidized or reduced forms.

[0142] In the embodiment, [ka] This is an oxidized form of the sterol described herein. In some embodiments, the oxidized form of the sterol is a modified parent sterol to include additional oxygen-containing groups. In embodiments, the oxidized form of the sterol includes one or more (e.g., 1, 2, 3, or 4) additional hydroxyl groups and / or carbonyl-containing groups (e.g., ketone, aldehyde, carboxylic acid, or carboxylic acid ester moieties) compared to the parent sterol. In some embodiments, the oxidized form of the sterol is a modified parent sterol to include unsaturated carbon-carbon bonds (e.g., carbon-carbon double bonds). In embodiments, the oxidized form of the sterol includes one or more (e.g., 1, 2, or 3) additional carbon-carbon double bonds compared to the parent sterol.

[0143] In the embodiment, [ka] is a reduced form of sterol as described herein. In some embodiments, the reduced form of sterol is a modified parent sterol that contains fewer oxygen-containing groups. In embodiments, the reduced form of sterol contains fewer hydroxyl groups and / or carbonyl-containing groups (e.g., ketone, aldehyde, carboxylic acid, or carboxylic acid ester moieties) compared to the parent sterol. In some embodiments, the reduced form of sterol is a modified parent sterol that contains fewer unsaturated carbon-carbon bonds (e.g., carbon-carbon double bonds). In embodiments, the reduced form of sterol contains a reduction in the number of carbon-carbon double bonds (e.g., fewer than 1, 2, or 3) compared to the parent sterol.

[0144] In this embodiment, the substituents are not optionally substituted.

[0145] In embodiments, the cationic lipid of the present invention has any one of the structures in Table A, or a pharmaceutically acceptable salt thereof.

[0146] In the embodiments, the present invention comprises a cationic lipid, and the following: (i) One or more noncationic lipids, (ii) One or more cholesterol-based lipids, (iii) One or more PEG-modified lipids, A composition comprising the above is provided herein.

[0147] In the embodiment, the cationic lipid of the present invention (for example, compound III or compound IV) and further, (i) One or more noncationic lipids, (ii) A composition comprising one or more PEG-modified lipids is provided herein.

[0148] In embodiments, the composition is lipid nanoparticles, optionally liposomes. In embodiments, one or more cationic lipids constitute about 30 mol% to 60 mol% of the lipid nanoparticles. In embodiments, one or more non-cationic lipids account for 10 mol% to 50 mol% of the lipid nanoparticles. In embodiments, one or more PEG-modified lipids account for 1 mol% to 10 mol% of the lipid nanoparticles. In embodiments, cholesterol-based lipids account for 10 mol% to 50 mol% of the lipid nanoparticles.

[0149] In embodiments, lipid nanoparticles encapsulate nucleic acids, optionally mRNA encoding peptides or proteins. In embodiments, the peptide is an antigen. In embodiments, lipid nanoparticles encapsulate mRNA encoding peptides or proteins. As used herein, the term “encapsulation rate” refers to the proportion of the therapeutic agent (e.g., mRNA) effectively encapsulated within a liposome-based vehicle (e.g., lipid nanoparticles) relative to the initial proportion of the therapeutic agent present in the lipid phase. In embodiments, lipid nanoparticles have an encapsulation rate of at least 50% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 55% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 60% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 65% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 70% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 75% mRNA. In embodiments, lipid nanoparticles have an encapsulation rate of at least 80% mRNA. In the embodiments, the lipid nanoparticles have an encapsulation rate of at least 85% mRNA. In the embodiments, the lipid nanoparticles have an encapsulation rate of at least 90% mRNA. In the embodiments, the lipid nanoparticles have an encapsulation rate of at least 95% mRNA. In the embodiments, the encapsulation rate is calculated by performing a Ribogreen assay (Invitrogen) with or without the presence of 0.1% Triton-X 100.

[0150] In the embodiment, the composition of the present invention is for use in therapeutic purposes.

[0151] In embodiments, the compositions of the present disclosure are for use in methods of treating or preventing diseases that are suitable for treatment or prevention with mRNA-encoded peptides or proteins, and which optionally include (a) protein deficiencies affecting the liver, lungs, brain or muscles, (b) autoimmune diseases, (c) infectious diseases, or (d) cancer.

[0152] In embodiments, a method for treating or preventing a disease is provided, the method comprising administering a composition of the present invention to a subject in need thereof, the disease being suitable for treatment or prevention with a peptide or protein encoded by mRNA, and optionally, the disease being (a) an optionally selected protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infection, or (d) cancer.

[0153] In the embodiments, the composition is administered optionally by spray, either intranasally, intravenously, intrathecally, intramuscularly, or pulmonaryly. In the embodiments, the composition is administered nasally. In the embodiments, the composition is administered optionally by spray, either pulmonaryly or pulmonaryly.

[0154] Exemplary Compounds In embodiments, the cationic lipid of the present invention comprises a compound selected from those shown in Table A, or a pharmaceutically acceptable salt thereof.

[0155] Examples of exemplary compounds include those listed in Table A, or their pharmaceutically acceptable salts.

[0156] [Table 1]

[0157] Any of the compounds specified in Table A above (I-II and V-VI) can be provided in the form of a pharmaceutically acceptable salt, and such salts are intended to be included by the present invention.

[0158] 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.

[0159] nucleic acid The compounds of the present invention described herein can be used to prepare compositions useful for nucleic acid delivery.

[0160] 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 carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain 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 inhibitor. The exact conditions vary depending on the specific application.

[0161] In some embodiments, the DNA template is transcribed in vitro for mRNA preparation according to the present invention. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, mutant T7, or SP6 promoter, followed by the desired mRNA and a desired nucleotide sequence for terminal signaling.

[0162] The present invention allows for the determination of a desired mRNA sequence and its incorporation 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 a degenerate genetic code. An optimization algorithm can then be used to select suitable codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content, while simultaneously taking into account the frequency of tRNA according to the codon usage. 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 structure analysis can also be used to calculate stabilization and destabilization properties, or regions of RNA, respectively.

[0163] modified mRNA In some embodiments, the mRNA according to the present invention may be synthesized as unmodified or modified mRNA. Modified mRNA includes nucleotide modifications in RNA. Therefore, modified mRNA according to the present invention may include nucleotide modifications, such as skeletal modifications, sugar modifications, or base modifications. In some embodiments, the mRNA may be derived from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) containing purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as from modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, and 2-methylthio-N-6-isopentenyl -Adenine, N-6-methyl-adenine, N-6-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 Syl, 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-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonyl These can be synthesized as methyluracil, 5-methoxyuracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetate(v), 1-methylpseuduracil, quaosin, β-D-mannosylquaosin, wybutoxosine, and phospholamides, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.The preparation of such analogues is known to those skilled in the art from, for example, U.S. Patent Nos. 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, U.S. Patent No. 5,262,530 and U.S. Patent No. 5,700,642, and these disclosures are incorporated by reference as a whole.

[0164] Cationic lipids and nucleic acid pharmaceutical formulations In certain embodiments, the compounds of the present invention described herein, and pharmaceutical and liposome compositions comprising 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 the subsequent transfection of one or more target cells. For example, in certain embodiments, the cationic lipids described herein (and liposome compositions comprising such lipids, etc.) are characterized by providing one or more release properties that give such compounds advantages over receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and macropinocytosis, fusion, endosomal or lysosome disruption, and other similarly classified lipids.

[0165] According to the present invention, nucleic acids described herein, for example, mRNA encoding proteins described herein (e.g., full-length, fragment, or partial protein), can be delivered via a delivery vehicle containing the compounds of the present invention described herein.

[0166] As used herein, the terms “delivery vehicle,” “transport vehicle,” “nanoparticles,” or grammatical synonyms are to be used interchangeably.

[0167] 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) is further: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) One or more cholesterol-based lipids and / or (iv) May contain one or more PEG-modified lipids.

[0168] For example, the present invention provides a composition (e.g., a pharmaceutical composition) comprising a compound described herein (e.g., compound III or compound IV) and one or more polynucleotides. The composition (e.g., a pharmaceutical composition) is further described below: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) One or more PEG-modified lipids, It may include.

[0169] In certain embodiments, the compositions exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally include the step of contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a compound described herein that encapsulates one or more polynucleotides), thereby transfecting one or more target cells with the encapsulated material therein (e.g., one or more polynucleotides). As used herein, the terms “transfect” or “transfection” refer to the intracellular introduction of one or more encapsulation materials (e.g., nucleic acids and / or polynucleotides) into a cell (e.g., a target cell). The introduced polynucleotides may be maintained stably or transiently within the target cell. The term “transfection efficiency” refers to the relative amount of such encapsulation 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 target cells 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 pathological site and subsequently expressed, while minimizing potential systemic adverse effects or toxicity associated with the compound or its encapsulated contents.

[0170] For example, after transfection of one or more target cells with polynucleotides encapsulated in one or more lipid nanoparticles containing a pharmaceutical or liposome composition disclosed herein, the production of products encoded by such polynucleotides (e.g., polypeptides or proteins) may be stimulated, and the ability of such target cells to express polynucleotides and produce, for example, the polypeptide or protein of interest may be enhanced. For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of proteins or enzymes encoded by such mRNA.

[0171] 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, and spleen. In embodiments, the delivery vehicle described herein (e.g., liposome delivery vehicle) may be prepared to preferentially distribute to the lungs. In 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 can be delivered to and / or transfected to target cells or tissues. In some embodiments, the encapsulated polynucleotides (e.g., mRNA) may be expressed by target cells and produce (and possibly excreted) functional polypeptide products, 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.

[0172] Liposome delivery medium In some embodiments, the composition is a suitable delivery vehicle. In some embodiments, the suitable delivery vehicle is a liposome delivery vehicle, such as lipid nanoparticles.

[0173] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0174] Enriching a liposome composition with one or more cationic lipids disclosed herein may be used as a means to improve the safety profile or 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). Accordingly, pharmaceutical compositions, particularly liposome compositions, comprising one or more cationic lipids disclosed herein are also intended.

[0175] 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 an encapsulation material (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membrane of such target cells).

