Cationic lipids based on phenolic acid lipids

Abstract

The present invention provides, in part, phenolic acid lipid compounds of Formula (I) and subformulae thereof, or pharmaceutically acceptable salts thereof. The compounds provided herein can be used to deliver and express mRNA and the encoded protein, for example as components of liposome delivery vehicles, and thus can be used to treat various diseases, disorders, and conditions, such as those associated with the deficiency of one or more proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 003,698, filed April 1, 2020, which is incorporated by reference in its entirety. Background Art

[0003] The delivery of nucleic acids has been widely explored as a potential treatment option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for treating a variety of diseases, including those associated with the deficiency of one or more proteins.

[0004] The effective delivery of liposome-encapsulated nucleic acids remains an active area of ​​research. Cationic lipid components play an important role in promoting the effective encapsulation of nucleic acids during the loading period of liposomes. In addition, cationic lipids can play an important role in the effective release of nucleic acid cargo from liposomes into the cytoplasm of target cells. Various cationic lipids suitable for in vivo use have been found. However, there is still a need to identify lipids that can be effectively and cheaply synthesized without forming potential toxic byproducts.

[0005] Phenolic acids possess many favorable properties, making them a good starting point for the synthesis of cationic lipids for use in vivo. For example, they exhibit no toxicity, are readily available in large quantities, and are easily derivatized. Broadly speaking, phenolic acids can be divided into two groups: benzoic acid and cinnamic acid, and their derivatives.

[0006] Examples of benzoic acids that can be used to synthesize the cationic lipids of the present invention include:

[0007]

[0008]

[0009] Examples of cinnamic acids that can be used to synthesize the cationic lipids of the present invention include:

[0010]

[0011] In some embodiments, examples of cinnamic acids that can be used to synthesize the cationic lipids of the present invention include:

[0012]

[0013] In some embodiments, examples of cinnamic acids that can be used to synthesize the cationic lipids of the present invention include:

[0014]

[0015] In some embodiments, examples of cinnamic acids that can be used to synthesize the cationic lipids of the present invention include:

[0016]

[0017] In some embodiments, examples of cinnamic acids that can be used to synthesize the cationic lipids of the present invention include:

[0018] Summary of the Invention

[0019] The present invention provides, among other things, a novel class of cationic lipid compounds for use in the in vivo delivery of therapeutic agents such as nucleic acids. These compounds are expected to be able to be efficiently delivered in vivo while maintaining a favorable toxicity profile.

[0020] Cationic lipids of the present invention can be synthesized from readily available starting reagents such as phenolic acid, benzoic acid, and cinnamic acid. Cationic lipids of the present invention also have unexpectedly high encapsulation efficiency. Cationic lipids of the present invention also include cleavable groups (e.g., esters and disulfides), which are believed to improve biodegradability and therefore contribute to their favorable toxicity profile.

[0021] In one aspect, a cationic lipid having a structure according to formula (I) is provided:

[0022]

[0023] wherein L1 is a bond, (C1-C6)alkyl or (C2-C6)alkenyl;

[0024] Wherein X is O or S;

[0025] where R 1 、R 2 、R 3 、R 4 and R 5 are each independently selected from H, OH, optionally substituted (C1-C6)alkyl, optionally substituted (C2-C6)alkenyl, optionally substituted (C2-C6)alkynyl, optionally substituted (C1-C6)alkoxy, and -OC(O)R';

[0026] where R 1 、R 2 、R 3 、R 4 or R 5 At least one of them is -OC(O)R';

[0027] Where R' is

[0028]

[0029] where R 6 for

[0030]

[0031] wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

[0032] where R 7 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A ;

[0033] where R 8 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B ;

[0034] where R 9 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C ;

[0035] where R 10 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D ;

[0036] wherein k, n, q and r are each independently 1, 2, 3, 4 or 5;

[0037] or (i) R 7 and R 8 or (ii) R9 and R 10 together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, wherein the heterocycloalkyl or heteroaryl includes 1 to 3 heteroatoms selected from N, O and S;

[0038] where R 11 、R 12 、R 13 and R 14 Each is independently selected from H, methyl, ethyl or propyl;

[0039] where R A 、R B 、R C and R D Each independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 )acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, -NH2;

[0040] where R 7 、R 8 、R 9 、R 10 At least one of them includes R A 、R B 、R C or R D part, wherein the R A 、R B 、R C or R D are independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)(C6-C 20 )alkyl or optionally substituted -OC(O)(C6-C 20 )alkenyl;

[0041] or a pharmaceutically acceptable salt thereof.

[0042] In one aspect, provided herein is a cationic lipid that is a pharmaceutically acceptable salt of Formula (I).

[0043] On the one hand, the present invention provides a composition comprising a cationic lipid of the present invention, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. On the one hand, the composition is a lipid nanoparticle, optionally a liposome.

[0044] In one aspect, the compositions comprising the cationic lipids of the invention can be used for therapeutic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 In vivo protein expression following intratracheal administration of lipid nanoparticles comprising one of the cationic lipid compounds 1-12 is demonstrated. Based on positive luciferase activity, lipid nanoparticles comprising the cationic lipids described herein effectively delivered FFL mRNA in vivo. DETAILED DESCRIPTION

[0046] definition

[0047] To make the present invention more easily understood, certain terms are first defined below. Additional definitions of the following and other terms are set forth throughout this specification. The publications and other reference materials cited herein to describe the background technology of the present invention and to provide additional details about its implementation are incorporated herein by reference.

[0048] Amino Acid: 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, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; and in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and / or substitutions. Amino acids, including the carboxyl 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 circulating half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. An amino acid may comprise one or more post-translational modifications, for example, conjugation to one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, and a biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. Whether the term refers to a free amino acid or a residue of a peptide will be apparent from the context in which the term is used.

[0049] Animal: As used herein, the term "animal" refers to any member of the kingdom Animalia. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal can be a transgenic animal, a genetically engineered animal, and / or a clone.

[0050] Approximately or approximately: As used herein, when applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the range in either direction (greater than or less than) of the value, unless otherwise specified or otherwise apparent from the context (unless the number exceeds 100% of the possible values).

[0051] Bioactive: As used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system, particularly an organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered bioactive.

[0052] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and retained in the target tissue (also referred to as "local distribution" or "local delivery"), as well as situations where the mRNA is delivered to the target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum) and then distributed systemically and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0053] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into polypeptides, the assembly of multiple polypeptides into complete proteins (e.g., enzymes), and / or post-translational modifications of polypeptides or fully assembled proteins (e.g., enzymes). In this application, the terms "expression" and "production" and their grammatical equivalents are used interchangeably.

[0054] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form that exhibits the properties and / or activities that characterize it.

[0055] Half-life: As used herein, the term "half-life" is the time required for the concentration or activity of a nucleic acid or protein, for example, to drop to half its value measured at the beginning of a time period.

[0056] Helper lipid: As used herein, the term "helper lipid" refers to any neutral or zwitterionic lipid material including cholesterol. Without wishing to be bound by a particular theory, helper lipids can increase stability, rigidity, and / or fluidity within the lipid bilayer / nanoparticle.

[0057] Improve, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents thereof, refer to values ​​relative to a baseline measurement, such as that measured in the same individual prior to initiation of a treatment described herein, or in a control subject (or multiple control subjects) in the absence of a treatment described herein. A "control individual" is an individual having the same form of disease as the individual being treated and who is approximately the same age as the individual being treated.

[0058] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0059] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0060] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that is (1) separated from at least some components with which it was originally associated when produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which it was originally associated. In some embodiments, the purity of the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, the calculation of the purity percentage of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0061] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Generally, as used herein, liposomes can be formed by mixing one or more lipids or by mixing one or more lipids and a polymer. In some embodiments, liposomes suitable for the present invention contain one or more cationic lipids and optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and / or optionally one or more PEG-modified lipids.

[0062] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA includes modified RNA and unmodified RNA. The term "modified mRNA" relates to an mRNA that comprises at least one chemically modified nucleotide. An mRNA may comprise one or more coding and non-coding regions. An mRNA may be purified from a natural source, produced using a recombinant expression system, and optionally purified, chemically synthesized, and the like. Where appropriate, for example, in the case of a chemically synthesized molecule, the mRNA may comprise nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, and the like. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' direction. In some embodiments, the mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0063] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain by a phosphodiester bond. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" encompasses 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 any one or more of 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), polyencoding nucleic acid (MCNA), polymeric encoding nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses RNA and DNA. In embodiments, the DNA can be in the form of antisense DNA, plasmid DNA, a portion of a plasmid DNA, pre-condensed DNA, a product of a polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or a derivative of these groups.In embodiments, RNA can be messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transactional siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K RNA, retrotransposon, viral genome, viroid, satellite RNA, or derivatives thereof. In some embodiments, the nucleic acid is an mRNA encoding a protein such as an enzyme.

[0064] Patient: As used herein, the term "patient" or "subject" refers to any organism to which the provided compositions can be administered, e.g., for experimental, diagnostic, prophylactic, 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 a human. Humans include both prenatal and postnatal forms.

[0065] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0066] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge 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 with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with 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 adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconoheptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1-4 alkyl) salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, and the like. Where appropriate, additional 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. Additional pharmaceutically acceptable salts include salts formed by quaternization of amines using appropriate electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0067] Systemic distribution or delivery: As used herein, the term "systemic distribution" or "systemic delivery" or grammatical equivalents thereof refers to a delivery or distribution mechanism or method that affects the entire body or entire organism. Typically, systemic distribution or delivery is accomplished via the body's circulatory system (e.g., bloodstream). This is in contrast to the definition of "local distribution or delivery."

[0068] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the individual is a human. The individual can be a patient, which refers to a person who presents to a medical provider for diagnosis or treatment of a disease. The term "subject" can be used interchangeably herein with "individual" or "patient." An individual can have or be susceptible to a disease or disorder, but may or may not show symptoms of the disease or disorder.

[0069] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. Those of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0070] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, target tissues include those tissues that exhibit disease-associated pathology, symptoms, or characteristics.

[0071] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of the disease, disorder, and / or condition. One of ordinary skill in the art will recognize that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0072] Treatment: As used herein, the term "treatment" refers to any method used to partially or completely alleviate, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of disease and / or who exhibit only early signs of disease in order to reduce the risk of developing pathology associated with the disease.

[0073] Chemical definition

[0074] Acyl: As used herein, the term "acyl" refers to an R Z -(C=O)-, where R Z is, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene group.

[0075] Aliphatic: As used herein, the term aliphatic refers to C 1- C 40Hydrocarbons include saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be straight chain, branched chain or cyclic. For example, C1-C 20 Aliphatic can include C1-C 20 Alkyl (e.g., straight or branched C1-C 20 saturated alkyl), C2-C 20 Alkenyl (e.g., straight or branched C4-C 20 Dienyl, linear or branched C6-C 20 triene, etc.) and C2-C 20 Alkynyl (e.g., straight-chain or branched C2-C 20 C1-C 20 Aliphatic can include C3-C 20 Cyclic aliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl or C8-C 20 In certain embodiments, the aliphatic group may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. The aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -C0H, -C0R", -CN, -OH, -OR", -OCOR', -OC0R", -NH, -NHR", -N(R"), -SR", or -S0R", wherein each instance of R" is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R" is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 alkyl or C1-C3 alkyl). In embodiments, R" is independently unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms. Alkyl: As used herein, the term "alkyl" means non-cyclic straight and branched hydrocarbon groups, for example, "C1-C 30"Alkyl" refers to an alkyl group having 1 to 30 carbon atoms. The alkyl group can be straight-chain 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-pentyl, hexyl, isohexyl, and the like. The term "lower alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R", wherein each instance of R" is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R" is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R" is independently unsubstituted C1-C3 alkyl. In some embodiments, the alkyl group is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In some embodiments, the alkyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkyl," where the prefix indicates an -OH group and "alkyl" is as described herein.

[0076] As used herein, the term "alkyl" also refers to a free radical of a straight or branched chain saturated hydrocarbon group having 1 to 50 carbon atoms ("C1-C 50 In some embodiments, the alkyl group has 1 to 40 carbon atoms ("C1-C 40 In some embodiments, the alkyl group has 1 to 30 carbon atoms ("C1-C 30 In some embodiments, the alkyl group has 1 to 20 carbon atoms ("C1-C 20 In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C1-C 10In 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, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each example of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents. In certain embodiments, an alkyl group is an unsubstituted C1-C6 alkyl group. 50 In certain embodiments, the alkyl group is a substituted C1-C 50 alkyl.

[0077] Prefixing a group with "ene" indicates that the group is a divalent moiety, for example, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0078] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight or branched hydrocarbon radical, and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, as used herein, the term "alkenylene" refers to an unsaturated divalent straight or branched hydrocarbon radical having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point along the chain, and the term "alkynylene" refers herein to an unsaturated divalent straight or branched hydrocarbon radical having one or more unsaturated carbon-carbon triple bonds, which may be present at any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene radical may contain one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. For example, an alkylene, alkenylene, or alkynylene group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCOR", -NH2, -NHR", -N(R")2, -SR", or -SO2R", wherein each instance of R" is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R" is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R" is independently unsubstituted C1-C3 alkyl. In some embodiments, the alkylene, alkenylene, or alkynylene groups are unsubstituted. In some embodiments, the alkylene, alkenylene, or alkynylene groups do not include any heteroatoms. Alkenyl: As used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may occur at any stable point along the chain, for example: "C2-C 30"Alkenyl" refers to an alkenyl group having 2-30 carbon atoms. For example, alkenyl includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In embodiments, the alkenyl group contains 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group 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 replaced by one For example, alkenyl can be substituted with one or more of the following (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents): halogen, -COR", -C0H, -C0R", -CN, -OH, -OR", -OCOR", -OC0R", -NH, -NHR", -N(R"), -SR", or -S0R", wherein each instance of R" is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In an embodiment, R" is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In some embodiments, R" is independently unsubstituted C1-C3 alkyl. In some embodiments, alkenyl is unsubstituted. In some embodiments, alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In some embodiments, alkenyl groups are substituted with -OH groups and may also be referred to herein as "hydroxyalkenyl," where the prefix indicates an -OH group and "alkenyl" is as described herein.

[0079] As used herein, "alkenyl" also refers to a straight or branched chain hydrocarbon radical ("C2-C 50 In some embodiments, an alkenyl group has 2 to 40 carbon atoms ("C2-C 40 In some embodiments, an alkenyl group has 2 to 30 carbon atoms ("C2-C 30 In some embodiments, an alkenyl group has 2 to 20 carbon atoms ("C2-C 20 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C2-C 10In some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C2-C9 alkenyl"). In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C2-C7 alkenyl"). In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C2-C5 alkenyl"). In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C2-C4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C2-C3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds can be located internally ( Examples of C2-C4 alkenyl groups include, but are not limited to, vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl groups include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each example of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl group") or substituted (a "substituted alkenyl group") with one or more substituents. In certain embodiments, an alkenyl group is an unsubstituted C2-C4 alkenyl group. 50 In certain embodiments, alkenyl is a substituted C2-C 50 Alkenyl.

[0080] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain in a straight or branched configuration having one or more carbon-carbon triple bonds occurring at any stable point along the chain, for example, "C2-C 30 "Alkynyl" refers to an alkynyl group having 2-30 carbon atoms. 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, and the like. In embodiments, the alkynyl group contains one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R", wherein each instance of R" is independently C1-C 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C15 Alkyl, C1-C 10 In an embodiment, R" is independently unsubstituted alkyl (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently unsubstituted C1-C3 alkyl. In embodiments, alkynyl is unsubstituted. In embodiments, alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).

[0081] As used herein, "alkynyl" also refers to a straight or branched chain hydrocarbon radical ("C2-C 50 Alkynyl groups with one or more triple bonds and one or more double bonds are also known as "ene-ynes." In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("C2-C 40 In some embodiments, an alkynyl group has 2 to 30 carbon atoms ("C2-C 30 In some embodiments, an alkynyl group has 2 to 20 carbon atoms ("C2-C 20 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-C 10In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C2-C9 alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-C7 alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C2 alkynyl”). The carbon-carbon triple bond or bonds may be located internally (as in 2-butynyl) or terminally (as 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), 2-butynyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl groups include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each example of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl group") or substituted (a "substituted alkynyl group") with one or more substituents. In certain embodiments, an alkynyl group is an unsubstituted C2-C4 alkynyl group. 50 In certain embodiments, the alkynyl group is a substituted C2-C 50 Alkynyl.

[0082] Aryl: The term "aryl" used alone or as part of a larger moiety as in "aralkyl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic, and wherein each ring in the system contains 4 to 7 ring members. In embodiments, aryl has 6 ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, aryl has 10 ring carbon atoms ("C 10 In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, wherein the radical or point of attachment is on the aromatic ring, and in such cases, the number of carbon atoms continues to refer to the number of carbon atoms in the aromatic ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

[0083] As used herein, "aryl" also refers to a radical ("C6-C 14 In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl" also includes ring systems in which an aromatic ring, as defined above, is fused to one or more carbocyclic or heterocyclic groups, wherein the radical or point of attachment is on the aromatic ring, and in such cases, the number of carbon atoms continues to refer to the number of carbon atoms in the aromatic ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl group") with one or more substituents. In certain embodiments, an aryl group is an unsubstituted C6-C 14 In certain embodiments, aryl is a substituted C6-C 14 Aryl.

