Piperazine-based cationic lipids

Abstract

The present invention provides, in part, piperazine-based lipid compounds of formula (I) and its subformulas: or pharmaceutically acceptable salts thereof. The compounds provided herein may be useful, for example, as components of liposome delivery vehicles, for the delivery and expression of mRNA and the encoded protein, and thus may be useful in treating various diseases, disorders, and conditions, such as those associated with deficiencies of one or more proteins. [Formula 1] TIFF2023542944000125.tif50154

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 082,101, filed September 23, 2020, the disclosure of which is hereby incorporated by reference. [Background technology]

[0002] Nucleic acid delivery has been widely explored as a promising therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for the treatment of various diseases, including those associated with deficiencies of one or more proteins. Summary of the Invention [Means for solving the problem]

[0003] The effective delivery of liposome-encapsulated nucleic acids remains an active area of ​​research and investigation. Cationic lipid components play an important role in facilitating the effective encapsulation of nucleic acids during loading into liposomes. In addition, cationic lipids may play an important role in the effective release of nucleic acid cargo from liposomes into the cytoplasm of target cells. A variety of cationic lipids suitable for in vivo use have been discovered. However, there remains a need to identify lipids that can be synthesized effectively and inexpensively without the formation of potentially toxic by-products.

[0004] The present invention provides, inter alia, cationic lipid compounds for the in vivo delivery of therapeutic agents, such as nucleic acids. It is contemplated that these compounds are capable of highly effective in vivo delivery while maintaining a favorable toxicity profile.

[0005] The cationic lipid of the present invention can be synthesized from easily available starting reagents.The cationic lipid of the present invention also has unexpectedly high encapsulation efficiency.The cationic lipid of the present invention also comprises cleavable groups (such as ester and disulfide), which are intended to improve biodegradability and therefore contribute to their favorable toxicity profile.

[0006] In one embodiment, a compound of formula (I'): [ka] Provided herein are cationic lipids having a structure according to the formula:

[0007] A 1 teeth, [ka] and -SS-, wherein the left hand side of each depicted structure is selected from (CH) a -bonded;

[0008] Z 1 teeth, [ka] and -SS-, wherein the right hand side of each depicted structure is selected from -(CH) a -bonded; Each R is independently selected from the following:

[0009] (i) [ka] (In the formula, each R 1are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -optionally substituted alkyl-(C=O)-O-optionally substituted alkyl, and -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl; and

[0010] (ii) [ka] (In the formula, each R 2 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted acyl; each a is independently selected from 2, 3, 4, and 5; Each b is independently selected from 2, 3, 4, 5, 6 and 7.

[0011] In one embodiment, a compound of formula (I): [ka] Provided herein are cationic lipids having a structure according to the formula:

[0012] A 1 teeth, [ka] and -SS-, wherein the left hand side of each depicted structure is selected from -(CH) a -bonded;

[0013] Z 1 teeth, [ka] and -SS-, wherein the right hand side of each depicted structure is selected from -(CH) a -bonded; Each R is independently selected from the following:

[0014] (iii) [ka] (In the formula, each R 1 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -optionally substituted alkyl-(C=O)-O-optionally substituted alkyl, and -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl; and

[0015] (iv) [ka] (In the formula, each R 2 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted acyl; Each a is independently selected from 2, 3, 4 and 5.

[0016] In one embodiment, provided herein is a cationic lipid that is a pharmaceutically acceptable salt of formula (I').

[0017] In one embodiment, provided herein is a cationic lipid that is a pharmaceutically acceptable salt of formula (I):

[0018] In one embodiment, 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.In one embodiment, the composition is a lipid nanoparticle, optionally a liposome.

[0019] In one embodiment, compositions comprising the cationic lipids of the present invention can be used in therapy. [Brief explanation of the drawings]

[0020] [Figure 1]

[0023] Figure 1 illustrates in vivo protein production resulting from delivery of mRNA (i.e., FFL mRNA) using lipid nanoparticles containing compounds D23, D21, C9, C17, A9, A11, A17, A15, A18, or A19 as described herein. As shown in this figure, the use of these compounds can result in high levels of in vivo protein production (i.e., FFL protein) following administration. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference.

[0022] 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 HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an l-amino acid. A "standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is synthetically produced or obtained from a natural source. As used herein, a "synthetic amino acid" encompasses chemically modified amino acids, including, but not limited to, salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including the carboxy- and / or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting their activity. Amino acids can participate in disulfide bonds. Amino acids can include self- or post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and / or amino acid residues of peptides. Whether it refers to a free amino acid or a residue of a peptide is clear from the context in which the term is used.

[0023] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. 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 may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0024] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about," unless otherwise specified or clear from the context, refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) the stated reference value (except where such number exceeds 100% of possible values).

[0025] Biologically active: As used herein, the term "biologically active" refers to a characteristic of any agent that has activity on a biological system, particularly an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be biologically active.

[0026] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, the delivery of mRNA encompasses the situation in which the mRNA is delivered to a target tissue, and the encoded protein is expressed and retained within the target tissue (also referred to as "local distribution" or "local delivery"), as well as the situation in which the mRNA is delivered to a target tissue, and the encoded protein is expressed, secreted into the patient's circulatory system (e.g., serum), distributed systemically, and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

[0027] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into an intact protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "production" and their grammatical equivalents are used interchangeably.

[0028] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0029] Half-life: As used herein, the term "half-life" is the time required for a quantity, such as the concentration or activity of a nucleic acid or protein, to fall to half of its value measured at the beginning of the time period.

[0030] Helper lipid: The term "helper lipid," as used herein, refers to any neutral or zwitterionic lipid material, including cholesterol. Without wishing to be bound by any particular theory, the helper lipid may add stability, rigidity, and / or fluidity within the lipid bilayer / nanoparticle.

[0031] Improve, increase, or reduce: As used herein, the terms "improve," "increase," or "reduce," or grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or more control subjects) in the absence of a treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated, and is about the same age as the subject being treated.

[0032] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., a test tube or reaction vessel, such as in cell culture, rather than within a multicellular organism.

[0033] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal. In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, for example, an in vitro system).

[0034] Isolated: As used herein, the term "isolated" refers to a substance and / or entity that (1) has been separated from at least some of the components that accompanied it when originally produced (in practice and / or in an experimental setting) and / or (2) has been produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities 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 the other components with which they were originally associated. In some embodiments, 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 greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculations of percent purity of isolated substances and / or entities should not include excipients (e.g., buffers, solvents, water, etc.).

[0035] Liposome: As used herein, the term "liposome" refers to any lamellar, multi-layered or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids, or by mixing one or more lipids and polymer(s). In some embodiments, liposomes suitable for the present invention contain cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and / or optionally PEG-modified lipid(s).

[0036] 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 encompasses both modified and unmodified RNA. The term "modified mRNA" refers to an mRNA that contains at least one chemically modified nucleotide. An mRNA can contain one or more coding and non-coding regions. An mRNA can be purified from a natural source, produced using a recombinant expression system, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, an mRNA can contain nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is designed to contain natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcyt ...bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C 6-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0037] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" refers to 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), micro-RNA (miRNA), multimeric coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" refers to 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" refers to both RNA and DNA. In embodiments, the DNA may be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a product of polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups.In embodiments, the RNA is a messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL 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), splice leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding RNA (lncRNA), micro-RNA (miRNA), piwi-binding RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K It may be in the form of RNA, retrotransposon, viral genome, viroid, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA that encodes a protein, such as an enzyme.

[0038] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, for example, 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. Human includes prenatal and postnatal forms.

[0039] Pharmaceutically acceptable: The term "pharmaceutically acceptable," as used herein, refers to materials that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0040] 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 this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed 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, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethylethanesulfonate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-butyl salts. + (C 1~4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and aryl sulfonates, where appropriate. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, e.g., alkyl halides, to form quaternized alkylated amino salts.

[0041] Systemic distribution or delivery: As used herein, the term "systemic distribution" or "systemic delivery," or grammatical equivalents thereof, refers to a mechanism or method of delivery or distribution that affects the entire body or organism. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with the definition of "local distribution or delivery."

[0042] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human who presents to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject is afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0043] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0044] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, the target tissue includes tissues that exhibit disease-associated pathologies, symptoms, or features.

[0045] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means 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 a symptom(s) of the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0046] Treating: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, relieve, relieve, 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 the disease and / or who exhibit only early signs of the disease, with the intent of reducing the risk of developing pathologies associated with the disease.

[0047] chemical definition Acyl: As used herein, the term “acyl” refers to RZ -(C=O)-, where R Z is, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene.

[0048] Aliphatic: As used herein, the term aliphatic refers to C1-C 50 Aliphatic refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic groups may be linear, branched, or cyclic. For example, C1-C 20 Aliphatic: C1-C 20 Alkyl (e.g., linear or branched C1-C 20 saturated alkyl), C2-C 20 Alkenyl (e.g., linear or branched C4-C 20 Dienyl, linear or branched, C6-C 20 trienyl, etc.), and C2-C 20 Alkynyl (e.g., linear or branched C2-C 20 alkynyl). 20 Aliphatic: C3 to C 20 Cycloaliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20 In certain embodiments, an aliphatic group can include one or more cycloaliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. An aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, an aliphatic group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR″, —COH, —COR″, —CN, —OH, —OR″, —OCOR′, —OCOR″, —NH, —NHR″, —N(R″), —SR″, or —SOR″, where each instance of R″ is independently C1-C6.20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an 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" refers to acyclic linear and branched hydrocarbon groups, e.g., "C1-C 30 "Alkyl" refers to an alkyl group having 1 to 30 carbon atoms. The alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentyl hexyl, isohexyl, and the like. The term "lower alkyl" refers to a straight chain alkyl group or a branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be readily apparent to one of ordinary skill 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, an alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR″, —COH, —COR″, —CN, —OH, —OR″, —OCOR′, —OCOR″, —NH, —NHR″, —N(R″), —SR″, or —SOR″, where each instance of R″ is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkyl group is substituted with an -OH group, and may also be referred to herein as a "hydroxyalkyl" group, where the prefix indicates the -OH group and "alkyl" is as described herein.

[0049] As used herein, "alkyl" also refers to a group of straight-chain or branched saturated hydrocarbon groups having from 1 to 50 carbon atoms ("C1-C 50 In some embodiments, an alkyl group has 1 to 40 carbon atoms ("C1-C 40 In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-C 30 In some embodiments, an alkyl group has 1 to 20 carbon atoms ("C1-C 20 In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-C 10In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C1-C6 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1-C5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, an 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, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). 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 It is alkyl.

[0050] The addition of the suffix "-ene" to a base indicates that the group is a divalent moiety, for example, arylene is a divalent moiety of an aryl, and heteroarylene is a divalent moiety of a heteroaryl.

[0051] Alkylene: The term "alkylene," as used herein, represents a saturated divalent straight- or branched-chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, the term "alkenylene," as used herein, represents an unsaturated divalent straight- or branched-chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, and the term "alkynylene," as used herein, represents an unsaturated divalent straight- or branched-chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur at any stable point along the chain. In certain embodiments, alkylene, alkenylene, or alkynylene groups can contain one or more cycloaliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. For example, an alkylene, alkenylene, or alkynylene can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR″, —COH, —COR″, —CN, —OH, —OR″, —OCOR″, —OCOR″, —NH2, —NHR″, —N(R″), —SR″, or —SOR″, where each instance of R″ is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10In certain embodiments, R" is independently an unsubstituted C1-C3 alkyl. In certain embodiments, the alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene does not contain any heteroatoms. Alkenyl: As used herein, "alkenyl" refers to any linear or branched hydrocarbon chain with one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, e.g., "C2-C 30 "Alkenyl" refers to an alkenyl group having 2 to 30 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, an alkenyl contains one, two, or three carbon-carbon double bonds. In embodiments, an alkenyl contains a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., two or three) are conjugated. An alkenyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkenyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR″, —COH, —COR″, —CN, —OH, —OR″, —OCOR″, —OCOR″, —NH2, —NHR″, —N(R″), —SR″, or —SOR″, where each instance of R″ is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkenyl group is substituted with an -OH group, and may also be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates an -OH group and "alkenyl" is as described herein.

[0052] As used herein, "alkenyl" also refers to a group of straight-chain or branched hydrocarbon groups having 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-C 50 In some embodiments, an alkenyl group has 2 to 40 carbon atoms (“C-C 40 In some embodiments, an alkenyl group has 2 to 30 carbon atoms (“C-C 30 In some embodiments, an alkenyl group has 2 to 20 carbon atoms (“C-C 20 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C-C 10In some embodiments, an alkenyl group has from 2 to 9 carbon atoms ("C2-C9 alkenyl"). In some embodiments, an alkenyl group has from 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has from 2 to 7 carbon atoms ("C2-C7 alkenyl"). In some embodiments, an alkenyl group has from 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has from 2 to 5 carbon atoms ("C2-C5 alkenyl"). In some embodiments, an alkenyl group has from 2 to 4 carbon atoms ("C2-C4 alkenyl"). In some embodiments, an alkenyl group has from 2 to 3 carbon atoms ("C2-C3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (e.g., 2-butenyl) or terminal (e.g., 1-butenyl). Examples of C2-C4 alkenyl groups include, without limitation, ethenyl (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 C2-C4 alkenyl groups described above, as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, an alkenyl group is an unsubstituted C2-C6 alkyl group. 50 In certain embodiments, the alkenyl group is a substituted C-C 50 It is alkenyl.

[0053] Alkynyl: As used herein, "alkynyl" means any hydrocarbon chain of either linear or branched configuration with one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "C2-C 30 "Alkynyl" refers to an alkynyl group having 2 to 30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, an alkynyl contains one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, —COR″, —COH, —COR″, —CN, —OH, —OR″, —OCOR″, —OCOR″, —NH2, —NHR″, —N(R″), —SR″, or —SOR″, where each instance of R″ is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently unsubstituted C1-C3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0054] As used herein, "alkynyl" also refers to a group of straight-chain or branched hydrocarbon groups having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., one, two, three, or four triple bonds) and optionally one or more double bonds (e.g., one, two, three, or four double bonds) ("C2-C 50 Alkynyl groups having one or more triple bonds and one or more double bonds are also referred to as "ene-ynes." In some embodiments, alkynyl groups have 2 to 40 carbon atoms ("C2-C 40 In some embodiments, an alkynyl group has 2 to 30 carbon atoms ("C-C 30 In some embodiments, an alkynyl group has 2 to 20 carbon atoms ("C-C 20 In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C-C 10In some embodiments, an alkynyl group has from 2 to 9 carbon atoms ("C2-C9 alkynyl"). In some embodiments, an alkynyl group has from 2 to 8 carbon atoms ("C2-C8 alkynyl"). In some embodiments, an alkynyl group has from 2 to 7 carbon atoms ("C2-C7 alkynyl"). In some embodiments, an alkynyl group has from 2 to 6 carbon atoms ("C2-C6 alkynyl"). In some embodiments, an alkynyl group has from 2 to 5 carbon atoms ("C2-C5 alkynyl"). In some embodiments, an alkynyl group has from 2 to 4 carbon atoms ("C2-C4 alkynyl"). In some embodiments, an alkynyl group has from 2 to 3 carbon atoms ("C2-C3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-triple bonds may be internal (e.g., 2-butynyl) or terminal (e.g., 1-butynyl). Examples of C2-C4 alkynyl groups include, without limitation, 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 C2-C4 alkynyl groups described above, as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents (a "substituted alkynyl"). In certain embodiments, an alkynyl group is an unsubstituted C2-C6 alkynyl group. 50 In certain embodiments, the alkynyl group is a substituted C-C 50 It is alkynyl.

