CN115667207B

CN115667207B - Cationic lipids based on phenolic acid lipids

Info

Publication number: CN115667207B
Application number: CN202180039080.XA
Authority: CN
Inventors: S·卡夫; F·德罗萨; A·旺威拉翁; S·卡马卡
Original assignee: Translation Bio Co
Current assignee: Translation Bio Co
Priority date: 2020-04-01
Filing date: 2021-03-31
Publication date: 2025-09-16
Anticipated expiration: 2041-03-31
Also published as: JP2023520047A; CN115667207A; EP4126813A1; WO2021202694A1; JP7791103B2; CN121318756A

Abstract

The present invention provides, in part, phenolic acid lipid compounds of formula (I) and sub-formulae thereof, or pharmaceutically acceptable salts thereof. The compounds provided herein are useful for delivering and expressing mRNA and encoded proteins, e.g., as components of liposome delivery vehicles, and thus are useful in the treatment of various diseases, disorders, and conditions, such as diseases, disorders, and conditions associated with the lack of one or more proteins.

Description

Cationic lipids based on phenolic acid lipids

Cross reference to related applications

The present application claims priority from U.S. provisional patent application 63/003,698 filed on 1 month 4 2020, which is incorporated by reference in its entirety.

Background

Delivery of nucleic acids has been widely explored as a potential therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapies have become an increasingly important option for the treatment of a variety of diseases, including those associated with the deficiency of one or more proteins.

Efficient delivery of liposome-encapsulated nucleic acids remains an active area of research. The cationic lipid component plays an important role in facilitating efficient encapsulation of nucleic acids during loading of liposomes. In addition, cationic lipids can play an important role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. Various cationic lipids have been found suitable for in vivo use. However, there remains a need to identify lipids that can be synthesized efficiently and inexpensively without the formation of potentially toxic byproducts.

Phenolic acids have many advantageous properties that make them good starting points for the synthesis of cationic lipids for use in vivo environments. For example, phenolic acids do not show toxicity, are available in large quantities, and are readily derivable. Phenolic acids can be broadly divided into two groups, benzoic acid and cinnamic acid, and derivatives thereof.

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

examples of cinnamic acids that can be used to synthesize the cationic lipids of the invention include:

in some embodiments, examples of cinnamic acids that can be used to synthesize the cationic lipids of the invention include:

Disclosure of Invention

The present invention provides, among other things, a novel class of cationic lipid compounds useful for in vivo delivery of therapeutic agents such as nucleic acids. These compounds are expected to be able to be delivered with high efficacy in vivo while maintaining favorable toxicity profiles.

The cationic lipids of the present invention can be synthesized from readily available starting reagents such as phenolic acid, benzoic acid, and cinnamic acid. The cationic lipids of the present invention also have unexpectedly high encapsulation efficiency. The cationic lipids of the present invention also include cleavable groups (e.g., esters and disulfides) that are believed to enhance biodegradability and thus contribute to its favorable toxicity profile.

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

Wherein L ₁ is a bond, (C ₁-C₆) alkyl or (C ₂-C₆) alkenyl;

Wherein X is O or S;

Wherein R ¹、R²、R³、R⁴ and R ⁵ are each independently selected from H, OH, optionally substituted (C ₁-C₆) alkyl, optionally substituted (C ₂-C₆) alkenyl, optionally substituted (C ₂-C₆) alkynyl, optionally substituted (C ₁-C₆) alkoxy and-OC (O) R';

wherein at least one of R ¹、R²、R³、R⁴ or R ⁵ is-OC (O) R';

wherein R' is

Wherein R ⁶ is

Wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

Wherein R ⁷ is selected from H, optionally substituted (C ₁-C₆) alkyl, optionally substituted (C ₂-C₆) alkenyl, optionally substituted (C ₂-C₆) alkynyl, optionally substituted (C ₁-C₆) acyl, - (CH ₂)_kR^A or- (CH ₂)_kCH(OR¹¹)R^A);

Wherein R ⁸ is selected from H, optionally substituted (C ₁-C₆) alkyl, optionally substituted (C ₂-C₆) alkenyl, optionally substituted (C ₂-C₆) alkynyl, optionally substituted (C ₁-C₆) acyl, - (CH ₂)_nR^B or- (CH ₂)_nCH(OR¹²)R^B);

wherein R ⁹ is selected from H, optionally substituted (C ₁-C₆) alkyl, optionally substituted (C ₂-C₆) alkenyl, optionally substituted (C ₂-C₆) alkynyl, optionally substituted (C ₁-C₆) acyl, - (CH ₂)_qR^C or- (CH ₂)_qCH(OR¹³)R^C);

Wherein R ¹⁰ is selected from H, optionally substituted (C ₁-C₆) alkyl, optionally substituted (C ₂-C₆) alkenyl, optionally substituted (C ₂-C₆) alkynyl, optionally substituted (C ₁-C₆) acyl, - (CH ₂)_rR^D or- (CH ₂)_rCH(OR¹⁴)R^D);

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

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

Wherein R ¹¹、R¹²、R¹³ and R ¹⁴ are each independently selected from H, methyl, ethyl or propyl;

Wherein R ^A、R^B、R^C and R ^D are each independently selected from optionally substituted (C ₆-C₂₀) alkyl, optionally substituted (C ₆-C₂₀) alkenyl, optionally substituted (C ₆-C₂₀) alkynyl, optionally substituted (C ₆-C₂₀) acyl, optionally substituted-OC (O) alkyl, optionally substituted-OC (O) alkenyl, optionally substituted (C ₁-C₆) monoalkylamino, optionally substituted (C ₁-C₆) dialkylamino, optionally substituted (C ₁-C₆) alkoxy, -OH, -NH ₂;

Wherein at least one of R ⁷、R⁸、R⁹、R¹⁰ comprises a R ^A、R^B、R^C or R ^D moiety, respectively, wherein the R ^A、R^B、R^C or R ^D is independently selected from optionally substituted (C ₆-C₂₀) alkyl, optionally substituted (C ₆-C₂₀) alkenyl, optionally substituted (C ₆-C₂₀) alkynyl, optionally substituted (C ₆-C₂₀) acyl, optionally substituted-OC (O) (C ₆-C₂₀) alkyl, or optionally substituted-OC (O) (C ₆-C₂₀) alkenyl;

Or a pharmaceutically acceptable salt thereof.

In one aspect, provided herein are cationic lipids as pharmaceutically acceptable salts of formula (I).

In one aspect, provided herein are compositions comprising a cationic lipid of the invention, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In one aspect, the composition is a lipid nanoparticle, optionally a liposome.

In one aspect, the composition comprising the cationic lipid of the present invention may be used for treatment.

Drawings

Figure 1 shows in vivo protein expression after intratracheal administration of lipid nanoparticles comprising one of the cationic lipid compounds 1-12. Lipid nanoparticles comprising the cationic lipids described herein are effective in delivering FFL mRNA in vivo based on positive luciferase activity.

Detailed Description

Definition of the definition

In order to make the invention easier to understand, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification. Publications and other references cited herein to describe the background of the invention and to provide additional details concerning its practice are incorporated herein by reference.

Amino acids As used herein, the term "amino acid" is used in its broadest sense to refer to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, the amino acid has the general structure H ₂ N-C (H) (R) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid, in some embodiments the amino acid is a d-amino acid, in some embodiments the amino acid is an l-amino acid. "Standard amino acid" refers to any of the twenty standard I-amino acids commonly found in naturally occurring peptides. "non-standard amino acid" refers to any amino acid other than a standard amino acid, whether synthetically prepared or obtained from natural sources. As used herein, "synthetic amino acids" encompass chemically modified amino acids, including but not limited to salts, amino acid derivatives (e.g., amides), and/or substitutions. Amino acids, including carboxyl and/or amino terminal amino acids in peptides, may be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can alter the circulating half-life of the peptide without adversely affecting its activity. Amino acids may participate in disulfide bonds. The amino acid may comprise one or more post-translational modifications, for example, that are bound to one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl groups, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, and the like). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to the amino acid residue of a free amino acid and/or peptide. Whether the term refers to a free amino acid or a residue of a peptide, it will be apparent from the context in which the term is used.

Animal as used herein, the term "animal" refers to any member of the kingdom animalia. In some embodiments, an "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., 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.

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

Bioactive as used herein, the term "bioactive" refers to the characteristic of any agent that is active in a biological system, particularly in 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.

Delivery as used herein, the term "delivery" encompasses local delivery and systemic delivery. For example, delivering mRNA encompasses situations in which 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"), and situations in which mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient's circulatory system (e.g., serum), then distributed systemically and taken up by other tissues (also referred to as "systemic distribution" or "systemic delivery").

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

Functional "biomolecules, as used herein, are biomolecules that exhibit a form that characterizes their properties and/or activity.

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

Helper lipid as used herein, the term "helper lipid" refers to any neutral or zwitterionic lipid material that includes cholesterol. Without wishing to be bound by a particular theory, the helper lipid may increase stability, rigidity, and/or fluidity within the lipid bilayer/nanoparticle.

Improvement, increase, or decrease as used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents thereof, mean a value relative to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment as described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment as described herein. A "control individual" is an individual having the same form of disease as the individual being treated, and having about the same age as the individual being treated.

In vitro the term "in vitro" as used herein refers to events that occur in an artificial environment, e.g., in a tube or reaction vessel, in a cell culture, etc., rather than in a multicellular organism.

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

Isolated, as used herein, the term "isolated" refers to (1) a substance and/or entity that is separated from at least some of the components associated therewith at the time of initial production (whether natural and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by man. The isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, the purity of the isolated reagent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than 99%. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of the percent purity of an isolated substance and/or entity should not include excipients (e.g., buffers, solvents, water, etc.).