[0084] Arylene: As used herein, the term "arylene" refers to a divalent aromatic group (ie, having two points of attachment to the molecule). Exemplary arylene groups include phenylene (eg, unsubstituted phenylene or substituted phenylene).

[0085] Carbocyclyl: As used herein, "carbocyclyl" or "carbocycle" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, the carbocyclyl group has 3 to 8 ring carbon atoms (“C3-C8 carbocyclyl”). In some embodiments, the carbocyclyl group has 3 to 7 ring carbon atoms (“C3-C7 carbocyclyl”). In some embodiments, the carbocyclyl group has 3 to 6 ring carbon atoms (“C3-C6 carbocyclyl”). In some embodiments, the carbocyclyl group has 4 to 6 ring carbon atoms (“C4-C6 carbocyclyl”). In some embodiments, the carbocyclyl group has 5 to 6 ring carbon atoms (“C5-C6 carbocyclyl”). In some embodiments, the carbocyclyl group has 5 to 10 ring carbon atoms (“C5-C 10Carbocyclyl”). Exemplary C3-C6 carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 carbocyclyl groups include, but are not limited to, the aforementioned C3-C6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), and the like. Exemplary C3-C 10 Carbocyclic groups include but are not limited to the aforementioned C3-C8 carbocyclic groups and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9), 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As shown in the foregoing examples, in certain embodiments, a carbocyclyl is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spirocyclic ring system, such as a bicyclic ring system ("bicyclic carbocyclyl") or a tricyclic ring system ("tricyclic carbocyclyl")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in such cases, the number of carbons always refers to the number of carbons in the carbocyclyl system. Unless otherwise specified, each instance of a carbocyclyl is independently unsubstituted ("unsubstituted carbocyclyl") or substituted ("substituted carbocyclyl") with one or more substituents. In certain embodiments, a carbocyclyl is an unsubstituted C3-C 10 In certain embodiments, the carbocyclyl group is a substituted C3-C 10 Carbocyclic group.

[0086] In some embodiments, "carbocyclyl" or "carbocycle" refers to a "cycloalkyl," i.e., a monocyclic saturated carbocyclyl having 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, cycloalkyl groups have 3 to 8 ring carbon atoms (“C3-C8 cycloalkyl”). In some embodiments, cycloalkyl groups have 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, cycloalkyl groups have 4 to 6 ring carbon atoms (“C4-C6 cycloalkyl”). In some embodiments, cycloalkyl groups have 5 to 6 ring carbon atoms (“C5-C6 cycloalkyl”). In some embodiments, cycloalkyl groups have 5 to 10 ring carbon atoms (“C5-C 10Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the aforementioned C5-C6 cycloalkyl groups, cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl group") or substituted (a "substituted cycloalkyl group") with one or more substituents. In certain embodiments, a cycloalkyl group is an unsubstituted C3-C6 cycloalkyl group. 10 In certain embodiments, the cycloalkyl group is a substituted C3-C 10 Cycloalkyl.

[0087] Halogen: As used herein, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0088] Assorted alkyl: the term "assorted alkyl" means a side chain or unbranched alkyl, alkenyl or alkynyl group having from 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. Assorted alkyl includes tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiester, phosphoramidates, sulfonamides and disulfides. Assorted alkyl groups can optionally include monocycles, dicycles or tricycles, wherein each ring ideally has three to six members. Examples of assorted alkyl include polyethers, such as methoxymethyl and ethoxyethyl.

[0089] Heteroalkylene: As used herein, the term "heteroalkylene" refers to a divalent form of a heteroalkyl group as described herein.

[0090] Heteroaryl: As used herein, the term "heteroaryl" is a fully unsaturated heteroatom-containing ring wherein at least one ring atom is a heteroatom such as, but not limited to, nitrogen and oxygen.

[0091] As used herein, "heteroaryl" also refers to a radical of a 5- to 14-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14π electrons shared in the ring array) having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein 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 point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in such cases, the number of ring members always refers to the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members refers to the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups where one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring with the heteroatom (e.g., 2-indolyl) or the ring without the heteroatom (e.g., 5-indolyl).

[0092] In some embodiments, heteroaryl is a 5- to 10-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 or more (e.g., 1, 2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5- to 10-membered heteroaryl”). In some embodiments, heteroaryl is a 5- to 8-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 or more (e.g., 1, 2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5- to 8-membered heteroaryl”). In some embodiments, heteroaryl is a 5- to 6-membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and 1 or more (e.g., 1, 2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5- to 6-membered heteroaryl”). In some embodiments, the 5- to 6-membered heteroaryl group has one or more (e.g., one, two, or three) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heteroaryl group has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heteroaryl group has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5- to 14-membered heteroaryl group. In certain embodiments, the heteroaryl group is a substituted 5- to 14-membered heteroaryl group.

[0093] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepanyl, oxepinyl, and thienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazole, benzothiazolyl, benzisothiazole, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, piperidinyl, quinolyl, isoquinolyl, quinolyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0094] As used herein, "heterocyclyl" or "heterocycle" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., one, two, three, or four) ring heteroatoms, wherein 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 point of attachment can be a carbon atom or a nitrogen atom, as valence permits. A heterocyclyl group can be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged, or spirocyclic ring system, such as a bicyclic ring system ("bicyclic heterocyclyl") or a tricyclic ring system ("tricyclic heterocyclyl")), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more carbocyclyl groups, wherein the point of attachment is on the carbocyclyl or heterocyclyl ring, or a ring system in which a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases, the number of ring members always refers to the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of a heterocyclyl group is independently unsubstituted (an "unsubstituted heterocyclyl group") or substituted (a "substituted heterocyclyl group") with one or more substituents. In certain embodiments, a heterocyclyl group is an unsubstituted 3- to 14-membered heterocyclyl group. In certain embodiments, a heterocyclyl group is a substituted 3- to 14-membered heterocyclyl group.

[0095] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heterocyclyl”). In some embodiments, the 5- to 6-membered heterocyclyl has 1 or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heterocyclyl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0096] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiolyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thiolyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocanyl, oxepanyl, and thiepanyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthypyridinyl, decahydro-1,8-naphthypyridinyl, decahydropyrrolo[3,2-b]pyrrole, indolyl, phthalimidyl, naphthylimidyl, chromanyl, benzothiophenyl, 1H-benzo[e][1,4]diazapyridinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl , 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, etc.

[0097] Heterocycloalkyl: As used herein, the term "heterocycloalkyl" is a non-aromatic ring in which at least one atom is a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocycloalkyl groups may be substituted or unsubstituted.

[0098] As will be appreciated from the foregoing, alkyl, alkenyl, alkynyl, acyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups as defined herein are, in certain embodiments, optionally substituted. Optionally substituted refers to groups that may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). In general, the term "substituted" refers to a group in which at least one hydrogen present is replaced by a permitted substituent, e.g., a substituent that produces a stable compound upon substitution, e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reactions. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is the same or different at each position. It is envisioned that the term "substituted" includes substitution with all permissible substituents of an organic compound, any substituent described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations to obtain a stable compound. For the purposes of the present invention, a heteroatom (e.g., nitrogen) may have a hydrogen substituent and / or any suitable substituent as described herein that satisfies the valence of the heteroatom and forms a stable moiety.

[0099] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2, -S03H, -OH, -ORaa, -ON(Rbb)2, -N(Rbb)2, -N(Rbb)3+X-, -N(ORcc)Rbb, -SeH, -SeRaa, -SH, -SRaa, -SSRcc, -C(=O)Raa, -CO2H, -CHO, -C(ORcc)2, -CO2Raa, -OC(=O)Raa, -OCO2Raa, -C(=O)N(Rbb)2, -OC(=O)N(Rbb)2, -NRbbC(=O)Raa, -NR bbCO2Raa, -NRbbC(=O)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)ORaa, -OC(=NRbb)Raa, -OC(=NRbb)ORaa, -C(=NRbb)N(Rbb)2, -OC(=NRbb)N(Rbb )2, -NRbbC(=NRbb)N(Rbb)2, -C(=O)NRbbSO2Raa, -NRbbSO2Raa, -SO2N(Rbb)2, -SO2Raa, -SO2ORaa, -OSO2Raa, -S(=O)Raa, -OS(=O)Raa, -Si( Raa)3, -OSi(Raa)3, -C(=S)N(Rbb)2, -C(=O)SRaa, -C(=S)SRaa, -SC(=S)SRaa, -SC(=O)SRaa, -OC(=O)SRaa, -SC(=O)ORaa, -SC(=O)Raa, -P( =O)2Raa, -OP(=O)2Raa, -P(=O)(Raa)2, -OP(=O)(Raa)2, -OP(=O)(ORcc)2, -P(=O)2N(Rbb)2, -OP(=O)2N(Rbb)2, -P(=O)(NRbb)2, -OP(=O)(N Rbb)2, -NRbbP(=O)(ORcc)2, -NRbbP(=O)(NRbb)2, -P(Rcc)2, -P(Rcc)3, -OP(Rcc)2, -OP(Rcc)3, -B(Raa)2, -B(ORcc)2, -BRaa(ORcc), C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C14 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

[0100] or two geminal hydrogen atoms on a carbon atom are replaced by groups =0, =S, =NN(Rbb)2, =NNRbbC(=O)Raa, =NNRbbC(=O)ORaa, =NNRbbS(=O)2Raa, =NRbb or =NORcc;

[0101] each instance of Raa is independently selected from C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Raa groups are linked to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

[0102] Each instance of Rbb is independently selected from hydrogen, -OH, -ORaa, -N(Rcc)2, -CN, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRcc)ORaa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, -C(=S)SRcc, -P(=O)2Raa, -P(=O )(Raa)2, -P(=O)2N(Rcc)2, -P(=O)(NRcc)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl and 5-14 membered heteroaryl, or two Rbb groups and the heteroatom to which they are attached form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rdd groups;

[0103] each instance of Rcc is independently selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rcc groups, taken together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl group is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

[0104] Each instance of Rdd is independently selected from halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORee, -ON(Rff)2, -N(Rff)2, -N(Rff)3+X-, -N(ORee)Rff, -SH, -SRee, -SSRee, -C(=O)Ree, -CO2H, -CO2Ree, -OC(=O)Ree, -OCO2Ree, -C(=O)N( Rff)2, -OC(=O)N(Rff)2, -NRffC(=O)Ree, -NRffCO2Ree, -NRffC(=O)N(Rff)2, -C(=NRff)ORee, -OC (=NRff)Ree, -OC(=NRff)ORee, -C(=NRff)N(Rff)2, -OC(=NRff)N(Rff)2, -NRffC(=NRff)N(Rff)2, -NRffSO2Ree, -SO2N(Rff)2, -SO2Ree, -SO2ORee, -OSO2Ree, -S(=O)Ree, -Si(Ree)3, -OSi(Ree)3, -C(=S)N(Rff)2, -C(=O)SRee, -C(=S)SRee, -SC(=S)SRee, -P(=O)2Ree, -P(=O)(Ree)2, -OP(=O)(Re e) 2, -OP(=O)(ORee)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rgg groups, or two geminal Rdd substituents can be linked to form =O or =S;

[0105] Each instance of Ree is independently selected from C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;

[0106] each instance of R is independently selected from hydrogen, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl, or two R groups, taken together with the heteroatom to which they are attached, form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups; and

[0107] Each instance of Rgg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C50 alkyl, -ON(C1-C50 alkyl)2, -N(C1-C50 alkyl)2, -N(C1-C50 alkyl)3+X-, -NH(C1-C50 alkyl)2+X-, -NH2(C1-C50 alkyl)+X-, -NH3+X-, -N(OC1-C50 alkyl)(C1-C50 alkyl), -N(OH)(C1- C50 alkyl), -NH(OH), -SH, -SC1-C50 alkyl, -SS(C1-C50 alkyl), -C(=O)(C1-C50 alkyl), -CO2H, -CO2(C1-C50 alkyl), -OC(=O)(C1-C50 alkyl), -OCO2(C1-C50 alkyl), -C(=O)NH2, -C(=O)N(C1-C50 alkyl)2, -OC(=O)NH(C1-C50 alkyl), -NHC(=O)(C1-C5 0 alkyl), -N(C1-C50 alkyl)C(=O)(C1-C50 alkyl), -NHCO2(C1-C50 alkyl), -NHC(=O)N(C1-C50 alkyl)2, -NHC(=O)NH(C1-C50 alkyl), -NHC(=O)NH2, -C(=NH)O(C1-C50 alkyl), -OC(=NH)(C1-C50 alkyl), -OC(=NH)OC1-C50 alkyl, -C(=NH)N(C1-C50 alkyl)2 , -C(=NH)NH(C1-C50 alkyl), -C(=NH)NH2, -OC(=NH)N(C1-C50alkyl)2, -OC(NH)NH(C1-C50 alkyl), -OC(NH)NH2, -NHC(NH)N(C1-C50 alkyl)2, -NHC(=NH)NH2, -NHSO2(C1-C50 alkyl), -SO2N(C1-C50 alkyl)2, -SO2NH(C1-C50 alkyl), -SO2NH2,-SO2(C1-C50 alkyl), -SO2O(C1-C50 alkyl), -OSO2(C1-C6 alkyl), -SO(C1-C6 alkyl), -Si(C1-C50 alkyl)3, -OSi(C1-C6 alkyl)3, -C(=S)N(C1-C50 alkyl)2, C(=S)NH(C1-C50 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-C50 alkyl), -P(=O)(C1-C50 alkyl)2, -OP(=O)(C1-C50 alkyl)2, -OP(=O)(OC1-C50 alkyl)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents may be linked to form =O or =S; wherein X- is a counterion.

[0108] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0109] As used herein, a "counter ion" is a negatively charged group associated with a positively charged quaternary amine to maintain electronic neutrality. Exemplary counter ions include halides (e.g., F-, Cl-, Br-, I-), NO3-, ClO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.), and carboxylate ions (e.g., acetate, acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0110] Where valence permits, the nitrogen atoms may be substituted or unsubstituted and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -ORaa, -N(Rcc)2, -CN, -C(=O)Raa, -C(=O)N(Rcc)2, -CO2Raa, -SO2Raa, -C(=NRbb)Raa, -C(=NRcc)ORaa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -SO2ORcc, -SORaa, -C(=S)N(Rcc)2, -C(=O)SRcc, -C(=S)SRcc, -P(=O)2Raa, -P(=O)(Raa) 2, -P(=O)2N(Rcc)2, -P(=O)(NRcc)2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl and 5-14 membered heteroaryl, or two Rcc groups together with the N atom to which they are attached form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are as defined above.

[0111] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as 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 PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0112] For example, nitrogen protecting groups such as amide groups (e.g., -C(=O)Raa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyamido)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylphenoxy)propionamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyl)benzamide.

[0113] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)ORaa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthogen)]methylcarbamate (DBD-Tmoc), 4-methoxybenzamidocarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Tmoc ... Ethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-ethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl) carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl) carbamate (Pyoc ), ethyl 2-(N,N-dicyclohexylcarbamate), tert-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, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, Benzyl chloride, 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-(dihydroxyformyl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-bromomethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrobenzyl)carbamate phenyl) methylcarbamate, tert-amylcarbamate, S-benzylthiocarbamate, p-cyanobenzylcarbamate, cyclobutylcarbamate, cyclohexylcarbamate, cyclopentylcarbamate, cyclopropylmethylcarbamate, p-decyloxybenzylcarbamate, 2,2-dimethoxyacylvinylcarbamate, o-(N,N-dimethylformamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propylcarbamate methylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furylmethylcarbamate, 2-iodoethylcarbamate, isobutylcarbamate, isonicotinoylcarbamate, 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, phenyl carbamate, benzyl p-(phenylazo)carbamate, 2,4,6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate and 2,4,6-trimethylbenzylcarbamate.

[0114] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)2Raa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), and the like. ), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylsulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylsulfonamide.