[0055] 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 6 to 14 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 each ring in the system contains 4 to 7 ring members. In 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," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C 14 "Aryl," e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the group or point of attachment is on the aryl ring; in such instances, the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Illustrative aryls include phenyl, naphthyl, and anthracene.

[0056] As used herein, "aryl" also refers to a group of monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring systems (e.g., having 6, 10, or 14 pi electrons shared in the cyclic array) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-C 14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 Aryl"; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl"; e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the group or point of attachment is on the aryl ring; in such instances, the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, an aryl group is an unsubstituted C6-C6 14 In certain embodiments, the aryl group is a substituted C-C 14 It is aryl.

[0057] Arylene: The term "arylene," as used herein, refers to an aryl group that is divalent (i.e., has two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted or substituted phenylene).

[0058] Carbocyclyl: As used herein, "carbocyclyl" or "carbocyclic" refers to a ring system having from 3 to 10 ring carbon atoms ("C3-C6"). 10 "C-C carbocyclyl" refers to a group of non-aromatic cyclic hydrocarbon groups having zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C-C carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C-C carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C-C carbocyclyl"). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C-C carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms ("C-C carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C-C 10carbocyclyl). Exemplary C3-C6 carbocyclyl groups include, without limitation, 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, without limitation, the C3-C6 carbocyclyl groups described above, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Illustrative C3-C 10 Carbocyclyl groups include, but are not limited to, the C3-C8 carbocyclyl groups described above, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10As the foregoing examples illustrate, in certain embodiments, a carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring system, such as a bicyclic ring system ("bicyclic carbocyclyl") or a tricyclic ring system ("tricyclic carbocyclyl")) and can be saturated or 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, and wherein the point of attachment is on the carbocyclyl ring; in such instances, the number of carbons continues to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is an unsubstituted C-C 10 In certain embodiments, the carbocyclyl group is a substituted C3-C 10 It is a carbocyclyl.

[0059] In some embodiments, a "carbocyclyl" or "carbocyclic" is referred to as a "cycloalkyl," i.e., a monocyclic saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, a cycloalkyl group has from 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). In some embodiments, a cycloalkyl group has from 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 4 to 6 ring carbon atoms ("C4-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 5 to 6 ring carbon atoms ("C5-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). 10Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the C5-C6 cycloalkyl groups described above, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the C3-C6 cycloalkyl groups described above, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, a cycloalkyl group is an unsubstituted C3-C6 cycloalkyl group. 10 In certain embodiments, the cycloalkyl group is a substituted C-C 10 It is cycloalkyl.

[0060] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0061] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyl groups can optionally contain monocyclic, bicyclic, or tricyclic rings, where each ring desirably has 3 to 6 members. Examples of heteroalkyls include polyethers such as methoxymethyl and ethoxyethyl.

[0062] Heteroalkylene: The term "heteroalkylene," as used herein, represents a divalent form of a heteroalkyl group, as described herein.

[0063] Heteroaryl: The term "heteroaryl," as used herein, refers to a fully unsaturated heteroatom-containing ring in which at least one ring atom is a heteroatom, such as, but not limited to, nitrogen and oxygen.

[0064] As used herein, "heteroaryl" also refers to a 5-14-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π-electrons shared in the cyclic array) having ring carbon atoms and one or more ring heteroatoms (e.g., 1, 2, 3, or 4 ring heteroatoms) provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, if 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, where the point of attachment is on the heteroaryl ring; in such cases, the number of ring members continues to designate 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, where the point of attachment is on either the aryl or heteroaryl ring; in such cases, the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on a ring containing a heteroatom (e.g., 2-indolyl) or on a ring without a heteroatom (e.g., 5-indolyl).

[0065] In some embodiments, a heteroaryl group is a 5- to 10-membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 10-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 8-membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 8-membered heteroaryl"). In some embodiments, a heteroaryl group is a 5- to 6-membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 6-membered heteroaryl"). In some embodiments, a 5- to 6-membered heteroaryl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5- to 6-membered heteroaryl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5- to 6-membered heteroaryl 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, a heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, a heteroaryl group is a substituted 5-14 membered heteroaryl.

[0066] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. 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, 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, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0067] As used herein, "heterocyclyl" or "heterocyclic" refers to a group of 3- to 14-membered non-aromatic ring systems having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic (a "monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems, such as a bicyclic system (a "bicyclic heterocyclyl") or a tricyclic system (a "tricyclic heterocyclyl")), and may be saturated or contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can contain 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, where the point of attachment is on either the carbocyclyl ring or the heterocyclyl ring, or a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring; in such instances, the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 14-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

[0068] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5- to 6-membered heterocyclyl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5- to 6-membered heterocyclyl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0069] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, aziridinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.Illustrative bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepin ... Examples include 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, and 1,2,3,4-tetrahydro-1,6-naphthyridinyl.

[0070] Heterocycloalkyl: The term "heterocycloalkyl," as used herein, refers to 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 can be substituted or unsubstituted.

[0071] As understood above, alkyl groups, alkenyl groups, alkynyl groups, acyl groups, carbocyclyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups as defined herein are, in certain embodiments, optionally substituted. Optionally substituted refers to a group that may be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl group, a "substituted" or "unsubstituted" alkenyl group, a "substituted" or "unsubstituted" alkynyl group, a "substituted" or "unsubstituted" heteroalkyl group, a "substituted" or "unsubstituted" heteroalkenyl group, a "substituted" or "unsubstituted" heteroalkynyl group, a "substituted" or "unsubstituted" carbocyclyl group, a "substituted" or "unsubstituted" heterocyclyl group, a "substituted" or "unsubstituted" aryl group, or a "substituted" or "unsubstituted" heteroaryl group). Generally, the term "substituted" means that at least one hydrogen atom on a group is replaced with an acceptable substituent, e.g., a substituent that results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction. 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 substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all acceptable substituents of organic compounds, and any of the substituents described herein will result in the formation of a stable compound. The present invention contemplates any and all such combinations to arrive at a stable compound. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any suitable substituents as described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0072] Illustrative carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb)2、-N(R bb )3+X - 、-N(OR cc )R bb 、-SeH、-SeR aa 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3-OSi(R aa )3-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa, -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 14 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 and 5- to 14-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group; Or two geminal hydrogens on a carbon atom can be =O group, =S group, =NN(R bb )2 units, =NNR bb C(=O)R aa base,=NNR bb C(=O)OR aa base,=NNR bb S(=O)2Raa base, =NR bb Group or =NOR cc has been replaced with a radical;

[0073] R aa Each example is independently C1 to C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R aa The groups are joined to form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently has 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0074] R bb Each instance of is independently hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, C1~C 50 Alkyl, C2-C 50Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R bb The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0075] R cc Each example is independently hydrogen, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R cc The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0076] R dd Each example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3+X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee, -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff ) OR ee , -OC(=NR ff )R ee , -OC(=NR ff ) OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-10 membered heterocyclyl, C6-C 10aryl, and 5- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group or two geminal R dd The substituents can be joined to form =O or =S;

[0077] R ee Each example is independently C1 to C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, C6-C 10 aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group;

[0078] R ff Each example is independently hydrogen, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-10 membered heterocyclyl, C6-C 10 aryl, and 5- to 10-membered heteroaryl, or two R ff The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R gg substituted with a group;

[0079] R ggEach example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C 50 Alkyl, -ON(C1-C 50 alkyl)2, -N(C1-C 50 alkyl)2, -N(C1-C 50 Alkyl)3+X - , -NH(C1~C 50 alkyl)2+X - , -NH2(C1~C 50 alkyl) + X - , -NH3+X - , -N(OC1-C 50 Alkyl) (C1-C 50 alkyl), -N(OH)(C1-C 50 alkyl), -NH(OH), -SH, -SC1-C 50 Alkyl, -SS(C1-C 50 alkyl), -C(=O)(C1-C 50 alkyl), -CO2H, -CO2(C1-C 50 alkyl), -OC(=O)(C1-C 50 alkyl), -OCO2(C1-C 50 alkyl), -C(=O)NH2, -C(=O)N(C1-C 50 alkyl)2, -OC(=O)NH(C1-C 50 alkyl), -NHC(=O)(C1-C 50 alkyl), -N(C1-C 50 Alkyl)C(=O)(C1-C 50 alkyl), -NHCO2(C1-C 50 alkyl), -NHC(=O)N(C1-C 50 alkyl)2, -NHC(=O)NH(C1-C 50 alkyl), -NHC(=O)NH2, -C(=NH)O(C1-C 50 alkyl), -OC(=NH)(C1-C 50 alkyl), -OC(=NH)OC1-C 50 Alkyl, -C(=NH)N(C1-C 50 alkyl)2, -C(=NH)NH(C1-C 50 alkyl), -C(=NH)NH2, -OC(=NH)N(C1-C50 alkyl)2, -OC(NH)NH(C1-C 50 alkyl), -OC(NH)NH2, -NHC(NH)N(C1-C 50 alkyl)2, -NHC(=NH)NH2, -NHSO2(C1-C 50 alkyl), -SO2N(C1-C 50 alkyl)2, -SO2NH(C1-C 50 alkyl), -SO2NH2, -SO2(C1-C 50 alkyl), -SO2O(C1-C 50 alkyl), -OSO2(C1-C6 alkyl), -SO(C1-C6 alkyl), -Si(C1-C 50 alkyl)3, -OSi(C1-C6 alkyl)3, -C(=S)N(C1-C 50 alkyl)2, C(=S)NH(C1-C 50 alkyl), C(=S)NH2, -C(=O)S(C1-C6 alkyl), -C(=S)S(C1-C6 alkyl), -SC(=S)S(C1-C6 alkyl), -P(=O)2(C1-C 50 alkyl), -P(=O)(C1-C 50 alkyl)2, -OP(=O)(C1-C 50 alkyl)2, -OP(=O)(OC1~C 50 Alkyl)2, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, C6-C 10 aryl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl; or two geminal R gg Substituents can be joined to form =O or =S; where X - is the counter ion.

[0080] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0081] As used herein, a "counterion" is a negatively charged group associated with a positively charged quaternary amine to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F - , Cl - , Br - , I - ), NO3 - , ClO4 - , O.H. - , 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, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0082] Nitrogen atoms may be substituted or unsubstituted, where valence allows, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Illustrative nitrogen atom substituents include, but are not limited to, hydrogen, —OH, —OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa, -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R cc The groups, together with the N atom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently contains 0, 1, 2, 3, 4, or 5 R dd is substituted with an R aa , R bb , R cc and R dd is as defined above.

[0083] 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, T.W. Greene and P.G.M. Butts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0084] For example, a nitrogen protecting group such as an amide group (e.g., —C(═O)R aa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0085] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR aa), but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- Trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate ( t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chloro Monylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2, 2-Dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1 -methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, 1-methyl-1(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0086] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)R aa ), 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), 2,4,6-trimethylbenzenesulfonamide amide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0087] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyl Disilyl azacyclopentane adducts (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-ones, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-ones, 1-substituted 3,5-dinitro-4-pyridones, N-methylamines, N-allylamines, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3)- ...methylamines, N-allylamines, N-methylamines, N-allylamines, N-methylamines, N-allylamines, N-methylamines, N-allylamines, N-methylamine -yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethyl Thiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidate, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

[0088] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group).Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0089] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl, and the like. Dimethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]phenanthroline [Nyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoro ... ethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-Dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)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-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate Acetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzyl carbonate, Benzene sulfonate, 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, monosuccinoate , (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0090] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P.G.M. Butts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0091] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxide, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosuberyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbomethyl, methyl ... nylaminomethyl, phenylacetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carboethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylprop-2-yl, Examples of suitable alkyl esters include acetyl, benzoyl, trifluoroacetyl, N-[[(p-biphenylyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, sulfenylthiocarbonate, 3-nitro-2-pyridinesulfenyl sulfide, and oxathiolane.

[0092] Compounds of the Invention Liposome-based vehicles are considered to be attractive carriers for therapeutic agents, and are still under development.Although liposome-based vehicles that contain certain lipid components have shown promising results in terms of encapsulation, stability and site localization, there is still a great need for the improvement of liposome-based delivery systems.For example, the significant drawback of liposome delivery systems is the construction of liposomes that have sufficient cell culture or in vivo stability to reach desired target cells and / or intracellular compartments, and the ability of these liposome delivery systems to effectively release their encapsulated materials into these target cells.

[0093] In particular, there remains a need for improved lipid compounds that demonstrate improved pharmacokinetic properties and can deliver macromolecules, such as nucleic acids, to a wide variety of cell types and tissues with enhanced efficiency. Importantly, there also remains a particular need for novel lipid compounds that are characterized by reduced toxicity and can effectively deliver encapsulated nucleic acids and polynucleotides to targeted cells, tissues, and organs.

[0094] Described herein is a cationic lipid compound for improving in vivo delivery of therapeutic agents such as nucleic acids.In particular, the cationic lipids described herein can be used, optionally together with other lipids, to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA and / or microRNA) for therapeutic use.

[0095] In embodiments, the compounds of the present invention as described herein can provide one or more desirable features or characteristics. That is, in certain embodiments, the compounds of the present invention as described herein can be characterized by one or more properties that provide them with advantages over other similarly classified lipids. For example, the compounds disclosed herein can allow for control and adjustment of the properties of the liposome compositions (e.g., lipid nanoparticles) of which they are components. In particular, the compounds disclosed herein can be characterized by enhanced transfection efficiency and their ability to induce specific biological results. Such results can include, for example, enhanced cellular uptake, endosome / lysosome disruption ability, and / or accelerated release of encapsulated materials (e.g., polynucleotides) within cells. Additionally, the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution, and efficacy (e.g., due to different dissociation rates of the polymer groups used).

[0096] This application demonstrates that the cationic lipids of the present invention are not only synthetically tractable from readily available starting materials, but also that they have unexpectedly high encapsulation efficiency.