[0176] As used herein, liposome delivery vehicles, such as lipid nanoparticles, are typically characterized as microscopic vesicles having an internal aqueous space isolated from an external medium by one or more bilayer membranes. The liposome bilayer is typically formed by amphiphilic molecules containing spatially separated hydrophilic and hydrophobic domains, such as synthetic or naturally occurring lipids (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, liposome delivery vehicles are typically used to deliver desired mRNA to target cells or tissues.

[0177] In certain embodiments, such compositions (e.g., liposome compositions) are loaded with or encapsulated with materials such as one or more biologically active polynucleotides (e.g., mRNA).

[0178] In the embodiment, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a peptide or protein encapsulated within a liposome. In the embodiment, the liposome is as follows: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) One or more cholesterol-based lipids and (iv) comprising one or more PEG-modified lipids, wherein one or more cationic lipids are compounds of the present invention as described herein.

[0179] In the embodiment, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a peptide or protein encapsulated within a liposome. In the embodiment, the liposome is as follows: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) comprising one or more PEG-modified lipids, At least one cationic lipid is one of the compounds of the present invention as described herein.

[0180] In the embodiment, the composition comprises mRNA encoding a peptide or protein (e.g., any peptide or protein described herein). In the embodiment, the composition comprises mRNA encoding a peptide (e.g., any peptide described herein). In the embodiment, the composition comprises mRNA encoding a protein (e.g., any protein described herein).

[0181] In the embodiments, the composition (e.g., a pharmaceutical composition) comprises nucleic acids encapsulated within liposomes, and the liposomes contain compounds described herein.

[0182] In the embodiments, the nucleic acid is mRNA encoding a peptide or protein. In the embodiments, the mRNA encodes a peptide or protein for use in delivery to or treatment of the target lung or lung cells. In the embodiments, the mRNA encodes a peptide or protein for use in delivery to or treatment of the target liver or liver cells. Further exemplary mRNAs are described herein.

[0183] In the embodiment, the liposome delivery vehicle (e.g., lipid nanoparticles) may have a net positive charge.

[0184] In the embodiment, the liposome delivery vehicle (e.g., lipid nanoparticles) may have a net negative charge.

[0185] In the embodiment, the liposome delivery vehicle (e.g., lipid nanoparticles) may have a net neutral charge.

[0186] In the 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.

[0187] 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).

[0188] In 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 (for example, about 0.5% to about 20% by weight) of the total dry weight of all lipids present in the composition (e.g., liposome composition).

[0189] In 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 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.

[0190] In 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).

[0191] In embodiments, the amount of the compounds of the present invention described herein is present in an amount of about 5% by weight, about 10% by weight, about 15% by weight, about 20% by weight, about 25% by weight or less of the total dry weight of the total lipids in the composition (e.g., liposome composition), and is present in an amount of 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 or less.

[0192] In 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 specified herein. In 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 specified herein. In 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, about 10% by weight, about 15% by weight, or up to about 20% by weight of the compound specified herein. In embodiments, this percentage results in an improvement in beneficial effects (e.g., improved delivery to target tissues such as the liver or lungs).

[0193] The amount of the compounds of the present invention described herein in a composition may also be expressed as a percentage ("mol%) of the total molar amount of total lipids in the composition (for example, the total molar amount of all lipids present in the liposome delivery vehicle).

[0194] In 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% (for example, 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.

[0195] In 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 embodiments, the compounds of the present invention described herein are present in amounts of about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 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.

[0196] In certain embodiments, the compounds of the present invention described herein may constitute about 0.1 mol% to about 50 mol%, or 0.5 mol% to about 50 mol%, or about 1 mol% to about 50 mol%, or about 5 mol% to about 50 mol%, or about 10 mol% to about 50 mol%, or about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol%, or about 25 mol% to about 50 mol%, or about 30 mol% to about 50 mol%, of the total amount of lipids in a composition (e.g., a liposome delivery vehicle).

[0197] In certain embodiments, the compounds of the present invention described herein may constitute more than 0.1 mol%, or about 0.5 mol%, or about 1 mol%, about 5 mol%, about 10 mol%, about 20 mol%, about 30 mol%, or about 40 mol% of the total amount of lipids in the lipid nanoparticles.

[0198] In certain embodiments, the compounds described may constitute 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).

[0199] In embodiments, the amount of the compound of the present invention described herein is present in an amount that is 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).

[0200] In embodiments, the amount of the compound of the present invention described herein is present in an amount that is 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% or less of the total molar amount of total lipids in the composition (e.g., liposome composition).

[0201] In this embodiment, this percentage results in improved beneficial effects (e.g., improved delivery to target tissues such as the liver or lungs, optionally the lungs).

[0202] In a typical embodiment, the composition of the present invention (e.g., a liposome composition) is as follows: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) One or more cholesterol-based lipids, (iv) comprising one or more PEG-modified lipids, One or more cationic lipids are compounds of the present invention as described herein.

[0203] In a typical embodiment, the composition of the present invention (e.g., a liposome composition) is as follows: (i) One or more cationic lipids, (ii) One or more noncationic lipids, (iii) comprising one or more PEG-modified lipids, One or more cationic lipids are compounds of the present invention as described herein (e.g., compound III or compound IV).

[0204] For example, a composition suitable for carrying out the present invention has four lipid components, including the compound of the present invention described herein as a cationic lipid component, and further: (i) Noncationic lipids and (ii) Cholesterol-based lipids, (iii) PEG-modified lipids, and

[0205] For example, a composition suitable for carrying out the present invention comprises a compound of the present invention described herein (e.g., compound III or compound IV) as a cationic lipid component, and further comprises three lipid components including the following: (i) Noncationic lipids and (ii) Contains PEG-modified lipids.

[0206] Noncationic lipids may be DOPE or DEPE. Cholesterol-based lipids may be cholesterol. PEG-modified lipids may be DMG-PEG2K.

[0207] 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 a 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 further embodiments, such a pharmaceutical (e.g., liposome) composition comprises: one or more PEG-modified lipids and one or more cholesterol lipids.

[0208] In further embodiments, the pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids and non-cationic lipids. In other embodiments, such a pharmaceutical (e.g., liposome) composition comprises: one or more PEG-modified lipids; and one or more non-cationic lipids. In further embodiments, such a pharmaceutical (e.g., liposome) composition comprises: one or more PEG-modified lipids.

[0209] In the 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.

[0210] In 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, including the compounds of the present invention as described herein as cationic lipid components, and further comprises: (i) Noncationic lipids (e.g., DOPE) (ii) Cholesterol-based lipids (e.g., cholesterol) (iii) comprising a PEG-modified lipid (e.g., DMG-PEG2K).

[0211] In embodiments, a composition (e.g., lipid nanoparticles) that encapsulates nucleic acids (e.g., mRNA encoding peptides or proteins) comprises one or more of the compounds of the present invention described herein (e.g., compound III or compound IV); and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids. Typically, such a composition comprises one of the compounds of the present invention described herein (e.g., compound III or compound IV) as a cationic lipid component, and further comprises three lipid components: (i) noncationic lipids (e.g., DOPE), and (ii) Contains a PEG-modified lipid (e.g., DMG-PEG2K).

[0212] In the embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) are one or more compounds of the present invention as described herein, and the following: (i) Cationic lipids, (ii) Noncationic lipids, (iii) PEGylated lipids, and (iv) comprising one or more lipids selected from the group consisting of cholesterol-based lipids.

[0213] In the embodiments, the lipid nanoparticles that encapsulate nucleic acids (e.g., mRNA encoding peptides or proteins) are one or more compounds of the present invention as described herein (e.g., Compound III or Compound IV), and one or more lipids selected from the group consisting of: (i) Cationic lipids, (ii) noncationic lipids, and (iii) Contains PEGylated lipids

[0214] According to various embodiments, the cationic lipids, non-cationic lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, and their relative molar ratios, are based on the characteristics of the selected lipids, the properties of the target cells, and the characteristics of the nucleic acids to be delivered. Further considerations include, for example, alkyl chain saturation, as well as the size, charge, pH, pKa, membrane fusion activity, and toxicity of the selected lipids. Therefore, the molar ratios can be adjusted as appropriate.

[0215] Cationic lipids In addition to any of the compounds of the present invention described herein, the composition may comprise one or more additional cationic lipids.

[0216] 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 described in the literature, and many are commercially available.

[0217] Suitable additional cationic lipids for use in the composition include those described in the literature.

[0218] 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, zwitterionic, 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), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoyl Examples of noncationic or helper lipids suitable for carrying out the present invention include, but are not limited to, phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (phosphatidyethanolamine) (SOPE), or mixtures thereof. A suitable noncationic or helper lipid for carrying out the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-dielucoyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as the noncationic or helper lipid.

[0219] In some embodiments, the noncationic lipid is a neutral lipid, i.e., a lipid that has no net charge under the conditions under which the composition is formulated and / or administered.

[0220] In some embodiments, noncationic lipids may be present in the composition 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 some embodiments, total noncationic lipids may be present in the composition 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 some embodiments, the percentage of non-cationic 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 non-cationic 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 non-cationic 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 non-cationic 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%.

[0221] 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 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 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.

[0222] Cholesterol-based lipids In some embodiments, a composition comprising the cationic lipid of the present invention (e.g., a liposome composition) further comprises one or more cholesterol-based lipids. “Cholesterol-based lipids” as used herein are distinct from the one or more cationic lipids of the present invention (i.e., formulas (I), (Ia), (Ia1), (Ib), or (Ib1)). 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), β-sitosterol, or the following structures [ka] Examples include imidazole cholesterol esters (ICE) that have [specific properties].

[0223] In some embodiments, cholesterol-based lipids may be present in a molar ratio (mol%) of about 10% to about 60%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30% of the total lipids present in the liposomes. In some embodiments, the proportion of cholesterol-based lipids in lipid nanoparticles may be greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, greater than about 40 mol%, or greater than about 50 mol%. In some embodiments, the proportion of cholesterol-based lipids in lipid nanoparticles may be less than or equal to about 20 mol%, less than or equal to about 30 mol%, less than or equal to about 40 mol%, less than or equal to about 50 mol%, or less than or equal to about 60 mol%.

[0224] In some embodiments, cholesterol-based 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-based 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-based 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.

[0225] 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).

[0226] For example, the use of derivatized lipids such as polyethylene glycol (PEG)-modified phospholipids and derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide) is also intended by the present invention in combination with one or more compounds of the present invention, and in some embodiments in combination with other lipids that together constitute liposomes. 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 )).