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

Messenger RNA (mRNA) As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA includes modified RNA and unmodified RNA. The term "modified mRNA" relates to an mRNA comprising at least one chemically modified nucleotide. An mRNA may comprise one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, and the like. Where appropriate, for example in the case of chemically synthesized molecules, the mRNA may comprise nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, and the like. Unless otherwise indicated, the mRNA sequences are shown in 5 'to 3'. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deadenosine, 8-oxoguanosine, O (6) -methylguanosine, and 2-thiocytidine), a chemically modified base (e.g., a methylated base), an inserted base, a modified sugar (e.g., 2' -fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose), and/or a modified phosphate group (e.g., a phosphorothioamide, N ' -phosphoramidite, and a phosphorothioate).

Nucleic acid As used herein, the term "nucleic acid" is used in its broadest sense to refer to any compound and/or substance that can be incorporated into a polynucleotide strand. In some embodiments, the nucleic acid is a compound and/or substance that is incorporated by a phosphodiester linkage or that can be incorporated by a phosphodiester linkage into a polynucleotide strand. 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 strand comprising a single nucleic acid residue. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double stranded DNA and/or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acid (RNA), including, but not limited to, any one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aana), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microrna (miRNA), poly-coding nucleic acid (MCNA), poly-coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, but not limited to, any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, the DNA may be in the form of antisense DNA, plasmid DNA, portions of plasmid DNA, pre-condensed DNA, products of the Polymerase Chain Reaction (PCR), vectors (e.g., P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In embodiments, the RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), microRNA (snRNA), micronucleolar RNA (snorRNA), 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 RNAs (lncRNA), micrornas (miRNA), RNAs that interact with piwi (piRNA), small interfering RNAs (siRNA), transaction siRNA (tasiRNA), repeat-related siRNA (rasiRNA), 73K RNA, retrotransposons, viral genomes, viroids, satellite RNAs, or derivatives of these groups. in some embodiments, the nucleic acid is an mRNA encoding a protein, such as an enzyme.

Patient as used herein, the term "patient" or "subject" refers to any organism to which the provided compositions can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, the patient is a human. Humans include prenatal and postnatal forms.

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

Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in J.pharmaceutical Sciences, S.M. Berge et al, (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic 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, camphorite, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodite, 2-hydroxyethanesulfonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N ⁺(C_1-4 alkyl group ₄ salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Where appropriate, additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulphates, phosphates, nitrates, sulphonates and arylsulphonates. Additional pharmaceutically acceptable salts include salts formed from quaternization of amines with suitable electrophiles (e.g., alkyl halides) to form quaternized alkylated amino salts.

Systemic distribution or delivery as used herein, the term "systemic distribution" or "systemic delivery" or grammatical equivalents thereof refers to a delivery or distribution mechanism or method that affects the whole body or whole organism. Typically, systemic distribution or delivery is accomplished via the circulatory system of the body (e.g., blood flow). In contrast to the definition of "localized distribution or delivery".

Subject the term "subject" as used herein refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the individual is a human. An individual may be a patient who is directed to a person who is a medical provider for diagnosis or treatment of a disease. The term "individual" is used interchangeably herein with "individual" or "patient". An individual may or may not be suffering from a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

Basically, as used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of a target feature or characteristic. Those of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, complete and/or continue to complete or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

Target tissue as used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, the target tissue includes those tissues that exhibit a disease-related pathology, symptom, or feature.

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

Treatment as used herein, the term "treatment" refers to any method for partially or completely alleviating, commensurating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. To reduce the risk of developing a pathology associated with a disease, treatment may be administered to subjects that do not exhibit signs of disease and/or exhibit only early signs of disease.

Chemical definition

Acyl as used herein, the term "acyl" refers to R ^Z - (c=o) -, wherein R ^Z is, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene.

Aliphatic as used herein, the term aliphatic refers to C _1-C₄₀ hydrocarbons and includes saturated hydrocarbons and unsaturated hydrocarbons. The aliphatic groups may be straight chain, branched or cyclic. For example, the C ₁-C₂₀ aliphatic group may include a C ₁-C₂₀ alkyl group (e.g., a straight or branched C ₁-C₂₀ saturated alkyl group), a C ₂-C₂₀ alkenyl group (e.g., a straight or branched C ₄-C₂₀ dienyl group), Linear or branched C ₆-C₂₀ trialkenyl, etc.) and C ₂-C₂₀ alkynyl (e.g., linear or branched C ₂-C₂₀ alkynyl). The C ₁-C₂₀ aliphatic may include C ₃-C₂₀ cycloaliphatic (e.g., C ₃-C₂₀ cycloalkyl, C ₄-C₂₀ cycloalkenyl, or C ₈-C₂₀ cycloalkynyl). In certain embodiments, the aliphatic groups may comprise one or more cyclic aliphatic groups and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. Aliphatic groups are unsubstituted or substituted with one or more substituents as described herein. for example, the aliphatic group may be one or more of (e.g., 1,2, 3, 4, 5 or 6 independently selected substituents) halogen 、-COR"、-CO₂H、-CO₂R"、-CN、-OH、-OR"、-OCOR'、-OCO₂R"、-NH₂、-NHR"、-N(R")₂、-SR" or-SO ₂ R ", wherein each instance of R" is independently C ₁-C₂₀ aliphatic (e.g., C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl or C ₁-C₃ alkyl). in embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted C ₁-C₃ alkyl. In embodiments, the aliphatic group is unsubstituted. In embodiments, the aliphatic group does not include any heteroatoms. Alkyl as used herein, the term "alkyl" means acyclic straight and branched chain hydrocarbon groups, e.g., "C ₁-C₃₀ alkyl" means an alkyl group having 1 to 30 carbons. 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 or branched chain alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art in view of the benefit of this disclosure. Alkyl groups may be unsubstituted or substituted with one or more substituents as described herein. For example, the alkyl group may be one or more of (e.g., 1, 2, 3, 4, 5 or 6 independently selected substituents) halogen 、-COR"、-CO₂H、-CO₂R"、-CN、-OH、-OR"、-OCOR'、-OCO₂R"、-NH₂、-NHR"、-N(R")₂、-SR" or-SO ₂ R ", wherein each instance of R" is independently C ₁-C₂₀ aliphatic (e.g., C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl or C ₁-C₃ alkyl). in embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted C ₁-C₃ alkyl. In embodiments, alkyl is substituted (e.g., with 1,2, 3, 4, 5, or 6 substituent groups 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, wherein the prefix represents an-OH group, and "alkyl" is as described herein.

As used herein, the term "alkyl" also refers to a radical ("C ₁-C₅₀ alkyl") of a straight or branched chain saturated hydrocarbon group having 1 to 50 carbon atoms. In some embodiments, the alkyl group has 1 to 40 carbon atoms ("C ₁-C₄₀ alkyl"). In some embodiments, the alkyl group has 1 to 30 carbon atoms ("C ₁-C₃₀ alkyl"). in some embodiments, the alkyl group has 1 to 20 carbon atoms ("C ₁-C₂₀ alkyl"). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C ₁-C₁₀ alkyl"). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C ₁-C₉ alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C ₁-C₈ alkyl"). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C ₁-C₇ alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C ₁-C₆ alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C ₁-C₅ alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C ₁-C₄ alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C ₁-C₃ alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C ₁-C₂ alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C ₁ alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C ₂-C₆ alkyl"). Examples of C ₁-C₆ alkyl groups include, but are not limited to, methyl (C ₁), ethyl (C ₂), n-propyl (C ₃), Isopropyl (C ₃), n-butyl (C ₄), tert-butyl (C ₄), sec-butyl (C ₄), Isobutyl (C ₄), n-pentyl (C ₅), 3-pentyl (C ₅), pentyl (C ₅), neopentyl (C ₅), 3-methyl-2-butyl (C ₅), tert-amyl (C ₅) and n-hexyl (C ₆). Further examples of alkyl groups include n-heptyl (C ₇), n-octyl (C ₈), and the like. Unless otherwise indicated, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents. In certain embodiments, the alkyl is unsubstituted C ₁-C₅₀ alkyl. In certain embodiments, the alkyl is a substituted C ₁-C₅₀ alkyl.

The prefix "arylene" is appended to a group to indicate that the group is a divalent moiety, e.g., arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

Alkylene As used herein, the term "alkylene" means a saturated divalent straight or branched chain hydrocarbon group, and is exemplified by methylene, ethylene, isopropylidene, and the like. Also, as used herein, the term "alkenylene" refers to an unsaturated divalent straight or branched hydrocarbon radical having one or more unsaturated carbon-carbon double bonds that may be present at any stable point along the chain, and the term "alkynylene" refers herein to an unsaturated divalent straight or branched hydrocarbon radical having one or more unsaturated carbon-carbon triple bonds that may be present at any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene group may contain one or more cycloaliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halogen, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. for example, an alkylene, alkenylene, or alkynylene group may be substituted with one or more of (e.g., 1, 2,3, 4, 5 or 6 independently selected substituents) halogen 、-COR"、-CO₂H、-CO₂R"、-CN、-OH、-OR"、-OCOR"、-OCO₂R"、-NH₂、-NHR"、-N(R")₂、-SR" or-SO ₂ R ", wherein each instance of R" is independently C ₁-C₂₀ aliphatic (e.g., C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl or C ₁-C₃ alkyl). in embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted C ₁-C₃ alkyl. In certain embodiments, the alkylene, alkenylene, or alkynylene group is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene group does not include any heteroatoms. Alkenyl As used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may occur at any stable point along the chain, e.g. "C ₂-C₃₀ alkenyl" means alkenyl having 2 to 30 carbons. For example, alkenyl includes prop-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-5-enyl, 2, 3-dimethylbut-2-enyl, and the like. In embodiments, alkenyl groups contain 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group comprises a single carbon-carbon double bond. In an embodiment, multiple double bonds (e.g., 2 or 3) are conjugated. Alkenyl groups may be unsubstituted or substituted with one or more substituents described herein. For example, alkenyl groups may be substituted with one or more of (e.g., 1, 2, 3, 4, 5 or 6 independently selected substituents) halogen 、-COR"、-CO₂H、-CO₂R"、-CN、-OH、-OR"、-OCOR"、-OCO₂R"、-NH₂、-NHR"、-N(R")₂、-SR" or-SO ₂ R ", wherein each instance of R" is independently C ₁-C₂₀ aliphatic (e.g., C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl or C ₁-C₃ alkyl). in embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted C ₁-C₃ alkyl. In embodiments, alkenyl groups are unsubstituted. In embodiments, alkenyl is substituted (e.g., with 1,2,3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkenyl group is substituted with an-OH group, and may also be referred to herein as a "hydroxyalkenyl" group, wherein the prefix represents an-OH group, and the "alkenyl" group is as described herein.