[0115] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiopheneimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilazidecyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5- -substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-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-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzofuranamine, N-tritylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorofluorenamine (PhF), N-2,7-dichloro- 9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-pyridylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)-mesitylmethyl]methyleneamine, N-(N', N'-dimethylaminomethylene)amine, N, N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosulfenamide, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylphosphinothioamide (Mpt), diphenylphosphinothioamide (Ppt), dialkylphosphoramidates, dibenzylphosphoramidates, diphenylphosphoramidates, benzenesulfonamides, o-nitrobenzenesulfonamide (Nps), 2,4-dinitrobenzenesulfonamide, pentachlorobenzenesulfonamide, 2-nitro-4-methoxybenzenesulfonamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

[0116] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxy 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 PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0117] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyrrolidone, thiopyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiophenyl, 2,3,3a,4,5,6,7,7α-octahydro-7,8,8-trimethyl-4,7-methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl 1-Benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl ester, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylseleno)ethyl, tert-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-pyridylmethyl, 4-pyridylmethyl, 3-methyl-2-pyridylmethyl N-oxyanion, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzosuberyl, trityl, α-naphthyl phenylmethyl, p-methoxyphenyldiphenylmethyl, bis(p-methoxyphenyl)phenylmethyl, tris(p-methoxyphenyl)methyl, 4-(4'-bromophenacylphenoxy)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinyloxyphenyl)methyl, 4,4',4"-tris(benzyloxy)methyl, 3-(imidazol-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-Benzodithiofuran-2-yl, benzisothiazolyl S, S-dioxanyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropyl (IPDMS), diethylisopropyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoyl, acetate, chloroacetate, dichloroacetic acid, trichloroacetic acid, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, Phenoxy, p-chlorophenoxy, 3-phenylpropionate, 4-oxopentanoate (levulinic acid ester), 4,4-(ethylene)pentanoate (levorotatory diacyl dithioacetal), pivalate, adamantane, crotonate, 4-crotonate methyl ester, benzoate, p-phenyl, 2,4,6-trimethylbenzoate (2,4,6-trimethylbenzoate or mesitoate), alkyl methyl carbonate, 9-fluorenyl ester (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonyl) ) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl ethylene carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoic acid, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6- Dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monomalate, (E)-2-methyl-2-butenoate, o-(methoxy)benzoate, alpha-naphthalene, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphine, alkyl 2,4-dinitrophenylsulfinate, sulfate, methanesulfonate (methanesulfonate or mesylate), benzylsulfonate, and toluenesulfonate (Ts).

[0118] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protective Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999, which reference is incorporated herein by reference.

[0119] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolylmethyl, 2-picolyl N-oxyanion, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosuccinyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, tert-butyl, 1-adamantyl, methoxymethyl (MOM), isobutyloxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, Allyloxycarbonylaminomethyl, phenylacetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(ethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylpropyl-2-yl, acetyl, benzoyl, trifluoroacetyl, N-[[(p-biphenyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, thiocarbonate, 3-nitro-2-pyridylthiosulfide, oxythiophene.

[0120] Compounds of the present invention

[0121] Liposome-based vehicles are considered to be attractive carriers of therapeutic agents, and still need continuous development efforts. Although the liposome-based vehicles comprising some lipid components show promising results in terms of encapsulation, stability and site location, there is still a great need to improve the delivery system based on liposomes. For example, the significant shortcoming of liposome delivery systems relates to the structure of the liposomes with enough cell cultures or in vivo stability to reach desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to effectively release the material of their encapsulation to such target cells.

[0122] In particular, there remains a need for improved lipid compounds that exhibit improved pharmacokinetic properties and are capable of delivering macromolecules (such as nucleic acids) to a variety of cell types and tissues with increased efficiency. Importantly, there remains a particular need for novel lipid compounds characterized by reduced toxicity and capable of effectively delivering encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0123] A novel cationic lipid compound is described herein for use in vivo for the improved delivery of therapeutic agents such as nucleic acids. Specifically, cationic lipids as described herein can be optionally used together with other lipids to formulate lipid-based nanoparticles (e.g., liposomes) for the encapsulation of therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic use.

[0124] In an embodiment, the compounds of the present invention as described herein can provide one or more desired characteristics or properties. That is, in certain embodiments, the compounds of the present invention as described herein can be characterized as having one or more properties, which provide advantages of such compounds relative to other similarly classified lipids. For example, the compounds disclosed herein can allow control and customization of the characteristics of the liposome compositions (e.g., lipid nanoparticles) of which they are components. Specifically, the characteristics of the compounds disclosed herein can be enhanced transfection efficiency and the ability to cause specific biological results thereof. Such results can include, for example, enhanced cellular uptake, endosome / lysosome destruction ability and / or promote the release of intracellular encapsulated materials (e.g., polynucleotides). In addition, the compounds disclosed herein have favorable pharmacokinetic properties, biodistribution and efficiency (e.g., due to the different dissociation rates of the polymer groups used).

[0125] This application demonstrates that the cationic lipids of the present invention are not only synthetically processible from readily available starting materials, but they also possess unexpectedly high encapsulation efficiencies.

[0126] In addition, the cationic lipids of the present invention have cleavable groups, such as ester groups and disulfides. These cleavable groups (e.g., esters and disulfides) are believed to increase biodegradability and thus contribute to their favorable toxicity characteristics.

[0127] Compounds of the present invention

[0128] Provided herein are compounds that are cationic lipids. For example, the cationic lipids of the present invention include compounds having a structure according to formula (I):

[0129]

[0130] wherein L1 is a bond, (C1-C6)alkyl or (C2-C6)alkenyl;

[0131] Wherein X is O or S;

[0132] where R 1 、R 2 、R 3 、R 4 and R 5 are each independently selected from H, OH, optionally substituted (C1-C6)alkyl, optionally substituted (C2-C6)alkenyl, optionally substituted (C2-C6)alkynyl, optionally substituted (C1-C6)alkoxy, and -OC(O)R';

[0133] where R 1 、R 2 、R 3 、R 4 or R 5 At least one of them is -OC(O)R';

[0134] Where R' is

[0135]

[0136] where R 6 for

[0137]

[0138] wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

[0139] where R 7 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A ;

[0140] where R 8 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B ;

[0141] where R 9is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C ;

[0142] where R 10 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D ;

[0143] wherein k, n, q and r are each independently 1, 2, 3, 4 or 5;

[0144] or (i) R 7 and R 8 or (ii) R 9 and R 10 together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, wherein the heterocycloalkyl or heteroaryl includes 1 to 3 heteroatoms selected from N, O and S;

[0145] where R 11 、R 12 、R 13 and R 14 Each is independently selected from H, methyl, ethyl or propyl;

[0146] where R A 、R B 、R C and R D Each independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 )acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, -NH2;

[0147] where R 7 、R 8 、R9 、R 10 At least one of them includes R A 、R B 、R C or R D part, wherein the R A 、R B 、R C or R D are independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)(C6-C 20 )alkyl or optionally substituted -OC(O)(C6-C 20 )alkenyl;

[0148] or a pharmaceutically acceptable salt thereof.

[0149] In embodiments, any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of: (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)acyl, (C1-C6)alkoxy, halogen, -COR, -CO2H, -CO2R, -CN, -OH, -OR, -OCOR, -OCOR, -NH2, -NHR, -N(R)2, -SR, or -S02R, or two geminal hydrogens on a carbon atom are replaced by a group =NH, wherein each instance of R is independently a C1-C10 aliphatic alkyl.

[0150] In an embodiment, L1 is independently a bond.

[0151] In an embodiment, L1 is (C1-C6)alkyl.

[0152] In an embodiment, L1 is (C2-C6)alkenyl.

[0153] In an embodiment, L1 is C2 alkenyl.

[0154] In an embodiment, RA and RB are the same. In an embodiment, RC and RD are the same. In an embodiment, RA and RB are the same, and RC and RD are the same.

[0155] In an embodiment, RA and RB are different. In an embodiment, RC and RD are different. In an embodiment, RA and RB are different, and RC and RD are different.

[0156] In an embodiment, RA, RB, RC, and RD are the same.

[0157] In an embodiment, RA, RB, RC, and RD are different.

[0158] In embodiments, RA, RB, RC, or RD are each independently selected from optionally substituted (C6-C20)alkyl, optionally substituted (C6-C20)alkenyl, optionally substituted (C6-C20)alkynyl, optionally substituted (C6-C20)acyl, optionally substituted -OC(O)(C6-C20)alkyl, or optionally substituted -OC(O)(C6-C20)alkenyl.

[0159] In embodiments, RA, RB, RC, or RD are the same and are selected from optionally substituted (C6-C20)alkyl, optionally substituted (C6-C20)alkenyl, optionally substituted (C6-C20)alkynyl, optionally substituted (C6-C20)acyl, optionally substituted -OC(O)(C6-C20)alkyl, or optionally substituted -OC(O)(C6-C20)alkenyl.

[0160] In an embodiment, RA and RB are each independently selected from optionally substituted (C6-C20)alkyl, optionally substituted (C6-C20)alkenyl, optionally substituted (C6-C20)alkynyl.

[0161] In an embodiment, RA and RB are the same and are selected from optionally substituted (C6-C20)alkyl, optionally substituted (C6-C20)alkenyl, optionally substituted (C6-C20)alkynyl.

[0162] In an embodiment, RA and RB are each independently optionally substituted (C6-C20)alkyl.

[0163] In an embodiment, RA and RB are the same and are optionally substituted (C6-C20)alkyl.

[0164] In embodiments, RA and RB are each independently optionally substituted (C6-C20)alkenyl.

[0165] In an embodiment, RA and RB are the same and are optionally substituted (C6-C20)alkenyl.

[0166] In embodiments, RA and RB are each independently optionally substituted (C6-C20)alkynyl.

[0167] In an embodiment, RA and RB are the same and are optionally substituted (C6-C20)alkynyl.

[0168] In embodiments, RA and RB are each independently optionally substituted (C6-C20)acyl.

[0169] In an embodiment, RA and RB are the same and are optionally substituted (C6-C20)acyl.

[0170] In embodiments, RA and RB are each independently optionally substituted -OC(O)(C6-C20)alkyl.

[0171] In an embodiment, RA and RB are the same and are optionally substituted -OC(O)(C6-C20)alkyl.

[0172] In embodiments, RA and RB are each independently optionally substituted -OC(O)(C6-C20)alkenyl.

[0173] In an embodiment, RA and RB are the same and are optionally substituted -OC(O)(C6-C20)alkenyl.

[0174] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are each independently selected from:

[0175]

[0176] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are the same and are selected from:

[0177]

[0178]

[0179] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are both C8H17.

[0180] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and both RA and RB are C10H21.

[0181] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are both C12H25.

[0182] In an embodiment, R7=-(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and RA and RB are

[0183] In an embodiment, X is O.

[0184] In an embodiment, X is S.

[0185] In an embodiment, only one of R1, R2, R3, R4, and R5 is -OC(O)R'. In an embodiment, only one of R1, R2, R3, R4, and R5 is -OC(O)R', and none of R1, R2, R3, R4, or R5 is OH.

[0186] In an embodiment, two of R1, R2, R3, R4, and R5 are -OC(O)R'. In an embodiment, two of R1, R2, R3, R4, and R5 are -OC(O)R', and none of R1, R2, R3, R4, or R5 is OH.

[0187] In an embodiment, three of R1, R2, R3, R4, and R5 are -OC(O)R'.

[0188] In an embodiment, R1 is -OC(O)R'. In an embodiment, R5 is -OC(O)R'. In an embodiment, both R1 and R5 are -OC(O)R'.

[0189] In an embodiment, R2 is -OC(O)R'. In an embodiment, R4 is -OC(O)R'. In an embodiment, both R2 and R4 are -OC(O)R'.

[0190] In an embodiment, R3 is -OC(O)R'.

[0191] In an embodiment, R3 is -OC(O)R', and R2 is OMe.

[0192] In an embodiment, L1 is a bond, R3 is -OC(O)R', and R2 is OMe.

[0193] In an embodiment, R3 is -OC(O)R', and R2 and R4 are OMe.

[0194] In an embodiment, L1 is a bond, R3 is -OC(O)R', and R2 and R4 are OMe.

[0195] In an embodiment, L1 is (C2-C6)alkenyl, R3 is -OC(O)R', and R2 and R4 are OMe.

[0196] In an embodiment, L1 is C2 alkenyl, R3 is -OC(O)R', and R2 and R4 are OMe.

[0197] In an embodiment, R7 is -(CH2)kCH(OR11)RA.

[0198] In an embodiment, R7 is -(CH2)1CH(OR11)RA.

[0199] In an embodiment, R7 is -(CH2)1CH(OH)RA.

[0200] In an embodiment, R8 is -(CH2)nCH(OR12)RB.

[0201] In an embodiment, R8 is -(CH2)1CH(OR12)RB.

[0202] In an embodiment, R8 is -(CH2)1CH(OH)RB.

[0203] In an embodiment, R7 is -(CH2)kCH(OR11)RA, and R8 is -(CH2)nCH(OR12)RB.

[0204] In an embodiment, R7 is -(CH2)1CH(OR11)RA, and R8 is -(CH2)1CH(OR12)RB.

[0205] In an embodiment, R7 is -(CH2)1CH(OH)RA, and R8 is -(CH2)1CH(OH)RB.

[0206] In an embodiment, R7 and R8 are each optionally substituted (C1-C6)alkyl, for example, with -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In an embodiment, R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 40 In an embodiment, R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 30 In an embodiment, R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 20 alkyl.

[0207] In an embodiment, R7 and R8 are the same and each is an optionally substituted (C1-C6) alkyl group, for example, a (C1-C6) alkyl group substituted with -CO2Raa, wherein Raa is a C1-C50 alkyl group. In an embodiment, R7 and R8 are the same and each is an optionally substituted (C1-C6) alkyl group, for example, a (C1-C6) alkyl group substituted with -CO2Raa, wherein Raa is a C1-C40 alkyl group. In an embodiment, R7 and R8 are the same and each is an optionally substituted (C1-C6) alkyl group substituted with -CO2Raa, wherein Raa is a C1-C30 alkyl group. In an embodiment, R7 and R8 are each an optionally substituted (C1-C6) alkyl group substituted with -CO2Raa, wherein Raa is a C1-C20 alkyl group.

[0208] In an embodiment, R7 and R8 are each

[0209] In an embodiment, R7 and R8 are each

[0210] In an embodiment, R9 and R10 are each independently selected from H, optionally substituted (C1-C6)alkyl, optionally substituted (C2-C6)alkenyl, optionally substituted (C2-C6)alkynyl.

[0211] In an embodiment, R9 and R10 are each independently optionally substituted (C1-C6)alkyl or optionally substituted (C2-C6)alkenyl.

[0212] In an embodiment, R9 and R10 are both optionally substituted (C1-C6)alkyl or optionally substituted (C2-C6)alkenyl.

[0213] In an embodiment, R9 and R10 are both optionally substituted (C1-C6)alkyl.

[0214] In an embodiment, R9 and R10 are both -CH3.

[0215] In an embodiment, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, and R9 and R10 are both -CH3.

[0216] In an embodiment, R7 is -(CH2)1CH(OR11)RA, R8 is -(CH2)1CH(OR12)RB, and R9 and R10 are both -CH3.

[0217] In an embodiment, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, and R9 and R10 are both -CH3.

[0218] In an embodiment, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, RA and RB are both C8H17, and R9 and R10 are both -CH3.

[0219] In an embodiment, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, RA and RB are both C8H17, and R9 and R10 are both -CH3.

[0220] In an embodiment, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, RA and RB are both C10H21, and R9 and R10 are both -CH3.

[0221] In an embodiment, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, RA and RB are both C10H21, and R9 and R10 are both -CH3.

[0222] In an embodiment, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, RA and RB are both C12H25, and R9 and R10 are both -CH3.

[0223] In an embodiment, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, RA and RB are both C12H25, and R9 and R10 are both -CH3.

[0224] In an embodiment, R7 is -(CH2)kCH(OR11)RA, R8 is -(CH2)nCH(OR12)RB, RA and RB are both C16H29, and R9 and R10 are both -CH3.

[0225] In an embodiment, R7 is -(CH2)1CH(OH)RA, R8 is -(CH2)1CH(OH)RB, RA and RB are both C16H29, and R9 and R10 are both -CH3.

[0226] In an embodiment, p, g, and r are the same. In an embodiment, one or more of p, q, and r are different. In an embodiment, q and r are the same, and p is different. In an embodiment, p and q are the same, and r is different. In an embodiment, p and r are the same, and q is different. In an embodiment, p, q, and r are different.

[0227] In an embodiment, k, m, and n are the same. In an embodiment, one or more of k, m, and n are different. In an embodiment, k and m are the same, and n is different. In an embodiment, m and n are the same, and k is different. In an embodiment, k and n are the same, and m is different. In an embodiment, k, m, and n are different.

[0228] In an embodiment, m is 1, 2, 3, 4, or 5. In an embodiment, m is 0. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In an embodiment, m is 0, 1, 2, 3, or 4.

[0229] In an embodiment, p is 1, 2, 3, 4, or 5. In an embodiment, p is 0. In an embodiment, p is 1. In an embodiment, p is 2. In an embodiment, p is 3. In an embodiment, p is 4. In an embodiment, p is 5. In an embodiment, p is 0, 1, 2, 3, or 4.

[0230] In an embodiment, m is 2, and p is 2.

[0231] In an embodiment, m is 3 and p is 2.

[0232] In an embodiment, k and n=1, and m=2.

[0233] In an embodiment, k and n=1, and m=3.

[0234] In an embodiment, q and r=1, and p=2.

[0235] In an embodiment, k, n, q and r are each = 1, m = 2 or 3, and p = 2.

[0236] In an embodiment, R' is:

[0237]

[0238] In an embodiment, R' is And k and n=1, and m=2 or 3.

[0239] In an embodiment, R' is And k and n=1, and m=2.

[0240] In an embodiment, R' is And k and n=1, and m=3.

[0241] In an embodiment, R' is And R 11 and R 12 For H.

[0242] In an embodiment, R' is And k and n = 1, m = 2 or 3, and R 11 and R 12 For H.