[0227] Further PEG-modified lipids intended (also referred to as PEGylated lipids, a term interchangeable with PEG-modified lipids in this specification) include (C6~C 20Examples include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently bonded to a lipid having an alkyl chain of ) length. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means to increase circulating lifespan and increase delivery of the lipid-nucleic acid composition to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly replaced from the formulation in vivo (see U.S. Patent No. 5,885,613).

[0228] 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).

[0229] Pharmaceutical preparations and therapeutic use The compounds of the present invention described herein may be used to prepare compositions that facilitate or enhance the delivery and release of encapsulation materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells) (e.g., for constructing liposome compositions).

[0230] For example, if a liposome composition (e.g., lipid nanoparticles) contains one or more of the compounds disclosed herein or is concentrated with one or more of those compounds, a phase transition in the lipid bilayer of one or more target cells may facilitate the delivery of an encapsulation material (e.g., one or more therapeutic polynucleotides encapsulated in lipid nanoparticles) to one or more target cells.

[0231] 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, and therefore, small amounts of such compositions can be administered to a target to achieve a desired therapeutic response or outcome.

[0232] In certain embodiments, the compounds of the present invention described herein may be used to prepare liposome vehicles characterized by effective nasal delivery of mRNA. In certain embodiments, the compounds of the present invention described herein may be used to prepare liposome vehicles characterized by effective pulmonary delivery of mRNA. In certain embodiments, the compounds of the present invention described herein may be used to prepare liposome vehicles characterized by achieving high levels of peptide or protein expression when mRNA encoding a peptide or protein is delivered by pulmonary delivery (e.g., intratracheal delivery).

[0233] Accordingly, pharmaceutical formulations containing the compounds described herein and the nucleic acids provided by the present invention can be used for the treatment and / or prevention of a variety of diseases. To facilitate in vivo delivery of nucleic acids, the compounds and nucleic acids described herein can be formulated in combination with one or more additional pharmaceutical carriers, targeted ligands, or stabilizing reagents. In some embodiments, the compounds described herein can be formulated via a pre-mixed lipid solution. In other embodiments, compositions containing the compounds described herein can be formulated using post-implantation techniques for nanoparticles into lipid membranes. For drug formulation and administration techniques, refer to “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0234] Preferred routes of administration include, for example, intrapulmonary or intra-intestinal administration including oral, rectal, vaginal, mucosal, intratracheal, or inhalation; and parenteral delivery including intradermal, transdermal (topical), intramuscular, subcutaneous, intrathecal injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal delivery. In detailed embodiments, intramuscular administration is to muscles selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, administration results in delivery of nucleic acids to muscle cells. In some embodiments, administration results in delivery of nucleic acids to hepatocytes (i.e., liver cells).

[0235] 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. Alternatively, the liposomal composition of the present invention may be administered intranasally for vaccination. Diseases or disorders affecting the eye may be treated by administering the liposomal composition of the present invention intravitreously.

[0236] Alternatively, or in addition, the pharmaceutical formulations of the present invention may be administered topically rather than systemically, for example, by direct injection of the pharmaceutical formulation into the targeted tissue (e.g., in a sustained-release formulation). Topical delivery can be achieved in a variety of ways depending on the target tissue. Exemplary tissues to which 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 embodiments, the liver is the targeted tissue. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention can be injected, for example, into a site of injury, symptom onset of disease, or pain; the composition can be provided in lozenges for oral, tracheal, or esophageal application; it can be supplied in liquid, tablet, or capsule form for gastric or intestinal administration; it can be supplied in suppository form for rectal or vaginal application; or even in the eyes, it can be delivered using a cream, eye drops, or even injection.

[0237] Alternatively or additionally, the pharmaceutical formulation of the present invention may be administered intranasally. For example, the pharmaceutical formulation of the present invention may be administered as a nasal spray. Exemplary tissues to which mRNA can be delivered and / or expressed include, but are not limited to, the lungs, heart, liver, and spleen. In this embodiment, the target tissue is the lungs.

[0238] Alternatively, or in addition, the pharmaceutical formulations of the present invention may be administered by pulmonary delivery, optionally by spraying or inhalation of a dry powder. In embodiments, the pharmaceutical formulations of the present invention are administered by pulmonary delivery by spraying. In embodiments, the pharmaceutical formulations of the present invention are administered by pulmonary delivery by inhalation of a dry powder. Exemplary tissues to which mRNA can be delivered and / or expressed include, but are not limited to, the lungs, heart, liver, and spleen. In embodiments, the target tissue is the lungs.

[0239] The compositions described herein may include mRNA encoding peptides (e.g., polypeptides such as proteins) that are described herein.

[0240] In this embodiment, mRNA encodes a polypeptide.

[0241] In the embodiment, mRNA encodes a peptide. In the embodiment, the peptide is an antigen.

[0242] In this embodiment, mRNA encodes a protein.

[0243] The present invention provides a method for delivering a composition having a full-length mRNA molecule encoding a target 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.

[0244] 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 include intranasal, intratracheal, or pulmonary administration by aerosolization, spraying, or infusion of a composition containing mRNA encoding a therapeutic peptide or protein in a suitable transfection or lipid carrier vehicle as described above. In some embodiments, the peptide or protein is encapsulated in liposomes. In some embodiments, the liposomes contain lipids that are the compounds of the present invention. Where used herein, administration of the compounds of the present invention includes administration of a composition containing the compounds of the present invention.

[0245] While local cells and tissues of the lung are potential targets that can function as biological depots or reservoirs for the production and secretion of mRNA-encoded proteins, the applicants have found that by administering the compounds of the present invention to the lungs via aerosolization, spraying, or infusion, non-secretory proteins can be distributed even outside of lung cells. Although we do not wish to be constrained by any particular theory, the nanoparticle compositions of the present invention cross the airway-blood barrier, so it is intended that intact nanoparticles will be transferred to non-lung cells and tissues, such as the heart, liver, and spleen, and the encoded peptides or proteins will be produced in those non-lung 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-lung target cells and / or tissues. They are useful in the management and treatment of numerous diseases. 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 peptides or proteins in the liver, spleen, heart, and / or other non-lung cells. For example, administration of the compounds of the present invention by aerosolization, spraying, or infusion into the lungs would result in the composition itself and its peptide or protein products (e.g., antigens or functional proteins) being detectable in both local cells and tissues of the lungs, and in peripheral target cells, tissues, and organs as a result of the migration of mRNA and delivery vehicles to non-lung cells.

[0246] 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 pulmonary airway blood barrier and be distributed to cells other than local lung cells. Accordingly, the compounds disclosed herein are administered to a target by a pulmonary administration route using various approaches known to those skilled in the art (e.g., by inhalation) and are distributed to both local target cells and tissues of the lung, as well as peripheral non-lung cells and tissues (e.g., liver, spleen, kidney, heart, skeletal muscle cells, 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. Accordingly, the present invention is not limited to the treatment of lung diseases or conditions and can be used as a non-invasive means to promote the delivery of polynucleotides or the production of peptides or proteins encoded therein in terminal organs, tissues, and cells (e.g., hepatocytes) 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.

[0247] After administration of the composition to the subject, the mRNA-encoded peptide or protein product (e.g., functional protein or enzyme) is detectable in peripheral target tissue for at least about 1 to 7 days or longer after administration of the compound to the subject. The amount of peptide or protein product required to achieve the therapeutic effect will vary depending on the condition being treated, the encoded peptide or protein, and the patient's condition. For example, the peptide or protein product may be 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, 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, less The compound may be detectable in terminal target tissue for at least 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 to the subject, at concentrations of 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 (e.g., therapeutic concentration).

[0248] Nucleic acids have been demonstrated to be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and by inhalation or reference of an aerosol mist generated by a liquid sprayer, as described in U.S. Patent No. 5,780,014 incorporated herein.

[0249] In certain embodiments, the compounds of the present invention may be formulated to be delivered to a subject as an aerosol or as a particulate liquid or solid before or at the time of administration. Such compounds may be administered with the help 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 sprayers, ultrasonic sprayers, dry powder inhalers, propellant-type inhalers or ventilators) 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 of the compound and a suitable propellant. In certain embodiments, the compounds of the present invention may be formulated as particulate powders intended for inhalation (e.g., breathable dry particles). In certain embodiments, the compositions of the present invention, formulated as breathable particles, are breathable depending on the subject or delivered using a suitable device (e.g., average D50 or D90 particle sizes of approximately 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 2.5 μm or less). 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 The drug is administered to the subject in a single dose such that a concentration of 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight is administered. In some embodiments, the compounds of the present invention are administered to a subject in one or more doses, such 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]

[0250] While specific compounds, compositions, and methods of the present invention are described along with their specificity according to particular embodiments, the following examples are merely illustrative of the compounds of the present invention and are not intended to limit them.

[0251] List of abbreviations: DCM: Dichloromethane DMAP: 4-dimethylaminopyridine DMF: N,N-dimethylformamide EDC.HCl: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride MeOH: methanol NaH: Sodium hydride NaOH: Sodium hydroxide Na2SO4: Sodium sulfate Pd / C: Palladium Carbon TFA: Trifluoroacetic acid THF: Tetrahydrofuran TLC: Thin-layer chromatography

[0252] Example 1: Synthesis of Compound I For example, compound I can be prepared according to scheme 1 (as shown in Figure 1). Intermediate (3): [ka]

[0253] As shown in Scheme 1, (1) (10.0 g, 25.86 mmol, 1.0 equivalent), (2) (2.58 g, 25.86 mmol, 1.0 equivalent) and 100 ml of dried DCM were added to a 250 ml round-bottom flask equipped with a stirring bar. Then, DMAP (3.16 g, 25.86 mmol, 1.0 equivalent) was added, and the reaction mixture was stirred overnight. After that, the reaction mixture was concentrated, resuspended in ethyl acetate, and washed with 10% citric acid (3 times), followed by brine (3 times). The organic layer was dried over sodium sulfate, filtered, and concentrated. Using silica gel chromatography, the residue was loaded with chloroform and purified by a DCM:methanol gradient (0-2% methanol in DCM) to obtain (3) as a white solid (3.5 g, 40% yield). The structure was confirmed by NMR analysis.