As used herein, "alkenyl" also refers to a radical ("C ₂-C₅₀ alkenyl") of a straight or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1,2, 3, or 4 double bonds). In some embodiments, alkenyl groups have 2 to 40 carbon atoms ("C ₂-C₄₀ alkenyl"). In some embodiments, alkenyl groups have 2 to 30 carbon atoms ("C ₂-C₃₀ alkenyl"). In some embodiments, alkenyl groups have 2 to 20 carbon atoms ("C ₂-C₂₀ alkenyl"). In some embodiments, alkenyl groups have 2 to 10 carbon atoms ("C ₂-C₁₀ alkenyl"). In some embodiments, alkenyl groups have 2 to 9 carbon atoms ("C ₂-C₉ alkenyl"). In some embodiments, alkenyl groups have 2 to 8 carbon atoms ("C ₂-C₈ alkenyl"). In some embodiments, alkenyl groups have 2 to 7 carbon atoms ("C ₂-C₇ alkenyl"). In some embodiments, alkenyl groups have 2 to 6 carbon atoms ("C ₂-C₆ alkenyl"). In some embodiments, alkenyl groups have 2 to 5 carbon atoms ("C ₂-C₅ alkenyl"). In some embodiments, alkenyl groups have 2 to 4 carbon atoms ("C ₂-C₄ alkenyl"). In some embodiments, alkenyl groups have 2 to 3 carbon atoms ("C ₂-C₃ alkenyl"). In some embodiments, the alkenyl group has 2 carbon atoms ("C ₂ alkenyl"). One or more of the carbon-carbon double bonds may be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of C ₂-C₄ alkenyl groups include, but are not limited to, vinyl (C ₂), 1-propenyl (C ₃), 2-propenyl (C ₃), 1-butenyl (C ₄), 2-butenyl (C ₄), butadienyl (C ₄), and the like. Examples of C ₂-C₆ alkenyl groups include the aforementioned C ₂-C₄ alkenyl group, pentenyl (C ₅), pentadienyl (C ₅), hexenyl (C6), and the like. Further examples of alkenyl groups include heptenyl (C ₇), octenyl (C ₈), octenyl (C ₈), and the like. Unless otherwise indicated, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted ("substituted alkenyl") with one or more substituents. In certain embodiments, the alkenyl is unsubstituted C ₂-C₅₀ alkenyl. In certain embodiments, the alkenyl is a substituted C ₂-C₅₀ alkenyl.

Alkynyl As used herein, "alkynyl" means any hydrocarbon chain of straight or branched configuration having one or more carbon-carbon triple bonds present at any stable point along the chain, e.g., "C ₂-C₃₀ alkynyl" means an alkynyl group having 2-30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl and the like. In embodiments, the alkynyl group comprises one carbon-carbon triple bond. Alkynyl groups may 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"、-CO₂H、-CO₂R"、-CN、-OH、-OR"、-OCOR"、-OCO₂R"、-NH₂、-NHR"、-N(R")₂、-SR" or-SO ₂ R ", where each instance of R" is independently C ₁-C₂₀ aliphatic (e.g., C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C ₁-C₂₀ alkyl, C ₁-C₁₅ alkyl, C ₁-C₁₀ alkyl, or C ₁-C₃ alkyl). In embodiments, R "is independently unsubstituted C ₁-C₃ alkyl. In embodiments, alkynyl groups are unsubstituted. In embodiments, an alkynyl group is substituted (e.g., with 1,2, 3, 4, 5, or 6 substituent groups as described herein).

As used herein, "alkynyl" also refers to a radical ("C ₂-C₅₀ alkynyl") of a straight or branched hydrocarbon radical having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2,3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2,3, or 4 double bonds). 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 ("C ₂-C₄₀ alkynyl"). In some embodiments, alkynyl groups have 2 to 30 carbon atoms ("C ₂-C₃₀ alkynyl"). In some embodiments, alkynyl groups have 2 to 20 carbon atoms ("C ₂-C₂₀ alkynyl"). In some embodiments, alkynyl groups have 2 to 10 carbon atoms ("C ₂-C₁₀ alkynyl"). In some embodiments, alkynyl groups have 2 to 9 carbon atoms ("C ₂-C₉ alkynyl"). In some embodiments, alkynyl groups have 2 to 8 carbon atoms ("C ₂-C₈ alkynyl"). In some embodiments, alkynyl groups have 2 to 7 carbon atoms ("C ₂-C₇ alkynyl"). In some embodiments, alkynyl groups have 2 to 6 carbon atoms ("C ₂-C₆ alkynyl"). In some embodiments, alkynyl groups have 2 to 5 carbon atoms ("C ₂-C₅ alkynyl"). In some embodiments, alkynyl groups have 2 to 4 carbon atoms ("C ₂-C₄ alkynyl"). In some embodiments, alkynyl groups have 2 to 3 carbon atoms ("C ₂-C₃ alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C ₂ alkynyl"). The one or more carbon-triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of C ₂-C₄ alkynyl include, but are not limited to, ethynyl (C ₂), 1-propynyl (C ₃), 2-propynyl (C ₃), 1-butynyl (C ₄), 2-butynyl (C ₄), and the like. Examples of C ₂-C₆ alkenyl groups include the aforementioned C ₂-C₄ alkynyl groups, pentynyl groups (C ₅), hexynyl groups (C ₆), and the like. Further examples of alkynyl groups include heptynyl (C ₇), octynyl (C ₈), and the like. Unless otherwise indicated, each instance of an alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted ("substituted alkynyl") with one or more substituents. In certain embodiments, the alkynyl is unsubstituted C ₂-C₅₀ alkynyl. In certain embodiments, the alkynyl is a substituted C ₂-C₅₀ alkynyl.

Aryl the term "aryl" used alone or as part of a larger moiety as in "aralkyl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the remainder of the molecule, at least one ring in the system being aromatic, and wherein each ring in the system contains 4to 7 ring members. In an embodiment, aryl has 6 ring carbon atoms ("C ₆ aryl", e.g., phenyl). In some embodiments, aryl has 10 ring carbon atoms ("C ₁₀ aryl", for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C ₁₄ aryl", e.g., anthracenyl). "aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, wherein the linking radical or point of attachment is on the aromatic ring, and in which case the number of carbon atoms continues to represent the number of carbon atoms in the aromatic ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

As used herein, "aryl" also refers to a free radical ("C ₆-C₁₄ aryl") of a single or multiple ring (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a ring array) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system. In some embodiments, aryl has 6 ring carbon atoms ("C ₆ aryl"; e.g., phenyl). In some embodiments, the 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., anthracenyl). "aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups, wherein the linking radical or point of attachment is on the aromatic ring, and in which case the number of carbon atoms continues to represent the number of carbon atoms in the aromatic ring system. Unless otherwise indicated, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted ("substituted aryl") with one or more substituents. In certain embodiments, the aryl is an unsubstituted C ₆-C₁₄ aryl. In certain embodiments, the aryl is a substituted C ₆-C₁₄ aryl.

Arylene As used herein, the term "arylene" refers to a divalent aryl group (i.e., having two points of attachment to a molecule). Exemplary arylene groups include phenylene (e.g., unsubstituted phenylene or substituted phenylene).

Carbocyclyl As used herein, "carbocyclyl" or "carbocycle" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("C ₃-C₁₀ carbocyclyl") and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C ₃-C₈ carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C ₃-C₇ carbocyclyl"). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C ₃-C₆ carbocyclyl"). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C ₄-C₆ carbocyclyl"). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms ("C ₅-C₆ carbocyclyl"). in some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C ₅-C₁₀ carbocyclyl"). Exemplary C ₃-C₆ carbocyclyl groups include, but are not limited to, cyclopropyl (C ₃), cyclopropenyl (C ₃), cyclobutyl (C ₄), Cyclobutenyl (C4), cyclopentyl (C ₅), cyclopentenyl (C ₅), cyclohexyl (C ₆), cyclohexenyl (C ₆), Cyclohexadienyl (C ₆), and the like. exemplary C ₃-C₈ carbocyclyl groups include, but are not limited to, the aforementioned C ₃-C₆ carbocyclyl group, cycloheptyl (C ₇), cycloheptenyl (C ₇), Cycloheptadienyl (C ₇), cycloheptatrienyl (C ₇), cyclooctyl (C ₈), cyclooctenyl (C ₈), Bicyclo [2.2.1] heptyl (C ₇), bicyclo [2.2.2] octyl (C ₈), and the like. Exemplary C ₃-C₁₀ carbocyclyl groups include, but are not limited to, the aforementioned C ₃-C₈ carbocyclyl group, cyclononyl (C ₉), cyclononenyl (C ₉), Cyclodecyl (C ₁₀), cyclodecyl (C ₁₀), octahydro-1H-indenyl (C ₉), decalinyl (C ₁₀), spiro [4.5] decyl (C ₁₀), etc. As shown in the foregoing examples, in certain embodiments, carbocyclyl is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring system, such as a bicyclic system ("bicyclic carbocyclyl") or tricyclic system) ("tricyclic carbocyclyl"), and may be saturated or may contain one or more carbon-carbon double or triple bonds. "carbocyclyl" also includes ring systems in which a carbocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in such cases the number of carbons always indicates the number of carbons in the carbocyclyl system. Unless otherwise indicated, each instance of a carbocyclyl is independently unsubstituted ("unsubstituted carbocyclyl") or substituted by one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl is an unsubstituted C ₃-C₁₀ carbocyclyl. In certain embodiments, the carbocyclyl is a substituted C ₃-C₁₀ carbocyclyl.