[0097] Additionally, 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 contemplated to improve biodegradability and therefore contribute to their favorable toxicity profile.

[0098] Compounds that are cationic lipids are provided herein. For example, cationic lipids of the present invention include compounds having the formula (I'): [ka] or a pharmaceutically acceptable salt thereof, wherein:

[0099] A 1 teeth, [ka] and -SS-, wherein the left hand side of each depicted structure is selected from -(CH) a -bonded;

[0100] Z 1 teeth, [ka] and -SS-, wherein the right hand side of each depicted structure is selected from -(CH) a -bonded; Each R is independently selected from the following:

[0101] (i) [ka] (In the formula, each R 1 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -optionally substituted alkyl-(C=O)-O-optionally substituted alkyl, and -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl; and

[0102] (ii) [ka] (In the formula, each R 2 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted acyl; each a is independently selected from 2, 3, 4, and 5; Each b is independently selected from 2, 3, 4, 5, 6 and 7.

[0103] In embodiments, the cationic lipids of the present invention include those having formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein:

[0104] A 1 teeth, [ka] and -SS-, wherein the left hand side of each depicted structure is selected from -(CH) a -bonded;

[0105] Z 1 teeth, [ka] and -SS-, wherein the right hand side of each depicted structure is selected from -(CH) a -bonded; Each R is independently selected from the following:

[0106] (iii) [ka] (In the formula, each R 1 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -optionally substituted alkyl-(C=O)-O-optionally substituted alkyl, and -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl; and

[0107] (iv) [ka] (In the formula, each R 2are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted acyl; Each a is independently selected from 2, 3, 4 and 5.

[0108] In embodiments, the cationic lipids of the present invention include those having formula (Ia): [ka] or a pharmaceutically acceptable salt thereof, wherein A 1 , Z 1 , R 2 and a is as defined for formula (I).

[0109] In embodiments, the cationic lipids of the present invention include those having formula (Ib): [ka] or a pharmaceutically acceptable salt thereof, wherein A 1 , Z 1 , R 1 and a is as defined for formula (I).

[0110] In an embodiment, the cationic lipid of the present invention includes a compound having formula (Ib'): [ka] or a pharmaceutically acceptable salt thereof, wherein A 1 , Z 1 , R 1 , a and b are as defined for formula (I').

[0111] In embodiments, the cationic lipids of the present invention include those having formula (Ic): [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 and a is as defined for formula (I).

[0112] In embodiments of the present invention (eg, of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4.

[0113] In embodiments of the present invention (eg, of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is the same.

[0114] In embodiments of the invention (eg, of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is 3.

[0115] In embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), R 2 is independently selected from optionally substituted alkyl. In an embodiment of the present invention, R 2 is an optionally substituted C5-C 50 In any of the above embodiments, R 2 is an optionally substituted C5-C 40 In any of the above embodiments, R 2 is an optionally substituted C5-C 30 In any of the above embodiments, R 2 is an optionally substituted C5-C 25 In any of the above embodiments, R 2 is an optionally substituted C5-C 20 alkyl.

[0116] In embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), R 2 is C8H 17 , C 10H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 and C 16 H 27 are independently selected from

[0117] In some embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted alkyl. In some embodiments, the optionally substituted alkyl is —OC(═O)R aa where each R aa is independently selected from optionally substituted alkyl. In some embodiments, the optionally substituted alkyl is —OC(═O)R aa C is replaced by 1~20 alkyl, where each R aa is an optionally substituted C1-C 50 In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~10 alkyl, where each R aa is an optionally substituted C1-C 50 In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~5 alkyl, where each R aa is an optionally substituted C1-C 50 alkyl.

[0118] In any of the above embodiments, R aa is an optionally substituted C1-C 40 In any of the above embodiments, Raa is an optionally substituted C1-C 30 In any of the above embodiments, R aa is an optionally substituted C1-C 25 In any of the above embodiments, R aa is an optionally substituted C1-C 20 In any of the above embodiments, R aa is an optionally substituted C1-C 15 In any of the above embodiments, R aa is an optionally substituted C1-C 10 In any of the above embodiments, R aa is independently selected from optionally substituted C2-C8 alkyl. In any of the above embodiments, R aa are independently selected from optionally substituted C3-C7 alkyl.

[0119] In embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each R 2 are the same.

[0120] In embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each R 2 is C 10 H 21 is.

[0121] In embodiments of the present invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each R 2 is -OC(=O)R aa C is replaced by 1~20 alkyl, where each R aa is an optionally substituted C1-C 20 alkyl.

[0122] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4; R 2 is independently selected from optionally substituted alkyl. In any of the above embodiments, R 2 is an optionally substituted C5-C 50 In any of the above embodiments, R 2 is an optionally substituted C5-C 40 In any of the above embodiments, R 2 is an optionally substituted C5-C 30 In any of the above embodiments, R 2 is an optionally substituted C5-C 25 In any of the above embodiments, R 2 is an optionally substituted C5-C 20 alkyl.

[0123] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4; R 2 is independently selected from optionally substituted alkyl, where optionally substituted alkyl is —OC(═O)R aa where each R aa is independently selected from optionally substituted alkyl. In some embodiments, the optionally substituted alkyl is —OC(═O)R aa C is replaced by 1~20 alkyl, where each R aa is an optionally substituted C1-C 50 In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~10 alkyl, where each R aa is an optionally substituted C1-C50 In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~5 alkyl, where each R aa is an optionally substituted C1-C 50 alkyl.

[0124] In any of the above embodiments, R aa is an optionally substituted C1-C 40 In any of the above embodiments, R aa is an optionally substituted C1-C 30 In any of the above embodiments, R aa is an optionally substituted C1-C 25 In any of the above embodiments, R aa is an optionally substituted C1-C 20 In any of the above embodiments, R aa is an optionally substituted C1-C 15 In any of the above embodiments, R aa is an optionally substituted C1-C 10 In any of the above embodiments, R aa is independently selected from optionally substituted C2-C8 alkyl. In any of the above embodiments, R aa are independently selected from optionally substituted C3-C7 alkyl.

[0125] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4; R 2 is C8H 17 , C 10 H 21 , C 12 H 25 , C14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 , and C 16 H 27 are independently selected from

[0126] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is 3 and R 2 is C8H 17 , C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 and C 16 H 27 are independently selected from

[0127] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is 3 and R 2 is independently selected from optionally substituted alkyl, where optionally substituted alkyl is —OC(═O)R aa where each R aa is independently selected from optionally substituted alkyl. In some embodiments, the optionally substituted alkyl is —OC(═O)R aa C is replaced by 1~20 alkyl, where each R aa is an optionally substituted C1-C 50 In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~10 alkyl, where each R aa is an optionally substituted C1-C 50In some embodiments, the optionally substituted alkyl is independently selected from -OC(=O)R aa C is replaced by 1~5 alkyl, where each R aa is an optionally substituted C1-C 50 alkyl.

[0128] In any of the above embodiments, R aa is an optionally substituted C1-C 40 In any of the above embodiments, R aa is an optionally substituted C1-C 30 In any of the above embodiments, R aa is an optionally substituted C1-C 25 In any of the above embodiments, R aa is an optionally substituted C1-C 20 In any of the above embodiments, R aa is an optionally substituted C1-C 15 In any of the above embodiments, R aa is an optionally substituted C1-C 10 In any of the above embodiments, R aa is independently selected from optionally substituted C2-C8 alkyl. In any of the above embodiments, R aa are independently selected from optionally substituted C3-C7 alkyl.

[0129] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is the same and each R 2 are the same.

[0130] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is 3 and each R2 is C 10 H 21 is.

[0131] In embodiments of the invention (e.g., of Formula (Ic), or a pharmaceutically acceptable salt thereof), each a is 3 and each R 2 is -OC(=O)R aa C is replaced by 1~20 alkyl, where each R aa is an optionally substituted C1-C 20 alkyl.

[0132] In embodiments, the cationic lipids of the present invention have the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0133] In embodiments, the cationic lipids of the present invention have the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0134] In embodiments, the cationic lipids of the present invention have the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0135] In embodiments, the cationic lipids of the present invention include those of formula (Id): [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 and a is as defined for formula (I).

[0136] In embodiments, the cationic lipids of the present invention include those having formula (Ie): [ka] or a pharmaceutically acceptable salt thereof, wherein R 2 and a is as defined in formula (I).

[0137] In embodiments, the cationic lipids of the present invention include those having the formula (If): [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 and a is as defined in formula (I).

[0138] In embodiments, the cationic lipids of the present invention include those having the formula (If'): [ka] or a pharmaceutically acceptable salt thereof, wherein R 1 , a and b are as defined in formula (I').

[0139] In embodiments of the present invention (eg, of Formula (If'), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4.

[0140] In embodiments of the present invention (eg, of formula (If'), or a pharmaceutically acceptable salt thereof), each a is the same.

[0141] In embodiments of the invention (eg, of Formula (If'), or a pharmaceutically acceptable salt thereof), each a is 3.

[0142] In embodiments of the invention (eg, of Formula (If'), or a pharmaceutically acceptable salt thereof), each b is independently selected from 2, 3, 4, 5, 6, and 7.

[0143] In embodiments of the present invention (eg, of formula (If'), or a pharmaceutically acceptable salt thereof), each b is the same.

[0144] In embodiments of the invention (eg, of Formula (If'), or a pharmaceutically acceptable salt thereof), each b is 3.

[0145] In an embodiment of the invention (eg, of Formula (If'), or a pharmaceutically acceptable salt thereof), each b is 7.

[0146] In embodiments of the present invention (e.g., of formula (If'), or a pharmaceutically acceptable salt thereof), R 1 is independently selected from optionally substituted alkyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1 is an optionally substituted C5-C 25 In any of the above embodiments, R 1 is an optionally substituted C5-C 20 alkyl.

[0147] In embodiments of the present invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each R 1 are the same.

[0148] In embodiments of the present invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C20 It is alkyl.

[0149] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4; each b is independently selected from 2, 3, 4, 5, 6, and 7; and R 1 is independently selected from optionally substituted alkyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1 is an optionally substituted C5-C 25 In any of the above embodiments, R 1 optionally substituted C5-C 20 alkyl.

[0150] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3 and each b is independently selected from 2, 3, 4, 5, 6, and 7; R 1 is independently selected from optionally substituted alkyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1is an optionally substituted C5-C 25 In any of the above embodiments, R 1 is an optionally substituted C5-C 20 alkyl.

[0151] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3, each b is independently selected from 2, 3, 4, 5, 6, and 7, and each R 1 is an optionally substituted C5-C 20 It is alkyl.

[0152] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3, each b is 3, and each R 1 is an optionally substituted C5-C 20 It is alkyl.

[0153] In one embodiment, the cationic lipids of the present invention have the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0154] In embodiments of the present invention (e.g., of formula (If'), or a pharmaceutically acceptable salt thereof), R 1 is independently selected from optionally substituted alkenyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1is an optionally substituted C5-C 25 In any of the above embodiments, R 1 is an optionally substituted C5-C 20 alkenyl.

[0155] In embodiments of the present invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each R 1 are the same.

[0156] In embodiments of the present invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 20 It is alkenyl.

[0157] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4; each b is independently selected from 2, 3, 4, 5, 6, and 7; and R 1 is independently selected from optionally substituted alkenyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1 is an optionally substituted C5-C 25 In any of the above embodiments, R 1 is an optionally substituted C5-C 20 alkenyl.

[0158] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3 and each b is independently selected from 2, 3, 4, 5, 6, and 7; R 1 is independently selected from optionally substituted alkenyl. In any of the above embodiments, R 1 is an optionally substituted C5-C 50 In any of the above embodiments, R 1 is an optionally substituted C5-C 40 In any of the above embodiments, R 1 is an optionally substituted C5-C 30 In any of the above embodiments, R 1 is an optionally substituted C5-C 25 In any of the above embodiments, R 1 is an optionally substituted C5-C 20 alkenyl.

[0159] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3, each b is independently selected from 2, 3, 4, 5, 6, and 7, and each R 1 is an optionally substituted C5-C 20 It is alkenyl.

[0160] In an embodiment of the invention (e.g., of Formula (If′), or a pharmaceutically acceptable salt thereof), each a is 3, each b is 7, and each R 1 is an optionally substituted C5-C 20 It is alkenyl.

[0161] In one embodiment, the cationic lipids of the present invention have the structure: [ka] or a pharmaceutically acceptable salt thereof.

[0162] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is independently selected from 2, 3, and 4.

[0163] In some embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is 2. In some embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is 3. In some embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is 4.

[0164] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is the same.

[0165] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ib), (Ib'), (Ic), (Id), (Ie), (If), (If'), or a pharmaceutically acceptable salt thereof), each a is different.

[0166] In any of the above embodiments (eg, compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is independently selected from 2, 3, 4, 5, 6, and 7.

[0167] In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 2. In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 3. In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 4. In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 5. In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 6. In some embodiments (e.g., compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is 7.

[0168] In any of the above embodiments (eg, compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is the same.

[0169] In any of the above embodiments (eg, compounds of Formula (I'), (Ib'), (If'), or a pharmaceutically acceptable salt thereof), each b is different.

[0170] In the embodiment, A 1 teeth, [ka] wherein the left hand side of each depicted structure is selected from -(CH) a - is bonded to

[0171] In the embodiment, A 1 teeth, [ka] where the left hand side of each depicted structure is -(CH) a In one embodiment, A 1 teeth, [ka] where the left hand side of each depicted structure is -(CH) a In one embodiment, A 1 is -SS-.

[0172] In an embodiment, Z 1 teeth, [ka] wherein the right hand side of each depicted structure is selected from -(CH) a - is bonded to

[0173] In an embodiment, Z 1 teeth, [ka] where the right hand side of each depicted structure is -(CH) a In an embodiment, Z 1 teeth, [ka] where the right hand side of each depicted structure is -(CH) a In an embodiment, Z 1 is -SS-.

[0174] In the embodiment, A 1 teeth, [ka] where the left hand side of each depicted structure is -(CH) a - and Z 1 teeth, [ka] where the right hand side of each depicted structure is -(CH) a - is bonded to

[0175] In the embodiment, A 1 teeth, [ka] where the left hand side of each depicted structure is -(CH) a - and Z 1 teeth, [ka] where the right hand side of each depicted structure is -(CH) a - is bonded to

[0176] In the embodiment, A 1 and Z 1 are -SS-, respectively.

[0177] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 are the same.

[0178] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is different.

[0179] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 are independently selected from: [ka]

[0180] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 are independently selected from: [ka]

[0181] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is independently selected from optionally substituted alkyl, optionally substituted alkenyl, -optionally substituted alkyl-(C=O)-O-optionally substituted alkyl, or -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl.