[0254] result: 1 H NMR(400MHz,CDCl3)~11.0(br,1H),5.37(d,1H),4.63(m,1H),2.67(m,2H),2.60(m,2H),2.31(d,2H),2.08 - 1.89(m,2H),1.88 - 1.72(m,3H),1.68-1.4(m,8H),1.39 - 1.20(m,4H),1.19 - 0.78(m,22H),0.67(s,3H).

[0255] (5) Synthesis of compound I: [ka] As shown in Scheme 1, (3) (0.500 g, 1.027 mmol, 1.0 equivalent), dried DCM (5 ml), and 5 drops of DMF were added to a 250 ml round-bottom flask equipped with a stirring bar. The reaction mixture was purged with nitrogen and cooled to 0°C. Next, oxalyl chloride (1.56 g, 12.327 mmol, 12.0 equivalents) was slowly added and stirred at room temperature for 3 hours. The reaction mixture was concentrated, dried DCM was added to the reaction flask, and it was concentrated again. The round-bottom flask was placed under high vacuum for 30 minutes. Meanwhile, in another 250 ml round-bottom flask, (4) (0.276 g, 1.027 mmol, 1.0 equivalent) and dried DCM (5 ml) were added, cooled to 0°C, and purged with nitrogen. Triethylamine (1.15 ml, 8.22 mmol, 8.0 equivalents) was added (until the reaction mixture became clear), then the acid chloride adduct from (3) was dissolved in dry DCM, and (5 ml) was added dropwise to a stirred round-bottom flask and stirred overnight at room temperature. The flask was then concentrated, suspended in ethyl acetate, water was added, and the layers were separated. The aqueous layer was washed again with ethyl acetate, and the combined organic layers were washed with brine (3 times). The layers were dried over sodium sulfate, filtered, concentrated, and the residue was purified by silica gel chromatography. The residue was loaded onto a column with its mobile phase being 100% hexane using DCM, and after loading the residue, it was run for 2 minutes. The impurities were removed to 100% ethyl acetate, and then compound I was obtained as an off-white sticky solid by switching to a methanol / DCM gradient (0-5%) (0.5 g, yield 66%).

[0256] result: 1 H NMR(400MHz,CDCl3),7.31(s,2H),5.36(d,1H),4.64(m,1H),4.44(t,2H),3.85(s,6H),2.96(m,2H),2.73(m,4H),2.36(s,6H),2.32(m,2H),2.05 - 1.91(m,2H),1.90 - 1.74(m,3H),1.70-1.41(m,8H),1.40 - 0.78(m,28H),0.67(s,3H). ESI-MS analysis: Calculated value C 44 H 67 NO8, [M+H] = 738.02, measured value = 738.08.

[0257] Example 2: Synthesis of Compound II For example, compound II can be prepared according to scheme 2 (as shown in Figure 2).

[0258] Intermediate (6): [ka] As shown in Scheme 2, to a stirred solution of cholesterol (1) (5.0 g, 12.95 mmol) in anhydrous THF (50 mL), NaH (60%, dispersed in mineral oil) (1.55 g, 38.8 mmol) was added to the reaction mixture at 0°C, and the mixture was stirred at the same temperature for 30 minutes. Subsequently, tert-butyl acrylate (4.97 g, 38.0 mmol) was added dropwise within 15 minutes. The resulting reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC, and the resulting reaction mixture was cooled to 0°C, quenched with ice water (50 mL), and extracted with ethyl acetate (3 × 100 mL). The total organic matter obtained was combined, dried over Na₂SO₄, and concentrated under reduced pressure. The resulting crude material was purified by column chromatography using 0-5% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain compound (6) (2.2 g, 33%) as a white solid.

[0259] result: 1 H-NMR(400MHz,CDCl3)- δ 5.33(t,J=2Hz,1H),3.76-3.68(m,2H),3.19-3.14(m,1H),2.46(t,J=6. 4Hz,2H),2.36-2.33(m,1H),2.20-2.14(m,1H),2.04-1.94(m,2H),1.89- 1.77(m,3H),1.53-1.48(m,4H),1.45-1.42(m,11H),1.39-1.21(m,6H), 1.18-1.10(m,8H),1.01(s,4H),0.91(d,J=6.4Hz,3H),0.87-0.85(dd,J= 1.6Hz, 6H), 0.67(s, 3H). ESI-MS analysis: Calculated value [M+H] = 515.4, Measured value [M+H] = 515.7

[0260] Intermediate (7): [ka] As shown in Scheme 2, TFA (8.0 ml) was added at 0°C to a stirred solution of compound (6) (1.95 g, 3.78 mmol) in anhydrous DCM (50 mL). The reaction mixture was stirred at room temperature for 4 hours. The progress of the reaction was monitored by TLC. The resulting mixture was concentrated under reduced pressure, and the crude product was crystallized with acetonitrile (20 mL) to obtain compound (7) (1.6 g, 92%) as a white solid.

[0261] result: 1 H-NMR(400MHz,CDCl3)- δ 5.37(d,J=5.2Hz,1H),3.78(t,J=6.4Hz,2H),3.26-3.21(m,1H),2.65(t,J=6Hz,2H),2.36(m,1H),2.22(m,1H),2.04-1.85(m ,6H),1.57-1.26(m,12H),1.20-1.05(m,8H),1.01(s,3H),0.93(d,J=6.4Hz,3H),0.88-0.86(dd,J=1.6Hz,6H),0.69(s,3H). ESI-MS analysis: Calculated value [M+H] = 458.3, Measured value [M+H] = 458.1

[0262] Intermediate (10): [ka] As shown in Scheme 2, 26 mL of oxyl chloride (303.03 mmol), followed by dimethylformamide (0.5 mL), was added to a suspension of syringic acid (20.0 g, 101.01 mmol) in 400 mL of dichloromethane at 0°C, and the resulting mixture was stirred at 20°C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 400 mL of dichloromethane. After cooling to 0°C, 31.0 g of 303.03 mol of 3-(dimethylamino)propan-1-ol was slowly added, and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was poured into a saturated NaHCO3 solution (1000 mL) and extracted with DCM (200 mL x 3). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. DCM:ethyl acetate (1:1, 200 mL) was added to the crude compound, and the mixture was stirred for 10 minutes. The precipitate was filtered, washed with ethyl acetate (20 mL x 2), and dried under vacuum to obtain compound (10) as a white solid (16.0 g, 56%).

[0263] result: 1 H NMR (400MHz, DMSO-d6): δ 9.24(br,1H),7.20(s,2H),4.24(t,J=6.8Hz,2H),3.80(s,6H),2.32(t,J=6.8Hz,2H),2.13(s,6H),1.85-1.78(m,2H). ESI-MS analysis: Calculated value [M+H] = 284.15, measured value = 284.4

[0264] (11) Synthesis of compound II: [ka] As shown in Scheme 2, DMAP (0.026 g, 0.218 mmol) was added to a stirred solution of compounds (7) (0.5 g, 1.09 mmol) and (10) (0.3 g, 1.09 mmol) in anhydrous dichloromethane (20 mL), followed by the single addition of EDC.HCl (0.410 g, 2.18 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (30 mL) and washed with 20 mL of aqueous solution. A sodium bicarbonate solution was extracted using DCM (3 × 50 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-2% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain compound II (0.260 g, 33%) as a white solid.

[0265] result: 1 H-NMR(400MHz,DMSO-d6)- δ 7.31(s,2H),5.34(t,J=4.8Hz,1H),4.37(t,J=6.4Hz,2H),3.87(t,J=6.4Hz,8H),3.26-3.20(m,1H),2.89(t,J=6.8Hz,2H),2.43-2.37(m,3H) ),2.25(s,7H),2.02-1.82(m,7H),1.53-1.03(m,18H),1.01-0.94(m,5H),0.91(d,J=6.4Hz,4H),0.87-0.85(dd,J=1.6Hz,6H),0.67(s,3H). ESI-MS analysis: Calculated value [M+H] = 724.5, measured value = 724.4

[0266] Example 3: Synthesis of Compound III For example, compound III can be prepared according to scheme 3 (as shown in Figure 3). Those skilled in the art will understand that compound V can be prepared by routine modifications of scheme 3.

[0267] Intermediate (6): [ka] As shown in Scheme 3, intermediate (6) was prepared as described in Example 2.

[0268] Intermediate (7): [ka] As shown in Scheme 3, intermediate (7) was prepared as described in Example 2.

[0269] Intermediate (12): [ka] As shown in Scheme 3, 4-hydroxy-3,5-dimethoxybenzoic acid (8) (21.0 g, 106.06 mmol) was stirred in anhydrous dichloromethane (210 mL), to which TEA (32.19 g, 318.18 mmol) was added, followed by the dropwise addition of benzyl bromide (27.2 g, 159.09 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The mixture was concentrated under reduced pressure, the residue was diluted with 10% aqueous NaOH (210 mL), and heated at 70°C for 2 hours. The resulting reaction mixture was cooled to room temperature, diluted with 2N HCl, and extracted with ethyl acetate (3 × 200 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-5% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain 4-(benzyloxy)-3,5-dimethoxybenzoic acid (12) (18.1 g, 59.2%) as a white solid.

[0270] result: 1 HNMR(400MHz,DMSOd6)- δ 12.97(bs,1H),7.44(d,J=7.2Hz,2H),7.38-7.27(m,3H),7.23(s,2H),4.98(s,2H),3.82(s,6H). ESI-MS analysis: Calculated value [M+H] = 288.3, ​​Measured value = 288.01

[0271] Intermediate (14): [ka] As shown in Scheme 3, 4-(benzyloxy)-3,5-dimethoxybenzoic acid (12.0 g, 41.6 mmol) and tert-butyl(2-hydroxyethyl) carbamate (10.1 g, 62.5 mmol) were stirred in anhydrous dichloromethane (240 mL). DMAP (7.62 g, 62.5 mmol) was added, followed by the one-time addition of EDC.HCl (11.98 g, 62.5 mmol) at 0°C. The resulting reaction mixture was allowed to stand at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with water (200 mL) and extracted with DCM (3 × 200 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-5% MeOH in DCM as the eluent. By combining the pure fractions and concentrating under reduced pressure, compound (14) (12.05 g, 66.5%) was obtained as a colorless liquid.