In some embodiments, "carbocyclyl" or "carbocycle" refers to "cycloalkyl", i.e., a monocyclic saturated carbocyclyl having 3 to 10 ring carbon atoms ("C ₃-C₁₀ cycloalkyl"). In some embodiments, cycloalkyl groups have 3 to 8 ring carbon atoms ("C ₃-C₈ cycloalkyl"). In some embodiments, cycloalkyl groups have 3 to 6 ring carbon atoms ("C ₃-C₆ cycloalkyl"). In some embodiments, cycloalkyl groups have 4 to 6 ring carbon atoms ("C ₄-C₆ cycloalkyl"). In some embodiments, cycloalkyl groups have 5 to 6 ring carbon atoms ("C ₅-C₆ cycloalkyl"). In some embodiments, cycloalkyl groups have 5 to 10 ring carbon atoms ("C ₅-C₁₀ cycloalkyl"). Examples of C ₅-C₆ cycloalkyl groups include cyclopentyl (C ₅) and cyclohexyl (examples of C ₅).C3-C₆ cycloalkyl groups include the aforementioned C ₅-C₆ cycloalkyl cyclopropyl (C ₃) and cyclobutyl (examples of C ₄).C₃-C₈ cycloalkyl groups include the aforementioned C ₃-C₆ cycloalkyl groups as well as cycloheptyl (C ₇) and cyclooctyl (C ₈). unless otherwise indicated, each instance of cycloalkyl is independently unsubstituted ("unsubstituted cycloalkyl") or substituted ("substituted cycloalkyl") with one or more substituents. In certain embodiments, the cycloalkyl is unsubstituted C ₃-C₁₀ cycloalkyl. In certain embodiments, the cycloalkyl is a substituted C ₃-C₁₀ cycloalkyl.

Halogen as used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine.

Heteroalkyl the term "heteroalkyl" means a branched or unbranched alkyl, alkenyl or alkynyl group having from 1 to 14 carbon atoms in addition to 1,2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S and P. Heteroalkyl groups include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. The heteroalkyl group may optionally include a single ring, a double ring, or a triple ring, where each ring desirably has three to six members. Examples of heteroalkyl groups include polyethers such as methoxymethyl and ethoxyethyl.

Heteroalkylene as used herein, the term "heteroalkylene" means a divalent form of heteroalkyl group as described herein.

Heteroaryl As used herein, the term "heteroaryl" is 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.

As used herein, "heteroaryl" also refers to a radical of a 5 to 14 membered monocyclic or multicyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a ring array) having ring carbon atoms and 1 or more (e.g., 1, 2,3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5 to 14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, where valency permits, the attachment point may be a carbon or nitrogen atom. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in such cases the number of ring members always represents 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 with one or more aryl groups, wherein the attachment point is on the aryl or heteroaryl ring, and in such cases the number of ring members represents the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. One ring in a polycyclic heteroaryl group does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring that does not contain a heteroatom (e.g., 5-indolyl).

In some embodiments, heteroaryl groups are 5-to 10-membered aromatic ring systems having a ring carbon atom provided in the aromatic ring system and 1 or more (e.g., 1,2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-to 10-membered heteroaryl"). In some embodiments, heteroaryl groups are 5-to 8-membered aromatic ring systems having a ring carbon atom provided in the aromatic ring system and 1 or more (e.g., 1,2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-to 8-membered heteroaryl"). In some embodiments, heteroaryl groups are 5-to 6-membered aromatic ring systems having a ring carbon atom provided in the aromatic ring system and 1 or more (e.g., 1,2, 3, 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-to 6-membered heteroaryl"). In some embodiments, the 5-to 6-membered heteroaryl has 1 or more (e.g., 1,2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-to 6-membered heteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-to 6-membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise indicated, each instance of heteroaryl is independently unsubstituted ("unsubstituted heteroaryl") or substituted by one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl is an unsubstituted 5-to 14-membered heteroaryl. In certain embodiments, the heteroaryl is a substituted 5-to 14-membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thienyl. Exemplary 5, 6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazole, benzothienyl, isobenzothienyl, benzofuranyl, benzisotofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, indolizinyl, and purinyl. Exemplary 6, 6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, piperidinyl, quinolinyl, isoquinolinyl, quinolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

As used herein, "heterocyclyl" or "heterocycle" refers to a radical of a 3-to 14-membered non-aromatic ring system having ring carbon atoms and 1 or more (e.g., 1,2, 3, or 4) ring heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3-to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, where valency permits, the point of attachment may be a carbon atom or a nitrogen atom. A heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a fused, bridged or spiro ring system, such as a bicyclic system ("bicyclic heterocyclyl") or tricyclic system ("tricyclic heterocyclyl")) and may be saturated or may contain one or more carbon-carbon double or triple bonds. The heterocyclyl-based multicyclic system may 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 rings, in which the attachment point is on a carbocyclyl or heterocyclyl ring, or ring systems in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, in which the attachment point is on a heterocyclyl ring, and in which case the number of ring members always indicates the number of ring members in the heterocyclyl ring system. Unless otherwise indicated, each instance of a heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") with one or more substituents. In certain embodiments, the heterocyclyl is an unsubstituted 3-to 14-membered heterocyclyl. In certain embodiments, the heterocyclyl is a substituted 3-to 14-membered heterocyclyl.

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

Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, thioalkenyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolane. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiolane, dioxanyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepinyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azacyclooctyl, oxacyclooctyl, and thiacyclooctyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochroenyl, octahydroisochroenyl, decahydronaphthyridinyl, decahydro-1, 8-naphthyridinyl, decahydro pyrrolo [3,2-b ] pyrrole, indolyl, phthalimidyl, naphthalimidyl, benzodihydropyranyl, benzothienyl, 1H-benzo [ e ] [1,4] naphthyridinyl, 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 [3,2-b ] pyrrolyl, 1H-benzo [3,4, 7-b ] pyrrolyl, 1,4, 7-tetrahydropyran [3,4-b ] pyrrolyl, 5, 6-dihydro-4H-furo [3,2-b ] pyrrolyl, 5, 7-dihydro-3, 4-b ] pyrrolyl, 3, 4-H-7-tetrahydropyran [3,4-b ] pyrrolyl, 3, 6, 4-dihydro-3, 4-b ] pyrrolyl, 6-dihydro-3, 4-b ] pyrrolyl, 6-dihydro-2, 3,4-b ] pyrrolyl, 4.

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

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

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

Or two twin hydrogens on carbon atoms are replaced with groups=o, =s, =nn (Rbb) 2, = NNRbbC (=o) Raa, = NNRbbC (=o) ORaa, = NNRbbS (=o) 2Raa, = NRbb or= NORcc;

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

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

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

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

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

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

Each instance Rgg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C50 alkyl, -ON (C1-C50 alkyl) 2, -N (C1-C50 alkyl) 3+X-, -NH (C1-C50 alkyl) 2+X-, -NH2 (C1-C50 alkyl) +X-, -NH3+X-, -N (OC 1-C50 alkyl) (C1-C50 alkyl), -N (OH) (C1-C50 alkyl), -NH (OH), -SH, -SC1-C50 alkyl, -SS (C1-C50 alkyl), -C (=o) (C1-C50 alkyl), -CO2H, -CO2 (C1-C50 alkyl), -OC (=o) (C1-C50 alkyl), -OCO2 (C1-C50 alkyl), -C (=o) NH2, -C (=o) N (C1-C50 alkyl) 2, -OC (=o) NH (C1-C50 alkyl), -NHC (=o) (C1-C50 alkyl), -N (C1-C50 alkyl) C (=o) (C1-C50 alkyl), -NHCO2 (C1-C50 alkyl), -NHC (=o) N (C1-C50 alkyl) 2, -NHC (=o) NH (C1-C50 alkyl), -NHC (=o) NH2, -C (=nh) O (C1-C50 alkyl), -OC (=nh) OC1-C50 alkyl, -C (=nh) N (C1-C50 alkyl) 2, -C (=nh) NH (C1-C50 alkyl), -C (=nh) NH2, -OC (=nh) N (C1-C50 alkyl) 2, -OC (NH) NH (C1-C50 alkyl), -OC (NH) NH2, -NHC (NH) N (C1-C50 alkyl) 2, -NHC (=nh) NH2, -NHSO2 (C1-C50 alkyl), -SO2N (C1-C50 alkyl) 2, -SO2NH (C1-C50 alkyl), -SO2NH2, -SO2 (C1-C50 alkyl), -SO2O (C1-C50 alkyl), -OSO2 (C1-C6 alkyl), -SO (C1-C6 alkyl), -Si (C1-C50 alkyl) 3, -OSi (C1-C6 alkyl) 3, -C (=s) N (C1-C50 alkyl) 2, C (=s) NH (C1-C50 alkyl), C (=s) NH2, -C (=o) S (C1-C6 alkyl), -C (=s) S (C1-C6 alkyl), -SC (=s) S (C1-C6 alkyl), -P (=o) 2 (C1-C50 alkyl), -P (=o) (C1-C50 alkyl) 2, -OP (=o) (OC 1-C50 alkyl) 2, C1-C50 alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, or two geminal Rgg substituents may be linked to form =o or =s, wherein X "is a counterion.

As used herein, the term "halo" or "halogen" refers to fluoro (fluoro, -F), chloro (chloro, -Cl), bromo (bromo, -Br) or iodo (iodo, -I).

As used herein, a "counterion" is a negatively charged group associated with the positively charged quaternary amine to maintain electron neutrality. Exemplary counterions include halide ions (e.g., F-, cl-, br-, I-), NO3-, clO4-, OH-, H2PO4-, HSO4-, sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethyl-1-sulfonic acid-2-sulfonate, and the like) and carboxylate ions (e.g., acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, and the like).

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

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 organic synthesis (Protecting Groups in Organic Synthesis), t.w. greene and p.g. m.wuts, 3 rd edition, john wili father company (John Wiley & Sons), 1999, which references are incorporated herein by reference.