[0182] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 50 Alkyl, optionally substituted C5-C 50 Alkenyl, optionally substituted C5-C 50 Alkynyl, optionally substituted C-C 25 Alkyl-(C=O)-O-optionally substituted C2-C 25 Alkyl, or optionally substituted C-C 25 Alkyl-O-(C=O)-optionally substituted C2-C 25 alkyl.

[0183] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 40Alkyl, optionally substituted C5-C 40 Alkenyl, optionally substituted C5-C 40 Alkynyl, optionally substituted C-C 20 Alkyl-(C=O)-O-optionally substituted C2-C 20 Alkyl, or optionally substituted C-C 20 Alkyl-O-(C=O)-optionally substituted C2-C 20 alkyl.

[0184] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 30 Alkyl, optionally substituted C5-C 30 Alkenyl, optionally substituted C5-C 30 Alkynyl, optionally substituted C-C 15 Alkyl-(C=O)-O-optionally substituted C2-C 15 Alkyl, or optionally substituted C-C 15 Alkyl-O-(C=O)-optionally substituted C2-C 15 alkyl.

[0185] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 25 Alkyl, optionally substituted C5-C 25 Alkenyl, optionally substituted C5-C 25 Alkynyl, optionally substituted C-C 15 Alkyl-(C=O)-O-optionally substituted C2-C 15 Alkyl, or optionally substituted C-C 15Alkyl-O-(C=O)-optionally substituted C2-C 15 alkyl.

[0186] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 20 Alkyl, optionally substituted C5-C 20 Alkenyl, optionally substituted C5-C 20 Alkynyl, optionally substituted C-C 10 Alkyl-(C=O)-O-optionally substituted C2-C 10 Alkyl, or optionally substituted C-C 10 Alkyl-O-(C=O)-optionally substituted C2-C 10 alkyl.

[0187] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 20 alkyl.

[0188] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 25In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 20 alkenyl.

[0189] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is an optionally substituted C5-C 20 alkynyl.

[0190] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is independently selected from -optionally substituted alkyl-(C═O)—O-optionally substituted alkyl. In any of the above embodiments (e.g., a compound of Formula (I′), (I), (Ib), (Ib′), (If), (If′), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 25 Alkyl-(C=O)-O-optionally substituted C2-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 20 Alkyl-(C=O)-O-optionally substituted C2-C 20 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 15 Alkyl-(C=O)-O-optionally substituted C2-C 15 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 10 Alkyl-(C=O)-O-optionally substituted C2-C 10 alkyl.

[0191] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is independently selected from -optionally substituted alkyl-O-(C=O)-optionally substituted alkyl. In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 25 Alkyl-O-(C=O)-optionally substituted C2-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 20 Alkyl-O-(C=O)-optionally substituted C2-C 20 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 15 Alkyl-O-(C=O)-optionally substituted C2-C 15 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 -Optionally substituted C2-C 10 Alkyl-O-(C=O)-optionally substituted C2-C 10 alkyl.

[0192] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), each R 1 is C8H 17, C 10 H 21 , C 12 H 25 , C 14 H 29 , C 16 H 33 , C 18 H 37 , C 18 H 35 , C 18 H 33 and C 18 H 31 are independently selected from

[0193] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C8H 17 is.

[0194] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 10 H 21 is.

[0195] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 12 H 25 is.

[0196] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 14 H 29 is.

[0197] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1is C 16 H 33 is.

[0198] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 18 H 37 is.

[0199] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 18 H 35 is.

[0200] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 18 H 33 is.

[0201] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ib), (Ib'), (If), (If'), or a pharmaceutically acceptable salt thereof), R 1 is C 18 H 31 is.

[0202] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), each R 2 are the same.

[0203] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), each R 2 is different.

[0204] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), each R 2 are independently selected from: [ka]

[0205] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), each R 2 are independently selected from: [ka]

[0206] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 are independently selected from optionally substituted alkyl, optionally substituted alkenyl, optionally substituted .

[0207] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is independently selected from optionally substituted alkyl. In embodiments (e.g., compounds of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 40In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 25 In any of the above embodiments, R 2 is an optionally substituted C5-C 20 alkyl.

[0208] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2is an optionally substituted C5-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 20 alkenyl.

[0209] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is independently selected from optionally substituted alkynyl. In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 20 alkynyl.

[0210] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is independently selected from optionally substituted acyl. In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 50 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 40 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 30 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 25 In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is an optionally substituted C5-C 20 acyl.

[0211] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C8H 17 , C 10 H 21 , C 12 H25 , C 14 H 29 , C 16 H 33 , C 16 H 31 , C 16 H 29 and C 16 H 27 are independently selected from

[0212] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C8H 17 is.

[0213] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 10 H 21 is.

[0214] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 12 H 25 is.

[0215] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 14 H 29 is.

[0216] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 16 H 33 is.

[0217] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 16 H 31 is.

[0218] In any of the above embodiments (e.g., compounds of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R2 is C 16 H 29 is.

[0219] In any of the above embodiments (e.g., a compound of Formula (I'), (I), (Ia), (Ic), (Id), (Ie), or a pharmaceutically acceptable salt thereof), R 2 is C 16 H 27 is.

[0220] In embodiments, the cationic lipids of the present invention include a compound selected from those depicted in Tables AD, or a pharmaceutically acceptable salt thereof.

[0221] In embodiments, a composition is provided comprising the cationic lipid of any one 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 lipid(s) comprise about 30 mol% to 60 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipid(s) comprise 10 mol% to 50 mol% of the lipid nanoparticle. In embodiments, the one or more PEG-modified lipid(s) comprise 1 mol% to 10 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipids comprise 10 mol% to 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 lipid nanoparticle has an encapsulation percentage of at least 70% for the mRNA. In embodiments, the lipid nanoparticle has an encapsulation percentage of at least 75% for the mRNA. In embodiments, the lipid nanoparticles have an encapsulation percentage of at least 80% for mRNA. In embodiments, the lipid nanoparticles have an encapsulation percentage of at least 85% for mRNA. In embodiments, the lipid nanoparticles have an encapsulation percentage of at least 90% for mRNA. In embodiments, the lipid nanoparticles have an encapsulation percentage of at least 95% for mRNA.

[0222] In an embodiment, the composition of any one of the preceding embodiments is for use in therapy.

[0223] In an embodiment, the composition of any one of the preceding embodiments is for use in a method of treating or preventing a disease treatable or preventable 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, lung, brain, or muscle; (b) an autoimmune disease; (c) an infectious disease; or (d) cancer.

[0224] In embodiments, the compositions are administered intravenously, intrathecally or intramuscularly, or by pulmonary delivery, optionally via inhalation administration.

[0225] Illustrative Compounds Illustrative compounds include those set forth in Tables AD, or pharmaceutically acceptable salts thereof.

[0226] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0227] [Table 2-1] [Table 2-2] [Table 2-3] Table 2-4 Table 2-5 Table 2-6

[0228] Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7

[0229] Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5

[0230] Any of the compounds identified in Tables A through D above may be provided in the form of a pharmaceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0231] The compounds of the present invention as described herein can be prepared according to methods known in the art, including the illustrative syntheses of the examples provided herein.

[0232] nucleic acid The compounds of the invention as described herein can be used to prepare compositions useful for the delivery of nucleic acids.

[0233] Nucleic acid synthesis The nucleic acid according to the present invention can be synthesized according to any known method.For example, the mRNA according to the present invention can be synthesized through in vitro transcription (IVT).Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can contain DTT and magnesium ions, and a suitable RNA polymerase (for example, T3, T7, mutant T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.The exact conditions vary depending on the specific application.

[0234] In some embodiments, for the preparation of mRNA according to the present invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, mutant T7, or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and a stop signal.

[0235] The desired mRNA sequence(s) according to the present invention can be determined using standard methods and incorporated into a DNA template. For example, starting from the desired amino acid sequence (e.g., an enzyme sequence), virtual reverse translation is performed based on the degeneracy of the genetic code. An optimization algorithm can then be used to select appropriate codons. Typically, the G / C content can be optimized, on the one hand, to achieve the highest possible G / C content, and, on the other hand, to maximize the frequency of tRNAs according to codon usage. The optimized RNA sequence can be established and displayed, for example, using a suitable display device, and compared with the original (wild-type) sequence. The secondary structure can then be analyzed to calculate the stabilizing and destabilizing properties, or regions of the RNA, respectively.

[0236] modified mRNA In some embodiments, mRNA according to the present invention can be synthesized as unmodified or modified mRNA. Modified mRNA includes nucleotide modifications in RNA. Modified mRNA according to the present invention can therefore include nucleotide modifications, such as backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA is 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 nucleotides such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopropyl-N ... Sopentenyl-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 N-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 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, queosine, beta-D-mannosyl-queosine, wybutoxosine, and modified nucleotide analogs or derivatives of purines and pyrimidines, such 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 from, for example, 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 U.S. Pat. No. 5,700,642, the disclosures of which are incorporated by reference in their entireties.

[0237] Pharmaceutical Formulations of Cationic Lipids and Nucleic Acids In certain embodiments, the compounds of the present invention as described herein, as well as pharmaceutical and liposomal compositions comprising such lipids, can be used in formulations to facilitate the delivery of encapsulated material (e.g., one or more polynucleotides, e.g., mRNA) to one or more target cells and their subsequent transfection. For example, in certain embodiments, the cationic lipids described herein (and compositions, such as liposomal compositions, comprising such lipids) are characterized as providing one or more of receptor-mediated endocytosis, clathrin- and caveolae-mediated endocytosis, phagocytosis and macropinocytosis, fusogenicity, endosomal or lysosomal disruption, and / or releasability properties that provide such compounds with advantages over other similarly classified lipids.

[0238] According to the present invention, a nucleic acid as described herein, e.g., an mRNA encoding a protein (e.g., a full-length, fragment, or portion of a protein), can be delivered via a delivery vehicle comprising a compound of the invention as described herein.

[0239] As used herein, the terms "delivery vehicle," "implantation vehicle," "nanoparticle," or grammatical equivalents thereof, are used interchangeably.

[0240] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a compound described herein and one or more polynucleotides. The compositions (e.g., pharmaceutical compositions) can further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and / or one or more PEG-modified lipids.

[0241] In certain embodiments, the composition exhibits enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, methods for transfecting one or more target cells are also provided herein. These methods generally involve contacting one or more target cells with the cationic lipids and / or pharmaceutical compositions disclosed herein (e.g., liposome formulations containing compounds described herein encapsulating one or more polynucleotides), thereby transfecting the one or more target cells with encapsulated material (e.g., one or more polynucleotides). As used herein, the terms "transfect" or "transfection" refer to the intracellular introduction of one or more encapsulated material (e.g., nucleic acids and / or polynucleotides) into a cell (e.g., 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 encapsulated material (e.g., polynucleotides) taken up by, introduced into, and / or expressed by the target cell undergoing transfection. In fact, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by target cells following transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein demonstrate high transfection efficiency, thereby improving the likelihood that an appropriate dosage of encapsulated material (e.g., one or more polynucleotides) will be delivered to the site of a pathology and subsequently expressed, while simultaneously minimizing potential systemic side effects or toxicity associated with the compound or its encapsulated content.

[0242] For example, following transfection of one or more target cells with a polynucleotide encapsulated in one or more lipid nanoparticles, including the pharmaceutical compositions or liposome compositions disclosed herein, the production of the product (e.g., polypeptide or protein) encoded by such polynucleotide is stimulated, enhancing the ability of such target cells to express the polynucleotide and produce, for example, the polypeptide or protein of interest. For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating mRNA enhances (i.e., increases) the production of the protein or enzyme encoded by such mRNA.

[0243] Furthermore, the delivery vehicles (e.g., liposomal delivery vehicles) described herein can be prepared to preferentially distribute to other target tissues, cells, or organs, such as the heart, lungs, kidneys, and spleen. In embodiments, the lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical compositions and liposomal compositions described herein can be delivered to and / or transfected into targeted cells or tissues. In some embodiments, the encapsulated polynucleotide (e.g., mRNA) can be expressed, and a functional polypeptide product can be produced (and in some instances, excreted) by the target cell, thereby conferring beneficial properties to the target cell or tissue, for example. Such encapsulated polynucleotides (e.g., mRNA) can encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest.

[0244] Liposomal Delivery Vehicles In some embodiments, the composition is a suitable delivery vehicle, hi embodiments, the composition is a liposome delivery vehicle, for example, a lipid nanoparticle.

[0245] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0246] Enriching liposome compositions with one or more of the cationic lipids disclosed herein can be used as a means of improving (e.g., reducing) toxicity or otherwise imparting one or more desirable properties to such enriched liposome compositions (e.g., improving delivery of encapsulated polynucleotides to one or more target cells and / or reducing toxicity of the liposome composition in vivo). Accordingly, also contemplated are pharmaceutical compositions, and particularly liposome compositions, comprising one or more of the cationic lipids disclosed herein.

[0247] Thus, in certain embodiments, the compounds of the present 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 penetrating or fusing with the lipid membranes of such target cells).

[0248] As used herein, liposome delivery vehicles, e.g., lipid nanoparticles, are typically characterized as microscopic vesicles with an internal aqueous space separated from the external medium by one or more bilayer membranes. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). In the context of the present invention, liposome delivery vehicles typically serve to transport desired mRNA to target cells or tissues.

[0249] In certain embodiments, such compositions (e.g., liposomal compositions) carry or otherwise encapsulate material such as, for example, one or more biologically active polynucleotides (e.g., mRNA).

[0250] In embodiments, a composition (e.g., a pharmaceutical composition) comprises an mRNA encoding a protein encapsulated in a liposome. In embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the present invention as described herein. In embodiments, a composition comprises an mRNA encoding a protein (e.g., any protein described herein). In embodiments, a composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In embodiments, a composition comprises an mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0251] In embodiments, a composition (eg, a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, wherein the liposome comprises a compound described herein.

[0252] In some embodiments, the nucleic acid is an mRNA that encodes a peptide or protein. In some embodiments, the mRNA encodes a peptide or protein that is delivered to or used in the treatment of a subject's lung or lung cells (for example, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein). In some embodiments, the mRNA encodes a peptide or protein that is delivered to or used in the treatment of a subject's liver or liver cells (for example, the mRNA encodes the ornithine transcarbamylase (OTC) protein). Other exemplary mRNAs are described herein.

[0253] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net positive charge.

[0254] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net negative charge.

[0255] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) can have a net neutral charge.

[0256] In embodiments, lipid nanoparticles encapsulating nucleic acids (eg, mRNA encoding a peptide or protein) comprise one or more compounds of the invention as described herein.

[0257] For example, the amount of a compound of the present invention as described herein in a composition 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).

[0258] In embodiments of the pharmaceutical compositions described herein, a compound of the invention as described herein is present in an amount that is from about 0.5 wt% to about 30 wt% (e.g., from 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).