[0243] In an embodiment, R' is And k and n = 1, m = 2, and R 11 and R 12 For H.

[0244] In an embodiment, R' is And k and n = 1, m = 3, and R 11 and R 12 For H.

[0245] In an embodiment, R' is:

[0246]

[0247] In which R' has the following structure In any of the above embodiments, R A and R B May be as defined in any of paragraphs

[0104] to

[0129] .

[0248] In an embodiment, R 6 for:

[0249]

[0250] In an embodiment, R 6 for q and r=1, and p=2.

[0251] In an embodiment, R 6 for And R 13 and R 14 For H.

[0252] In an embodiment, R 6 for q and r = 1, p = 2, and R 13 and R 14 For H.

[0253] In an embodiment, R 6 Selected from the group consisting of:

[0254]

[0255] In an embodiment, R 6 Selected from the group consisting of:

[0256]

[0257] In an embodiment, R 6 Selected from the group consisting of:

[0258] In an embodiment, R 6 for:

[0259]

[0260] In an embodiment, R 6 for:

[0261]

[0262] In an embodiment, R 6 for:

[0263]

[0264] In an embodiment, R 6 Selected from the group consisting of:

[0265] In an embodiment, R 6 for:

[0266]

[0267] In an embodiment, R 6 for:

[0268]

[0269] In an embodiment, R 6 for:

[0270]

[0271] In an embodiment, R 6 for:

[0272]

[0273] In an embodiment, R 6 for:

[0274]

[0275] In an embodiment, R 6 Same as R'.

[0276] In an embodiment, R 6 for R' is m is 2, and p is 2.

[0277] In an embodiment, R 6 for R' is m is 3, and p is 2.

[0278] In the embodiment, L1 key, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0279] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0280] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0281] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 1 and R 5 H, R 6 for And R' is

[0282]

[0283] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0284] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 1 and R 5 H, R 6for And R' is

[0285]

[0286] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for And R' is

[0287] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for And R' is

[0288] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 1 、R 4 and R 5 H, R 6 for And R' is

[0289]

[0290] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for And R' is

[0291] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for And R' is

[0292] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 1 、R 4 and R 5 H, R 6 for And R' is

[0293] In an embodiment, L1 is C2 alkenyl, R3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0294] In an embodiment, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0295] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0296] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 1 and R 5 H, R 6 for And R' is

[0297]

[0298] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for And R' is

[0299] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 1 and R 5 H, R 6 for And R' is

[0300]

[0301] In an embodiment, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl.

[0302] In an embodiment, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, m is 2, and p is 2. In some embodiments, R 7 and R 8 In some embodiments, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 For OMe.

[0303] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 same.

[0304] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0305] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 In some embodiments, m is 2.

[0306] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 1 and R 5 H, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 In some embodiments, m is 2.

[0307] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0308] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 and R4 OMe, R 1 and R 5 H, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0309] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 In some embodiments, m is 2.

[0310] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 In some embodiments, m is 2.

[0311] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 1 、R 4 and R 5 H, R 6 for R' is And R 7 and R8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 In some embodiments, m is 2.

[0312] In an embodiment, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0313] In the embodiment, X=O, L1 is a bond, R 3 -OC(O)R', R 2 OMe, R 1 、R 4 and R 5 H, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0314] In an embodiment, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R8 same.

[0315] In an embodiment, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0316] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R 4 OMe, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 same.

[0317] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R',R 2 and R 4 OMe, R 1 and R 5 H, R 6 for R' is And R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In some embodiments, R 7 and R 8 same.

[0318] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R', R 2 and R4 OMe, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0319] In the embodiment, X=O, L1 is C2 alkenyl, R 3 -OC(O)R',R 2 and R 4 OMe, R 1 and R 5 H, R 6 for R' is R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 alkyl, and m is 2. In some embodiments, R 7 and R 8 same.

[0320] In which R 7 and R 8 For -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In any of the above embodiments of alkyl, R aa Can alternatively be C1-C 40 alkyl.

[0321] In which R 7 and R 8 For -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In any of the above embodiments of alkyl, R aa Can alternatively be C1-C 30 alkyl.

[0322] In which R 7 and R 8 For -CO2R aa Substituted (C1-C6) alkyl, wherein R aaC1-C 50 In any of the above embodiments of alkyl, R aa Can alternatively be C1-C 20 alkyl.

[0323] In which R 7 and R 8 For -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In any of the embodiments of the alkyl group, R 7 and R 8 Can each

[0324]

[0325] In which R 7 and R 8 Each is -CO2R aa Substituted (C1-C6) alkyl, wherein R aa C1-C 50 In any of the embodiments of the alkyl group, R 7 and R 8 Can each

[0326]

[0327] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (II):

[0328]

[0329] or a pharmaceutically acceptable salt thereof, wherein R 1 -R 6 and X are as defined herein.

[0330] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIA):

[0331]

[0332] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0333] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIB), (IIC), (IID), (IIE), (IIJ), or (IIK):

[0334]

[0335]

[0336]

[0337] or a pharmaceutically acceptable salt thereof.

[0338] In an embodiment, the cationic lipid of the invention has the structure:

[0339] or a pharmaceutically acceptable salt thereof.

[0340] In an embodiment, the cationic lipid of the invention has the structure:

[0341] or a pharmaceutically acceptable salt thereof.

[0342] In an embodiment, the cationic lipid of the invention has the structure:

[0343]

[0344] or a pharmaceutically acceptable salt thereof.

[0345] In an embodiment, the cationic lipid of the invention has the structure:

[0346]

[0347] or a pharmaceutically acceptable salt thereof.

[0348] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIF):

[0349]

[0350] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0351] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIG):

[0352]

[0353] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0354] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to Formula (IIH):

[0355]

[0356] wherein one of Y and Z is OH and the other is -OC(O)R', or wherein both Y and Z are each independently -OC(O)R', and wherein R', R 6 and X are as defined herein.

[0357] In an embodiment, the cationic lipid of the present invention comprises a compound having a structure according to formula (III):

[0358]

[0359] or a pharmaceutically acceptable salt thereof, wherein R 1 -R 6 and X are as defined herein.

[0360] In an embodiment, the cationic lipid of the invention comprises a compound having a structure according to Formula (IIIA):

[0361]

[0362] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0363] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIIB):

[0364]

[0365] or a pharmaceutically acceptable salt thereof, wherein R A 、R B and p are as defined herein. In an embodiment, the cationic lipid of the present invention has the structure:

[0366]

[0367] or a pharmaceutically acceptable salt thereof, wherein R A and R B As defined herein. In an embodiment, the cationic lipid of the present invention has the structure:

[0368]

[0369] or a pharmaceutically acceptable salt thereof, wherein R A and R B As defined herein. In an embodiment, the cationic lipid of the present invention has the structure:

[0370]

[0371] or a pharmaceutically acceptable salt thereof.

[0372] In an embodiment, the cationic lipid of the invention has the structure:

[0373]

[0374] or a pharmaceutically acceptable salt thereof.

[0375] In an embodiment, the cationic lipid of the present invention comprises a compound having a structure according to Formula (IIID):

[0376]

[0377] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0378] In embodiments, the cationic lipids of the invention comprise compounds having a structure according to Formula (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ), or (IIIK):

[0379]

[0380]

[0381]

[0382] or a pharmaceutically acceptable salt thereof.

[0383] In an embodiment, the cationic lipid of the invention has the structure:

[0384] In an embodiment, the cationic lipid of the invention has the structure:

[0385] In an embodiment, the cationic lipid of the invention has the structure:

[0386] In an embodiment, the cationic lipid of the invention has the structure:

[0387] In an embodiment, the cationic lipid of the invention has the structure:

[0388] In an embodiment, the cationic lipid of the invention has the structure:

[0389]

[0390] In an embodiment, the cationic lipid of the invention has the structure:

[0391]

[0392] In an embodiment, the cationic lipid of the invention comprises a compound having a structure according to Formula (IIIL):

[0393]

[0394] or a pharmaceutically acceptable salt thereof, wherein R', R 6 and X are as defined herein.

[0395] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (IV):

[0396]

[0397] wherein M is selected from H, OH, OMe or Me,

[0398] or a pharmaceutically acceptable salt thereof, wherein R A 、R B , m and p are as defined herein.

[0399] In embodiments the cationic lipid of the invention comprises a compound having a structure according to Formula (VI), (VII), (VIII), (IX) or (X):

[0400]

[0401]

[0402] or a pharmaceutically acceptable salt thereof,

[0403] wherein one of Y and Z is OH and the other is -OC(O)R', or wherein both Y and Z are each independently -OC(O)R', and wherein R', R 6 and X are as already defined herein, or a pharmaceutically acceptable salt thereof.

[0404] In an embodiment, one of Y and Z is OH and the other is -OC(O)R'.

[0405] In an embodiment, Y is OH, and Z is -OC(O)R'.

[0406] In an embodiment, Y is -OC(O)R', and Z is OH.

[0407] In an embodiment, both Y and Z are -OC(O)R'.

[0408] In embodiments, a composition is provided that includes the cationic lipid of any of the preceding embodiments, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In embodiments, the composition is a lipid nanoparticle. In embodiments, the one or more cationic lipids constitute approximately 30-60 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids constitute 10-50 mol% of the lipid nanoparticle. In embodiments, the one or more PEG-modified lipids constitute 1-10 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipids constitute 10-50 mol% of the lipid nanoparticle. In embodiments, the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticle is at least 70%. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticle is at least 75%. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticle is at least 80%. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticle is at least 85%. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticle is at least 90%. In embodiments, the mRNA encapsulation percentage of the lipid nanoparticles is at least 95%.

[0409] In embodiments, the composition according to any one of the preceding embodiments is used for use in therapy.

[0410] In an embodiment, the composition according to any of the preceding embodiments is used in a method of treating or preventing a disease suitable for treatment or prevention by a peptide or protein encoded by an mRNA, optionally wherein the disease is: (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain or muscle; (b) an autoimmune disease; (c) an infectious disease; or (d) cancer.

[0411] In embodiments, the composition is administered intravenously, intrathecally, or intramuscularly, or delivered pulmonary, optionally by aerosolization.

[0412] Exemplary compounds

[0413] Exemplary compounds include those described in Tables 1-8

[0414] Table 1

[0415]

[0416]

[0417]

[0418]

[0419] Table 2

[0420]

[0421]

[0422]

[0423]

[0424] Table 3

[0425]

[0426]

[0427]

[0428]

[0429] Table 4

[0430]

[0431]

[0432]

[0433]

[0434] Table 5

[0435]

[0436]

[0437]

[0438]

[0439] Table 6

[0440]

[0441]

[0442]

[0443]

[0444] Table 7

[0445]

[0446]

[0447] Table 8

[0448]

[0449] Any of the compounds identified in Tables 1 to 8 above may be provided in the form of a pharmaceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0450] Unless otherwise stated, R = C 16 H 29 With structure:

[0451]

[0452] Unless otherwise stated, R = C 16 H 31 With structure:

[0453]

[0454] The compounds of the present invention as described herein can be prepared according to methods known in the art, including the exemplary syntheses of the examples provided herein.

[0455] Nucleic Acids

[0456] The compounds of the invention as described herein can be used to prepare compositions useful for the delivery of nucleic acids.

[0457] Nucleic acid synthesis

[0458] Nucleic acids according to the present invention can be synthesized according to any known method. For example, mRNA according to the present invention can be synthesized by in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, a mutant T7 or SP6 RNA polymerase), DNAse I, a pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application.

[0459] In certain embodiments, to prepare mRNA according to the present invention, the DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription, such as T3, T7, a mutated T7 or SP6 promoter, followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0460] Can use standard method to measure according to the expectation mRNA sequence of the present invention and mix it in the DNA template.For example, starting from the expected amino acid sequence (for example enzyme sequence), carry out virtual reverse translation based on degenerate genetic code.Then can use optimization algorithm to select suitable codon.Usually, can optimize G / C content on the one hand, to realize G / C content as high as possible, on the other hand, use the frequency of considering tRNA according to codon as far as possible.Can for example set up and display optimized RNA sequence by suitable display device, and compare it with original (wild type) sequence.Can also analyze secondary structure to calculate respectively stable and destabilizing characteristic or the zone of RNA.

[0461] Modified mRNA

[0462] In certain embodiments, the mRNA according to the present invention can be synthesized as unmodified mRNA or modified mRNA. The modified mRNA includes nucleotide modifications in the RNA. Therefore, the modified mRNA according to the present invention may include nucleotide modifications, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNAs can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5- uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetate, 5-methylaminomethyl-uracil , 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, quercetin, β-D-mannosyl-quercetin, whibutoxoside, as well as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. The preparation of such analogs is known to those skilled in the art, for example, according to U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0463] Cationic lipid and nucleic acid pharmaceutical preparations

[0464] In certain embodiments, the compounds of the invention as described herein, and pharmaceuticals and liposome compositions comprising such lipids, can be used in formulations to facilitate delivery of encapsulated materials (e.g., one or more polynucleotides, such as mRNA) to one or more target cells and subsequent transfection. For example, in certain embodiments, the cationic lipids described herein (and compositions comprising such lipids, such as liposome compositions) are characterized by causing one or more of receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and macropinocytosis, fusogenicity, endosomal or lysosomal disruption, and / or releasable properties, which provide advantages of such compounds over other similarly classified lipids.

[0465] According to the present invention, a nucleic acid as described herein, eg, mRNA encoding a protein (eg, full length, fragment, or portion of a protein), can be delivered via a delivery vehicle comprising a compound of the invention as described herein.

[0466] As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle," or their grammatical equivalents are used interchangeably.

[0467] For example, the invention provides compositions (e.g., pharmaceutical compositions) comprising a compound as described herein and one or more polynucleotides. Compositions (e.g., pharmaceutical compositions) can also include one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and / or one or more PEG-modified lipids.

[0468] In certain embodiments, the composition shows enhanced (e.g., improved) ability to transfect one or more target cells. Therefore, a method for transfecting one or more target cells is also provided herein. Such methods generally include 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), so that one or more target cells are transfected with a material encapsulated therein (e.g., one or more polynucleotides). As used herein, the term "transfection" refers to introducing one or more encapsulating materials (e.g., nucleic acids and / or polynucleotides) into a cell intracellularly, or preferably into a target cell. The introduced polynucleotides can be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulating materials (e.g., polynucleotides) taken up, introduced and / or expressed by the target cells undergoing transfection. In fact, transfection efficiency can be estimated by the amount of the reporter polynucleotide product produced by the target cell after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the likelihood that an appropriate dose of the encapsulated material (e.g., one or more polynucleotides) will be delivered to the site of pathology and subsequently expressed, while minimizing potential systemic side effects or toxicity associated with the compound or its encapsulated contents.

[0469] After transfection of one or more target cells by, for example, polynucleotides encapsulated in one or more lipid nanoparticles constituting a pharmaceutical composition or liposome composition disclosed herein, the production of a product (e.g., a polypeptide or protein) encoded by such polynucleotides can be preferably stimulated, and the ability of such target cells to express the polynucleotides and produce, for example, a polypeptide or protein of interest can be enhanced. For example, transfection of target cells by one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of a protein or enzyme encoded by such mRNA.

[0470] In addition, delivery vehicle as herein described (for example, liposome delivery vehicle) can be prepared to preferentially be distributed to other target tissues, cells or organs, such as heart, lung, kidney, spleen.In an embodiment, lipid nanoparticle of the present invention can be prepared to realize the delivery of the enhancement to target cell and tissue.For example, the polynucleotide (for example, mRNA) encapsulated in one or more compounds as herein described or pharmaceutical composition and liposome composition can be delivered to and / or transfected target cell or target tissue.In certain embodiments, the polynucleotide (for example, mRNA) of encapsulation can be expressed by target cell and produced (and secreted in some cases) functional polypeptide product by target cell, thereby giving the characteristic that for example target cell or target tissue are useful.The polynucleotide (for example, mRNA) of this type of encapsulation can encode for example hormone, enzyme, receptor, polypeptide, peptide or other protein of interest.

[0471] Liposomal delivery vehicles

[0472] In some embodiments, the composition is a suitable delivery vehicle. In some embodiments, the composition is a liposomal delivery vehicle, e.g., a lipid nanoparticle.

[0473] The terms "liposomal delivery vehicle" and "liposomal composition" are used interchangeably.

[0474] Enriching liposome compositions using one or more cationic lipids among the cationic lipids disclosed herein can be used as a means to improve (e.g., reduce) toxicity or otherwise impart one or more desired properties to such enriched liposome compositions (e.g., improving the delivery of encapsulated polynucleotides to one or more target cells and / or reducing the in vivo toxicity of the liposome composition). Thus, pharmaceutical compositions, particularly liposome compositions, comprising one or more cationic lipids disclosed herein are also contemplated.