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

Nitrogen protecting groups such as urethane groups (e.g., -C (=o) ORaa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), 9- (2-sulfo) fluorenylmethylcarbamate, 9- (2, 7-dibromo) fluoroalkenylmethylcarbamate, 2, 7-di-tert-butyl- [9- (10, 10-dioxo-10, 10-tetrahydrothioxanthogen) ] methylcarbamate (DBD-Tmoc), 4-methoxybenzoyl carbamate (Phenoc), 2-trichloroethylcarbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1-methylethyl carbamate (Adpoc), 1-dimethyl-2-ethylcarbamate, 1-dimethyl-2, 2-dibromoethylcarbamate (DB-t-BOC), 1-dimethyl-2, 2-Trichloroethylcarbamate (TCBOC), 1-methyl-1- (4-biphenylyl) carbamate (Bpoc), 1- (3, 5-di-tert-butylphenyl) -1-methylethyl carbamate (t-Bumeoc), Ethyl 2- (2 '-and 4' -pyridyl) carbamate (Pyoc), ethyl 2- (N, N-dicyclohexylcarboxamido) carbamate, tert-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-quinolinyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithio carbamate, 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-methylsulfinylethyl carbamate, 2- (p-toluenesulfonyl) carbamate, [2- (1, 3-dithianyl) ] methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2, 4-dimethylthiophenyl carbamate (Bmpc), 2-Phosphonoethyl carbamate (Peoc), 2-triphenylphosphine isopropyl carbamate (Ppoc), 1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyformyl) benzyl carbamate, 5-benzisoxazolylmethylcarbamate, 2- (trifluoromethyl) -6-bromomethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3, 5-dimethoxybenzyl carbamate, nitrobenzyl orthocarbamate, 3, 4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methylcarbamate, Tertiary amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2-dimethoxyacyl vinyl carbamate, benzyl o (N, N-dimethylformamide) carbamate, 1-dimethyl-3- (N, N-dimethylformamide) propyl carbamate, 1-dimethylpropynyl carbamate, di (2-pyridyl) methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, Isobutyl carbamate, isonicotinyl carbamate, p- (p' -methoxyphenylazo) benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1 (3, 5-dimethoxyphenyl) ethyl carbamate, 1-methyl-1- (p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1- (4-pyridinyl) carbamate, phenyl carbamate, benzyl p (phenylazo) carbamate, 2,4, 6-tri-tert-butylphenyl carbohydrate, 4- (trimethylammonium) benzyl carbamate and 2,4, 6-trimethylbenzyl carbamate.

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

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-dithio-phenemide (Dts), N-2, 3-diphenylmaleimide, N-2, 5-dimethylpyrrole, N-1, 4-tetramethyldisilylcyclopentane adducts (STABASE), 5-substituted 1, 3-dimethyl-1, 3, 5-triazacyclohexan-2-one, 5-substituted 1, 3-dibenzyl-1, 3, 5-triazacyclohexan-2-one, 1-substituted 3, 5-dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2- (trimethylsilyl) ethoxy ] methylamine (SEM), N-3-acetyloxypropylamine, N- (1-isopropyl-2-nitro-2-methoxyaniline), N-benzylbenzamide (N-4-methoxy) benzyl-2-one, 5-trimethyl-2-one, 5-nitro-N-phenylamine (N-methoxy) N-4-phenylamine (N-methoxy) N-phenylTr-4-methoxy-phenylamine (N-methoxy) N-4-phenylamine), N-9-phenylfluorofluorenamine (PhF), N-2, 7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamine (Fcm), N-2-pyridylamino N '-oxide, N-1, 1-dimethylthiomethyleneamine, N-benzylyleneamine, np-methoxybenzylyleneamine, N-diphenylmethyleneamine, N- [ (2-pyridyl) trimesaminyl ] methyleneamine, N- (N', N '-dimethylaminomethyleneamine, N, N' -isopropylenediamine, np-nitrobenzyleneamine, N-salicylyleneamine, N-5-chlorosulfinamide, N- (5-chloro-2-hydroxyphenyl) phenylmethyleneamine N-cyclohexyleneamine, N- (5, 5-dimethyl-3-oxo-1-cyclohexenyl) amine, N-borane derivatives, N-diphenylboronic acid derivatives, N- [ phenyl (penta-acyl-chrome-or tungsten) acyl ] amines, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosoamines, amine N-oxides, diphenylphosphamides (dppp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylphosphoramidates, dibenzylaminophosphate, diphenylphosphoramidates, benzenesulfonamides, o-nitrobenzenesulfonamides (Nps), 2, 4-dinitrobenzenesulfonamides, pentachlorobenzenesulfonamide, 2-nitro-4-methoxybenzenesulfonamide, triphenylmethyl sulfinamide, and 3-nitropyridine sulfinamide (Npys).

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 organic Synthesis (Protecting Groups in Organic Synthesis), T.W.Greene and P.G.M.Wuts, 3 rd edition, john Willi parent, 1999, which references are incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxymethyl (p-AOM), guaiacomethyl (GUM), t-butoxymethyl, 4-Pentenoxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), Tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1- [ (2-chloro-4-methyl) phenyl ] -4-methoxypiperidin-4-yl (CTMP), 1, 4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothienyl, 2, 3a,4,5,6,7,7α -octahydro-7, 8-trimethyl-4, 7-methylenebenzofuran-2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl) ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2, 4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3, 4-dimethoxybenzyl, o-nitrobenzyl, p-halobenzyl, 2, 6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxo-anion, Diphenylmethyl, p '-dinitrobenzhydryl, 5-dibenzocycloheptyl, trityl, alpha-naphthylbenzhydryl, p-methoxyphenyl-diphenylmethyl, di (p-methoxyphenyl) phenylmethyl, tri (p-methoxyphenyl) methyl, 4- (4' -bromobenzoylmethylphenoxy) diphenylmethyl, 4', 4' -tris (4, 5-dichlorophthalimidophenyl) methyl, 4 '-tris (levulinyloxy) phenyl) methyl, 4',4 '-tris (benzyloxy) methyl, 3- (imidazol-1-yl) bis (4', 4 '-dimethoxyphenyl) methyl, 1-bis (4-methoxyphenyl) -1' -pyrenylmethyl, 9-anthryl, 9- (9-phenyl) xanthyl, 9- (9-phenyl-10-oxo) anthryl, 1, 3-benzodithiofuran-2-yl, benzisothiazolyl S, S-dioxanyl, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropyl (IPDMS), diethylisopropyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), Formate, benzoyl, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxy, p-chlorophenoxy, 3-phenylpropionate, 4-oxovalerate (levulinate), 4- (ethylene) valerate (levodiacyldithioacetal), pivalate, adamantane, crotonate, methyl 4-crotonate, benzoate, p-phenyl, 2,4,6-trimethylbenzoate (2, 4,6-trimethylbenzoate or mesitoate), methyl alkyl carbonate, 9-fluorenyl (Fmoc), Alkyl ethyl carbonate, alkyl 2, 2-trichloroethyl carbonate (Troc), 2- (trimethylsilyl) ethyl carbonate (TMSEC), 2- (phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphine) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, p-nitrophenyl carbonate, alkyl benzyl carbonate, p-methoxybenzyl alkyl carbonate, alkyl 3, 4-dimethoxybenzyl carbonate, o-nitrobenzyl alkyl carbonate, p-nitrobenzyl alkyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthalene carbonate, and, Methyl dithiocarbonate, 2-iodobenzoic acid, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2- (methylthiomethoxymethyl) benzoate, 2, 6-dichloro-4-methylphenoxyacetate, 2, 6-dichloro-4- (1, 3-tetramethylbutyl) phenoxyacetate, 2, 4-bis (1, 1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monomalic acid ester, (E) -2-methyl-2-butenoic acid ester, O- (methoxy) benzoate, alpha naphthalene, nitrate, alkyl N, N, N ', N' -tetramethyl phosphoryl diamine, alkyl N-phenyl carbamate, borate, dimethyl thiophosphine, alkyl 2, 4-dinitrophenyl sulfinate, sulfate, mesylate (methanesulfonate or mesylate), benzyl sulfonate and tosylate (Ts).

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 organic Synthesis, T.W.Greene and P.G.M.Wuts, 3 rd edition, john Willi parent, 1999, which references are incorporated herein by reference.

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-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxo-group, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis (4-methoxyphenyl) methyl, 5-dibenzosuccinyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2, 4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM) isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbonylaminomethyl, phenylacetamide methyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl) ethyl, 2- (2, 4-dinitrophenyl) ethyl, 2-cyanoethyl, 2- (trimethylsilyl) ethyl, 2-bis (ethoxy) ethyl, (1-m-nitrophenyl-2-benzoyl) ethyl, 2-benzenesulfonylethyl, 2- (4-methylbenzenesulfonyl) -2-methylpropyl-2-yl, acetyl, benzoyl, trifluoroacetyl, N- [ [ (p-biphenyl) isopropoxy ] carbonyl ] -N-methyl ] -gamma-aminothiobutyrate, 2-trichloroethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, thiocarbonate, 3-nitro-2-pyridylthiosulfide, and oxythiophene.

Compounds of the invention

Liposome-based vehicles are considered attractive carriers for therapeutic agents and there is still a continuing development effort. Although liposome-based vehicles that include certain lipid components show promising results in terms of encapsulation, stability, and site-positioning, there remains a great need for improved liposome-based delivery systems. For example, a significant disadvantage of liposome delivery systems relates to the construction of liposomes with sufficient cell culture or in vivo stability to achieve the desired target cells and/or intracellular compartments, and the ability of such liposome delivery systems to effectively release their encapsulated material to such target cells.

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

Described herein are a novel class of cationic lipid compounds for improved in vivo delivery of therapeutic agents such as nucleic acids. In particular, the cationic lipids described herein can optionally be used with other lipids to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents, such as nucleic acids (e.g., DNA, siRNA, mRNA, micrornas) for therapeutic use.

In embodiments, the compounds of the invention as described herein may provide one or more desired characteristics or properties. That is, in certain embodiments, the compounds of the invention described herein may be characterized as having one or more properties that provide advantages of such compounds over other similarly classified lipids. For example, the compounds disclosed herein may allow for control and tailoring of the properties of the liposome compositions (e.g., lipid nanoparticles) of which they are a component. In particular, the compounds disclosed herein may be characterized by enhanced transfection efficiency and their ability to elicit specific biological results. Such results may include, for example, enhanced cellular uptake, endosomal/lysosomal disrupting ability, and/or facilitating release of intracellular encapsulated material (e.g., polynucleotide). In addition, the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution and efficiency (e.g., due to the different dissociation rates of the polymer groups used).