[0259] In embodiments, the compound of the present invention as described herein is present in an amount that is 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., liposome composition).In embodiments, the compound of the present invention as described herein is present in an amount that is 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, such as a liposome delivery vehicle.

[0260] In embodiments, the amount of a compound of the present invention as described herein is present in an amount that is 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., liposome composition).

[0261] In embodiments, the amount of a compound of the present invention as described herein is present in an amount that is less than or equal to 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., liposome composition).

[0262] In embodiments, the composition (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.1 wt% to about 20 wt% (e.g., about 0.1 wt% to about 15 wt%) of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of a compound described herein. In embodiments, the percentage results in improved beneficial effects (e.g., improved delivery to targeted tissues such as the liver or lungs).

[0263] The amount of a compound of the invention as described herein in a composition can also be described as a percentage ("mol %) of the combined molar amount of the total lipid of the composition (e.g., the combined molar amount of all lipids present in a liposome delivery vehicle).

[0264] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in an amount that is from about 0.5 mol % to about 50 mol % (e.g., from about 0.5 mol % to about 20 mol %) of the combined molar amount of all lipids present in the composition, such as a liposome delivery vehicle.

[0265] In embodiments, a compound of the invention as described herein is present in an amount that is 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 a composition, such as a liposome delivery vehicle. In embodiments, a compound of the invention as described herein is present in an amount that is from 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 a composition, such as a liposome delivery vehicle.

[0266] In certain embodiments, the compounds of the invention as described herein can comprise from about 0.1 mol% to about 50 mol%, or 0.5 mol% to about 50 mol%, or from about 1 mol% to about 50 mol%, or from about 5 mol% to about 50 mol%, or from about 10 mol% to about 50 mol%, or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol% of the total amount of lipid in a composition (e.g., a liposome delivery vehicle).

[0267] In certain embodiments, the compounds of the present invention as described herein can comprise more than about 0.1 mol%, or more than about 0.5 mol%, or more than about 1 mol%, or more than about 5 mol%, or more than about 10 mol%, or more than about 20 mol%, or more than about 30 mol%, or more than about 40 mol% of the total amount of lipid in the lipid nanoparticles.

[0268] In certain embodiments, the compound as described can comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, or less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipid in the composition (e.g., liposomal delivery vehicle).

[0269] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined molar amount of total lipids in the composition (e.g., liposome composition).

[0270] In embodiments, the amount of a compound of the invention as described herein is present in an amount that is less than or equal to 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., liposome composition).

[0271] In embodiments, the percentage results in improved beneficial effects (eg, improved delivery to targeted tissues such as the liver or lungs).

[0272] In a typical embodiment, a composition of the present invention (e.g., a liposome composition) comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is 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 the 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.

[0273] In further embodiments, pharmaceutical (e.g., liposomal) compositions comprise one or more of a PEG-modified lipid, a non-cationic lipid, and a cholesterol lipid. In other embodiments, such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids. In yet further embodiments, such pharmaceutical (e.g., liposomal) compositions comprise: one or more PEG-modified lipids, and one or more cholesterol lipids.

[0274] In embodiments, a composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprises one or more compounds of the invention as described herein and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids.

[0275] In an embodiment, a composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more compounds of the present invention as described herein; one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids; and further comprises a cholesterol-based lipid. Typically, such a composition has four lipid components, including a compound of the present invention as described herein as the cationic lipid component, a non-cationic lipid (e.g., dope), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K).

[0276] In embodiments, lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding a peptide or protein) comprise one or more compounds of the invention as described herein and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, PEGylated lipids, and cholesterol-based lipids.

[0277] According to various embodiments, the selection of cationic lipid, non-cationic lipid and / or PEG-modified lipid that constitutes lipid nanoparticles, and the relative molar ratio of these lipids to each other are based on the characteristics of selected lipid(s), the nature of intended target cell, and the characteristics of mRNA to be delivered.Additional considerations include, for example, the saturation of alkyl chain, and the size, charge, pH, pKa, membrane fusion property and toxicity of selected lipid(s).Therefore, molar ratio can be adjusted accordingly.

[0278] In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) can be between about 30-60:10 to 50:10 to 50:1 to 10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) can be between about 30-60:20 to 40:10 to 30:1 to 10, respectively.

[0279] cationic lipids In addition to any of the compounds of the present invention as described herein, the compositions can include one or more additional cationic lipids.

[0280] In some embodiments, liposome can contain one or more additional cationic lipids.As used herein, the phrase " cationic lipid " refers to any of a large number of lipid species that have net positive charge at selected pH, such as physiological pH.Some cationic lipids have been described in the literature, and many of them are commercially available.

[0281] Suitable additional cationic lipids for use in the compositions include cationic lipids as described in the literature.

[0282] Helper lipids The composition (e.g., liposome composition) can also contain one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a number of lipid species 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), dioleoylphosphatidylglycerin (DOPG), dipalmitoylphosphatidylglycerin (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidyl Examples of suitable non-cationic or helper lipids include dioleoylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. A suitable non-cationic or helper lipid for practicing the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as the non-cationic or helper lipid.

[0283] In some embodiments, the non-cationic lipid is a neutral lipid, ie, a lipid that carries no net charge in the conditions in which the composition is formulated and / or administered.

[0284] In some embodiments, non-cationic lipids can be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, non-cationic lipids can be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in liposomes 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 some embodiments, the percentage of total non-cationic lipids in liposomes 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 some embodiments, the percentage of total non-cationic lipids in liposomes can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in liposomes can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%.

[0285] In some embodiments, non-cationic lipids can be present in 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 lipids present in the composition. In some embodiments, non-cationic lipids can be present in 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 lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in liposomes 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 some embodiments, the percentage of total non-cationic lipids in liposomes 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 some embodiments, the percentage of total non-cationic lipids in liposomes can be less than about 5 wt%, less than about 10 wt%, less than about 20 wt%, less than about 30 wt%, or less than about 40 wt%. In some embodiments, the percentage of total non-cationic lipids in liposomes can be less than about 5 wt%, less than about 10 wt%, less than about 20 wt%, less than about 30 wt%, or less than about 40 wt%.

[0286] Cholesterol-based lipids In some embodiments, the composition (e.g., liposome composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al., BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), or a lipid having the following structure: [ka] Examples include imidazole cholesterol esters (ICEs) having the formula:

[0287] In some embodiments, the cholesterol-based lipid can be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid 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 some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle can be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0288] In some embodiments, cholesterol-based lipids can be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in liposomes.In some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles 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 some embodiments, the percentage of cholesterol-based lipids in lipid nanoparticles can be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0289] PEGylated lipids In some embodiments, the composition (e.g., liposome composition) comprises 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).

[0290] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, e.g., derivatized ceramides (PEG-ceramides), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (CPEG-2000 ceramide), is also contemplated by the present invention in combination with one or more of the compounds of the present invention as described herein, and in some embodiments, together with other lipids, including liposomes. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., CPEG-2000 ceramides). 14 or C 18 ) is a PEG-ceramide.

[0291] Additional PEG-modified lipids (also referred to herein as PEGylated lipids, which term is interchangeable with PEG-modified lipids) contemplated include, but are not limited to, C6-C 20 These include polyethylene glycol chains up to 5 kDa in length, covalently attached to lipids with long alkyl chain(s). In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of these components can prevent complex aggregation, and can also provide a means for increasing circulation life and delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected to rapidly exchange from the formulation in vivo (see U.S. Patent No. 5,885,613).

[0292] 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 lipid present in a composition (e.g., a liposome composition).

[0293] Pharmaceutical Formulations and Therapeutic Uses The compounds of the present invention as described herein can be used in the manufacture of compositions (e.g., to construct liposomal compositions) that facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) into one or more target cells (e.g., by penetrating or fusing with the lipid membranes of such target cells).

[0294] For example, when a liposome composition (e.g., lipid nanoparticle) comprises or is otherwise enriched with one or more of the 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) into one or more target cells.

[0295] Similarly, in certain embodiments, the compounds of the present invention as described herein can be used to prepare liposome vehicles characterized by their reduced toxicity in vivo. 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.

[0296] Therefore, pharmaceutical formulations containing the compounds described herein and nucleic acids provided by the present invention can be used for various therapeutic purposes.To facilitate in vivo nucleic acid delivery, the compounds described herein and nucleic acids can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands, or stabilizing reagents.In some embodiments, the compounds described herein can be formulated via a premixed lipid solution.In other embodiments, compositions containing the compounds described herein can be formulated using a post-insertion technique into the lipid membrane of nanoparticles.Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0297] Suitable administration routes include, for example, oral, rectal, intravaginal, transmucosal, intratracheal or pulmonary, including inhalation, or intestinal administration; intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In a specific embodiment, intramuscular administration is into a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, administration results in delivery of the nucleic acid to muscle cells. In some embodiments, administration results in delivery of the nucleic acid to hepatocytes (i.e., liver cells).

[0298] A common route for administering the liposome composition of the present invention can be intravenous delivery, especially when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency). Alternatively, depending on the disease or disorder being treated, the liposome composition can be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposome composition of the present invention is typically administered intramuscularly. Diseases or disorders affecting the eye can be treated by administering the liposome composition of the present invention intravitreally.

[0299] Alternatively or additionally, the pharmaceutical preparations of the present invention can be administered in a local rather than systemic manner, for example, by injecting the pharmaceutical preparation directly into the targeted tissue (e.g., in a sustained-release formulation). Local delivery can be affected in various ways depending on the targeted tissue. Illustrative tissues to 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 embodiments, the liver is the targeted tissue. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention can be injected, for example, into the site of injury, disease symptoms, or pain; the composition can be provided in a lozenge for oral, tracheal, or esophageal application; it can be provided in liquid, tablet, or capsule form for gastric or intestinal administration, or in suppository form for rectal or vaginal application; or it can even be delivered to the eye by using a cream, drops, or even injection.

[0300] The compositions described herein can include mRNA that encodes a peptide, including those described herein (e.g., a polypeptide such as a protein).

[0301] In an embodiment, the mRNA encodes a polypeptide.

[0302] In an embodiment, the mRNA encodes a protein.

[0303] Exemplary peptides encoded by mRNA (eg, exemplary proteins encoded by mRNA) are described herein.

[0304] The present invention provides methods for delivering compositions having full-length mRNA molecules encoding peptides or proteins of interest for use in treating a subject, e.g., a human subject or cells of a human subject, or cells that are treated and delivered to a human subject.

[0305] Delivery method The delivery route used in the method of the present invention allows for non-invasive self-administration of the compound of the present invention.In some embodiments, the method involves intratracheal or pulmonary administration by aerosolization, inhalation, or instillation of a 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 with liposomes.In some embodiments, the liposomes comprise lipids that are the compound of the present invention.As used herein below, administration of a compound of the present invention includes administration of a composition comprising a compound of the present invention.

[0306] While local lung cells and tissues represent potential targets that can function as biological depots or reservoirs for the production and secretion of proteins encoded by mRNA, Applicants have discovered that administering compounds of the present invention to the lungs via aerosolization, inhalation, or instillation further distributes non-secreted proteins outside of lung cells. Without wishing to be bound by any particular theory, it is contemplated that the nanoparticle compositions of the present invention pass through the pulmonary airway-blood barrier, resulting in translation of the intact nanoparticles in non-pulmonary cells and tissues, such as the heart, liver, and spleen, where it leads to the production of the encoded protein in these non-pulmonary tissues. Thus, the utility of the compounds and methods of the present invention extends beyond the production of therapeutic proteins in lung cells and tissues and can be used to deliver them to non-pulmonary target cells and / or tissues. They are useful in the management and treatment of numerous diseases, particularly peripheral diseases resulting from both secreted and non-secreted protein and / or enzyme deficiencies (e.g., one or more lysosomal storage disorders). In certain embodiments, the compounds of the present invention used in the methods of the present invention result in the distribution of mRNA-encapsulated nanoparticles and the production of the encoded protein in the liver, spleen, heart, and / or other non-pulmonary cells. For example, administration of the compounds of the present invention by aerosolization, inhalation, or instillation into the lungs results in the composition itself, and its protein product (e.g., functional beta-galactosidase protein), being detectable in both local lung cells and tissues, as well as in peripheral target cells, tissues, and organs as a result of the transfer of the mRNA and delivery vehicle to non-pulmonary cells.

[0307] In certain embodiments, the compounds of the present invention can be used in the methods of the present invention to specifically target peripheral cells or tissues. Following pulmonary delivery, it is intended that the compounds of the present invention cross the pulmonary airway-blood barrier and distribute to cells other than local lung cells. Thus, the compounds disclosed herein can be administered to a subject via the pulmonary route of administration using various methods known by those skilled in the art (e.g., by inhalation) and distributed to both local target cells and tissues in the lung and peripheral non-lung cells and tissues (e.g., cells of the liver, spleen, kidney, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both local lung cells and peripheral non-lung cells can serve as biological reservoirs or depots that can produce and / or secrete translation products encoded by one or more polynucleotides. Thus, the present invention is not limited to the treatment of pulmonary diseases or conditions, but can rather be used as a non-invasive means to facilitate the delivery of polynucleotides, or the production of enzymes and proteins encoded thereby, in peripheral organs, tissues, and cells (e.g., liver parenchymal cells) that would otherwise only be achieved by systemic administration. Illustrative peripheral non-pulmonary cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, nervous system cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac muscle cells, 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.

[0308] Following administration of the composition to a subject, the protein product (e.g., a functional protein or enzyme) encoded by the mRNA is detectable in peripheral target tissues for at least about 1 to 7 days or longer following administration of the compound to the subject. The amount of protein product necessary to achieve a therapeutic effect will vary depending on the condition being treated, the encoded protein, and the condition of the patient. For example, the protein product may be present in a concentration 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 1.0 μg / ml, at least 1.1 μg / ml, at least 1.2 μg / ml). The compound may be detectable in peripheral target tissues at a concentration (e.g., a therapeutic concentration) of at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days or more following administration of the compound to a subject.

[0309] It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and by inhalation of an aerosol mist generated by the use of a liquid nebulizer or a dry powder device such as that described in U.S. Pat. No. 5,780,014, incorporated herein by reference.

[0310] In certain embodiments, the compounds of the present invention can be formulated so that they are aerosolized or otherwise delivered as particulate liquids or solids before or upon administration to a subject. Such compounds can be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (such as aerosolized aqueous solutions or suspensions) to generate particles that can be easily respirable or inhaled by a subject. In some embodiments, such devices (e.g., metered-dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers, or insufflators) facilitate the administration of a predetermined mass, volume, or dose of the composition to a subject (e.g., about 0.5 mg / kg of mRNA per dose). For example, in certain embodiments, the compounds of the present invention are administered to a subject using a metered-dose inhaler containing a suspension or solution comprising the compound and a suitable propellant. In certain embodiments, the compounds of the present invention can be formulated as particulate powders intended for inhalation (e.g., respirable dry particles). In certain embodiments, compositions of the invention formulated as respirable particles are appropriately sized (e.g., 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 smaller) so that they can be respirable by a subject or delivered using a suitable device. In yet other embodiments, compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).In some embodiments, the compounds of the invention are administered at a concentration of at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least The subject is administered a single dose such that a concentration of 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight is administered. In some embodiments, a compound of the invention is administered to a subject such that a total amount of mRNA of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg is administered in one or more doses.