[0475] Thus, in certain embodiments, the compounds of the invention as described herein can be used as components of liposome compositions to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0476] As used herein, liposome delivery vehicles (for example, lipid nanoparticles) are generally characterized as microscopic vesicles with internal water space, and this internal water space is isolated from external medium by one or more double-layered films.The double-layer membrane of liposome is generally formed by amphipathic molecules, such as the synthesis of hydrophilic and hydrophobic domains separated in space or lipids of natural origin (Lasic, Trends Biotechnol., 16:307-321,1998).The double-layer membrane of liposome can also be formed by amphipathic polymers and surfactants (for example, polymer vesicles, noid liposomes etc.).In the context of the present invention, liposome delivery vehicles are generally used for transporting desired mRNA to target cells or tissues.

[0477] In certain embodiments, such compositions (eg, liposomal compositions) are loaded with or otherwise encapsulate a material, such as one or more biologically active polynucleotides (eg, mRNA).

[0478] In an embodiment, a composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein encapsulated within a liposome. In an embodiment, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, and wherein at least one cationic lipid is a compound of the invention as described herein. In an embodiment, the composition comprises an mRNA encoding a protein (e.g., any protein described herein). In an embodiment, the composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In an embodiment, the composition comprises an mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0479] In embodiments, a composition (eg, a pharmaceutical composition) includes a nucleic acid encapsulated in a liposome, wherein the liposome comprises a compound described herein.

[0480] In an embodiment, the nucleic acid is an mRNA encoding a peptide or protein. In an embodiment, the mRNA encodes a peptide or protein for delivery to a subject's lung or lung cells or for treatment thereof (e.g., mRNA encoding cystic fibrosis transmembrane conductance regulator (CFTR) protein). In an embodiment, the mRNA encodes a peptide or protein for delivery to a subject's liver or liver cells or for treatment thereof (e.g., mRNA encoding ornithine transcarbamylase (OTC) protein). Other exemplary mRNAs are also described herein.

[0481] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) can have a net positive charge.

[0482] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) can have a net negative charge.

[0483] In embodiments, the liposomal delivery vehicle (eg, lipid nanoparticle) can have a net neutral charge.

[0484] In embodiments, lipid nanoparticles encapsulating nucleic acids (eg, mRNA encoding a peptide or protein) include one or more compounds of the invention as described herein.

[0485] For example, the amount of a compound of the invention in a composition as described herein can be described as a percentage ("wt %") of the combined dry weight of all lipids of the composition (e.g., the combined dry weight of all lipids present in a liposome composition).

[0486] In embodiments of the pharmaceutical compositions described herein, the compound of the invention as described herein is present in an amount of about 0.5 wt % to about 30 wt % (e.g., about 0.5 wt % to about 20 wt %) of the combined dry weight of all lipids present in the composition (e.g., a liposomal composition).

[0487] In an embodiment, the compound of the invention as described herein is present in an amount of about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal composition). In an embodiment, the compound of the invention as described herein is present in an amount of about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in a composition (e.g., a liposomal delivery vehicle).

[0488] In embodiments, the amount of the compound of the invention as described herein is present in an amount of at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt% or about 99 wt% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0489] In embodiments, the amount of the compound of the invention as described herein is present in an amount of no more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt% or about 99 wt% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0490] In an embodiment, compositions (for example, liposome delivery vehicle, such as lipid nanoparticles) include about 0.1wt% to about 20wt% (for example, about 0.1wt% to about 15wt%) of a compound as described herein. In an embodiment, delivery vehicle (for example, liposome delivery vehicle, such as lipid nanoparticles) include about 0.5wt%, about 1wt%, about 3wt%, about 5wt% or about 10wt% of a compound as described herein. In an embodiment, delivery vehicle (for example, liposome delivery vehicle, such as lipid nanoparticles) include up to about 0.5wt%, about 1wt%, about 3wt%, about 5wt%, about 10wt%, about 15wt% or about 20wt% of a compound as described herein. In an embodiment, the percentage results in improved beneficial effects (for example, improved delivery to target tissues (such as liver or lungs).

[0491] The amount of a compound of the invention in a composition as described herein can also be described as a percentage ("mol %") of the combined moles of the total lipids of the composition (eg, the combined moles of all lipids present in a liposomal delivery vehicle).

[0492] In embodiments of the pharmaceutical compositions described herein, the compound of the invention as described herein is present in an amount of about 0.5 mol % to about 50 mol % (e.g., about 0.5 mol % to about 20 mol %) of the combined molar amount of all lipids present in the composition (e.g., liposomal delivery vehicle).

[0493] In embodiments, the compound of the invention as described herein is present in an amount 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 combined molar amount of all lipids present in the composition (e.g., liposomal delivery vehicle). In embodiments, the compound of the invention as described herein is present in an amount 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 combined molar amount of all lipids present in the composition (e.g., liposomal delivery vehicle).

[0494] In certain embodiments, the compounds of the invention as described herein may comprise from about 0.1 mol% to about 50 mol%, or from 0.5 mol% to about 50 mol%, or from about 1 mol% to about 25 mol%, or from about 1 mol% to about 10 mol% of the total amount of lipids in a composition (e.g., a liposomal delivery vehicle).

[0495] In certain embodiments, the compound of the invention as described herein may comprise greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, 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% of the total amount of lipid in the lipid nanoparticle.

[0496] In certain embodiments, the compounds as described may comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, 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 a composition (e.g., a liposomal delivery vehicle).

[0497] In embodiments, the amount of a compound of the invention as described herein is present in an amount of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined molar amount of the total lipids in the composition (e.g., a liposome composition).

[0498] In embodiments, the amount of the compound of the invention as described herein is present in an amount of no more than 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 combined molar amount of total lipids in the composition (e.g., a liposome composition).

[0499] In embodiments, this percentage results in an improved beneficial effect (eg, improved delivery to a target tissue such as the liver or lung).

[0500] In a typical embodiment, the composition of the present invention (e.g., liposome composition) includes one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the present invention as described herein. For example, a composition suitable for practicing the present invention has four lipid components, including a compound of the present invention as described herein as a cationic lipid component, a non-cationic lipid, a cholesterol-based lipid and a PEG-modified lipid. The non-cationic lipid can be DOPE or DEPE. The cholesterol-based lipid can be cholesterol. The PEG-modified lipid can be DMG-PEG2K.

[0501] In an embodiment, the composition of the present invention includes the cationic lipid of the present invention, DMG-PEG2000, cholesterol and DOPE, and the molar ratio of cationic lipid:DMG-PEG2000:cholesterol:DOPE is 40:5:25:30.

[0502] In another embodiment, the pharmaceutical (e.g., liposome) composition comprises one or more of a PEG-modified lipid, a non-cationic lipid, and a cholesterol lipid. In other embodiments, such pharmaceutical (e.g., liposome) composition comprises: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids. In yet another embodiment, such pharmaceutical (e.g., liposome) composition comprises: one or more PEG-modified lipids and one or more cholesterol lipids.

[0503] In an embodiment, a composition (e.g., a lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprises one or more compounds of the invention described herein and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, and a PEGylated lipid.

[0504] In an embodiment, the composition (e.g., lipid nanoparticle) of the encapsulated nucleic acid (e.g., mRNA encoding a peptide or protein) includes 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 includes a cholesterol-based lipid. Typically, such compositions have four lipid components, including a compound of the present invention as described herein, a non-cationic lipid (e.g., DOPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K) as a cationic lipid component.

[0505] In an embodiment, a composition (e.g., a lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprises one or more compounds of the invention described herein and one or more lipids selected from the group consisting of a cationic lipid, a non-cationic lipid, a PEGylated lipid, and a cholesterol-based lipid.

[0506] According to various embodiments, the selection of cationic lipids, non-cationic lipids and / or PEG-modified lipids comprising lipid nanoparticles, and the relative molar ratios of these lipids to each other are based on the characteristics of the selected lipids, the properties of the intended target cells, and the characteristics of the mRNA to be delivered. Other considerations include, for example, the degree of saturation of the alkyl chain and the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipids. Therefore, the molar ratio can be adjusted accordingly.

[0507] In some embodiments, the ratio of cationic lipids to non-cationic lipids to cholesterol-based lipids to PEG-modified lipids can be between about 30-60:20-40:20-30:1-10, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:30:20:10, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:30:25:5, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 40:32:25:3, respectively. In some embodiments, the ratio of one or more cationic lipids to one or more non-cationic lipids to one or more cholesterol-based lipids to one or more PEG-modified lipids is about 50:25:20:5.

[0508] Cationic lipids

[0509] In addition to any of the compounds of the invention as described herein, the compositions may include one or more additional cationic lipids.

[0510] In certain embodiments, liposomes may comprise one or more additional cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a variety of lipid species having a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

[0511] Suitable additional cationic lipids for use in the composition include those described in the literature.

[0512] Helper lipids

[0513] Composition (for example, liposome composition) can also include one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, phrase "non-cationic lipids" refers to any neutral, zwitterionic or anionic lipids. As used herein, phrase "anionic lipids" refers to any of a variety of lipid substances that carry a net negative charge at a selected pH such as physiological pH. Non-cationic lipids include but are not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-diethylene glycol-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (P

[0014] The present invention also provides a lipid composition comprising the following: a) 1,2-dioleoylphosphatidylethanolamine (DOPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, L-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a mixture thereof. A non-cationic or helper lipid suitable for practicing the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-diethylene glycol-sn-glycero-3-phosphoethanolamine (DEPE) can be used as a non-cationic or helper lipid.

[0514] In some embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that carries no net charge under the conditions under which the composition is formulated and / or administered.

[0515] In certain embodiments, the non-cationic lipid can exist in the composition at a mol 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 TL present in the non-cationic lipid. In certain embodiments, the total non-cationic lipid can exist in the composition at a mol 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 TL present in the non-cationic lipid. In certain embodiments, the percentage ratio of non-cationic lipid in the liposome can 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 certain embodiments, the percentage of total non-cationic lipid in the liposome can 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 certain embodiments, the percentage of non-cationic lipid in the liposome is no more than about 5 mol %, no more than about 10 mol %, no more than about 20 mol %, no more than about 30 mol % or no more than about 40 mol %. In certain embodiments, the percentage of total non-cationic lipid in the liposome can be no more than about 5 mol %, no more than about 10 mol %, no more than about 20 mol %, no more than about 30 mol % or no more than about 40 mol %.

[0516] In certain embodiments, the non-cationic lipid can exist in the composition at a weight ratio (wt %) 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 lipid present in the composition. In certain embodiments, the total non-cationic lipid can exist in the composition at a weight ratio (wt %) 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 lipid present in the composition. In certain embodiments, the percentage ratio of non-cationic lipid can 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 liposome. In certain embodiments, the percentage of total non-cationic lipid in the liposome can 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 certain embodiments, the percentage of non-cationic lipid in the liposome is no more than about 5 % by weight, no more than about 10 % by weight, no more than about 20 % by weight, no more than about 30 % by weight or no more than about 40 % by weight. In certain embodiments, the percentage of total non-cationic lipid in the liposome can be no more than about 5 % by weight, no more than about 10 % by weight, no more than about 20 % by weight, no more than about 30 % by weight or no more than about 40 % by weight.

[0517] Cholesterol-based lipids

[0518] In certain embodiments, compositions (for example, liposome compositions) comprises one or more cholesterol-based lipids.For example, cholesterol is suitable for putting into practice the present invention's lipid based on cholesterol.Other suitable lipid based on cholesterol comprises for example, DC-Chol (N, N-dimethyl-N-ethylcarboxylic acid amide cholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao et al. " Biochemistry and Biophysics Research Communications (Biochem.Biophys.Res.Comm.) " 179,280 (1991); Wolf et al. " Biotechnology (BioTechniques) " 23,139 (1997); U.S. Patent No. 5,744,335), or imidazole cholesterol ester (ICE) with following structure:

[0519]

[0520] In certain embodiments, the lipid based on cholesterol can be present in a mol ratio (mol %) of about 1% to about 30% or about 5% to about 20% of the total lipid present in the liposome. In certain embodiments, the percentage ratio of the lipid based on cholesterol in the lipid nanoparticle can 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 certain embodiments, the percentage ratio of the lipid based on cholesterol in the lipid nanoparticle can be no more than about 5 mol %, no more than about 10 mol %, no more than about 20 mol %, no more than about 30 mol % or no more than about 40 mol %.

[0521] In certain embodiments, the cholesterol-based lipid can be present in a weight ratio (wt %) of about 1% to about 30% or about 5% to about 20% of the total lipid present in the liposome. In certain embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be greater than about 5 wt %, greater than about 10 wt %, greater than about 20 wt %, greater than about 30 wt % or greater than about 40 wt %. In certain embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be no more than about 5 wt %, no more than about 10 wt %, no more than about 20 wt %, no more than about 30 wt % or no more than about 40 wt %.

[0522] PEGylated lipids

[0523] In some embodiments, the composition (e.g., liposome composition) includes one or more additional PEGylated lipids. A suitable PEG-modified or PEGylated lipid for practicing the present invention is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0524] For example, the present invention also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide) in combination with one or more of the compounds of the present invention described herein, and in some embodiments, other lipids including liposomes. In some embodiments, particularly useful exchangeable lipids are those with shorter acyl chains (e.g., C 14 or C 18 ) of PEG-ceramide.

[0525] Additional contemplated PEG-modified lipids (also referred to herein as PEGylated lipids, a term interchangeable with PEG-modified lipids) include, but are not limited to, lipids covalently linked to C6-C 20In some embodiments, the lipid or PEGylated lipid of the present invention is a PEGylated lipid having an alkyl chain of at most 5 kDa. In some embodiments, the PEG-modified lipid or PEGylated lipid is PEGylated cholesterol or PEG-2K. Adding such components can prevent complex aggregation, and can also provide a method for increasing circulation life and increasing the delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or the components can be selected to quickly swap out the formulation in vivo (see U.S. Patent No. 5,885,613).

[0526] The additional PEG-modified phospholipids and derivatized lipids of the present invention can 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., a liposome composition).

[0527] Pharmaceutical preparations and therapeutic uses

[0528] The compounds of the invention as described herein can be used to prepare compositions (e.g., to construct liposome compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membranes of such target cells).

[0529] For example, when a liposomal composition (e.g., lipid nanoparticle) contains or is otherwise enriched with one or more compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate delivery of the encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) to the one or more target cells.

[0530] Similarly, in certain embodiments, the compounds of the invention described herein can be used to prepare liposomal vehicles characterized by their reduced in vivo toxicity. In certain embodiments, the reduced toxicity is a function of the high transfection efficiency associated with the compositions disclosed herein, such that reduced amounts of such compositions can be administered to a subject to achieve a desired therapeutic response or outcome.

[0531] Therefore, pharmaceutical formulations comprising the described compounds and nucleic acids provided by the invention can be used for various therapeutic purposes. In order to promote the delivery of nucleic acids in vivo, the compounds described herein and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands or stabilizers. In certain embodiments, the compounds described herein can be formulated by premixed lipid solutions. In other embodiments, the compositions comprising the compounds described herein can be formulated into the lipid membrane of nanoparticles using post-insertion technology. The technology for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0532] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or enteral administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection; and intrathecal, directly intraventricular, intravenous, intraperitoneal or intranasal. In a specific embodiment, intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle and cardiac muscle. In certain embodiments, administration results in nucleic acid delivery to muscle cells. In certain embodiments, administration results in nucleic acid delivery to hepatocytes (i.e., liver cells).

[0533] A common route of administration of the liposome compositions of the present invention can 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 condition to be treated, the liposome compositions can be administered by pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposome compositions of the present invention are typically administered intramuscularly. Diseases or conditions affecting the eye can be treated by intravitreal administration of the liposome compositions of the present invention.

[0534] Alternatively or in addition, the pharmaceutical formulations of the present invention can be administered in a local rather than systemic manner, for example, by direct injection of the pharmaceutical formulation into the target tissue, preferably in the form of a sustained release formulation. Depending on the tissue to be targeted, local delivery can be affected in various ways. Exemplary tissues in which the delivered mRNA can be delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In an embodiment, the tissue to be targeted is in the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); for example, the composition of the present invention can be injected into the site of injury, disease manifestation, or pain; the composition can be provided in the form of a lozenge for oral, tracheal, or esophageal application; it can be supplied to the stomach or intestine in the form of a liquid, tablet, or capsule, or it can be supplied to the rectum or vagina in the form of a suppository; or it can even be delivered to the eye by using a cream, drops, or even an injection.

[0535] The compositions described herein can include mRNA encoding peptides, including peptides described herein (eg, polypeptides, such as proteins).

[0536] In embodiments, the mRNA encodes a polypeptide.

[0537] In embodiments, the mRNA encodes a protein.

[0538] Exemplary peptides encoded by mRNA (eg, exemplary proteins encoded by mRNA) are described herein.

[0539] The present invention provides methods for delivering compositions having full-length mRNA molecules encoding a peptide or protein of interest for use in treating a subject, such as a human subject or cells of a human subject or cells treated and delivered to a human subject.

[0540] Therefore, in certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising a full-length mRNA encoding a peptide or protein, the therapeutic composition being used to deliver to or treat a subject's lung or lung cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an ATP-binding cassette subfamily A member 3 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dynamin axon intermediate chain 1 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dynamin axon heavy chain 5 (DNAH5) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding an alpha-1-antitrypsin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a forkhead box P3 (FOXP3) protein. In certain embodiments, the present invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more surfactant proteins (e.g., one or more of surfactant protein A, surfactant protein B, surfactant protein C, and surfactant protein D).