The present application demonstrates that the cationic lipids of the present application can not only be synthetically processed from readily available starting materials, but they also have unexpectedly high encapsulation efficiencies.

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

Compounds of the invention

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

Wherein L ₁ is a bond, (C ₁-C₆) alkyl or (C ₂-C₆) alkenyl;

Wherein X is O or S;

wherein at least one of R ¹、R²、R³、R⁴ or R ⁵ is-OC (O) R';

wherein R' is

Wherein R ⁶ is

Wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

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

Or a pharmaceutically acceptable salt thereof.

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

In an embodiment, L1 is independently a bond.

In embodiments, L1 is (C1-C6) alkyl.

In embodiments, L1 is (C2-C6) alkenyl.

In embodiments, L1 is C2 alkenyl.

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

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

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

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

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

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

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

In embodiments, RA and RB are the same and are selected from optionally substituted (C6-C20) alkyl, optionally substituted (C6-C20) alkenyl, optionally substituted (C6-C20) alkynyl.

In embodiments, RA and RB are each independently optionally substituted (C6-C20) alkyl.

In embodiments, RA and RB are the same and are optionally substituted (C6-C20) alkyl.

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

In embodiments, RA and RB are the same and are optionally substituted (C6-C20) alkenyl.

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

In embodiments, RA and RB are the same and are optionally substituted (C6-C20) alkynyl.

In embodiments, RA and RB are each independently an optionally substituted (C6-C20) acyl group.

In embodiments, RA and RB are the same and are optionally substituted (C6-C20) acyl.

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

In embodiments, RA and RB are the same and are optionally substituted-OC (O) (C6-C20) alkyl.

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

In embodiments, RA and RB are the same and are optionally substituted-OC (O) (C6-C20) alkenyl.

In an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are each independently selected from:

In an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are the same and are selected from:

in an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C8H17.

In an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C10H21.

In an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C12H25.

In an embodiment, r7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both

In an embodiment, X is O.

In an embodiment, X is S.

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

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

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

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

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

In embodiments, R3 is-OC (O) R'.

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

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

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

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

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

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

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA.

In an embodiment, R7 is- (CH 2) 1CH (OR 11) RA.

In embodiments, R7 is- (CH 2) 1CH (OH) RA.

In an embodiment, R8 is- (CH 2) nCH (OR 12) RB.

In an embodiment, R8 is- (CH 2) 1CH (OR 12) RB.

In embodiments, R8 is- (CH 2) 1CH (OH) RB.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA and R8 is- (CH 2) nCH (OR 12) RB.

In an embodiment, R7 is- (CH 2) 1CH (OR 11) RA, and R8 is- (CH 2) 1CH (OR 12) RB.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, and R8 is- (CH 2) 1CH (OH) RB.

In embodiments, R7 and R8 are each optionally substituted (C1-C6) alkyl, e.g., (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In an embodiment, R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted with —co ₂R^aa, wherein R ^aa C₁-C₄₀ alkyl. In an embodiment, R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted with —co ₂R^aa, wherein R ^aa C₁-C₃₀ alkyl. In an embodiment, R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted with —co ₂R^aa, wherein R ^aa C₁-C₂₀ alkyl.

In embodiments, R7 and R8 are the same and are each optionally substituted (C1-C6) alkyl, e.g., (C1-C6) alkyl substituted with-CO 2Raa, wherein Raa is C1-C50 alkyl. In embodiments, R7 and R8 are the same and are each (C1-C6) alkyl substituted with-CO 2Raa, wherein Raa is C1-C40 alkyl. In embodiments, R7 and R8 are the same and are each (C1-C6) alkyl substituted with-CO 2Raa, wherein Raa is C1-C30 alkyl. In an embodiment, R7 and R8 are each (C1-C6) alkyl substituted with-CO 2Raa, wherein Raa is C1-C20 alkyl.

In embodiments, R7 and R8 are each

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

In embodiments, R9 and R10 are each independently optionally substituted (C1-C6) alkyl or optionally substituted (C2-C6) alkenyl.

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

In embodiments, R9 and R10 are both optionally substituted (C1-C6) alkyl.

In an embodiment, R9 and R10 are both-CH 3.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and R9 and R10 are both-CH 3.

In an embodiment, R7 is- (CH 2) 1CH (OR 11) RA, R8 is- (CH 2) 1CH (OR 12) RB, and R9 and R10 are both-CH 3.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, R8 is- (CH 2) 1CH (OH) RB, and R9 and R10 are both-CH 3.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, RA and RB are both C8H17, and R9 and R10 are both-CH 3.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, R8 is- (CH 2) 1CH (OH) RB, both RA and RB are C8H17, and both R9 and R10 are-CH 3.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, both RA and RB are C10H21, and both R9 and R10 are-CH 3.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, R8 is- (CH 2) 1CH (OH) RB, both RA and RB are C10H21, and both R9 and R10 are-CH 3.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, both RA and RB are C12H25, and both R9 and R10 are-CH 3.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, R8 is- (CH 2) 1CH (OH) RB, both RA and RB are C12H25, and both R9 and R10 are-CH 3.

In an embodiment, R7 is- (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, both RA and RB are C16H29, and both R9 and R10 are-CH 3.

In embodiments, R7 is- (CH 2) 1CH (OH) RA, R8 is- (CH 2) 1CH (OH) RB, both RA and RB are C16H29, and both R9 and R10 are-CH 3.

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

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

In embodiments, m is 1,2,3,4, or 5. In an embodiment, m is 0. In an embodiment, m is 1. In an embodiment, m is 2. In an embodiment, m is 3. In an embodiment, m is 4. In an embodiment, m is 5. In embodiments, m is 0, 1,2,3, or 4.

In embodiments, p is 1,2,3,4, or 5. In an embodiment, p is 0. In an embodiment, p is 1. In an embodiment, p is 2. In an embodiment, p is 3. In an embodiment, p is 4. In an embodiment, p is 5. In embodiments, p is 0, 1,2,3, or 4.

In an embodiment, m is 2 and p is 2.

In an embodiment, m is 3 and p is 2.

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

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

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

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

In an embodiment, R' is:

in embodiments, R' is And k and n=1, and m=2 or 3.

In embodiments, R' isAnd k and n=1, and m=2.

In embodiments, R' isAnd k and n=1, and m=3.

In embodiments, R' isAnd R ¹¹ and R ¹² are H.

In embodiments, R' isAnd k and n=1, m=2 or 3, and R ¹¹ and R ¹² are H.

In embodiments, R' isAnd k and n=1, m=2, and R ¹¹ and R ¹² are H.

In embodiments, R' isAnd k and n=1, m=3, and R ¹¹ and R ¹² are H.

In an embodiment, R' is:

wherein R' has the following structure In any of the above embodiments, R ^A and R ^B may be as defined in any of paragraphs [0104] to [0129 ].

In an embodiment, R ⁶ is:

in embodiments, R ⁶ is Q and r=1, and p=2.

In embodiments, R ⁶ isAnd R ¹³ and R ¹⁴ are H.

In embodiments, R ⁶ isQ and r=1, p=2, and R ¹³ and R ¹⁴ are H.

In embodiments, R ⁶ is selected from the group consisting of:

In an embodiment, R ⁶ is:

In embodiments, R ⁶ is selected from the group consisting of:

In an embodiment, R ⁶ is:

In embodiments, R ⁶ and R' are the same.

In embodiments, R ⁶ isR' isM is 2, and p is 2.

In embodiments, R ⁶ isR' isM is 3 and p is 2.

In an embodiment, the L ₁ bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isAnd R' is

In embodiments, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isAnd R' is

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isAnd R' is

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isAnd R' is

In an embodiment, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ⁶ isAnd R' is

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ⁶ isAnd R' is

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ¹、R⁴ and R ⁵ are H, R ⁶ isAnd R' is

In embodiments, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isAnd R' is

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isAnd R' is

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isAnd R' is

In embodiments, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl.

In embodiments, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, m is 2, and p is 2. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, L ₁ is a bond, R ³ is-OC (O) R', R ², and R ⁴ are OMe.

In embodiments, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, m is 2.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, m is 2.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In an embodiment, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, m is 2.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, m is 2.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ¹、R⁴ and R ⁵ are H, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same. In some embodiments, m is 2.

In an embodiment, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In an embodiment, x=o, L ₁ is a bond, R ³ is-OC (O) R', R ² is OMe, R ¹、R⁴ and R ⁵ are H, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isR' isAnd R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In embodiments, x=o, L ₁ is C ₂ alkenyl, R ³ is-OC (O) R', R ² and R ⁴ are OMe, R ¹ and R ⁵ are H, R ⁶ isR' isR ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted by-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl and m is 2. In some embodiments, R ⁷ and R ⁸ are the same.

In any of the above embodiments wherein R ⁷ and R ⁸ are (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, R ^aa can alternatively be C ₁-C₄₀ alkyl.

In any of the above embodiments wherein R ⁷ and R ⁸ are (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, R ^aa can alternatively be C ₁-C₃₀ alkyl.

In any of the above embodiments wherein R ⁷ and R ⁸ are (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, R ^aa can alternatively be C ₁-C₂₀ alkyl.

In any of the embodiments wherein R ⁷ and R ⁸ are (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, R ⁷ and R ⁸ may each be

In any of the embodiments wherein R ⁷ and R ⁸ are each (C ₁-C₆) alkyl substituted with-CO ₂R^aa, wherein R ^aa is C ₁-C₅₀ alkyl, R ⁷ and R ⁸ may each be

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (II):

Or a pharmaceutically acceptable salt thereof, wherein R ¹-R⁶ and X are as defined herein.

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIA):

Or a pharmaceutically acceptable salt thereof, wherein R', R ⁶ and X are as defined herein.