[0311] Synthesis of Compounds of the Invention Cationic lipid MC3 is currently the gold standard for the in vivo delivery of, for example, siRNA (see WO2010 / 144740).However, the synthesis of this lipid involves a six-step process and requires the handling of Grignard reagent.In contrast, the present invention provides cationic lipids that can be produced from readily available starting reagents.The compounds of the present invention as described herein can be produced according to methods known in the art, including the illustrative synthesis of the examples provided herein. [Example]

[0312] While certain compounds, compositions and methods of the present invention have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the compounds of the present invention and are not intended to limit it.

[0313] [Examples 1 to 11] Synthesis of HEP-based cationic lipids The HEP-based cationic lipids described herein can be prepared according to Scheme 1:

[0314] Scheme 1 - Synthesis of HEP-based cationic lipids HEP-E3-E10 [4] [ka]

[0315] Synthesis of [3] As presented in Scheme 1: To a solution containing HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E3-E10[2] (0.668 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (70% yield).

[0316] Synthesis of HEP-E3-E10[4] As presented in Scheme 1: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar, [3] (0.500 g, 0.344 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.76 mL, 67.86 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (60% yield). 1H NMR (400 MHz, CDCl3) 4.16 (m, 4H), 3.60 (m, 4H), 2.97 (m, 3H), 2.78 (d, 3H), 2.58 (m, 9H), 2.37 (m, 12H), 2.15 (m, 2H), 1.78 (m, 4H), 1.44 (m, 7H), 1.36 (m, 9H), 1.26 (br, 45H), 1.05 (d, 6H), 0.87 (t, 12H). Expected M / Z = 998.59, Observed = 998.0.

[0317] The HEP-based cationic lipids described herein can be prepared according to Scheme 2:

[0318] Scheme 2 - Synthesis of HEP-based cationic lipid HEP-E3-E18:2[7] [ka]

[0319] Synthesis of [6] As presented in Scheme 2: To a solution containing HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E3-E18:2[5] (0.893 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (45% yield).

[0320] Synthesis of HEP-E3-E18:2[7] As presented in Scheme 2: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar, [6] (0.418 g, 0.222 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.14 mL, 43.713 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (47% yield). 1H NMR (400 MHz, CDCl3) 5.35 (m, 16H), 4.16 (br, 4H), 3.62 (br, 4H), 2.96 (m, 2H), 2.77 (t, 12H), 2.55 (m, 9H), 2.37 (m, 14H), 2.15 (m, 2H), 2.04 (m, 16H), 1.79 (br, 4H), 1.44 (m, 6H), 1.30 (br, 64H), 1.05 (d, 6H), 0.89 (t, 12H). Expected M / Z = 1430.40, Observed = 1430.0.

[0321] The HEP-based cationic lipids described herein can be prepared according to Scheme 3:

[0322] Scheme 3 - Synthesis of HEP-based cationic lipids HEP-E3-E14

[10] [ka]

[0323] Synthesis of [9] As presented in Scheme 3: To a solution containing HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E3-E14[8] (0.785 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (60.3% yield).

[0324] Synthesis of HEP-E3-E14

[10] As presented in Scheme 3: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar, [9] (0.500 g, 0.297 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.53 mL, 58.766 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (55% yield). 1H NMR (400 MHz, CDCl3) 4.17 (m, 4H), 3.62 (m, 4H), 2.97 (m, 3H), 2.76 (d, 2H), 2.55 (m, 8H), 2.37 (m, 14H), 2.15 (m, 2H), 1.79 (m, 4H), 1.45 (m, 6H), 1.37 (m, 6H), 1.25 (br, 80H), 1.04 (d, 6H), 0.89 (t, 12H), Expected M / Z = 1222.02, Observed = 1222.0.

[0325] The HEP-based cationic lipids described herein can be prepared according to Scheme 4:

[0326] Scheme 4 - Synthesis of HEP-based cationic lipids HEP-E4-E10

[13] [ka]

[0327] Synthesis of

[12] As presented in Scheme 4: To a solution of HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E4-E10

[11] (0.683 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (63.3% yield).

[0328] Synthesis of HEP-E4-E10

[13] As presented in Scheme 4: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[12] (0.450 g, 0.303 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.55 mL, 59.920 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3%) as the mobile phase to give a colorless oil (48.4% yield). 1H NMR (400 MHz, CDCl3) 4.16 (t, 4H), 3.62 (br, 4H), 2.96 (q, 3H), 2.76 (d, 4H), 2.56 (m, 8H), 2.40 (m, 4H), 2.32 (t, 4H), 2.13 (t, 2H), 1.61 (m, 4H), 1.46 (m, 8H), 1.37 (m, 8H), 1.28 (br, 44H), 1.03 (d, 6H), 0.87 (t, 12H), 13C NMR (400 MHz, CDCl3) 173.65 (2C), 69.65 (2C), 68.04 (2C), 62.84 (2C), 61.82 (2C), 61.44 (2C), 60.89 (2C), 55.57 (4C), 51.55 (2C), 35.35 (4C), 34.20 (2C), 32.09 (7C), 30.00 (5C), 29.77 (6C), 29.47 (6C), 26.93 (2C), 25.84 (5C), 22.84 (9C), 17.77 (2C), 14.30 (7C). Expected M / Z = 1025.64, Observed = 1025.8.

[0329] The HEP-based cationic lipids described herein can be prepared according to Scheme 5:

[0330] Scheme 5 - Synthesis of HEP-based cationic lipids HEP-E4-E12

[16] [ka]

[0331] Synthesis of

[15] As presented in Scheme 5: To a solution of HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E4-E12

[14] (0.742 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (66% yield).

[0332] Synthesis of HEP-E4-E12

[16] As presented in Scheme 5: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[15] (0.520 g, 0.326 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.67 mL, 64.376 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (48.4% yield). 1H NMR (400 MHz, CDCl3) 4.17 (m, 4H), 3.63 (m, 4H), 2.95 (m, 3H), 2.76 (d, 4H), 2.56 (m, 8H), 2.39 (m, 9H), 2.32 (t, 4H), 2.13 (t, 2H), 1.61 (m, 4H), 1.46 (m, 8H), 1.37 (m, 12H), 1.25 (br, 61H), 1.04 (d, 6H), 0.87 (t, 12H), 13C NMR (400 MHz, CDCl3) 173.56 (2C), 69.65 (2C), 68.04 (2C), 62.84 (2C), 61.82 (4C), 60.89 (2C), 55.57 (4C), 53.61 (1C), 51.56 (2C), 35.36 (4C), 33.65 (2C), 32.12 (6C), 29.63 (31C), 25.85 (5C), 22.77 (9C), 14.31 (7C), Expected M / Z = 1137.86, Observed = 1138.0.

[0333] The HEP-based cationic lipids described herein can be prepared according to Scheme 6:

[0334] Scheme 6 - Synthesis of HEP-based cationic lipids HEP-E4-E14

[19] [ka]

[0335] Synthesis of

[18] As presented in Scheme 6: To a solution of HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv.), E4-E14

[17] (0.799 g, 1.038 mmol, 2.1 equiv.), 1 mL of dimethylformamide, 3 mL of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv.), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 h). The reaction mixture was then concentrated using a rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (70.1% yield).

[0336] Synthesis of HEP-E4-E14

[19] As presented in Scheme 6: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[18] (0.591 g, 0.346 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.77 mL, 68.322 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (50% yield). 1H NMR (400 MHz, CDCl3) 4.16 (m, 4H), 3.63 (m, 4H), 2.95 (m, 3H), 2.75 (d, 4H), 2.57 (m, 8H), 2.41 (m, 8H), 2.32 (t, 4H), 2.13 (t, 2H), 1.59 (m, 4H), 1.46 (m, 9H), 1.38 (m, 9H), 1.25 (br, 82H), 1.03 (d, 6H), 0.87 (t, 12H), 13C NMR (400 MHz, CDCl3) 171.77 (2C), 69.66 (3C), 67.25 (2C), 63.12 (3C), 61.42 (2C), 60.90 (2C), 55.90 (2C), 55.57 (2C), 55.14 (1C), 53.61 (1C), 51.15 (2C), 35.36 (5C), 34.21 (8C), 29.89 (48C), 26.93 (2C), 25.85 (5C), 23.28 (10C), 17.77 (3C), 14.31 (8C), Expected M / Z = 1250.07, Observed = 1250.01.

[0337] The HEP-based cationic lipids described herein can be prepared according to Scheme 7:

[0338] Scheme 7 - Synthesis of HEP-based cationic lipids HEP-E2-E10

[22] [ka]

[0339] Synthesis of

[21] As presented in Scheme 7: To a solution of HEP[1] (0.12 g, 0.59 mmol, 1.0 equiv), E2-E10

[20] (0.822 g, 1.31 mmol, 2.2 equiv), HOBT (0.240 g, 1.78 mmol, 3.0 equiv), DMAP (0.022 g, 0.178 mmol, 0.3 equiv), DIPEA (1.03 ml, 5.93 mmol, 10.0 equiv), and 12 ml of dimethylformamide, HBTU (0.675 g, 1.78 mmol, 3.0 equiv) was added and allowed to stir at 65 °C for 1 h and then at room temperature overnight. The reaction mixture was then diluted with ethyl acetate, extracted with saturated sodium chloride (3x), dried over sodium sulfate, filtered, and rotary evaporated to give an amber oil. The amber oil was purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (53.5% yield).

[0340] Synthesis of HEP-E2-E10

[22] As presented in Scheme 7: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[21] (0.450 g, 0.315 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (1.62 mL, 62.25 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (12.0% yield). Expected M / Z = 969.53, observed = 969.8.

[0341] The HEP-based cationic lipids described herein can be prepared according to Scheme 8:

[0342] Scheme 8 - Synthesis of HEP-based cationic lipids HEP-E2-E14

[25] [ka]

[0343] Synthesis of

[24] As presented in Scheme 8: To a solution of HEP[1] (0.150 g, 0.74 mmol, 1.0 equiv), E2-E14

[23] (0.840 g, 1.63 mmol, 2.2 equiv), HOBT (0.300 g, 2.22 mmol, 3.0 equiv), DMAP (0.027 g, 0.222 mmol, 0.3 equiv), DIPEA (1.30 ml, 7.40 mmol, 10.0 equiv), and 10 ml of dimethylformamide, HBTU (0.840 g, 2.22 mmol, 3.0 equiv) was added and allowed to stir at 65 °C for 1 h and then at room temperature overnight. The reaction mixture was then diluted with ethyl acetate, extracted with saturated sodium chloride (3x), dried over sodium sulfate, filtered, and rotary evaporated to give an amber oil. The amber oil was purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (18.0% yield).

[0344] Synthesis of HEP-E2-E14

[25] As presented in Scheme 8: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[24] (0.150 g, 0.091 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (0.465 mL, 17.93 mmol, 197.3 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (10.0% yield). Expected M / Z = 1193.96, observed = 1193.0.

[0345] The HEP-based cationic lipids described herein can be prepared according to Scheme 9:

[0346] Scheme 9 - Synthesis of HEP-based cationic lipids HEP-E3-E12

[28] [ka]

[0347] Synthesis of

[27] As presented in Scheme 9: To a solution containing HEP[1] (0.200 g, 0.988 mmol, 1.0 equiv.), E3-E12

[26] (1.6 g, 2.27 mmol, 2.3 equiv.), 20 mL of dichloroethane, diisopropylethylamine (0.860 mL, 4.94 mmol, 5.0 equiv.), and N,N-dimethylaminopyridine (0.036 g, 0.296 mmol, 0.3 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.568 g, 2.96 mmol, 3.0 equiv.) was added and allowed to react at room temperature overnight (18 hours). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a pale yellow oil (0.95 g, 61% yield). Calculation C 90 H 189 N4O8Si4 [M+H] = 1565.35, observed [M+H] = 1566.10.

[0348] Synthesis of HEP-E3-E12

[28] As presented in Scheme 9: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[27] (0.950 g, 0.607 mmol, 1.0 equiv.) was added along with 8 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (3.4 mL, 121.4 mmol, 200 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (381 mg, 56.6% yield). 1 H NMR (400 MHz, CDCl3): 4.14-4.20 (m, 4H), 3.63-3.66 (m, 4H), 2.95-3.00 (m, 3H), 2.77-2.80 (dd, 2H), 2.33-2.65 (m, 20H), 2.14-2.20 (m, 2H), 1.78-1.83 (m, 4H), 1.25-1.46 (m, 76H), 1.04-1.05 (d, 6H), 0.86-0.89 (m, 12H), Calculated C 66 H 133 N4O 8, M / Z = 1109.0, Observed = 1109.8.

[0349] The HEP-based cationic lipids described herein can be prepared according to Scheme 10:

[0350] Scheme 10 - Synthesis of HEP-based cationic lipids HEP-E3-E6+6

[31] [ka]

[0351] Synthesis of

[30] As presented in Scheme 10: To a solution containing HEP[1] (0.100 g, 0.49 mmol, 1.0 equiv.), AIM-E3-E6+6

[29] (0.86 g, 1.09 mmol, 2.2 equiv.), 10 mL of dichloroethane, diisopropylethylamine (0.86 mL, 4.94 mmol, 10.0 equiv.), and N,N-dimethylaminopyridine (0.06 g, 0.49 mmol, 1.0 equiv.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.237 g, 1.23 mmol, 2.5 equiv.) was added and allowed to react at room temperature overnight (18 h). The reaction mixture was then concentrated using rotavapor and purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a pale yellow oil (0.43 g, 50% yield). Calculated C94H189N4O16Si4 [M+H] = 1743.90, observed = 1743.10.

[0352] Synthesis of HEP-E3-E6+6

[31] As presented in Scheme 10: To a 20 mL polypropylene scintillation vial equipped with a PTFE stir bar,

[30] (0.430 g, 0.247 mmol, 1.0 equiv.) was added along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C on an ice bath, and HF / pyridine (2.4 mL, 49.36 mmol, 100 equiv.) was added dropwise. After the addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (230 mg, 72.5% yield). 1 H NMR (400 MHz, CDCl3): 4.15-4.18 (m, 4H), 4.04-4.07 (t, 8H), 3.62-3.66 (m, 4H), 2.94-3.04 (m, 2H), 2.76-2.80 (m, 2H), 2.32-2.64 (m, 20H), 2.26-2.30 (t, 8H), 2.12-2.20 (m, 2H), 1.17-1.83 (m, 4H), 1.53-1.66 (m, 21H), 1.39-1.43 (m, 12H), 1.26-1.32 (m, 26H), 1.03-1.06 (d, 6H), 0.86-0.89 (m, 12H). Expected C 70 H 132 N4O 16 M / Z = 1285.8, Observed = 1285.9.