[0541] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding peptides or proteins for delivery to or treatment of the liver or hepatocytes of a subject. Such peptides and polypeptides may include those associated with urea cycle disorders, lysosomal storage disorders, glycogen storage disorders, amino acid metabolism disorders, lipid metabolism or fibrosis disorders, methylmalonic acidemia, or any other metabolic disorder for which delivery to or treatment of the liver or hepatocytes with enriched full-length mRNA provides a therapeutic benefit.

[0542] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with urea cycle disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding ornithine transcarbamylase (OTC) proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate synthetase 1 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding carbamoyl phosphate synthetase 1 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate lyase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding argininosuccinate lyase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding arginase proteins.

[0543] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding proteins associated with lysosomal storage disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding α-galactosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding glucocerebrosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding isocyanate-2-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding iduronidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding N-acetyl-α-D-glucosaminidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding heparan N-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNAs encoding galactosamine-6-sulfatase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a β-galactosidase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a lysosomal lipase protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having a full-length mRNA encoding a transcription factor EB (TFEB).

[0544] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with glycogen storage disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding acid alpha-glucosidase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glucose-6-phosphatase (G6PC) proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding liver glycogen phosphorylase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding muscle phosphoglycerate mutant enzyme proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glycogen debranching enzymes.

[0545] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins related to amino acid metabolism. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding phenylalanine hydroxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding glutaryl-CoA dehydrogenase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding propionyl-CoA carboxylase. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding oxalase alanine-glyoxylate aminotransferase.

[0546] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding proteins associated with lipid metabolism or fibrotic disorders. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding mTOR inhibitors. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding ATPase phospholipid transporter 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding one or more NF-κB inhibitors, such as one or more of I-κBα, interferon-related developmental regulator 1 (IFRD1), and Sirtuin 1 (SIRT1). In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding PPAR-γ protein or active variants.

[0547] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a methylmalonyl-CoA mutant enzyme protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding a methylmalonyl-CoA epimerase protein.

[0548] In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA, for which delivery to or treatment of the liver can provide therapeutic benefits. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding ATP7B protein (also known as Wilson disease protein). In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding porphobilinogen deaminase. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding one or more coagulase, such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the present invention provides a method for preparing a therapeutic composition with full-length mRNA encoding human hemochromatosis (HFE) protein.

[0549] In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising a full-length mRNA encoding a peptide or protein for delivery to or treatment of a subject's cardiovascular system or cardiovascular cells. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising a full-length mRNA encoding a vascular endothelial growth factor A protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising a full-length mRNA encoding a relaxin protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising a full-length mRNA encoding a bone morphogenetic protein 9 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition comprising a full-length mRNA encoding a bone morphogenetic protein 2 receptor protein.

[0550] In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein, the therapeutic composition is used to deliver to or treat a subject's muscle or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a dystrophin. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein, the therapeutic composition is used to deliver to or treat a subject's myocardium or myocardial cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein that regulates one or both of potassium channels and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein that regulates Kv7.1 channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition having a full-length mRNA encoding a protein that regulates Nav1.5 channels in muscle tissue or muscle cells.

[0551] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein, the therapeutic composition being used to deliver to or treat a subject's nervous system or nervous system cells. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding survival motor neuron 2 protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an ATP-binding cassette subfamily D member 1 (ABCD1) protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a CLN3 protein.

[0552] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding peptides or proteins for delivery to or treatment of a subject's blood or bone marrow or blood or bone marrow cells. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding beta globin. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding Bruton's tyrosine kinase proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII, and Factor X.

[0553] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a peptide or protein for delivery to or treatment of a subject's kidney or renal cells. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA encoding type IV collagen α5 chain (COL4A5) protein.

[0554] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein, the therapeutic composition being used to deliver to or treat an eye or eye cell of a subject. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an ATP-binding cassette subfamily A member 4 (ABCA4) protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a retinal chitin protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a retinal pigment epithelium-specific 65kDa (RPE65) protein. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a centrosomal protein (CEP290) of 290kDa.

[0555] In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein, which is used to deliver a vaccine to a subject or a subject's cells or to treat with a vaccine. For example, in certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an infectious source such as a virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding an antigen from a respiratory syncytial virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides methods for producing a therapeutic composition having a full-length mRNA encoding an antigen from a rotavirus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from hepatitis viruses, such as hepatitis A, hepatitis B, or hepatitis C. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from human papillomaviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from herpes simplex viruses, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from human immunodeficiency viruses, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding antigens from human metapneumoviruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from human parainfluenza viruses, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from malaria viruses. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from Zika virus. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding antigens from Chikungunya virus.

[0556] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen associated with a subject's cancer or an antigen identified from a subject's cancer cells. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen identified from a subject's own cancer cells, i.e., providing a personalized cancer vaccine. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antigen expressed from a mutant KRAS gene.

[0557] In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against OX40. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against VEGF. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against tissue necrosis factor alpha. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against CD3. In certain embodiments, the present invention provides methods for preparing therapeutic compositions comprising full-length mRNA encoding an antibody against CD19.

[0558] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding immunomodulators. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin-12. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin-23. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding interleukin-36γ. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding constitutively active variants of one or more stimulator of interferon genes (STING) proteins.

[0559] In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding endonucleases. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding RNA-guided DNA endonucleases proteins, such as Cas 9 proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding meganucleases proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding transcription activator-like effector nuclease proteins. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNAs encoding zinc finger nuclease proteins.

[0560] Delivery Method

[0561] The delivery routes used in the methods of the present invention allow for non-invasive self-administration of the compounds of the present invention. In some embodiments, the methods involve intratracheal or pulmonary administration of a composition by atomization, vaporization, or instillation, the composition comprising mRNA encoding a therapeutic protein in a suitable transfection or lipid carrier vehicle, as described above. In some embodiments, the protein is encapsulated by liposomes. In some embodiments, the liposomes comprise lipids, which are compounds of the present invention. As used hereinafter, administration of a compound of the present invention comprises administration of a composition comprising a compound of the present invention.

[0562] Although local cells and tissues of the lung represent potential targets for biological reservoirs or reservoirs for the production and secretion of proteins encoded by mRNA, applicants have found that administering the compounds of the present invention to the lungs by nebulization, vaporization, or instillation results in the distribution of even non-secreted proteins outside the lung cells. Without wishing to be bound by any particular theory, it is expected that the nanoparticle compositions of the present invention pass through the lung airway-blood barrier to achieve complete nanoparticle transfer to non-lung cells and tissues, for example, heart, liver, spleen, which results in the production of encoded proteins in these non-lung tissues. Therefore, the uses of the compounds of the present invention and the methods of the present invention go 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 can be used to manage and treat a large number of diseases, and in particular peripheral diseases caused by secretory and non-secretory proteins and / or enzyme deficiencies (e.g., one or more lysosomal storage diseases). In certain embodiments, the compounds of the present invention used in the methods of the present invention achieve the distribution of mRNA-encapsulated nanoparticles in the liver, spleen, heart, and / or other non-lung cells and the production of encoded proteins in the liver, spleen, heart, and / or other non-lung cells. For example, administration of a compound of the invention to the lung by nebulization, infusion, or instillation will result in the composition itself and its protein product (e.g., functional β-galactosidase protein) being detectable in local cells and tissues of the lung, as well as in peripheral target cells, tissues, and organs, due to translocation of the mRNA and delivery vehicle to non-lung cells.

[0563] In certain embodiments, the compound of the present invention can be used in the method of the present invention to specifically target peripheral cells or tissues. After pulmonary delivery, it is envisioned that the compound of the present invention passes through the lung airway-blood barrier and is distributed to cells other than local lung cells. Therefore, various methods known to those skilled in the art (for example, by inhalation) can be used to administer compounds disclosed herein to subjects by pulmonary administration route, and are distributed to local target cells and tissues of the lung, and in peripheral non-lung cells and tissues (for example, liver, spleen, kidney, heart, skeletal muscle, lymph node, brain, cerebrospinal fluid and plasma cells). Therefore, both the local cells of the lung and peripheral non-lung cells can serve as biorepositories or depots that can produce and / or secrete translation products encoded by one or more polynucleotides. Therefore, the present invention is not limited to the treatment of lung disease or condition, but can be used as the non-invasive means of the production of the delivery of the promotion polynucleotides or the enzymes and proteins encoded therein in peripheral organs, tissues and cells (for example, hepatocytes) that can only be achieved by systemic administration. Exemplary peripheral non-lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, cardiac myocytes, 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.

[0564] After administration of the composition to a subject, the protein product (e.g., a functional protein or enzyme) encoded by the mRNA can be detected in peripheral target tissues for at least about one to seven days or longer after administration of the compound to the subject. The amount of protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the encoded protein, and the condition of the patient. For example, a protein product of at least 0.025-1.5 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 10 μg / ml, at least 11 μg / ml, at least 12 μg / ml, at least 13 μg / ml, at least 14 μg / ml, at least 15 μg / ml, at least 16 μg / ml, at least 17 μg / ml, at least 18 μg / ml, at least 19 μg / ml, at least 20 μg / ml, at least 21 μg / ml, at least 22 μg / ml, at least 23 μg / ml, at least 24 μg / ml, at least 25 μg / ml, at least 26 μg / ml, at least 27 μg / ml, at least 28 μg / ml, at least 29 μg / ml, at least 30 μg / ml, at least 31 μg / ml, at least 32 μg / ml, at least 33 μg / ml, at least 34 μg / ml, at least 35 μg / ml, at least 36 μg / ml, The protein product is detected in peripheral target tissues at a concentration (e.g., therapeutic concentration) of at least 1.2 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, 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.

[0565] It has been demonstrated that nucleic acids can be delivered to the lung by intratracheal administration of a liquid suspension of the compound and inhalation of an aerosol mist generated by a liquid nebulizer or using a dry powder device such as that described in US Patent 5,780,014, which is incorporated herein by reference.

[0566] In certain embodiments, the compound of the present invention can be formulated so that it can be atomized or otherwise delivered as a granular liquid or solid before or after being used to the subject. Such compounds can be used with the help of one or more suitable devices for using such solid or liquid particle compositions (for example, atomized aqueous solutions or suspensions) to produce particles that are easily breathed or inhaled by the subject. In certain embodiments, such devices (for example, metered dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, inhalers based on propellants or insufflators) help to administer a composition (for example, the mRNA of about 0.5mg / kg per dose) of predetermined mass, volume or dosage to the subject. For example, in certain embodiments, a metered dose inhaler is used to administer the compound of the present invention to the subject, the inhaler containing a suspension or solution comprising the compound and a suitable propellant. In certain embodiments, the compound of the present invention can be formulated into a granular powder (for example, respirable dry particles) for inhalation. In certain embodiments, the compositions of the present invention formulated into inhalable particles have an appropriate size so that they can be inhaled by a subject or delivered using a suitable device (e.g., an average D50 or D90 particle size of less than about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or less). In yet other embodiments, the compounds of the present invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, a compound of the invention is administered to a subject such that the concentration administered in a single dose is 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

[0014] In some embodiments, the dosage form of the present invention may be an oral dosage form of at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg or at least 100 mg / kg body weight.In some embodiments, a compound of the invention is administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of mRNA is administered in one or more doses.

[0567] Examples

[0568] Although certain compounds, compositions, and methods of the present invention have been specifically described with reference to certain examples, the following examples are merely illustrative of the compounds of the present invention and are not intended to limit the same.

[0569] Synthesis scheme of vanilloid lipids

[0570]

[0571] Synthesis of 3-(dimethylamino)propyl 4-hydroxy-3-methoxybenzoate (3)

[0572]

[0573] At 0 ℃, oxalyl chloride (2.0 mL, 23.8 mmol) was added to a suspension of vanillic acid 1 (1.0 g, 5.9 mmol) in 25 mL of dichloromethane, followed by dimethylformamide (1 drop), and the resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0 ℃, 3- (dimethylamino) propan-1-ol 2 (0.7 mL, 5.9 mmol) was slowly added, and the reaction mixture was stirred at room temperature overnight. The precipitate was filtered to produce 4- hydroxy -3- methoxybenzoic acid 3- (dimethylamino) propyl ester 3 (1.18 g, 79%) as a white solid.

[0574] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butyryl)oxy)-3-methoxybenzoate (4)

[0575]

[0576] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoic acid AIM-3-E12 (1.0 g, 1.43 mmol) in 20 mL of dichloromethane was added oxalyl chloride (0.15 mL, 1.72 mmol) at 0 ° C., dimethylformamide (1 drop) was then added, and the mixture was stirred at 0 ° C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0 ° C., 3-(dimethylamino)propyl 4-hydroxy-3-methoxybenzoate 3 (0.18 g, 0.7 mmol) was added, followed by pyridine (0.4 mL, 4.9 mmol), and the reaction mixture was stirred at room temperature overnight. Ice was added to quench the reaction, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to give 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3-methoxybenzoate 4 (330 mg, 50%) as a light yellow oil.

[0577] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butyryl)oxy)-3-methoxybenzoate (VA-3-E12-DMAPr)

[0578]

[0579] To a solution of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3-methoxybenzoate 4 (330 mg, 0.35 mmol) in 10 mL of tetrahydrofuran was added dropwise hydrofluoric acid in pyridine (70%, 2.5 mL) at 0° C., and the mixture was stirred at room temperature overnight. Saturated sodium bicarbonate solution was added to pH=7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse phase column chromatography (C18: 5-95% MeCN / water / 0.1% TFA) to give 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3-methoxybenzoate (120 mg, 48%) as a TFA salt. This compound was stored in 2-butanol to prevent decomposition.

[0580] All other lipids were prepared in similar yields following the representative procedure.

[0581] Synthesis scheme of syringic acid lipid

[0582]

[0583] Synthesis of 3-(Dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate (6)

[0584]

[0585] Oxalyl chloride (12.8 mL, 0.15 mol) was added to a suspension of syringic acid 5 (7.5 g, 0.04 mol) in 100 mL of dichloromethane at 0 ° C., followed by dimethylformamide (5 drops), and the resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 100 mL of dichloromethane. After cooling to 0 ° C., 3- (dimethylamino) propan-1-ol 2 (4.5 mL, 40 mmol) was slowly added, and the reaction mixture was stirred at room temperature overnight. The precipitate was filtered to produce 4-hydroxy-3,5-dimethoxybenzoic acid 3- (dimethylamino) propyl ester 6 (6.2 g, 58%) as a white solid.

[0586] Synthesis of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butyryl)oxy)-3,5-dimethoxybenzoate (7)

[0587]

[0588] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoic acid AIM-3-E12 (0.99 g, 1.41 mmol) in 20 mL of dichloromethane was added oxalyl chloride (0.15 mL, 1.72 mmol) at 0 ° C., dimethylformamide (1 drop) was then added, and the mixture was stirred at 0 ° C. for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0 ° C., 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate 6 (0.2 g, 0.7 mmol) was added, followed by pyridine (0.35 mL, 4.34 mmol), and the reaction mixture was stirred at room temperature overnight. Ice was added to quench the reaction, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to give 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate 7 (380 mg, 50%) as a light yellow oil.

[0589] Synthesis of 3-(Dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butyryl)oxy)-3,5-dimethoxybenzoate (SA-3-E12-DMAPr)

[0590]

[0591] To a solution of 3-(dimethylamino)propyl 4-((4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate 7 (380 mg, 0.39 mmol) in 10 mL of tetrahydrofuran was added dropwise hydrofluoric acid in pyridine (70%, 2.5 mL) at 0°C, and the mixture was stirred at room temperature overnight. Saturated sodium bicarbonate solution was added to pH = 7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse phase column chromatography (C18: 5-95% MeCN / water / 0.1% TFA) to give 3-(dimethylamino)propyl 4-((4-(bis(2-hydroxydodecyl)amino)butanoyl)oxy)-3,5-dimethoxybenzoate (94 mg, 32%) as a TFA salt.

[0592] All other lipids were prepared in similar yields following the representative procedure.

[0593] Synthesis scheme of sinapic acid lipids

[0594]

[0595] Synthesis of 3-(dimethylamino)propyl (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate (9)

[0596]

[0597] To a suspension of mustard acid 8 (5 g, 22 mmol) in 100 mL of dichloromethane was added oxalyl chloride (7.5 mL, 90 mmol) at 0 ° C., followed by dimethylformamide (5 drops), and the resulting mixture was stirred at this temperature for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 100 mL of dichloromethane. After cooling to 0 ° C., 3- (dimethylamino) propan-1-ol 2 (2.64 mL, 22 mmol) was slowly added, and the reaction mixture was stirred at room temperature overnight. The precipitate was filtered to produce (E) -3- (4- hydroxy -3,5- dimethoxyphenyl) acrylic acid 3- (dimethylamino) propyl ester 9 (2.66 g, 39%) as a light yellow solid.