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

Or a pharmaceutically acceptable salt thereof.

In an embodiment, the cationic lipids of the present invention have the structure:

Or a pharmaceutically acceptable salt thereof.

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIF):

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIG):

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIH):

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

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (III):

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIIA):

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IIIB):

Or a pharmaceutically acceptable salt thereof, wherein R ^A、R^B and p are as defined herein. In an embodiment, the cationic lipids of the present invention have the structure:

Or a pharmaceutically acceptable salt thereof, wherein R ^A and R ^B are as defined herein. In an embodiment, the cationic lipids of the present invention have the structure:

Or a pharmaceutically acceptable salt thereof.

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

In an embodiment, the cationic lipid of the present invention comprises a compound having a structure according to formula (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ) or (IIIK):

Or a pharmaceutically acceptable salt thereof.

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

In an embodiment, the cationic lipids of the present invention comprise a compound having a structure according to formula (IV):

wherein M is selected from H, OH, OMe or Me,

Or a pharmaceutically acceptable salt thereof, wherein R ^A、R^B, m, and p are as defined herein.

In embodiments the cationic lipids of the present invention comprise compounds having a structure according to formula (VI), (VII), (VIII), (IX) or (X):

Or a pharmaceutically acceptable salt thereof,

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

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

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

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

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

In an embodiment, 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 an embodiment, the composition is a lipid nanoparticle. In embodiments, the one or more cationic lipids comprise about 30mol% to 60mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids comprise 10mol% to 50mol% of the lipid nanoparticle. In embodiments, the one or more PEG-modified lipids constitute 1mol% to 10mol% of the lipid nanoparticle. In an embodiment, the cholesterol-based lipids constitute 10mol% to 50mol% of the lipid nanoparticle. In an embodiment, the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein. In an embodiment, the percentage of encapsulation of mRNA by the lipid nanoparticle is at least 70%. In an embodiment, the percentage of encapsulation of mRNA by the lipid nanoparticle is at least 75%. In embodiments, the percent encapsulation of mRNA by the lipid nanoparticle is at least 80%. In an embodiment, the percent encapsulation of mRNA by the lipid nanoparticle is at least 85%. In an embodiment, the percentage of encapsulation of mRNA by the lipid nanoparticle is at least 90%. In an embodiment, the percentage of encapsulation of mRNA by the lipid nanoparticle is at least 95%.

In an embodiment, the composition according to any one of the preceding embodiments is for use in therapy.

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

In embodiments, the composition is administered intravenously, intrathecally, or intramuscularly, or by pulmonary delivery, optionally by nebulization.

Exemplary Compounds

Exemplary compounds include exemplary compounds described in tables 1-8

TABLE 1

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

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

Unless otherwise stated, r=c ₁₆H₂₉ has the structure:

Unless otherwise stated, r=c ₁₆H₃₁ has the structure:

The compounds of the invention as described herein may be prepared according to methods known in the art, including exemplary syntheses of the examples provided herein.

Nucleic acid

The compounds of the invention as described herein may be used to prepare compositions useful for delivering nucleic acids.

Synthesis of nucleic acids

The nucleic acid according to the invention can be synthesized according to any known method. For example, mRNA according to the invention can be synthesized by In Vitro Transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template comprising a promoter, a pool of ribo-triphosphates, a buffer system possibly comprising DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, mutated T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase and/or RNAse inhibitor. The exact conditions will vary depending on the particular application.

In some embodiments, to prepare an mRNA according to the invention, a DNA template is transcribed in vitro. Suitable DNA templates typically have a promoter for in vitro transcription, e.g., a T3, T7, mutated T7 or SP6 promoter, followed by the desired nucleotide sequence and termination signal of the desired mRNA.

The desired mRNA sequence according to the invention can be determined using standard methods and incorporated into a DNA template. For example, starting from a desired amino acid sequence (e.g., an enzyme sequence), virtual reverse translation is performed based on the degenerate genetic code. An optimization algorithm can then be used to select the appropriate codon. In general, the G/C content can be optimized on the one hand to achieve as high a G/C content as possible, and on the other hand to take into account the frequency of tRNA as much as possible in terms of codon usage. The optimized RNA sequence can be created and displayed, for example, by means of a suitable display device, and compared to the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties or regions of RNA, respectively.

Modified mRNA

In some embodiments, an mRNA according to the invention may be synthesized as an unmodified mRNA or a modified mRNA. The modified mRNA contains nucleotide modifications in the RNA. Thus, modified mRNA according to the invention may include nucleotide modifications, e.g., backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), including, but not limited to, purine (adenine (A), guanine (G)) or pyrimidine (thymine (T), cytosine (C), uracil (U)), and derivatives of either purine and pyrimidine as modified nucleotide analogs, e.g., 1-methyl-adenine, 2-methylsulfanyl-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2, 6-diaminopurine, 1-methyl-guanine, 2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-cytosine, 2-methyl-uracil, 5-bromo-5-amino-uracil, 5-fluoro-carboxy-5-methyl-uracil, 5-bromo-amino-uracil, 5-fluoro-methyl-uracil, 5-bromo-amino-5-methyl-uracil, bromo-5-uracil, 5-methyl-uracil, and the like, 5-methyl-uracil, methyl N-uracil-5-oxoacetate, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5' -methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxoacetate, uracil-5-oxoacetic acid (v), 1-methyl-pseudouracil, pigtail glycoside, beta-D-mannosyl-pigtail glycoside, huai Dingyang glycoside, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. patent No. 4,373,071, U.S. patent No. 4,401,796, U.S. patent No. 4,415,732, U.S. patent No. 4,458,066, U.S. patent No. 4,500,707, U.S. patent No. 4,668,777, U.S. patent No. 4,973,679, U.S. patent No. 5,047,524, U.S. patent No. 5,132,418, U.S. patent No. 5,153,319, U.S. patent nos. 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

Pharmaceutical formulations of cationic lipids and nucleic acids

In certain embodiments, the compounds of the invention as described herein, as well as pharmaceutical and liposome compositions comprising such lipids, can be used in formulations to facilitate delivery of encapsulating material (e.g., one or more polynucleotides, such as mRNA) to one or more target cells and subsequent transfection. For example, in certain embodiments, the cationic lipids (and compositions comprising such lipids, such as liposome compositions) described herein are characterized by one or more of receptor-mediated endocytosis, clathrin-mediated and pit-mediated endocytosis, phagocytosis, and macropolytics, fusogenic, endosomal or lysosomal destruction, and/or releasable properties that provide advantages of such compounds over other similarly classified lipids.

According to the invention, nucleic acids as described herein, e.g., mRNA encoding a protein (e.g., full length, fragment, or portion of a protein) may be delivered by a delivery vehicle comprising a compound of the invention as described herein.

As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle," or grammatical equivalents thereof are used interchangeably.

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

In certain embodiments, the compositions exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Thus, also provided herein are methods of transfecting one or more target cells. Such methods generally include the step of contacting one or more target cells with a cationic lipid and/or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a compound described herein encapsulating one or more polynucleotides), such that the one or more target cells are transfected with a material (e.g., one or more polynucleotides) encapsulated therein. As used herein, the term "transfection" refers to the introduction of one or more encapsulating materials (e.g., nucleic acids and/or polynucleotides) into a cell, or preferably into a target cell, within a cell. The introduced polynucleotide may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulating material (e.g., polynucleotide) taken up, introduced and/or expressed by the target cells undergoing transfection. In fact, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cells after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the likelihood that an appropriate dose of encapsulating material (e.g., one or more polynucleotides) will be delivered to a pathological site and subsequently expressed, while minimizing potential systemic side effects or toxicity associated with the compound or its encapsulated content.

After transfection of one or more target cells by, for example, polynucleotides encapsulated in one or more lipid nanoparticles comprising the pharmaceutical or liposomal compositions disclosed herein, production of products (e.g., polypeptides or proteins) encoded by such polynucleotides may preferably be stimulated, and the ability of such target cells to express the polynucleotides, as well as produce, for example, polypeptides or proteins of interest, is enhanced. For example, transfection of a target cell with one or more compounds or pharmaceutical compositions that encapsulate mRNA will enhance (i.e., increase) the production of a protein or enzyme encoded by such mRNA.

In addition, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to preferentially distribute to other target tissues, cells, or organs, such as the heart, lung, kidney, 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 compounds or pharmaceutical compositions and liposome compositions described herein can be delivered to and/or transfected with a target cell or target tissue. In some embodiments, the encapsulated polynucleotide (e.g., mRNA) is capable of being expressed by and produced (and in some cases secreted) by a target cell, thereby imparting beneficial properties to, for example, the target cell or target tissue. Such encapsulated polynucleotides (e.g., mRNA) may encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest.

Liposome delivery vehicles

In some embodiments, the composition is a suitable delivery vehicle. In embodiments, the composition is a liposome delivery vehicle, e.g., a lipid nanoparticle.

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

The use of one or more cationic lipid-enriched liposome compositions of the cationic lipids disclosed herein can be used as a means to improve (e.g., reduce) toxicity or otherwise impart one or more desired properties to such enriched liposome compositions (e.g., improve delivery of the encapsulated polynucleotide to one or more target cells and/or reduce in vivo toxicity of the liposome composition). Thus, pharmaceutical compositions, particularly liposome compositions, comprising one or more of the cationic lipids disclosed herein are also contemplated.

Thus, in certain embodiments, the compounds of the invention as described herein may be used as components of liposome compositions to facilitate or enhance delivery and release of an encapsulating material (e.g., one or more therapeutic agents) to one or more target cells (e.g., by permeation through or fusion with the lipid membrane of such target cells).

As used herein, a liposome delivery vehicle (e.g., a lipid nanoparticle) is generally characterized as a microscopic vesicle having an internal aqueous space that is isolated from an external medium by one or more bilayer membranes. Bilayer membranes of liposomes are typically formed from amphiphilic molecules, such as synthetic or naturally derived lipids comprising spatially separated hydrophilic and hydrophobic domains (Lasic, trends biotechnol.,16:307-321,1998). The bilayer membrane of the liposome may also be formed from amphiphilic polymers and surface active substances (e.g., polymer vesicles, liposomes, etc.). In the context of the present invention, a liposome delivery vehicle is typically used to transport the desired mRNA to a target cell or tissue.