[0272] result: 1 H-NMR(400MHz,DMSOd6)- δ 7.44(d,J=7.2Hz,2H),7.38-7.31(m,3H),7.27(s,2H),7.08(t,J=6.0Hz,1H) ,4.99(s,2H),4.20(t,J=5.2Hz,2H),3.83(s,6H),3.30(m,2H),1.36(s,9H). ESI-MS analysis: Calculated value [M+H] = 432.19, Measured value = 432.08

[0273] Intermediate (15): [ka] As shown in Scheme 3, 5 g of Pd / C (10% and 50% wet) was added to a stirred solution of 2-((tert-butoxycarbonyl)amino)ethyl 4-(benzyloxy)-3,5-dimethoxybenzoate (11.6 g, 26.6 mmol) in methanol (250 mL). The resulting reaction mixture was stirred under an H2 atmosphere for 2 hours. The progress of the reaction was monitored by TLC, and the resulting reaction mixture was filtered on a Celite bed, washed with MeOH (20 mL), and the resulting filtrate was concentrated under reduced pressure to obtain (15) (9.1 g, 99%) as a pink solid.

[0274] result: 1 H-NMR(400MHz,DMSOd6)- δ 9.32(s,1H),7.24(s,2H),7.03(t,J=6.0Hz,1H),4.16(t,J=5.2Hz,2H),3.83(s,6H),3.30(m,2H),1.36(s,9H). ESI-MS analysis: Calculated value [M+H] = 342.15, Measured value = 342.07

[0275] Intermediate (16): [ka] As shown in Scheme 3, DMAP (0.561 g, 4.6 mmol) was added to a stirred solution of (7) (1.4 g, 3.1 mmol) and (15) (1.05 g, 3.1 mmol) in anhydrous dichloromethane (20 mL), followed by the single addition of EDC.HCl (0.88 g, 4.6 mmol) at 0°C. The resulting reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 × 50 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-2% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain compound (16) (1.3 g, 54%) as a colorless liquid.

[0276] result: 1 H-NMR(400MHz,DMSOd6)- δ 7.31(s,2H),5.34(t,J=2.4Hz,1H),4.80(br,1H),4.38(t,J=6Hz,2H),3.89(m,1H),3.86(s,6H) ,3.53(m,2H),3.24-3.20(m,1H),2.89(t,J=6.8Hz,2H),2.45-2.36(m,1H),2.28-2.18(m,1H),2. 05-1.78(m,6H),1.57(br,4H),1.54-1.45(m,4H),1.43(s,9H),1.39-1.30(m,3H),1.28-1.22(m, 2H),1.23-1.02(s,8H),1.01(s,3H),0.91(d,J=6.8Hz,3H),0.86(dd,J=5.2Hz,6H),0.67(s,3H). ESI-MS analysis: Calculated value [M+H] = 782.5, Measured value = 782.6

[0277] Intermediate (17): [ka] As shown in Scheme 3, compound (16) (0.7 g, 0.89 mmol) was stirred in anhydrous dichloromethane (35 mL), to which 4N HCl in 1,4-dioxane (14 mL) was added dropwise at 0°C. The resulting reaction mixture was stirred at room temperature for 6 hours, and the progress of the reaction was monitored by TLC. The resulting reaction mixture was concentrated under reduced pressure to obtain compound (17) (0.6 g crude product) as a colorless semi-solid, which was then carried over to the next step without further purification. ESI-MS analysis: Calculated value [M+H] = 682.4, Measured value = 682.4

[0278] Intermediate (19): [ka] As shown in Scheme 3, DIPEA (0.54 g, 4.17 mmol) was added at 0°C to a stirred solution of (17) (0.6 g, 0.835 mmol) in anhydrous dichloromethane (10 mL), followed by the simultaneous addition of 1,3-di(tert-butyloxycarbonyl)-2-(trifluoromethylsulfonyl)guanidine, intermediate (18) (0.294 g, 0.752 mmol). The reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography using 0-2% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain (19) (0.290 g, 27%) as a white solid.

[0279] result: 1 H-NMR(400MHz,DMSOd6)- δ 11.52(s,1H),8.80(brs,1H),7.34(s,2H),5.35(d,J=4.8Hz,1H),4.45(t,J=4.4Hz,2H ),3.87(s,6H),3.22(m,1H),2.89(t,J=6.4Hz,2H),2.41-2.37(m,1H),2.30-2.15(m,1 H),2.01-1.81(m,5H),1.53(s,11H),1.47(s,11H),1.40-1.2(m,10H),1.20-1.02(m,8 H),1.00(s,3H),0.92-0.86(m,5H),0.88-083(m,6H),0.82-0.74(m,1H),0.67(s,3H). ESI-MS analysis: Calculated value [M+H] = 924.5, measured value = 924.6

[0280] (20) Synthesis of compound III: [ka] As shown in Scheme 3, TFA (3 mL) was added dropwise at 0°C to a stirred solution of compound (19) (0.28 g, 0.29 mmol) in anhydrous dichloromethane (10 mL), and the resulting reaction mixture was stirred at room temperature for 6 hours. The progress of the reaction was monitored by TLC, and the resulting reaction mixture was concentrated under reduced pressure to obtain a semi-solid, which was triturated with ether / pentane, and after lyophilization, compound III (0.156 g, 70.9%) was obtained as a white solid (as the TFA salt).

[0281] result: 1 H-NMR(400MHz,DMSOd6)- δ 7.75(t,J= 5.6Hz,1H),7.31(s,2H),7.25-7.07(brs,3H),5.33(bs,1H),4.35(t,J=4.4Hz,2H),3.8 1(s,6H),3.75-3.71(m,2H),3.59-3.56(m,2H),3.19-3.14(m,1H),2.79-2.71(m,2H),2 .56-2.53(m,1H),2.35(m,1H),2.12-2.06(m,1H),1.97-1.73(m,4H),1.53-1.46(m,4H) ,1.41-1.30(m,5H),1.27-1.19(m,3H),1.17-1.03(m,6H),1.02-0.95(m,5H),0.89(d,J= 9.2Hz,3H),0.88-0.83(dd,J=1.6Hz,6H),0.65(s,3H). ESI-MS analysis: Calculated value [M+H] = 724.4, measured value = 724.2

[0282] Example 4: Synthesis of Compound IV For example, compound IV of the present invention can be prepared according to scheme 4 (as shown in Figure 4). Those skilled in the art will understand that compound VI can be prepared by routine modifications of scheme 4.

[0283] Intermediate (6): [ka] As shown in Scheme 4, intermediate (6) was prepared as described in Example 2.

[0284] Intermediate (7): [ka] As shown in Scheme 4, intermediate (7) was prepared as described in Example 2.

[0285] Intermediate (12): [ka] As shown in Scheme 4, 4-hydroxy-3,5-dimethoxybenzoic acid (8) (21.0 g, 106.06 mmol) was stirred in anhydrous dichloromethane (210 mL), to which TEA (32.19 g, 318.18 mmol) was added, followed by the dropwise addition of benzyl bromide (27.2 g, 159.09 mmol) at 0°C. The resulting reaction mixture was allowed to cool to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC. The mixture was concentrated under reduced pressure, the residue was diluted with 10% NaOH aqueous solution (210 mL), and heated at 70°C for 2 hours. The resulting reaction mixture was cooled to room temperature, diluted with water (200 mL), and extracted with ethyl acetate (3 × 200 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-5% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain 4-(benzyloxy)-3,5-dimethoxybenzoic acid (12) (18.1 g, 59.2%) as a white solid.

[0286] result: 1 H-NMR(400MHz,DMSOd6)- δ 12.97(bs,1H),7.44(d,J=7.2Hz,2H),7.38-7.27(m,3H),7.23(s,2H),4.98(s,2H),3.82(s,6H). ESI-MS analysis: Calculated value [M+H] = 287.3, Measured value = 287.01

[0287] Intermediate (22): [ka] As shown in Scheme 4, 4-(benzyloxy)-3,5-dimethoxybenzoic acid (12) (4.0 g, 13.8 mmol) and tert-butyl (3-hydroxypropyl) carbamate (3.77 g, 20.8 mmol) were stirred in anhydrous DMF (50 mL). DMAP (2.54 g, 20.8 mmol) was added, followed by the one-time addition of EDC.HCl (3.56 g, 20.8 mmol) at 0°C. The resulting reaction mixture was allowed to cool to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was diluted with water (50 mL) and extracted with DCM (3 × 50 mL). The organic layers were combined, dried over Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-5% MeOH in DCM as the eluent. By combining the pure fractions and concentrating under reduced pressure, compound (22) (2.8 g, 46%) was obtained as a colorless liquid.

[0288] result: 1 H-NMR(400MHz,DMSOd6)- δ 7.44(d,J=7.2Hz,2H),7.37-7.28(m,3H),7.25(s,2H),6.93(d,J=4Hz,1H),4.99(s,2H ),4.24(t,J=6Hz,2H),3.86(s,6H),3.10-3.05(m,2H),1.82-1.77(m,2H),1.35(s,9H). ESI-MS analysis: Calculated value [M+H] = 446.5, Measured value = 446.1

[0289] Intermediate (23): [ka] As shown in Scheme 4, 1.0 g of Pd / C (10% and 50% wet) was added to a stirred solution of compound (22) (2.1 g, 4.71 mmol) in methanol (50 mL). The reaction mixture was stirred under an H2 atmosphere for 2 hours. The progress of the reaction was monitored by TLC. The reaction mixture was filtered on a Celite bed and washed with MeOH (20 mL x 2). The filtrate was concentrated under reduced pressure to obtain compound (23) (1.51 g, 92.6%) as a pink solid.

[0290] result: 1 H-NMR(400MHz,DMSOd6)- δ 9.31(bs,1H),7.21(s,2H),6.90(s,1H),4.21(t,J=6Hz,2H),3.80(s,6H),3.09-3.04(m,2H),1.80(m,2H),1.35(s,9H). ESI-MS analysis: Calculated value [M+H] = 354.3, Measured value = 354.3

[0291] Intermediate (24): [ka] As shown in Scheme 4, to a stirred solution of compounds (7) (0.65 g, 1.41 mmol) and (23) (0.503 g, 1.41 mmol) in anhydrous dichloromethane (25 mL), DMAP (0.260 g, 2.12 mmol) was added, followed by the single addition of EDC.HCl (0.364 g, 2.12 mmol) at 0°C. The reaction mixture was allowed to cool to room temperature and stirred for 16 hours. The progress of the reaction was monitored by TLC. The mixture was diluted with water (50 mL) and extracted with DCM (3 x 50 mL). The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography using 0-5% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain compound (24) (0.6 g, 53%) as a colorless liquid.