[0598] Synthesis of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxopropyl-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate (10)

[0599]

[0600] To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoic acid AIM-3-E12 (1.8 g, 2.59 mmol) in 20 mL of dichloromethane was added oxalyl chloride (0.3 mL, 3.09 mmol) at 0 ° C., followed by dimethylformamide (1 drop), and the mixture was stirred at 0 ° C for 2 hours. The reaction mixture was evaporated to dryness, and the residue was dissolved in 20 mL of dichloromethane. After cooling to 0 ° C., (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate 3-(dimethylamino)propyl ester 9 (0.4 g, 1.29 mmol) was added, followed by pyridine (0.62 mL, 7.7 mmol), and the reaction mixture was stirred at room temperature overnight. Ice was added to quench the reaction, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by flash chromatography to give (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxopropyl-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate 10 (540 mg, 42%) as a light yellow oil.

[0601] Synthesis of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxopropyl-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-hydroxydodecyl)amino)butanoate (SI-3-E12-DMAPr)

[0602]

[0603] To a solution of (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxopropyl-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-((tert-butyldimethylsilyl)oxy)dodecyl)amino)butanoate 10 (540 mg, 0.55 mmol) in 10 mL of tetrahydrofuran was added dropwise hydrofluoric acid-containing pyridine (70%, 2.5 mL) at 0° C., and the mixture was stirred at room temperature overnight. Saturated sodium bicarbonate solution was added to pH=7-8, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by reverse phase column chromatography (C18: 5-95% MeCN / water / 0.1% TFA) to give (E)-4-(3-(3-(dimethylamino)propoxy)-3-oxopropyl-1-en-1-yl)-2,6-dimethoxyphenyl 4-(bis(2-hydroxydodecyl)amino)butanoate (330 mg, 80%) as a TFA salt.

[0604] All other lipids were prepared in similar yields following the representative procedure.

[0605] Synthesis of phenolic acid lipids Scheme A

[0606]

[0607] Synthesis of intermediate (3a) in Synthesis Scheme A:

[0608]

[0609] To a solution of (1) (2.00 g, 2.86 mmol) in anhydrous CHCl (10 mL) was added oxalyl chloride (0.98 mL, 4.0 equiv) dropwise at 0°C, and the reaction mixture was slowly warmed to room temperature and stirred for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was redissolved in anhydrous CHCl (30 mL). To the stirred acid chloride solution was added (2a) (555 mg, 1.0 equiv), DMAP (349 mg, 1.0 equiv), and then triethylamine (3.18 mL, 8.0 equiv) at 0°C. The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature for 16 hours. After 16 hours, the reaction mixture was diluted with CHCl and saturated NaHCO 3(溶液) The separated organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give a crude material. The crude material was first purified using 50% EtOAc in hexane and then repurified using 15% EtOAc in CH2Cl2 to obtain (3a) (623 mg, 25%) as a viscous oil. MS (ESI+) calculated for C 50 H 93 NO7Si2, [M+H] + =876.65, observed value =876.6.

[0610] Synthesis of intermediate (3b) in Synthesis Scheme A:

[0611]

[0612] The procedure of (3a) was followed using (2b) to afford (3b) as a viscous oil (557 mg, 23%). MS (ESI+) calculated for C 49 H 91 NO6Si2, [M+H] + =846.64, observed value =846.6.

[0613] Synthesis of intermediate (5a) in Synthesis Scheme A:

[0614]

[0615] To a solution of (3a) (308 mg, 0.351 mmol) in anhydrous CH2Cl2 (3 mL) was added oxalyl chloride (0.15 mL, 5.0 equiv) at room temperature and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was redissolved in anhydrous CH2Cl2 (3 mL). 3-dimethylaminopropanol (4) (109 mg, 3 equiv) was added to the stirred acyl chloride solution at 0 ° C., followed by triethylamine (0.10 mL, 2.0 equiv). The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature for 16 hours. After the reaction was completed by MS monitoring, the reaction mixture was concentrated to dryness under reduced pressure and purified using CH2Cl2 containing 0-10% MeOH to obtain (5a) (204 mg, 60%) as a viscous oil. MS (ESI+) calculated C 55 H 104 N2O7Si2, [M+H] + =961.74, observed value =961.7.

[0616] Synthesis of intermediate (5b) in Synthesis Scheme A:

[0617]

[0618] The procedure of (5a) was followed using (3b) to afford (5b) as a viscous oil (248 mg, 90%). MS (ESI+) calculated for C 54 H 102 N2O6Si2, [M+H] + =931.73, observed value =931.7.

[0619] Synthesis of TBL-0731 compound 355 (6a) in Synthesis Scheme A:

[0620]

[0621] To a stirred solution of (5a) (204 mg, 0.212 mmol) in anhydrous THF (3 mL) was added dropwise pyridine (1.09 mL, 197 equivalents) containing 70% hydrogen fluoride at 0 ° C, and then slowly warmed to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was completed by MS monitoring, the reaction mixture was cooled to 0 ° C and quenched by adding solid NaHCO3 in batches. After gas formation was minimized, the resulting mixture was diluted with EtOAc and quenched with saturated NaHCO 3(水溶液)The solution was neutralized. The separated organic layer was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to give a crude material. The crude material was purified using 0-20% MeOH in CH2Cl2 to obtain TBL-0731(6a) (132 mg, 85%) as a viscous oil. MS (ESI+) calculated for C 43 H 76 N2O7, [M+H] + =733.57, observed value =733.5. 1 H NMR(500MHz, CDCl3)δ7.64(d,J=15.9Hz,1H),7.14–7.08(m,2H),7.05(d,J=8.1, 3.5Hz,1H),6.37(d,J=16.0Hz,1H),4.27(t,J=6.4Hz,2H),3.86(s,3H),3.72–3.6 3(m,2H),2.78–2.70(m,2H),2.67–2.47(m,6H),2.46–2.40(m,2H),2.36(s,6H), 2.02–1.90(m,4H),1.49–1.35(m,4H),1.34–1.15(m,32H),0.87(t,J=6.9Hz,6H).

[0622] Synthesis of TBL-0750 compound 467 (6b) in Synthesis Scheme A:

[0623]

[0624] The procedure of (6a) was followed using (5b) to afford TBL-0750 (6b) (153 mg, 82%) as a viscous oil. MS (ESI+) calculated for C 42 H 74 N2O6, [M+H] + =703.55, observed value =703.6. 1HNMR(400MHz, CDCl3)δ7.66(d,J=16.0Hz,1H),7.53(d,J=8.7,1.3Hz,2H),7.12( d,J=8.6,1.5Hz,2H),6.38(d,J=16.0,0.9Hz,1H),4.26(t,J=6.4Hz,2H),3.70–3. 63(m,2H),2.73–2.64(m,2H),2.64–2.46(m,6H),2.45–2.40(m,2H),2.34(s,6H), 1.99–1.88(m,4H),1.43–1.34(m,4H),1.31–1.21(m,32H),0.87(t,J=6.8Hz,6H).

[0625] Synthesis of phenolic acid lipids Scheme B

[0626]

[0627] Synthesis of intermediate (3) in Synthesis Scheme B:

[0628]

[0629] To a solution of the acid intermediate (1) (4.58 g) in CHCl (30 mL, anhydrous) was added oxalyl chloride (1.04 mL, 2 eq) and stirred at room temperature for 2 hours. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CHCl (30 mL, anhydrous). To this solution was then added syringic acid (2) (1.32 g, 1.1 eq) followed by pyridine (2.93 mL, 6 eq) and the resulting mixture was stirred at room temperature overnight. After stirring overnight, the solvent was removed under reduced pressure and the remaining residue was dissolved with minimal CHCl and then filtered through a short silica gel plug using 100% EtOAc as eluent. The clear filtrate was concentrated to dryness to provide a crude material which was purified on MPLC using a 10-100% EtOAc / hexane gradient over 10CV to provide the acid product (3) (3.70 g, 78%). MS (ESI+) calculated C 53 H 101 NO8Si2, [M+H] + =936.7, observed value =936.7.

[0630] Synthesis of intermediate (5a) in Synthesis Scheme B:

[0631]

[0632] To a solution of benzoic acid (3) (400 mg) in CH2Cl2 (4 mL, anhydrous) was added oxalyl chloride (0.18 mL, 5 equivalents) and stirred at room temperature for 2 hours. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CH2Cl2 (3 mL, anhydrous). The resulting solution was cooled with an ice bath, and then a solution of 2-aminoethanol (4a) (76 mg) in CH2Cl2 (1 mL) was added. The reaction mixture was warmed to room temperature and stirred at the same temperature for 16 hours. After complete consumption of the starting material by LC-MS monitoring, the reaction mixture was concentrated to dryness, and the crude product material was purified on MPLC using 0-20% MeOH / CH2Cl2 gradient over 12CV to provide (5a) (225 mg, 52%) as a viscous oil. MS (ESI+) calculated C 57 H 110 N2O8Si2, [M+H] + =1007.8, observed value =1007.6.

[0633] Synthesis of intermediate (5b) in Synthesis Scheme B:

[0634]

[0635] The procedure for the synthesis of (5a) was followed using 4-aminobutanol (4b) to afford the product (5b) as a viscous oil (360 mg, 81%). MS (ESI+) calculated for C 59 H 114 N2O8Si2, [M+H] + =1035.8, observed value =1035.6.

[0636] Synthesis of intermediate (5c) in Synthesis Scheme B:

[0637]

[0638] The synthetic procedure of (5a) was followed using intermediate (3) (500 mg) and 3-morpholinopropanol (4c) to provide the product (5c) (350 mg, 62%). MS (ESI+) calculated for C 60 H 114 N2O9Si2, [M+H] + =1063.8, observed value =1063.6.

[0639] Synthesis of intermediate (5d) in Synthesis Scheme B:

[0640]

[0641] The synthetic procedure of (5a) was followed using intermediate (3) (500 mg) and 2-pyridinemethanol (4d) to provide the product (5d) (237 mg, 43%). MS (ESI+) calculated for C 60 H 108 N2O8Si2, [M+H] + =1041.8, observed value =1041.6.

[0642] Synthesis of intermediate (5e) in Synthesis Scheme B:

[0643]

[0644] The synthetic procedure of 5a was followed using intermediate (3) (843 mg) and 4-methylpiperazineethanol (4e) to provide the product (5e) (346 mg, 36%). MS (ESI+) calculated for C 60 H 115 N3O8Si2,[M+H] + =1062.8, observed value =1062.7.

[0645] Synthesis of TBL-0507 compound 48 (6a) in Synthesis Scheme B:

[0646]

[0647] To a stirred solution of TBS-protected intermediate (5a) (225 mg) in THF (3 mL, anhydrous) in a plastic polymer scintillation vial (not glass) was added triethylamine (0.16 mL, 5 eq.) and then triethylamine-3HF (0.36 mL, 10 eq.) dropwise. The reaction mixture was stirred at 50 ° C. overnight. The reaction mixture was dried over a mixture of EtOAc and NaHCO 3(水溶液) The reaction mixture was suspended between the layers for two drops to monitor the reaction and analyze the organic layer (TLC or LC-MS). Once the starting material is consumed, excess HF and volatiles are removed by blowing off with N in a fume hood, and the remaining material is diluted with EtOAc and neutralized with a saturated NaHCO aqueous solution (check with pH test paper). The separated organic layer is washed with salt water, dried over NaSO, and concentrated under reduced pressure to provide a crude product material. The crude product material is purified on MPLC using a 0-40% MeOH / CHCl gradient over 10CV to provide TBL-0507 (6a) (90 mg, 52%). MS (ESI+) calculated C 45 H 82 N2O8, [M+H] + =779.6, observed value =779.5.

[0648] Synthesis of TBL-0508 compound 49 (6b) in Synthesis Scheme B:

[0649]

[0650] The procedure of (6a) was followed using TBS protected intermediate (5b) (360 mg) to provide TBL-0508 (6b) (71 mg, 25%). MS (ESI+) calculated for C 47 H 86 N2O8, [M+H] + =807.6, observed value =807.5.

[0651] Synthesis of TBL-0517 compound 562 (6c) in Synthesis Scheme B:

[0652]

[0653] The procedure of (6a) was followed using the TBS-protected intermediate (5c) (350 mg) and purified with a 0-10% MeOH / CH2Cl2 gradient to provide TBL-0517 (6c) (164 mg, 60%). MS (ESI+) calculated for C 48 H 86 N2O9, [M+H] + =835.6, observed value =835.5.

[0654] Synthesis of TBL-0518 compound 563 (6d) in Synthesis Scheme B:

[0655]

[0656] The procedure of (6a) was followed using the TBS-protected intermediate (5d) (237 mg) and purified with a 0-10% MeOH / CH2Cl2 gradient to provide TBL-0518 (6d) (111 mg, 60%). MS (ESI+) calculated for C 48 H 80 N2O8, [M+H] + =813.6, observed value =813.5.

[0657] Synthesis of TBL-0535 compound 564 (6e) in Synthesis Scheme B:

[0658]

[0659] The procedure of (6a) was followed using the TBS-protected intermediate (5e) (346 mg) and purified with a 0-20% MeOH / CH2Cl2 gradient to provide TBL-0535 (6e) (88 mg, 32%). MS (ESI+) calculated for C 48 H 87 N3O8, [M+H] + =834.6, observed value =834.6.

[0660] Synthesis of Phenolic Acid Lipids Scheme C

[0661]

[0662] To a flask containing amino acid (7) (500 mg) and dodecyl acrylate (8a) (2.91 g, 2.5 eq) was added isopropyl alcohol (5 mL) and triethylamine (1.35 mL, 2 eq). The resulting mixture was heated at 90° C. for 3 hours. After completion of the reaction as monitored by MS, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The remaining material was purified on MPLC using 0-12% MeOH / CH2Cl2 to provide the product (9a) (987 mg, 35%). MS (ESI+) calculated C 34 H 65 NO6, [M+H] + =584.5, observed value =584.5.

[0663] Synthesis of intermediate (9b) in Synthesis Scheme C:

[0664]

[0665] The procedure of (9a) was followed using amino acid (7) (1.00 g), tetradecyl acrylate (8b) (6.51 g, 2.5 eq), isopropanol (10 mL) and triethylamine (2.70 mL, 2 eq) to provide the product (9b) (1.80 g, 29%). MS (ESI+) calculated for C 38 H 73 NO6, [M+H] + =640.5, observed value =640.5.

[0666] Synthesis of TBL-0484 compound 565 (11a) in Synthesis Scheme C:

[0667]

[0668] To a solution of intermediate (9a) (300 mg) in anhydrous CHCl (3 mL) was added oxalyl chloride (1.0 mL, 23 eq) dropwise at room temperature and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was redissolved in anhydrous CHCl (3 mL). Phenolic acid (10) (146 mg, 1.0 eq) and pyridine (0.21 mL, 5 eq) were added to the stirred acyl chloride solution at room temperature, and then stirred at the same temperature for 16 hours. After completion of the reaction, which was monitored by MS, the reaction mixture was concentrated under reduced pressure. The remaining crude material was purified on MPLC using 0-10% MeOH / CHCl to provide product TBL-0484 (11a) (90 mg, 21%). MS (ESI+) calculated C 48 H 84 N2O 10 , [M+H] + =849.6, observed value =849.5.

[0669] Synthesis of TBL-0485 compound 566 (11b) in Synthesis Scheme C:

[0670]

[0671] The procedure of (11a) was followed using intermediate (9b) (300 mg) to provide TBL-0485 (11b) (80 mg, 19%). MS (ESI+) calculated for C 52 H 92 N2O 10 , [M+H] + =905.7, observed value =905.6.

[0672] Example 1: Lipid Nanoparticle Formulation

[0673] According to methods known in the art, cationic lipids as described herein can be used to prepare lipid nanoparticles. For example, suitable methods include the methods described in International Publication No. WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[0674] An exemplary method of lipid nanoparticle formulation is method A of WO 2018 / 089801 (see, e.g., Examples 1 and 2 of WO 2018 / 089801). Figure 1). Method A ("A") relates to a conventional method for encapsulating mRNA by mixing mRNA with a lipid mixture without first preforming the lipids into lipid nanoparticles. In an exemplary method, an ethanol lipid solution and a buffered aqueous solution of mRNA are prepared separately. A solution of a lipid mixture (cationic lipids, helper lipids, zwitterionic lipids, PEG lipids, etc.) is prepared by dissolving the lipids in ethanol. The mRNA solution is prepared by dissolving the mRNA in a citrate buffer. The mixture is then heated to 65°C before mixing. The two solutions are then mixed using a pump system. In some cases, the two solutions are mixed using a gear pump system. In certain embodiments, the two solutions are mixed using a 'T' confluence (or "Y" confluence). The mixture is then purified by diafiltration using a TFF method. The resulting formulation is concentrated and stored at 2-8°C until further use.