[0353] Scheme 11 - Synthesis of AIM-E3-E6+6 intermediate

[29] [ka]

[0354] Synthesis of hex-5-en-1-ylheptanoate (34) [ka]

[0355] As presented in Scheme 11: To a solution of hex-5-en-1-ol (32) (20 g, 199.6 mmol) and heptanoic acid (33) (33.9 mL, 239.6 mmol) in 400 mL of dichloromethane, DMAP (4.9 g, 39.9 mmol), DIPEA (104.3 mL, 599.0 mmol), and EDC (57.4 g, 299.5 mmol) were added. The resulting mixture was stirred at room temperature overnight. MS and TLC (Rf: 0.6, 10% EtOAc / hexane) analysis indicated the reaction was complete. The reaction mixture was then diluted with DCM and washed with saturated NaHCO3 solution, water, and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified (0-1% ethyl acetate in hexanes) to give hex-5-en-1-ylheptanoate (34) (34.3 g, 81%). result: ESI-MS analysis: Calculated C13H25O2, [M+H]=213.19, Observed=213.3

[0356] Synthesis of 4-(oxiran-2-yl)butylheptanoate (35) [ka] As presented in Scheme 11: To a solution of hex-5-en-1-ylheptanoate (34) (5.0 g, 23.5 mmol) in 50 mL of dichloromethane was added 3-chloroperbenzoic acid (6.09 g, 35.3 mmol) at 0 °C. The resulting mixture was warmed to room temperature and stirred overnight. MS and TLC (Rf: 0.3, 10% EtOAc / hexane) analysis indicated the reaction was complete. The reaction mixture was then diluted with dichloromethane and washed with 10% sodium hydroxide solution and water. The organic layer was separated, dried over anhydrous sodium sulfate, and concentrated. The crude residue was purified (SiO: 4-5% ethyl acetate in hexane gradient) to give 4-(oxiran-2-yl)butylheptanoate (35) (4.5 g, 84%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C13H25O3, [M+H]=229.18, Observed=229.2

[0357] Synthesis of 4-(bis(6-(heptanoyloxy)-2-hydroxyhexyl)amino)butanoic acid (37) [ka] As presented in Scheme 11: A solution of 4-aminobutanoic acid (36) (1.15 g, 11.15 mmol), 4-(oxiran-2-yl)butylheptanoate (35) (5.09 g, 22.3 mmol), and diisopropylethylamine (4.85 mL, 27.88 mmol) in 75 mL of methanol was heated at 75 °C for 4 h. After concentration to dryness, the oily residue was purified by column chromatography (SiO: 7-8% methanol in dichloromethane gradient) to give 4-(bis(6-(heptanoyloxy)-2-hydroxyhexyl)amino)butanoic acid (37) (3.4 g, 54%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C30H58NO8, [M+H]=560.42, Observed=560.3

[0358] Synthesis of AIM-E3-E6+6(29) [ka] As presented in Scheme 11: To a solution of 4-(bis(6-(heptanoyloxy)-2-hydroxyhexyl)amino)butanoic acid (37) (2.0 g, 3.57 mmol) in 40 mL of dichloromethane was added 2,6-lutidine (1.25 mL, 10.71 mmol) and tert-butyldimethylsilyl trifluoromethanesulfonate (1.97 mL, 8.57 mmol) at 0 °C. The resulting mixture was warmed to room temperature and stirred for 1 h. MS analysis indicated the completion of the reaction and the formation of di- and tri-TBS products. The reaction mixture was diluted with DCM and washed with saturated NaHCO3 solution, water, and brine solution. The organic layer was dried over anhydrous Na2SO4. 。 After concentration to dryness, the oily residue was dissolved in DMF / HO (10 mL / 1 mL), and the resulting solution was heated at 50 °C for 6 h. MS analysis showed the reaction was complete. The reaction mixture was then diluted with DCM and washed with saturated NH4Cl solution, water, and brine solution. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was purified by column chromatography (SiO2: 36-40% ethyl acetate in hexane gradient) to give AIM-E3-E6+6 (29, 1.54 g, 55% over two steps), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C42H86NO8Si2, [M+H]=788.59, Observed=788.3

[0359] [Examples 12 to 13] Synthesis of TEP thioester-based cationic lipids The TEP-based cationic lipids described herein can be prepared according to Scheme 12:

[0360] Scheme 12-Synthesis of TEP-TE-3-E10

[13] : [ka]

[0361] TEP-TE-3-E10-TBS

[12] : As shown in Scheme 12: To a solution of [3] (0.125 g, 0.534 mmol) in DCM (2 mL),

[11] (0.722 g, 1.121 mmol), EDC (0.307 g, 1.602 mmol), DMAP (0.026 g, 0.213 mmol), and DIPEA (0.37 mL, 2.135 mmol) were added and stirred at room temperature for 16 h. The reaction was monitored for completion by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound

[12] as a pale yellow oil (0.44 g, 55%), which was confirmed by MS analysis. result: ESI-MS analysis: calculated for C82H173N4O6S2Si4, [M+H] = 1486.1, observed = 1486.0

[0362] TEP-TE-3-E10

[13] : As presented in Scheme 12: To a solution of

[12] (0.435 g, 0.293 mmol) in THF (4 mL), HF.Py (70% HF) (1.67 mL, 58.55 mmol) was slowly added at 0 °C and stirred for 5 minutes. The reaction mixture was then brought to room temperature and stirred for 6 hours. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the resulting residue was dissolved in ethyl acetate and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 3% MeOH in DCM) to give pure compound

[13] as a colorless oil (0.16 g, 53%), which was confirmed by NMR and MS analysis. result: 1H NMR (500 MHz, CDCl3) δ 3.70 - 3.58 (m, 4H), 3.07 - 2.73 (m, 10H), 2.66 - 2.36 (m, 19H), 2.20 - 2.11 (m, 1H), 1.87 - 1.77 (m, 4H), 1.50 - 1.34 (m, 12H), 1.34 - 1.21 (m, 48H), 1.07 (d, 6H), 0.87 (t, 12H). 13C NMR (100 MHz, CDCl3) δ 199.5, 199.3, 69.7, 68.0, 62.7, 61.2, 59.9, 55.2, 55.0, 54.3, 52.5, 41.7, 41.7, 35.3, 35.2, 32.1, 30.0, 29.8, 29.5, 25.9, 25.8, 25.3, 23.2, 23.0, 22.9, 17.6, 14.3. ESI-MS analysis: Calculated for C58H117N4O6S2, [M+H] = 1029.8; Observed = 1029.7

[0363] The TEP-based cationic lipids described herein can be prepared according to Scheme 13:

[0364] Scheme 13 Synthesis of TEP-TE-4-E10

[16] : [ka]

[0365] TEP-TE-4-E10-TBS

[15] : As shown in Scheme 13: To a solution of [3] (0.125 g, 0.534 mmol) in DCM (2 mL),

[14] (0.737 g, 1.121 mmol), EDC (0.307 g, 1.602 mmol), DMAP (0.026 g, 0.213 mmol), and DIPEA (0.37 mL, 2.135 mmol) were added and stirred at room temperature for 16 h. The reaction was monitored for completion by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound

[15] as a pale yellow oil (0.495 g, 61%), which was confirmed by MS analysis. result: ESI-MS analysis: calculated for C84H177N4O6S2Si4, [M+H] = 1514.2, observed = 1514.0

[0366] TEP-TE-4-E10

[16] : As presented in Scheme 13: To a solution of

[12] (0.49 g, 0.323 mmol) in THF (4 mL), HF.Py (70% HF) (1.85 mL, 64.73 mmol) was slowly added at 0 °C and stirred for 5 minutes. The reaction mixture was then brought to room temperature and stirred for 6 hours. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the resulting residue was dissolved in ethyl acetate and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified by silica gel chromatography (eluent: 3% MeOH in DCM) to give pure compound

[16] as a colorless oil (0.166 g, 49%), which was confirmed by NMR and MS analysis. result: 1H NMR (500 MHz, CDCl3) δ 3.70 - 3.58 (m, 4H), 3.07 - 2.73 (m, 10H), 2.64 - 2.35 (m, 19H), 2.20 - 2.12 (m, 1H), 1.75 - 1.58 (m, 4H), 1.57 - 1.35 (m, 16H), 1.34 - 1.17 (m, 48H), 1.07 (d, 6H), 0.87 (t, 12H). 13C NMR (100 MHz, CDCl3) δ 199.4, 199.3, 69.6, 68.0, 62.8, 61.4, 59.9, 55.8, 55.1, 55.0, 53.6, 52.6, 43.9, 43.8, 35.3, 35.2, 32.1, 30.0, 29.9, 29.8, 29.5, 26.5, 25.9, 25.8, 25.2, 23.49, 23.4, 22.9, 17.6, 14.3. ESI-MS analysis: Calculated for C60H121N4O6S2, [M+H] = 1057.8; Observed = 1057.8

[0367] [Examples 14 to 15] Synthesis of TEP disulfide-based cationic lipids The TEP-based cationic lipids described herein can be prepared according to Scheme 14:

[0368] Scheme 14-Synthesis of TEP-SS-3-E10[7]: [ka]

[0369] 2,2'-(2,5-dimethylpiperazine-1,4-diyl)bis(ethane-1-thiol) [3]: As shown in Scheme 14: To a solution of 2,5-dimethylpiperazine [1] (3.0 g, 26.27 mmol) in DCM (60 mL), ethylene sulfide [2] (6.25 mL, 105.08 mmol) was slowly added in small portions. The resulting mixture was stirred at room temperature for 6 hours. The completion of the reaction was monitored by MS. The reaction mixture was concentrated to give compound [3] as a pale yellow solid (6.1 g, 99%), which was confirmed by MS analysis. result: ESI-MS analysis: calculated for C10H23N2S2, [M+H] = 235.1; observed = 235.2

[0370] 1,1'-((3-(pyridin-2-yldisulfanyl)propyl)azanediyl)bis(decan-2-ol) [6]: As shown in Scheme 14: To a solution of [5] (0.027 g, 0.12 mmol) in MeOH (1.5 mL) was added [4] (0.050 g, 0.12 mmol) in MeOH (1.5 mL). The resulting mixture was stirred at room temperature for 18 hours. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the crude compound was purified by silica gel chromatography (eluent: 40% EtOAc in DCM) to give pure compound [6] as a pale yellow oil (0.030 g, 48%), which was confirmed by MS analysis. result: ESI-MS analysis: calculated for C28H53N2O2S2, [M+H] = 513.3; observed = 513.3

[0371] TEP-SS-3-E10[7]: As shown in Scheme 14: To a solution of [3] (0.030 g, 0.128 mmol) in DCM (2 mL), triethylamine (53 mL, 0.384 mmol) was added and stirred at room temperature for 3 minutes. To this was added [6] (0.144 g, 0.281 mmol) in DCM (3 mL) and stirred at room temperature for 18 hours. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the crude compound was purified by silica gel chromatography (eluent: 5% to 10% MeOH in DCM) to give pure compound [6] as a pale yellow oil (0.013 g, 59%), which was confirmed by 1 H NMR and MS analysis. 1H NMR (400 MHz, Chloroform-d) δ 3.87 - 3.68 (m, 4H), 3.20 - 2.38 (m, 25H), 2.31 - 1.86 (m, 5H), 1.66 - 1.37 (m, 12H), 1.37 - 1.17 (m, 48H), 1.13 (d, 6H), 0.87 (t, 12H). ESI-MS analysis: Calculated C56H117N4O4S4, [M+H] = 1037.8, Observed = 1037.7

[0372] The TEP-based cationic lipids described herein can be prepared according to Scheme 15:

[0373] Scheme 1: Synthesis of 5-TEP-SS-4-E14

[10] : [ka]

[0374] 1,1'-((4-(pyridin-2-yldisulfanyl)butyl)azanediyl)bis(tetradecan-2-ol) [9]: As shown in Scheme 15: To a solution of [5] (0.38 g, 1.75 mmol) in MeOH (15 mL) was added [8] (0.93 g, 1.75 mmol) in MeOH (15 mL). The resulting mixture was stirred at room temperature for 16 h. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the crude compound was purified by silica gel chromatography (eluent: 30%-40% EtOAc in DCM) to give pure compound [9] as a pale yellow oil (0.59 g, 53%), which was confirmed by MS analysis. result: ESI-MS analysis: calculated for C37H71N2O2S2, [M+H] = 639.5; observed = 639.5

[0375] TEP-SS-4-E14

[10] : As shown in Scheme 15: To a solution of [3] (0.1 g, 0.43 mmol) in DCM (6 mL), triethylamine (178 mL, 1.28 mmol) was added and stirred at room temperature for 3 min. To this, [9] (0.6 g, 0.94 mmol) in DCM (9 mL) was added and stirred at room temperature for 18 h. The completion of the reaction was monitored by TLC and MS. The reaction mixture was concentrated, and the crude compound was purified by silica gel chromatography (eluent: 4-5% MeOH in DCM) to give pure compound

[10] as a colorless oil (0.087 g, 16%), which was confirmed by 1 H NMR and MS analysis. 1H NMR (400 MHz, Chloroform-d) δ 3.77 - 3.61 (m, 4H), 3.12 - 2.99 (m, 3H), 2.88 - 2.40 (m, 29H), 2.21 - 2.08 (m, 2H), 1.80 - 1.66 (m, 4H), 1.50 - 1.36 (m, 12H), 1.36 - 1.18 (m, 80H), 1.09 (d, 6H), 0.88 (t, 12H). ESI-MS analysis: Calculated for C74H153N4O4S4, [M+H] = 1290.0; Observed = 1289.9

[0376] [Examples 16 to 23] Synthesis of further HEP-based cationic lipids Intermediates for the HEP-based cationic lipids described herein can be prepared according to Scheme 16:

[0377] Scheme 16 - Synthesis of acid intermediate [1]: [ka]

[0378] As presented in Scheme 16: To a solution of 5-aminovaleric acid (0.5 g, 4.27 mmol) dissolved in i-PrOH (5 mL) and EtN (1.2 mL) was added n-octyl acrylate (2.7 mL, 12.8 mmol). The reaction was heated to 90 °C for 3 hours. After completion of the reaction, the crude mixture was evaporated under reduced pressure. Finally, the crude material was purified using silica gel column chromatography (0-12% MeOH in CHCl) to give pure compound [1] as a colorless oil (0.56 g, 27%). Expected [M+H] = 486.4, observed = 486.4