[0292] result: 1H-NMR (400 MHz, CDCl3)-δ 7.31(s,2H),5.34(d,J=4.8Hz,1H),4.76(brs,1H),4.39(t,J=6Hz,2H),3.86 (brs,8H),3.27-3.19(m,3H),2.89(t,J=6.8Hz,2H),2.41(m,1H),2.22(m,1H) ),2.18-1.80(m,8H),1.57-1.43(m,6H),1.40(s,9H),1.35-1.03(m,13H),1. 00(s,3H),0.91(d,J=6.4Hz,3H),0.87-0.85(dd,J=1.6Hz,6H),0.67(s,3H). ESI-MS analysis: Calculated value [M+H] = 796.5, Measured value = 796.2

[0293] Intermediate (25): [ka] As shown in Scheme 4, 4N HCl in dioxane (12 mL) was added dropwise at 0°C to a stirred solution of compound (24) (0.6 g, 0.753 mmol) in anhydrous dichloromethane (30 mL). The reaction mixture was stirred at room temperature for 6 hours. The progress of the reaction was monitored by TLC. The resulting reaction mixture was concentrated under reduced pressure to obtain compound (25) (0.6 g, crude compound) as a colorless semi-solid (HCl salt), which was used in the next step without further purification.

[0294] result: ESI-MS analysis: Calculated value [M+H] = 696.4, Measured value = 696.4

[0295] Intermediate (26): [ka] As shown in Scheme 4, compound (25) (0.6 g, 0.82 mmol) was stirred in anhydrous dichloromethane (10 mL), to which DIPEA (0.42 g, 3.2 mmol) was added at 0°C, followed by the sudden addition of 1,3-di(tert-butyloxycarbonyl)-2-(trifluoromethylsulfonyl)guanidine (18) (0.29 g, 0.73 mmol). The reaction mixture was stirred at room temperature for 16 hours. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by column chromatography using 0-2% MeOH in DCM as the eluent. The pure fractions were combined and concentrated under reduced pressure to obtain the final compound (26) (0.28 g, 36.4%) as a white solid.

[0296] result: 1 H-NMR(CDCl3)- δ 11.49(s,1H),8.48(brs,1H),7.30(s,2H),5.34(d,J=5.2Hz,1H),4.40(t,J=6.0Hz,2H),3 .87(d,J=2.8Hz,8H),3.60(d,J=6.0Hz,2H),3.26-3.20(m,1H),2.89(t,J=6.8Hz,2H),2.4 1-2.37(m,2H),2.31-2.19(m,2H),2.14-1.80(m,10H),1.49(s,9H),1.47(s,9H),1.42-1. 04(m,16H),1.00(s,3H),0.91(d,J=6.4Hz,3H),0.87-0.85(dd,J=1.6Hz,6H),0.67(s,3H). ESI-MS analysis: Calculated value [M+H] = 938.6, Measured value = 938.6

[0297] (27) Synthesis of compound IV: [ka] As shown in Scheme 4, TFA (3 mL) was added dropwise at 0°C to a stirred solution of (26) (0.28 g, 0.298 mmol) in anhydrous dichloromethane (10 mL), and the resulting reaction mixture was stirred at room temperature for 6 hours. The progress of the reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure to obtain a semi-solid, which was pulverized with ether / pentane and freeze-dried to obtain the desired compound, compound IV (0.156 g, 70.9%), as a white solid (TFA salt).

[0298] result: 1 H-NMR(400MHz,DMSOd6)- δ 7.60(t,J= 5.6Hz,1H),7.29(s,2H),7.12-6.70(brs,3H),5.33(brs,1H),4.33(t,J=6.0Hz,2H),3.81(s,6H),3.73(t,J= 6.0Hz,2H),3.27(m,2H),3.18(m,1H),2.78(t,J= 5.6Hz,2H),2.09(m,1H),1.97-1.74(m,7H),1.56-1.42(m,4H),1.42-1.26(m,6H),1.23(s,2H) ,1.20-0.97(m,10H),0.95(s,3H),0.89(d,J=6.4Hz,3H),0.84(dd,J=5.2Hz,6H),0.65(s,3H). ESI-MS analysis: Calculated value [M+H] = 738.5, measured value = 738.4

[0299] Example 5: Lipid Nanoparticle Formulation The cationic lipids described herein can be used in the preparation of lipid nanoparticles by methods known in the art. For example, a preferred method is the one described in International Publication No. 2018 / 089801 (which is incorporated herein by reference in its entirety).

[0300] The lipid nanoparticles in the examples of the present invention were formulated using Process A of International Publication No. 2018 / 089801 (see, for example, Example 1 and Figure 1 of International Publication No. 2018 / 089801). Process A ("A") is a conventional method for encapsulating mRNA by mixing mRNA with a lipid mixture without first pre-forming the lipids into lipid nanoparticles. In the exemplary process, an ethanol-lipid solution and a buffered aqueous solution of mRNA were prepared separately. The solution of the lipid mixture (cationic lipids, helper lipids, zwitterionic lipids, PEG lipids, etc.) was prepared by dissolving the lipids in ethanol. The mRNA solution was prepared by dissolving mRNA in citrate buffer. These two solutions were then mixed using a pump system. In some examples, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixed using a "T" junction (or "Y" junction). The mixture was then purified by diafiltration with a TFF process. The resulting formulation was concentrated and stored at 2–8°C until further use.

[0301] The lipid nanoparticle formulations shown in Table 1 were prepared by process A. All lipid nanoparticle formulations contained lipids different from FFL mRNA (cationic lipids: DMG-PEG2000; cholesterol: DOPE) in the mol% ratios specified in Table 1.

[0302] The polydispersity index (PDI) of lipid nanoparticles can be determined by diluting the formulation with 10% trehalose to an mRNA concentration of approximately 0.1 mg / ml, and then measuring the size using a Malvern zetasizer. Lipid nanoparticle size can be obtained using the Malvern Zetasizer Nano-ZS.

[0303] Dynamic light scattering (DLS) measurements were performed using a Malvern Instruments Zetasizer with a 173° backscatter detector angle and a 4mW, 633nm He-Ne laser (Worcestershire, UK). Samples were diluted with 10% trehalose and analyzed by measuring size and polydispersity index (PDI) in optical-grade polystyrene cuvettes.

[0304] [Table 2]

[0305] Lipid nanoparticles containing compounds I and II tested in the examples have formulation number 1. Lipid nanoparticles containing compounds III and IV tested in the examples have formulation number 2.

[0306] Example 6: In vitro degradation test Lipid degradation by mouse / human lung S9 in vitro Assay format - 4 or 5 time points in a triple sequence. I. Assay Procedure: 1) Plan the experiment, compounds, and reagents. 2) Dissolve each lipid in DMSO or IPA to prepare a 5 mM stock solution, and then dilute it with IPA to a 200 μm working solution. 3) Thaw the mouse and human lung S9. 4) Prepare the pooled incubation mixture on ice according to the following reaction formula. 5) Dispense the 495 μL incubation mixture prepared in step 4 into each well of a 2 mL 96-well plate. 6) Add 5 μL of the compound to each well to start the reaction. Take a t0 sample (as in step 8). 7) Cover the plate with two layers of breathable seal and incubate the plate in a 37°C CO2 incubator on an orbital shaker at 150 rpm. 8) At each time point, pipette the incubation mixture five times, then transfer 70 μL of the incubation mixture to a new plate. Store immediately in a freezer at -20°C. 9) Add 210 μL (3 × volume) of ice-cooled stop solution to each well of the collected sample plate. Mix in an orbital shaker at 600 rpm for 15 minutes. 10) Centrifuge the quenched plate at 3800 rpm at 4°C for 10 minutes, and transfer the supernatant to a new plate. 11) Load the supernatant into a filtration plate and centrifuge again at 3800 rpm at 4°C for 5 minutes. Collect the final sample into a new plate for LC / MS.

[0307] II. Time progression and stopping solution: 4-5 time points (hours): For example, 0, 4, 8, 24, 48 hours Stop solution: 1:1:1 ACN / MeOH / IPA (v / v / v), containing propranolol and MC3 as internal standards. Store at 4°C.

[0308] III. Reactive components and preparation method: Mouse / Human Lung S9

[0309] [Table 3]

[0310] [Table 4]

[0311] Example 7: Delivery of firefly luciferase (FFL) mRNA by pulmonary administration Lipid nanoparticle formulations listed in Table 1, containing FFL mRNA, cationic lipids, DMG-PEG2000, cholesterol, and DOPE, were administered to male CD1 mice (6-8 weeks old) under anesthesia via a single intratracheal administration via Catheter® (50 μl / animal). Approximately 24 hours after administration, the animals were administered 150 mg / kg (60 mg / ml) of luciferin 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 5 shows that lipid nanoparticles containing cationic lipids as described herein are effective in delivering FFL mRNA in vivo based on positive luciferase activity.

[0312] Example 8: Results of a firefly luciferase (FFL) mRNA delivery test by intranasal administration Lipid nanoparticle formulation 3, listed in Table 1 and containing FFL mRNA, cationic lipids, DMG-PEG2000, cholesterol, and DOPE, was administered to mice by pipetting 10 μg / animal and 15 μl / nostril. On day 2, 24 hours (±5%) post-administration, all animals underwent a luminescence imaging session using IVIS with separate ROIs in the nose and lungs. Whole-body imaging was performed 10–15 minutes after D-luciferin administration. All animals were administered 0.2 mL of 15 mg / mL D-luciferin solution by intraperitoneal (IP) injection. The animals were anesthetized with isoflurane during the procedure and placed face down on the sternum. Intranasal vaccine drug products were administered via nasal spray.

[0313] [Table 5]

[0314] Example 8: Decomposition Test The cationic lipids of the present invention, derived from aromatic head groups having a hydroxyl group (e.g., phenol), exhibit improved degradability as the pKa of the hydroxyl group decreases. For example, the cationic lipid of the present invention derived from picolinic acid (phenol pKa 7.86) has improved degradability compared to nicotinic acid (phenol pKa 8.31) and syringic acid (phenol pKa 8.44) (see Table 4). Similarly, the cationic lipid of the present invention derived from nicotinic acid (phenol pKa 8.31) has improved degradability compared to syringic acid (phenol pKa 8.44).