[0675] A second exemplary method for lipid nanoparticle formulations is method B of WO 2018 / 089801 (see, for example, Example 2 and Figure 2 of WO 2018 / 089801). Method B ("B") refers to a process in which messenger RNA (mRNA) is encapsulated by mixing preformed lipid nanoparticles with mRNA. A range of different conditions can be used in method B, such as varying temperatures (i.e., heating or not heating the mixture), buffers, and concentrations. In an exemplary method, lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process. The resulting formulation mixture is empty lipid nanoparticles in a citrate buffer containing alcohol. The formulation is then subjected to a TFF purification process, in which buffer exchange occurs. The resulting suspension of preformed empty lipid nanoparticles is then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution after mixing results in a higher percentage of lipid nanoparticles containing mRNA and a higher total mRNA yield.

[0676] The lipid nanoparticle formulations of Table 5 were prepared by Method A or B. Each formulation included mRNA encoding firefly luciferase protein (FFL mRNA) and lipids (cationic lipid: DMG-PEG2000; cholesterol: DOPE) at the mol% ratios listed in Table 5.

[0677] Table 5. Exemplary lipid nanoparticle formulations for intratracheal administration

[0678]

[0679]

[0680] Delivery of FFL mRNA by intratracheal administration

[0681] By means of Anesthetized male CD1 mice (6-8 weeks old) were administered a single intratracheal aerosol administration of the lipid nanoparticle formulations (50 ul / animal) containing FFL mRNA listed in Table 5. Approximately 24 hours after administration, the animals were given 150 mg / kg (60 mg / ml) of luciferin by intraperitoneal injection at 2.5 ml / kg. 5-15 minutes later, all animals were imaged using an IVIS imaging system to measure luciferase production in the lungs. Figure 1 It was shown that lipid nanoparticles comprising the cationic lipids described herein effectively delivered FFL mRNA in vivo based on positive luciferase activity.

[0682] Numbered Examples

[0683] 1. A cationic lipid having a structure according to formula (I):

[0684]

[0685] wherein L1 is a bond, (C1-C6)alkyl or (C2-C6)alkenyl;

[0686] Wherein X is O or S;

[0687] where R 1 、R 2 、R 3 、R 4 and R 5 are each independently selected from H, OH, optionally substituted (C1-C6)alkyl, optionally substituted (C2-C6)alkenyl, optionally substituted (C2-C6)alkynyl, optionally substituted (C1-C6)alkoxy, and -OC(O)R';

[0688] where R 1 、R 2 、R 3 、R 4 or R 5 At least one of them is -OC(O)R';

[0689] Where R' is

[0690]

[0691] where R 6 for

[0692]

[0693] wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

[0694] where R7 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A ;

[0695] where R 8 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B ;

[0696] where R 9 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C ;

[0697] where R 10 is selected from H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D ;

[0698] wherein k, n, q and r are each independently 1, 2, 3, 4 or 5;

[0699] or (i) R 7 and R 8 or (ii) R 9 and R 10 together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, wherein the heterocycloalkyl or heteroaryl includes 1 to 3 heteroatoms selected from N, O and S;

[0700] where R 11 、R 12 、R13 and R 14 Each is independently selected from H, methyl, ethyl or propyl;

[0701] where R A 、R B 、R C and R D Each independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 )acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, -NH2;

[0702] where R 7 、R 8 、R 9 、R 10 At least one of them includes R A 、R B 、R C or R D part, wherein the R A 、R B 、R C or R D are independently selected from optionally substituted (C6-C 20 )alkyl, optionally substituted (C6-C 20 )alkenyl, optionally substituted (C6-C 20 )alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)(C6-C 20 )alkyl or optionally substituted -OC(O)(C6-C 20 )alkenyl;

[0703] or a pharmaceutically acceptable salt thereof.

[0704] 2. The cationic lipid of numbered embodiment 1, or a pharmaceutically acceptable salt thereof, wherein any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl group is optionally substituted with one or more substituents selected from the group consisting of: (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C1-C6)acyl, (C1-C6)alkoxy, halogen, -COR, -CO2H, -CO2R, -CN, -OH, -OR, -OCOR, -OCOR, -NH2, -NHR, -N(R)2, -SR, or -S02R, or two geminal hydrogens on a carbon atom are replaced with a group =NH, wherein each instance of R is independently C1-C6 10 Aliphatic alkyl group.

[0705] 3. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein:

[0706] i)R A and R B the same; and / or

[0707] ii) R C and R D same.

[0708] 4. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered embodiment 1 or 2, wherein:

[0709] i)R A and R B different; and / or

[0710] ii) R C and R D different.

[0711] 5. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of numbered embodiments 1 to 3, wherein R A 、R B 、R C and R D same.

[0712] 6. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of numbered embodiments 1 to 4, wherein R A 、R B 、R C and R D One or more of them are different.

[0713] 7. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein X is O.

[0714] 8. The cationic lipid or pharmaceutically acceptable salt thereof of any one of numbered embodiments 1 to 6, wherein X is S.

[0715] 9. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R 1 、R 2 、R 3 、R 4 and R 5 Only one of them is -OC(O)R'.

[0716] 10. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered embodiment 9, wherein R 1 、R 2 、R 3 、R 4 or R 5 Neither of them is OH.

[0717] 11. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of numbered embodiments 1 to 8, wherein R 1 、R 2 、R 3 、R 4 and R 5 Both of them are -OC(O)R'.

[0718] 12. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered embodiment 11, wherein R 1 、R 2 、R 3 、R 4 or R 5 Neither of them is OH.

[0719] 13. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of numbered embodiments 1 to 8, wherein R 1 、R 2 、R 3 、R 4 and R 5 The three in it are -OC(O)R'.

[0720] 14. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R 1 and / or R 5 It is -OC(O)R'.

[0721] 15. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R 2 and / or R 4It is -OC(O)R'.

[0722] 16. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R 3 It is -OC(O)R'.

[0723] 17. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein:

[0724] i) p, q and r are the same; or

[0725] ii) one or more of p, q and r are different; or

[0726] iii) q and r are the same, and p is different.

[0727] 18. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein:

[0728] i) k, m and n are the same; or

[0729] ii) one or more of k, m and n are different; or

[0730] iii) k and n are the same, and m is different.

[0731] 19. The cationic lipid or pharmaceutically acceptable salt thereof of any preceding numbered embodiment, wherein m is 1, 2, or 3.

[0732] 20. The cationic lipid of any one of the preceding numbered embodiments, or a pharmaceutically acceptable salt thereof, wherein p is 1, 2, or 3.

[0733] 21. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R' is:

[0734]

[0735] 22. The cationic lipid or pharmaceutically acceptable salt thereof of numbered embodiment 21, wherein:

[0736] i) k, m, and n = 1; or

[0737] ii) k, m and n = 1, and R 11 and R 12 =H; or

[0738] iii) k and n = 1, and m = 2; or

[0739] iv) k and n = 1, m = 2, and R11 and R 12 =H; or

[0740] v) k and n = 1, and m = 3; or

[0741] vi) k and n = 1, m = 3, and R 11 and R 12 =H.

[0742] 23. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein R 6 for:

[0743]

[0744] 24. The cationic lipid or pharmaceutically acceptable salt thereof of numbered embodiment 23, wherein:

[0745] i) p, q, and r = 1; or

[0746] ii) p, q and r = 1, and R 13 and R 14 is H; or

[0747] iii) q and r = 1, and p = 2; or

[0748] iv) q and r = 1, p = 2, and R 13 and R 14 For H.

[0749] 25. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of numbered embodiments 1 to 22, wherein R 6 Selected from the group consisting of:

[0750]

[0751] 26. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered embodiment 25, wherein R 6 for:

[0752]

[0753] 27. The cationic lipid of any preceding numbered embodiment, having a structure according to Formula (II):

[0754]

[0755] or a pharmaceutically acceptable salt thereof.

[0756] 28. The cationic lipid of numbered Example 27, having a structure according to Formula (IIA):

[0757]

[0758] or a pharmaceutically acceptable salt thereof.

[0759] 29. The cationic lipid of numbered embodiment 28, having a structure according to one of formulas (IIB), (IIC), (IID), or (IIE):

[0760]

[0761]

[0762] or a pharmaceutically acceptable salt thereof.

[0763] 30. The cationic lipid of numbered Example 27, having a structure according to Formula (IIF):

[0764]

[0765] or a pharmaceutically acceptable salt thereof.

[0766] 31. The cationic lipid of numbered Example 27, having a structure according to Formula (IIG):

[0767]

[0768] or a pharmaceutically acceptable salt thereof.

[0769] 32. The cationic lipid of numbered Example 27, having a structure according to Formula (IIH):

[0770]

[0771] wherein one of Y and Z is OH and the other is -OC(O)R', or wherein both Y and Z are each independently -OC(O)R', or a pharmaceutically acceptable salt thereof.

[0772] 33. The cationic lipid of any one of numbered embodiments 1 to 26, having a structure according to formula (III):

[0773]

[0774] or a pharmaceutically acceptable salt thereof.

[0775] 34. The cationic lipid of numbered embodiment 33, having a structure according to formula (IIIA):

[0776]

[0777] or a pharmaceutically acceptable salt thereof.

[0778] 35. The cationic lipid of Numbered Example 33 or Numbered Example 34, having a structure according to Formula (IIIB):

[0779]

[0780] or a pharmaceutically acceptable salt thereof.

[0781] 36. The cationic lipid of numbered embodiment 35, having a structure according to formula (IIIC):

[0782]

[0783] or a pharmaceutically acceptable salt thereof.

[0784] 37. The cationic lipid of numbered Example 33, having a structure according to Formula (IIID):

[0785]

[0786] or a pharmaceutically acceptable salt thereof.

[0787] 38. The cationic lipid of numbered embodiment 37, having a structure selected from Formula (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ), or (IIIK):

[0788]

[0789]

[0790]

[0791] or a pharmaceutically acceptable salt thereof.

[0792] 39. The cationic lipid of numbered Example 33, having a structure according to Formula (IIIL):

[0793]

[0794] or a pharmaceutically acceptable salt thereof.

[0795] 40. The cationic lipid of numbered embodiment 33, having a structure according to formula (IV):

[0796]

[0797] wherein M is selected from H, OH, OMe or Me, or a pharmaceutically acceptable salt thereof.

[0798] 41. The cationic lipid of numbered embodiment 33, having a structure according to Formula (VI), (VII), (VIII), (IX), or (X):

[0799]

[0800]

[0801] wherein one of Y and Z is OH and the other is -OC(O)R', or wherein both Y and Z are each independently -OC(O)R', or a pharmaceutically acceptable salt thereof.

[0802] 42. The cationic lipid of numbered embodiment 41, or a pharmaceutically acceptable salt thereof, wherein one of Y and Z is OH and the other is -OC(O)R'.

[0803] 43. The cationic lipid of numbered embodiment 42, or a pharmaceutically acceptable salt thereof, wherein Y is OH and Z is -OC(O)R'.

[0804] 44. The cationic lipid of numbered embodiment 42, or a pharmaceutically acceptable salt thereof, wherein Y is -OC(O)R' and Z is OH.

[0805] 45. The cationic lipid of numbered embodiment 41, or a pharmaceutically acceptable salt thereof, wherein both Y and Z are -OC(O)R'.

[0806] 46. ​​A compound selected from the group consisting of the compounds listed in Tables 1 to 8, or a pharmaceutically acceptable salt thereof.

[0807] 47. A composition comprising the cationic lipid of any one of the preceding numbered embodiments, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

[0808] 48. The composition of numbered embodiment 47, wherein the composition is a lipid nanoparticle, optionally a liposome.

[0809] 49. The composition of numbered embodiment 48, wherein the one or more cationic lipids comprise about 30 mol%-60 mol% of the lipid nanoparticles.

[0810] 50. The composition of any of numbered embodiments 48 or 49, wherein the one or more non-cationic lipids comprise 10 mol% to 50 mol% of the lipid nanoparticles.

[0811] 51. The composition of any one of numbered embodiments 48 to 50, wherein the one or more PEG-modified lipids constitute 1 mol%-10 mol% of the lipid nanoparticles.

[0812] 52. The composition of any one of numbered embodiments 48 to 51, wherein the cholesterol-based lipid comprises 10 mol%-50 mol% of the lipid nanoparticle.

[0813] 53. The composition of any one of numbered embodiments 48 to 52, wherein the lipid nanoparticles encapsulate a nucleic acid, optionally an mRNA encoding a peptide or protein.

[0814] 54. The composition of any one of numbered embodiments 48 to 52, wherein the lipid nanoparticles encapsulate mRNA encoding a peptide or protein.

[0815] 55. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 70%.

[0816] 56. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 75%.

[0817] 57. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 80%.

[0818] 58. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 85%.

[0819] 59. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 90%.

[0820] 60. The composition of numbered embodiment 54, wherein the lipid nanoparticles have an mRNA encapsulation percentage of at least 95%.

[0821] 61. The composition of any one of numbered embodiments 54 to 60 for use in therapy.

[0822] 62. The composition of any one of numbered embodiments 54 to 60, for use in a method of treating or preventing a disease amenable to treatment or prevention by a peptide or protein encoded by an mRNA, optionally wherein the disease is: (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lung, brain, or muscle; (b) an autoimmune disease; (c) an infectious disease; or (d) cancer.

[0823] 63. The composition for use of numbered embodiment 61 or 62, wherein the composition is administered intravenously, intrathecally, or intramuscularly, or is delivered pulmonary, optionally by nebulization.

[0824] 64. A method for treating or preventing a disease, wherein the method comprises administering to a subject in need thereof the composition of any one of numbered Examples 54 to 60, and wherein the disease is amenable to treatment or prevention by a peptide or protein encoded by the mRNA, optionally wherein the disease is: (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain, or muscle; (b) an autoimmune disease; (c) an infectious disease; or (d) cancer.

[0825] 65. The method of numbered embodiment 64, wherein the composition is administered intravenously, intrathecally, or intramuscularly, or delivered pulmonary, optionally by aerosolization.

Claims

1. A cationic lipid having a structure according to formula (I): Wherein L1 is a bond, C1-C6 alkyl or C2-C6 alkenyl; Where X is O; where R 1 、R 2 、R 4 and R 5 Each independently selected from H and C1-C6 alkoxy; where R 3 is -OC(O)R'; Where R' is where R 6 for wherein m and p are each independently 0, 1, 2, 3, 4 or 5; where R 9 is an optionally substituted C1-C6 alkyl group; where R 10 is an optionally substituted C1-C6 alkyl group; wherein k and n are each independently 1; where R 11 and R 12 Each is H; where R A and R B Each independently selected from optionally substituted C6-C 20 Alkyl and optionally substituted C6-C 20 alkenyl; in, "Optionally substituted" refers to substituents selected from -CO2R", OH and -OCOR", wherein R" is C1-C 20 alkyl; or a pharmaceutically acceptable salt thereof.

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

3. The cationic lipid or pharmaceutically acceptable salt thereof according to claim 1, wherein p is 1, 2 or 3.

4. The cationic lipid according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 6 for:

5. The cationic lipid of claim 1 , having a structure according to: (a) Formula (IIIA): or a pharmaceutically acceptable salt thereof, (c) Formula (IIID): or a pharmaceutically acceptable salt thereof, (d) Formula (IIA): or a pharmaceutically acceptable salt thereof, (e) Formula (IIF): or a pharmaceutically acceptable salt thereof, or (f) Formula (IIG): or a pharmaceutically acceptable salt thereof.

6. The cationic lipid according to claim 1, having a structure selected from compounds 1-12: or a pharmaceutically acceptable salt thereof.

7. The cationic lipid according to claim 6, having the structure of compound 5: or a pharmaceutically acceptable salt thereof.

8. A cationic lipid selected from the group consisting of the cationic lipids listed in Tables 2 to 8, or a pharmaceutically acceptable salt thereof.

9. A composition comprising the cationic lipid of any one of claims 1-8, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

10. The composition of claim 9, wherein the composition is a lipid nanoparticle.

11. The composition of claim 10, wherein the lipid nanoparticles encapsulate nucleic acids.

12. The composition of claim 11, wherein the nucleic acid is an mRNA encoding a peptide or protein.

13. The composition of claim 12, wherein the lipid nanoparticles have an encapsulation percentage of at least 70% for mRNA.

14. A composition according to any one of claims 9 to 13 for use in therapy.

15. A method for preparing a therapeutic composition, wherein the therapeutic composition comprises the composition of claim 10 and a full-length mRNA encoding a peptide or protein, wherein the therapeutic composition is used to deliver a vaccine to a subject or a cell of a subject or to treat with a vaccine.

16. The method of claim 15, wherein the full-length mRNA encodes an antigen from an infectious source.

17. A method for preparing a therapeutic composition for treating a human patient in need thereof, wherein the therapeutic composition comprises an mRNA encoding a peptide or protein encapsulated within a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the lipid nanoparticles further comprise one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

19. The method of claim 17 or claim 18, wherein the therapeutic composition is used to deliver a vaccine.

20. Use of lipid nanoparticles comprising mRNA in the preparation of a pharmaceutical composition for treating or preventing a disease, the disease being amenable to treatment or prevention by a peptide or protein encoded by the mRNA, and wherein the lipid nanoparticles comprise the cationic lipid according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

21. The use according to claim 20, wherein the disease is: (a) protein deficiency; (b) autoimmune disease; (c) infectious disease; or (d) cancer.

22. The use according to claim 21, wherein the protein deficiency affects the liver, lungs, brain or muscle.

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