[0379] The HEP-based cationic lipids described herein can be prepared according to Scheme 17:

[0380] Scheme 17 - Synthesis of HEP-E4-O8[3]: [ka]

[0381] As shown in Scheme 17: To a solution of [2] (0.100 g, 0.494 mmol) in DCE (2 mL), [1] (0.530 g, 1.09 mmol), EDC (0.284 g, 1.48 mmol), DMAP (0.12 g, 0.99 mmol), and DIPEA (0.86 mL, 4.94 mmol) were added in DCE (8 mL) and stirred at room temperature for 16 hours. The completion of the reaction was monitored by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound [3] as a pale yellow oil (0.15 g, 27%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C64H121N4O12, [M+H]=1137.90, Observed=1137.85

[0382] Intermediates for the HEP-based cationic lipids described herein can be prepared according to Scheme 18:

[0383] Scheme 18 - Synthesis of acid intermediate [4]: [ka]

[0384] As presented in Scheme 18: To a solution of 5-aminovaleric acid (0.5 g, 4.27 mmol) dissolved in i-PrOH (5 mL) and EtN (1.2 mL) was added isodecyl acrylate (3.12 mL, 12.8 mmol). The reaction was heated to 90 °C for 3 h. After completion of the reaction, the crude mixture was evaporated under reduced pressure. Finally, the crude material was purified using silica gel column chromatography (0-12% MeOH in CHCl) to give pure compound [4] as a colorless oil (0.420 g, 18%). Expected [M+H] = 542.4, observed = 542.4

[0385] The HEP-based cationic lipids described herein can be prepared according to Scheme 19:

[0386] Scheme 19-Synthesis of HEP-E4-Oi10[5]: [ka]

[0387] As shown in Scheme 19: To a solution of [2] (0.065 g, 0.321 mmol) in DCE (2 mL), [4] (0.382 g, 0.707 mmol), EDC (0.184 g, 0.964 mmol), DMAP (0.079 g, 0.642 mmol), and DIPEA (0.56 mL, 3.21 mmol) were added in DCE (6 mL) and stirred at room temperature for 16 h. Completion of the reaction was monitored by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound [5] as a pale yellow oil (95 mg, 24%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C72H137N4O12, [M+H]=1250.02, Observed=1250.0

[0388] Intermediates for the HEP-based cationic lipids described herein can be prepared according to Scheme 20:

[0389] Scheme 20 - Synthesis of acid intermediate [6]: [ka]

[0390] As presented in Scheme 20: To a solution of 5-aminovaleric acid (0.5 g, 4.27 mmol) dissolved in i-PrOH (5 mL) and EtN (1.2 mL) was added tetradecyl acrylate (3.08 g, 12.8 mmol). The reaction was heated to 90 °C for 3 h. After completion of the reaction, the crude mixture was evaporated under reduced pressure. Finally, the crude material was purified using silica gel column chromatography (0-12% MeOH in CHCl) to give pure compound [6] as a colorless oil (0.720 g, 28%). Expected [M+H] = 598.5, observed = 598.5

[0391] The HEP-based cationic lipids described herein can be prepared according to Scheme 21:

[0392] Scheme 21-Synthesis of HEP-E4-O12[7]: [ka]

[0393] As shown in Scheme 21: To a solution of [2] (0.100 g, 0.494 mmol) in DCE (2 mL), [6] (0.650 g, 1.09 mmol), EDC (0.284 g, 1.48 mmol), DMAP (0.12 g, 0.99 mmol), and DIPEA (0.86 mL, 4.94 mmol) were added in DCE (8 mL) and stirred at room temperature for 16 h. Completion of the reaction was monitored by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound [7] as a colorless oil (0.20 g, 30%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C80H153N4O12, [M+H]=1362.15, Observed=1362.09

[0394] Intermediates for the HEP-based cationic lipids described herein can be prepared according to Scheme 22:

[0395] Scheme 22 - Synthesis of acid intermediate [8]: [ka]

[0396] As presented in Scheme 22: To a solution of 5-aminovaleric acid (0.5 g, 4.27 mmol) dissolved in i-PrOH (5 mL) and EtN (1.2 mL) was added tetradecyl acrylate (3.4 g, 12.8 mmol). The reaction was heated to 90 °C for 3 h. After completion of the reaction, the crude mixture was evaporated under reduced pressure. Finally, the crude material was purified using silica gel column chromatography (0-12% MeOH in CHCl) to give the pure compound [8] as a colorless oil (0.680 g, 24%). Expected [M+H] = 654.6, observed = 654.6

[0397] The HEP-based cationic lipids described herein can be prepared according to Scheme 23:

[0398] Scheme 2: Synthesis of 3-HEP-E4-O14[9]: [ka]

[0399] As shown in Scheme 23: To a solution of [2] (0.100 g, 0.494 mmol) in DCE (2 mL), [8] (0.711 g, 1.09 mmol), EDC (0.284 g, 1.48 mmol), DMAP (0.12 g, 0.99 mmol), and DIPEA (0.86 mL, 4.94 mmol) were added in DCE (8 mL) and stirred at room temperature for 16 h. Completion of the reaction was monitored by TLC and MS. The reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude compound was purified by silica gel chromatography (eluent: 4% EtOAc in hexane) to give pure compound [9] as a pale yellow oil (0.22 g, 30%), which was confirmed by MS analysis. result: ESI-MS analysis: Calculated C88H169N4O12, [M+H]=1474.27, Observed=1474.20

[0400] [Example 24] Lipid nanoparticle formulation The cationic lipids described herein can be used in the preparation of lipid nanoparticles according to methods known in the art. For example, suitable methods include those described in International Publication No. WO2018 / 089801, the entire contents of which are hereby incorporated by reference.

[0401] One exemplary process for lipid nanoparticle formulation is Process A of WO2018 / 089801 (see, for example, Example 1 and Figure 1 of WO2018 / 089801). Process A ("A") relates to a conventional method of encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles. In the exemplary process, an ethanolic lipid solution and a buffered aqueous solution of mRNA were separately prepared. A solution of a mixture of lipids (such as cationic lipids, helper lipids, zwitterionic lipids, PEG lipids, etc.) was prepared by dissolving the lipids in ethanol. An mRNA solution was prepared by dissolving the mRNA in a citrate buffer. These two solutions were then mixed using a pump system. In some instances, the two solutions were mixed using a gear pump system. In certain embodiments, the two solutions were mixed using a "T" junction (or "Y" junction). The mixture was then purified by diafiltration in a TFF process. The resulting formulation was concentrated and stored at 2-8°C until further use.

[0402] A second exemplary process for lipid nanoparticle formulation is Process B of WO2018 / 089801 (see, for example, Example 2 and Figure 2 of WO2018 / 089801). Process B ("B") refers to a process of encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with the mRNA. A range of different conditions can be used in Process B, such as various temperatures (i.e., heating or not heating the mixture), buffers, and concentrations. In the exemplary process, a pump system was used to mix lipids dissolved in ethanol and citrate buffer. Instantaneous mixing of the two streams resulted in the formation of empty lipid nanoparticles, a self-assembly process. The resulting formulation mixture was empty lipid nanoparticles in a citrate buffer containing alcohol. The formulation was then subjected to a TFF purification process, in which buffer exchange occurred. The resulting suspension of preformed empty lipid nanoparticles was then mixed with the mRNA using a pump system. For certain cationic lipids, heating the solution after mixing resulted in a higher percentage of lipid nanoparticles containing mRNA and a higher overall yield of mRNA.

[0403] [Example 25] Delivery of firefly luciferase (FFL) mRNA by intratracheal administration Lipid nanoparticle formulations prepared using Process A containing FFL mRNA, cationic lipid, DMG-PEG2000, cholesterol, and dope (40:5:25:30 or 45:5:20:30 mol% ratio) were administered to male CD1 mice (6-8 weeks old) under anesthesia by a single intratracheal aerosol administration (50 μl / animal) via a Microsprayer®. Approximately 24 hours after dosing, animals were dosed with luciferin at 150 mg / kg (60 mg / ml) via intraperitoneal injection at 2.5 ml / kg. Five to 15 minutes later, all animals were imaged using an IVIS imaging system to measure luciferase production in the lungs. Figure 1 demonstrates that lipid nanoparticles containing the cationic lipids described herein are effective in delivering FFL mRNA in vivo based on positive luciferase activity.

[0404] From the foregoing description, those skilled in the art can easily ascertain the basic features of this invention, and can make various changes and modifications to the invention to adapt it to various usages and conditions without departing from the spirit and scope thereof.

[0405] All U.S. or foreign references, patents, or applications cited herein are hereby incorporated by reference as if written in their entirety herein. In the event of any conflict, the material disclosed herein by letter will control.

Claims

1. Formula (I'): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein: A 1 teeth, 【Chemistry 2】 and -S-S-, wherein the left hand side of each depicted structure is selected from -(CH 2 ) a - is bonded to; Z 1 teeth, 【Transformation 3】 and -S-S-, wherein the right hand side of each depicted structure is selected from -(CH 2 ) a - is bonded to; Each R is independently selected from the following: (i) 【Chemistry 4】 (In the formula, each R 1 is independently selected from C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, —C 1 -C 20 alkyl-(C═O)—O—C 1 -C 20 alkyl, and —C 1 -C 20 alkyl-O—(C═O)—C 1 -C 20 alkyl; and (ii) 【Transformation 5】 (In the formula, each R 2 are independently selected from C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, and acyl; wherein C 1 -C 20 alkyl is optionally substituted with —CO 2 R″ or —OCOR″, where R″ is C 1 -C 20 alkyl; each a is independently selected from 2, 3, 4, and 5; each b is independently selected from 2, 3, 4, 5, 6, and 7; Acyl is R Z —(C═O)—, where R Z is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, or C 2 -C 20 alkynyl.

2. Formula (I): 【Transformation 6】 2. The compound of claim 1, having a structure according to the formula: Middle: A 1 teeth, 【Transformation 7】 and -S-S-, wherein the left hand side of each depicted structure is selected from -(CH 2 ) a - is bonded to; Z 1 teeth, 【Transformation 8】 and -S-S-, wherein the right hand side of each depicted structure is selected from -(CH 2 ) a - is bonded to; Each R is independently selected from the following: (i) 【Chemistry 9】 (In the formula, each R 1 is independently selected from C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, —C 1 -C 20 alkyl-(C═O)—O—C 1 -C 20 alkyl, and —C 1 -C 20 alkyl-O—(C═O)—C 1 -C 20 alkyl; and (ii) 【Chemistry 10】 (In the formula, each R 2 are independently selected from C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl, and acyl; wherein C 1 -C 20 alkyl is optionally substituted with —CO 2 R″ or —OCOR″, where R″ is C 1 -C 20 alkyl; each a is independently selected from 2, 3, 4 and 5].

3. Formula (Ia): 【Chemistry 11】 3. The compound of claim 1 or 2, having a structure according to: or a pharmaceutically acceptable salt thereof.

4. Formula (Ib): 【Chemistry 12】 3. The compound of claim 1 or 2, having a structure according to: Or formula (Ib'): 【Chemistry 13】 3. The compound of claim 1 or 2, having a structure according to: or a pharmaceutically acceptable salt thereof.

5. Formula (Ic): 【Chemistry 14】 4. The compound of any one of claims 1 to 3, having a structure according to: or a pharmaceutically acceptable salt thereof.

6. Formula (Id): 【Chemistry 15】 4. The compound of any one of claims 1 to 3, having a structure according to: or a pharmaceutically acceptable salt thereof.

7. Formula (Ie): 【Chemistry 16】 4. The compound of any one of claims 1 to 3, having a structure according to: or a pharmaceutically acceptable salt thereof.

8. Formula (If): 【Chemistry 17】 10. The compound of claim 1, 2 or 4, having a structure according to: Or formula (If'): [Chemistry 18] 10. The compound of claim 1, 2 or 4, having a structure according to: or a pharmaceutically acceptable salt thereof.

9. The compound of any one of claims 1 to 8, wherein each a is independently selected from 2, 3 and 4.

10. In the compound of formula (I'), (I), (Ia), (Ib) or (Ib'), A 1 teeth, 【Chemistry 19】 where the left hand side of the depicted structure is -(CH 2 ) a is bonded to -; or Z 1 teeth, 【Chemistry 20】 where the right hand side of the depicted structure is —(CH 2 ) a 10. The compound of any one of claims 1 to 4 and 9, or a pharmaceutically acceptable salt thereof, wherein the compound is bonded to -.

11. In the compound of formula (I'), (I), (Ia), (Ib) or (Ib'), A 1 teeth, 【Chemistry 21】 where the left hand side of the depicted structure is -(CH 2 ) a is bonded to -; or Z 1 teeth, 【Chemistry 22】 where the right hand side of the depicted structure is —(CH 2 ) a 10. The compound of any one of claims 1 to 4 and 9, or a pharmaceutically acceptable salt thereof, wherein the compound is bonded to -.

12. In the compound of formula (I'), (I), (Ia), (Ib) or (Ib'), A 1 is -S-S- or Z 1 The compound according to any one of claims 1 to 4 and 9, wherein is -SS-, or a pharmaceutically acceptable salt thereof.

13. In the compounds of formula (I'), (I), (Ib), (Ib'), (If) or (If'), each R 1 The compound of any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein is C 1 -C 20 alkyl or C 2 -C 20 alkenyl.

14. In the compounds of formula (I'), (I), (Ib), (Ib'), (If) or (If'), each R 1 is selected from: 【Chemistry 23】 13. The compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof.

15. In the compounds of formula (I'), (I), (Ia), (Ic), (Id) or (Ie), each R 2 The compound of any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein is C 1 -C 20 alkyl or C 2 -C 20 alkenyl.

16. In the compounds of formula (I'), (I), (Ia), (Ic), (Id) or (Ie), each R 2 is selected from: 【Chemistry 24】 13. The compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof.

17. A compound selected from those listed in the following table: Table 1 or a pharmaceutically acceptable salt thereof.

18. A composition comprising the compound of any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

19. 19. The composition of claim 18, which is a lipid nanoparticle.

20. The composition of claim 18, which is a liposome.

21. 20. The composition of claim 19, wherein the lipid nanoparticles encapsulate nucleic acids.

22. 20. The composition of claim 19, wherein the lipid nanoparticles encapsulate mRNA encoding a peptide or protein.

23. 23. The composition of claim 22 for use in therapy.

24. 23. The composition of claim 22, for use in a method for treating or preventing a disease amenable to treatment or prevention by a peptide or protein encoded by the mRNA.

25. The composition described in claim 24, wherein the disease is (a) a protein deficiency, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

26. 25. The composition for use according to claim 23 or 24, which is administered intravenously, intrathecally or intramuscularly, or by pulmonary delivery.

27. A composition for use as described in claim 23 or 24, delivered via inhalation administration.

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