[0315] The decomposition test was carried out as described in Example 6.

[0316] [Table 6]

[0317] Example 9: RiboGreen Assay The encapsulation efficiency of mRNA in lipid nanoparticles can be determined using the Invitrgen RiboGreen assay kit. Unencapsulated mRNA was detected directly. Total mRNA was measured after lysis of lipid nanoparticles in the presence of Triton X-100. Encapsulation efficiency was calculated as (total mRNA - unencapsulated mRNA) / total mRNA × 100%.

[0318] The RiboGreen assay is a fluorescence-based method for determining mRNA concentration (total and free) and % inclusion in mRNA containing lipid nanoparticles, using the Quant-iT® RiboGreen® RNA reagent.

[0319] Materials / Reagents Triton-X, 98%, for molecular biology, DNAse, RNAse, and protease free, Acros Organics, catalog AC327371000 • Ultrapure DNase / RNase-free distilled water, Life Technologies, Catalog 10977-023 • RNaseZap (registered trademark) RNase decontamination solution, Life Technologies, catalog AM9784 • Quant-iT® RiboGreen® RNA reagent, Life Technologies, catalog R11491 or Quant-iT® RiboGreen® RNA assay kit, Life Technologies, catalog R11490 • RNase-free 20X TE buffer, Life Technologies, Catalog T11493 • RNaseZap (registered trademark) RNase decontamination solution, Life Technologies, catalog AM9784

[0320] device • Molecular Devices Gemini EM Microplate Reader • RNase-free microcentrifuge tube (2.0 mL) • RNase-free flacon tubes (15 and 50 mL) Vortex Mixer • Corning® 96-well special optical microplate with transparent background (catalog number 3615)

[0321] Preparation of an mRNA standard

[0322] [Table 7]

[0323] [Table 8]

[0324] Sample preparation

[0325] [Table 9]

[0326] [Table 10]

[0327] Preparation of 200x RiboGreen Dye

[0328] [Table 11]

[0329] procedure Add 1.0 mL of 200-fold Ribogreen reagent solution to each of the standards (blank, mRNA-1, mRNA-2, mRNA-3, mRNA-4, mRNA-5) and samples (free mRNA and total mRNA), and gently mix by inversion. This is a 2-fold dilution. Using the reverse pipetting technique, triple-ply 200 μL of each standard and sample into a 96-well Costar Black plate with a clear background. Ensure that there are no air bubbles in the plate before taking fluorescence readings. • Read the fluorescence signal using the following instrument parameters: • Reading type: Fluorescent, bottom lead Excitation: 485nm; Cutoff: 515nm; Emission: 530nm • Plate type: 96-well Costar Black with transparent background

[0330] Data Analysis The average fluorescence from each calibration standard is plotted against concentration, and a linear calibration curve is generated using MS Excel software. The coefficient of determination (R) of the calibration curve is calculated. 2 ) is R 2 It must be >0.99. The resulting linear equation can be interpreted as follows: Y=mx+c During the ceremony, Y = Average fluorescence value M: Slope x: Concentration (μg / mL) c:y intercept Using a linear equation, the free mRNA concentration and total mRNA concentration in the test sample are calculated by replacing the y value in the equation with the average fluorescence value of each sample. Once the concentration is determined, the actual concentration in the sample can be calculated in reverse by multiplying the concentration in the test sample by the dilution factor (DF) as follows: Total mRNA concentration = Total mRNA concentration in the test sample × 800 (DF) Total mRNA concentration = Total mRNA concentration in the test sample × 4000 (DF) The concentration of encapsulated mRNA can be determined by subtracting the concentration of free mRNA from the total mRNA concentration. Next, the inclusion rate can be calculated by taking the ratio of inclusion mRNA to total mRNA and multiplying the result by 100.

[0331] From the above description, those skilled in the art will readily be able to identify the essential features of the present invention and make various changes and modifications to the invention to suit various uses and conditions without departing from its spirit and scope.

[0332] All references, patents, or applications (U.S. or foreign) cited herein are incorporated herein by reference as if they were written herein in their entirety. In the event of any conflict, the material literally disclosed herein shall be used for comparison.

[0333] Numbered Embodiments 1. Equation (I): [ka] Cationic lipids having a structure such as or a pharmaceutically acceptable salt thereof During the ceremony, [ka] This is selected from optionally substituted arylenes or optionally substituted heteroarylenes. In the formula, L2 is selected from a bond, an optionally substituted (C1-C6) alkylene, or an optionally substituted (C2-C6) alkenylene. In the formula, X 1 It is selected from O, In the formula, R 1 teeth, [ka] And, In the formula, a is selected from 0, 1, 2, 3, 4, or 5. In the formula, R 2 and R 3 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, R 4 and R 5 Each is independently selected from H or optionally substituted (C1-C6) alkyl groups. In the formula, L1 is selected from D or E-L3-C(=O)O-, and the right side of the cited structure is, [ka] It is connected, In the formula, D is selected from -(C1~C3)alkyl-O-, -OC(=O)O-, -SC(=O)O-, -OC(=O)S-, -(C1~C3)alkyl-OC(=O)O-, or -C(=O)O-, where the right side of each specified structure is: [ka] It is connected, In the formula, E is selected from -O- or -OC(=O)-, and the right side of each structure is bonded to L3. In the formula, L3 is selected from (C1-C6) alkylenes that are optionally substituted, or from (C2-C6) alkenylenes that are optionally substituted, and [ka] These are naturally occurring sterols or non-naturally occurring sterols. 2. Cationic lipids, [ka] The cationic lipid according to numbered embodiment 1, which is selected from optionally substituted phenylene, optionally substituted pyridinylene, optionally substituted pyrrolylene, optionally substituted thiophenylene, optionally substituted furanylene, optionally substituted thiazolyene, optionally substituted imidazolyene, optionally substituted indolylene, optionally substituted tetrazolyene, optionally substituted piperidinylene, or optionally substituted pyrrolidinylene. 3. Cationic lipids, [ka] The following can be selected: [ka] (The right-hand side of each illustrated structure is connected to L2, In the formula, X 2 is N or -C(R 16 )- and, In the formula, X 3 is N or -C(R 17 )- and, In the formula, X 4 is NH, O, or S, In the formula, X 5 is N, and In the formula, R 13 , R 14 , R 15 , R 16 , R 17 、 R 18 , R 19 , R 20 and R 21If present, each is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy, as described in numbered embodiment 1 or 2. 4. Cationic lipids, [ka] but, [ka] And in the formula, R 13 ~R 15 , R 17 or R 18 Two of the substituents are absent, one of the absent substituents is substituted by a bond with L1, and the other absent substituent is substituted by a bond with L2. In the formula, X 2 is N or -C(R 16 )- and In the formula, R 13 , R 14 , R 15 , R 16 , R 17 and R 18 The cationic lipid according to numbered embodiment 1 or 2, wherein, if present, each is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy. 5. Cationic lipids, [ka] but, [ka] The cationic lipid according to any one of the numbered embodiments 1 to 4, wherein the right-hand side of the illustrated structure is bonded to L2. 6. Equation (Ia): [ka] A cationic lipid having the structure described in any one of the numbered embodiments 1 to 5, or a pharmaceutically acceptable salt thereof. 7. Equation (Ia1): [ka] A cationic lipid of the numbered embodiment 6 having the structure of, or a pharmaceutically acceptable salt thereof. 8. Formula (Ib): [ka] A cationic lipid having the structure described in any one of the numbered embodiments 1 to 5, or a pharmaceutically acceptable salt thereof. 9. Formula (Ib1): [ka] A cationic lipid having the structure of the above, as described in numbered embodiment 8, or a pharmaceutically acceptable salt thereof. 10. Cationic lipids, [ka] but, Formula (II): [ka] It has a structure that follows the formula, where R 6 and R 7 Each of these is independently selected from H, OH, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, and optionally substituted (C1-C6) alkoxy; In the formula, R 8 (C1~C 30 ) alkyl, optionally substituted (C2~C 30) Alkenyl, optionally substituted (C2~C 30 ) Alkinyl, optionally substituted (C1~C 30 )alkoxy, optionally substituted (C1~C 10 )Alkylene-C(O)O-Optionally substituted (C1~C 20 ) Selected from alkyl groups; and optionally substituted (C1~C 10 ) is alkylene-C(O)OH, In the formula, R 9 H is R 10 Is it OH, or R 9 and R 10 Both are H, or neither is present, R 9 and R 10 If R does not exist, 9 and R 10 A C=C double bond exists between the carbon atoms to which it is bonded, and In the formula, R 11 OH is R 12 Is H or R 11 and R 12 Both are H, or neither is present, R 11 and R 12 If it does not exist, the C=C double bond is R 11 and R 12 A cationic lipid according to any one of the numbered embodiments 1 to 6 or 8, which is present between the carbon atoms to which it is bonded. 11. Cationic lipids, [ka] However, equation (IIa): [ka] A cationic lipid according to the numbered embodiment 10, having the structure described herein. 12. Cationic lipids, [ka] but, Selected from animal sterols, or their oxidized or reduced forms; phytosterols, or their oxidized or reduced forms; synthetic sterols, or their oxidized or reduced forms; bile acids, or their alkyl esters, or their oxidized or reduced forms; [ka] (Cholesterol) or its oxidized or reduced form; [ka] And in the formula, R 22 (C1~C 20 ) Alkyl (alkyl lithocholate) or its oxidized or reduced form; [ka] However, it is (stigmasterol) or its oxidized or reduced form; [ka] However, it is (stigma mastolon) or its oxidized or reduced form; [ka] However, it is (campesterol) or its oxidized or reduced form; [ka] However, it is (ergosterol) or its oxidized or reduced form; [ka] However, (sitosterol) or its oxidized or reduced form; or [ka] And in the formula, R 23 is replaced by H or optionally (C1~C 20) A cationic lipid according to any one of the numbered embodiments 1 to 6, 8, 10, or 11, which is alkyl (cholic acid) or its oxidized or reduced form. 13. Cationic lipids, [ka] However, the cationic lipid described in any one of the numbered embodiments 1 to 6, 8, or 10 to 12 is cholesterol. 14. Cationic lipids, [ka] However, equation (IIb): [ka] A cationic lipid having the structure described in any one of the numbered embodiments 1 to 6, 8, or 10 to 13. 15. Cationic lipids, [ka] but, Formula (IIc): [ka] A cationic lipid having the structure described in any one of the numbered embodiments 1 to 6, 8, or 10 to 14. 16. A cationic lipid according to any one of the numbered embodiments 1 to 15, wherein a is 1. 17. A cationic lipid according to any one of the numbered embodiments 1 to 15, wherein a is 2. 18. R 4 and R 5 A cationic lipid according to any one of the numbered embodiments 1 to 17, wherein the molecule is methyl. 19. Cationic lipids have the structure shown in formula (I) below, R 1 but, [ka] A cationic lipid according to any one of the numbered embodiments 1 to 18. 20. A cationic lipid has the structure shown in the following formula (I), where R 1 but, [ka] A cationic lipid as described in one of the numbered embodiments 1 to 18. 21. The cationic lipid has a structure according to one of the following formulas: (I), (Ib), or (Ib1), where R 2 , R 3 , R 4 and R 5 A cationic lipid according to any one of the numbered embodiments 1 to 17, wherein is hydrogen. 22. A cationic lipid has the structure shown in the following formula (I), where R 1 but, [ka] A cationic lipid according to any one of the numbered embodiments 1 to 17 or 21. 23. A cationic lipid has the structure shown in the following formula (I), where R 1 but, [ka] A cationic lipid according to any one of the numbered embodiments 1 to 17 or 21. 24. Cationic lipids, wherein L1 is [ka] A cationic lipid according to any one of the numbered embodiments 1 to 23. 25. A cationic lipid, wherein the cationic lipid has a structure according to formula (Ia) or formula (Ia1), and in the formula L1 is [ka] A cationic lipid according to any one of the numbered embodiments 1 to 23. 26. L1 is [ka] And here, the right side of the described structure is [ka] A cationic lipid according to any one of the numbered embodiments 1 to 23, which is bonded to a. 27. The cationic lipid has a structure that follows any one of formulas (Ia), (Ia1), (Ib), or (Ib1), where L1 is [ka] And here, the right side of the described structure is [ka] A cationic lipid according to any one of the numbered embodiments 1 to 23, which is bonded to a. 28. A compound selected from those listed in Table A, or a pharmaceutically acceptable salt thereof. 29. A composition comprising a cationic lipid as described in any one of the numbered embodiments 1 to 28, and further comprising the following: (i) One or more noncationic lipids, (ii) one or more cholesterol-based lipids, and (iii) One or more PEG-modified lipids. 30. A composition comprising a cationic lipid as described in any one of the numbered embodiments 1 to 28, and further comprising the following: (i) one or more noncationic lipids, and (iii) One or more PEG-modified lipids. 31. Compositions of numbered embodiments 29 or 30, wherein lipid nanoparticles, optionally liposomes. 32. The composition of the numbered embodiment 31, wherein one or more cationic lipids constitute approximately 30 mol% to 60 mol% of the lipid nanoparticles. 33. The composition according to the numbered embodiment 31 or 32, wherein one or more noncationic lipids constitute 10 mol% to 50 mol% of the lipid nanoparticles. 34. The composition according to any one of the numbered embodiments 31 to 33, wherein one or more PEG-modified lipids constitute 1 mol% to 10 mol% of the lipid nanoparticles. 35. The composition according to any one of the numbered embodiments 31 to 34, wherein cholesterol-based lipids constitute 10 mol% to 60 mol% of the lipid nanoparticles. 36. The composition according to any one of the numbered embodiments 31 to 35, wherein the lipid nanoparticles encapsulate nucleic acids, optionally mRNA encoding peptides or proteins. 37. The composition according to any one of the numbered embodiments 31 to 36, wherein lipid nanoparticles encapsulate mRNA encoding a peptide or protein. 38. Lipid nanoparticles, (a) at least 70%; (b) at least 75%; (c) at least 80%; (d) at least 85%; (e) at least 90%; or (f) at least 95% The composition according to the numbered embodiment 37, having the encapsulation rate. 39. A composition according to any one of the numbered embodiments 36 to 38, for use in treatment. 40. The composition according to numbered embodiment 37 or 38 for use in a method of treating or preventing a disease suitable for treatment or prevention with a peptide or protein encoded by mRNA, which optionally is (a) a protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer. 41. A composition for use according to numbered embodiment 39 or 40, which is administered via spray therapy, optionally by intranasal, intravenous, subarachnoid, or intramuscular administration, or by intrapulmonary delivery. 42. The composition for use according to the numbered embodiment 39 or 40, wherein the composition is administered intranasally. 43. The composition for use according to the numbered embodiment 39 or 40, wherein the composition is administered optionally by pulmonary delivery by spray. 44. A method for treating or preventing a disease, the method comprising administering a composition described in numbered embodiment 37 or 38 to a subject in need thereof, wherein the disease is suitable for treatment or prevention with a peptide or protein encoded by mRNA, and optionally the disease is (a) an optionally selected protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer. 45. The method according to the numbered embodiment 44, wherein the composition is administered optionally by spraying, either intranasally, intravenously, intrathecally, intramuscularly, or pulmonaryly. 46. ​​The method according to the numbered embodiment 44 or 45, wherein the composition is administered intranasally. 47. The method according to the numbered embodiment 44 or 45, wherein the composition is administered by optional pulmonary delivery by spray.

Claims

1. Equation (I): 【Chemistry 1】 A cationic lipid having the structure, or a pharmaceutically acceptable salt thereof, During the ceremony, 【Chemistry 2】 This is selected from optionally substituted arylenes or optionally substituted heteroarylenes. In the ceremony, L ２ is joined, optionally substituted (C １ ~C ６ ) alkylene or optionally substituted (C ２ ~C ６ ) Selected from alkenylenes, In the formula, X １ It is selected from O, In the formula, R １ teeth, 【Transformation 3】 And, In the formula, a is selected from 0, 1, 2, 3, 4, or 5. wherein, R ２ and R ３ are each independently selected from H or optionally substituted (C １ -C ６ ) alkyl, In the formula, R ４ and R ５ Each is independently of H or optionally substituted with (C). １ ~C ６ ) Selected from alkyl groups, In the ceremony, L １ is D or E-L ３ Selected from -C(=O)O-, the right side of the aforementioned cited structure is 【Chemistry 4】 It is connected, In the formula, D is -(C １ ~C ３ ) Alkyl-O-, -OC(=O)O-, -SC(=O)O-, -OC(=O)S-, -(C １ ~C ３ ) Selected from alkyl-OC(=O)O- or -C(=O)O-, where the right side of each specified structure is, 【Transformation 5】 It is connected, In the formula, E is selected from -O- or -OC(=O)-, and the right side of each specified structure is L ３ It is connected, In the ceremony, L ３ (C) is optionally replaced １ ~C ６ ) alkylene, or optionally substituted (C ２ ~C ６ ) Selected from alkenylenes, and 【Transformation 6】 This refers to cationic lipids, which are naturally occurring or non-naturally occurring sterols, or pharmaceutically acceptable salts thereof.

2. Equation (Ia): 【Transformation 7】 A cationic lipid according to claim 1 having the structure, or a pharmaceutically acceptable salt thereof.

3. Equation (Ia1): 【Transformation 8】 A cationic lipid according to claim 2 having the structure, or a pharmaceutically acceptable salt thereof.

4. Formula (Ib): 【Chemistry 9】 A cationic lipid according to claim 1 having the structure, or a pharmaceutically acceptable salt thereof.

5. Formula (Ib1): 【Chemistry 10】 A cationic lipid according to claim 4 having the structure, or a pharmaceutically acceptable salt thereof.

6. Cationic lipids, 【Chemistry 11】 Equation (II): 【Chemistry 12】 It has the structure, in the formula R ６ and R ７ These are H, OH, and (C) which are substituted (C) independently of each other. １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) Alkenyl, optionally substituted (C ２ ~C ６ ) Alkinyl and optionally substituted (C １ ~C ６ ) Selected from alkoxy; In the formula, R ８ (C) is optionally replaced １ ~C ３０ ) alkyl, optionally substituted (C ２ ~C ３０ ) Alkenyl, optionally substituted (C ２ ~C ３０ ) Alkinyl, optionally substituted (C １ ~C ３０ ) alkoxy, optionally substituted (C １ ~C １０ ) Alkylene-C(O)O-Optionally substituted (C １ ~C ２０ ) selected from alkyl groups; and optionally substituted (C １ ~C １０ ) is alkylene-C(O)OH, In the formula, R ９ H is R １０ Is it OH, or R ９ and R １０ Both are H, or neither is present, R ９ and R １０ If R does not exist, ９ and R １０ A C=C double bond exists between the carbon atoms to which it is bonded, and In the formula, R １１ OH and R １２ is H or R １１ and R １２ Both are H, or neither is present, R １１ and R １２ If it does not exist, the C=C double bond is R １１ and R １２ A cationic lipid according to any one of claims 1, 2, or 4, which is present between the carbon atoms to which it is bonded.

7. A compound selected from those listed in Table A, or a pharmaceutically acceptable salt thereof.

8. The following comprises a cationic lipid as described in any one of claims 1 to 6: (i) One or more noncationic lipids, (ii) One or more cholesterol-based lipids, and (iii) One or more PEG-modified lipids and A composition further comprising:

9. The following comprises a cationic lipid as described in any one of claims 1 to 7: (i) one or more noncationic lipids, and (iii) One or more PEG-modified lipids and A composition further comprising:

10. The composition according to claim 8 or 9, wherein the composition is lipid nanoparticles, optionally liposomes.

11. The composition according to claim 10, wherein the lipid nanoparticles encapsulate nucleic acids, optionally peptides, or mRNA encoding proteins.

12. The composition according to claim 10 or 11, wherein the lipid nanoparticles encapsulate mRNA encoding a peptide or protein.

13. The composition according to claim 11 or 12 for use in therapeutic purposes.

14. The composition according to claim 12 for use in a method of treating or preventing a disease suitable for treatment or prevention by the peptide or protein encoded by the mRNA, the disease being optionally (a) a protein deficiency affecting the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

15. The composition for use according to claim 13 or 14, which is optionally administered via spray therapy by intravenous, intranasal, subarachnoid, or intramuscular administration, or by intrapulmonary delivery.

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