In certain embodiments, such compositions (e.g., liposome compositions) load or otherwise encapsulate materials, such as one or more bioactive polynucleotides (e.g., mRNA).

In embodiments, the composition (e.g., pharmaceutical composition) comprises an mRNA encoding a protein encapsulated within 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, and wherein at least one cationic lipid is a compound of the invention as described herein. In embodiments, the composition comprises mRNA encoding a protein (e.g., any of the proteins described herein). In an embodiment, the composition comprises an mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In an embodiment, the composition comprises an mRNA encoding an Ornithine Transcarbamylase (OTC) protein.

In embodiments, a composition (e.g., a pharmaceutical composition) includes a nucleic acid encapsulated in a liposome, wherein the liposome comprises a compound described herein.

In embodiments, the nucleic acid is an mRNA encoding a peptide or protein. In embodiments, the mRNA encodes a peptide or protein (e.g., the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein) for delivery to or treatment of a lung or lung cell of a subject. In embodiments, the mRNA encodes a peptide or protein (e.g., the mRNA encodes an Ornithine Transcarbamylase (OTC) protein) for delivery to or treatment of a liver or hepatocyte of a subject. Other exemplary mRNAs are also described herein.

In embodiments, the liposome delivery vehicle (e.g., lipid nanoparticle) can have a net positive charge.

In embodiments, the liposome delivery vehicle (e.g., lipid nanoparticle) can have a net negative charge.

In embodiments, the liposome delivery vehicle (e.g., lipid nanoparticle) can have a net neutral charge.

In embodiments, lipid nanoparticles encapsulating a nucleic acid (e.g., mRNA encoding a peptide or protein) comprise one or more compounds of the invention as described herein.

For example, the amount of a compound of the invention in a composition as described herein can be described as a percentage 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) ("wt%").

In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in an amount of about 0.5wt% to about 30wt% (e.g., about 0.5wt% to about 20 wt%) of the combined dry weight of all lipids present in the composition (e.g., liposome composition).

In embodiments, the compounds of the invention as described herein are present in an amount of about 1wt% to about 30wt%, about 1wt% to about 20wt%, about 1wt% to about 15wt%, about 1wt% to about 10wt%, or about 5wt% to about 25wt% of the combined dry weight of all lipids present in the composition (e.g., liposome composition). In embodiments, the compounds of the invention as described herein are present in an amount of about 0.5wt% to about 5wt%, about 1wt% to about 10wt%, about 5wt% to about 20wt%, or about 10wt% to about 20wt% of the combined dry weight of all lipids present in the composition (e.g., liposome delivery vehicle).

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

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

In embodiments, the composition (e.g., liposome delivery vehicle, such as lipid nanoparticle) comprises about 0.1wt% to about 20wt% (e.g., about 0.1wt% to about 15 wt%) of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposome delivery vehicle, such as a lipid nanoparticle) comprises about 0.5wt%, about 1wt%, about 3wt%, about 5wt%, or about 10wt% of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposome delivery vehicle, such as a lipid nanoparticle) comprises up to about 0.5wt%, about 1wt%, about 3wt%, about 5wt%, about 10wt%, about 15wt%, or about 20wt% of a compound described herein. In embodiments, this percentage results in improved beneficial effects (e.g., improved delivery to the target tissue (such as liver or lung)).

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

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

In embodiments, the compounds of the invention as described herein are present in an amount of about 0.5mol% to about 5mol%, about 1mol% to about 10mol%, about 5mol% to about 20mol%, about 10mol% to about 20mol%, about 15mol% to about 30mol%, about 20mol% to about 35mol%, about 25mol% to about 40mol%, about 30mol% to about 45mol%, about 35mol% to about 50mol%, about 40mol% to about 55mol%, or about 45mol% to about 60mol% of the combined molar amount of all lipids present in the composition (e.g., liposome delivery vehicle). In embodiments, the compounds of the invention as described herein are present in an amount of about 1mol% to about 60mol%, 1mol% to about 50mol%, 1mol% to about 40mol%, 1mol% to about 30mol%, about 1mol% to about 20mol%, about 1mol% to about 15mol%, about 1mol% to about 10mol%, about 5mol% to about 55mol%, about 5mol% to about 45mol%, about 5mol% to about 35mol%, or about 5mol% to about 25mol% of the combined molar amount of all lipids present in the composition (e.g., liposome delivery vehicle).

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

In certain embodiments, the compounds of the present invention as described herein may comprise greater than about 0.1mol%, or greater than about 0.5mol%, or greater than about 1mol%, greater than about 5mol%, greater than about 10mol%, greater than about 20mol%, greater than about 30mol%, or greater than about 40mol% of the total amount of lipids in the lipid nanoparticle.

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

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

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

In embodiments, this percentage results in improved beneficial effects (e.g., improved delivery to the target tissue (such as liver or lung)).

In a typical embodiment, the compositions (e.g., liposome compositions) of the present invention comprise 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, compositions suitable for practicing the present invention have four lipid components, including the compounds of the present invention described herein as cationic lipid components, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids. The non-cationic lipid may be DOPE or DEPE. The cholesterol-based lipid may be cholesterol. The PEG modified lipid may be DMG-PEG2K.

In an embodiment, the composition of the invention comprises the cationic lipid of the invention, DMG-PEG2000, cholesterol and DOPE, and the molar ratio of cationic lipid to DMG-PEG2000 to cholesterol to DOPE is 40:5:25:30.

In further embodiments, the pharmaceutical (e.g., liposome) composition comprises one or more of a PEG-modified lipid, a non-cationic lipid, and a cholesterol lipid. In other embodiments, such pharmaceutical (e.g., liposome) compositions include 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., liposome) compositions include one or more PEG-modified lipids and one or more cholesterol lipids.

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

In embodiments, compositions (e.g., lipid nanoparticles) encapsulating nucleic acids (e.g., mRNA encoding a peptide or protein) include one or more compounds of the invention described herein, one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and pegylated lipids, and further include cholesterol-based lipids. Typically, such compositions have four lipid components, including as cationic lipid components the compounds of the invention as described herein, non-cationic lipids (e.g., DOPE), cholesterol-based lipids (e.g., cholesterol), and PEG-modified lipids (e.g., DMG-PEG 2K).

In embodiments, a composition (e.g., a lipid nanoparticle) encapsulating a nucleic acid (e.g., mRNA encoding a peptide or protein) includes one or more compounds of the invention described herein, and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, pegylated lipids, and cholesterol-based lipids.

According to various embodiments, the selection of cationic lipids, non-cationic lipids, and/or PEG-modified lipids comprising the lipid nanoparticle, and the relative molar ratio of these lipids to each other is based on the characteristics of the selected lipids, the properties of the intended target cell, the characteristics of the mRNA to be delivered. Other considerations include, for example, the saturation of the alkyl chain, the size, charge, pH, pKa, fusibility, and toxicity of the lipid selected. Thus, the molar ratio can be adjusted accordingly.

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

Cationic lipids

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

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

Suitable additional cationic lipids for use in the composition comprise the cationic lipids described in the literature.

Helper lipids

The composition (e.g., liposome composition) can also include 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 variety of lipid materials that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), 1, 2-diglycol-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, L-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. A non-cationic or auxiliary lipid suitable for use in the practice of the present invention is dioleoyl phosphatidylethanolamine (DOPE). Alternatively, 1, 2-diethylene glycol-sn-glycerol-3-phosphoethanolamine (DEPE) may be used as a non-cationic or auxiliary lipid.

In some embodiments, the non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge under the conditions of formulation and/or administration of the composition.

In some embodiments, the non-cationic lipid may be present in a molar ratio (mole%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipid present in the composition. In some embodiments, the total non-cationic lipids can be present in a molar ratio (mole%) 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 the liposomes can be greater than about 5 mole%, greater than about 10 mole%, greater than about 20 mole%, greater than about 30 mole%, or greater than about 40 mole%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be greater than about 5 mole%, greater than about 10 mole%, greater than about 20 mole%, greater than about 30 mole%, or greater than about 40 mole%. In some embodiments, the percentage of non-cationic lipids in the liposomes is no more than about 5 mole%, no more than about 10 mole%, no more than about 20 mole%, no more than about 30 mole%, or no more than about 40 mole%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be no more than about 5 mole%, no more than about 10 mole%, no more than about 20 mole%, no more than about 30 mole%, or no more than about 40 mole%.

In some embodiments, the non-cationic lipid may 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 lipid present in the composition. In some embodiments, the total 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 the 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 the 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 non-cationic lipids in the liposomes is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

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-ethylcarboxamide cholesterol), 1, 4-bis (3-N-oleylamino-propyl) piperazine (Gao et al, "Biochem. Biophys. Res. Comm.)" (179, 280 (1991); wolf et al, "biotechnology (Biotechnology); 23,139 (1997); U.S. Pat. No. 5,744,335), or Imidazole Cholesterol Ester (ICE) having the following structure:

In some embodiments, the cholesterol-based lipids may be present in a molar ratio (mole%) of about 1% to about 30% or about 5% to about 20% of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticle may be greater than about 5 mole%, greater than about 10 mole%, greater than about 20 mole%, greater than about 30 mole%, or greater than about 40 mole%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticle may be no more than about 5 mole%, no more than about 10 mole%, no more than about 20 mole%, no more than about 30 mole%, or no more than about 40 mole%.

In some embodiments, the cholesterol-based lipids may 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 the liposome. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticle may be greater than about 5wt%, 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 the lipid nanoparticle may be no more than about 5wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

Pegylated lipids

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

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

Contemplated additional PEG-modified lipids (also referred to herein as pegylated lipids, which term is interchangeable with PEG-modified lipids) include, but are not limited to, polyethylene glycol chains up to 5kDa in length covalently attached to lipids having alkyl chains of C ₆-C₂₀ length. In some embodiments, the PEG-modified lipid or pegylated lipid is pegylated cholesterol or PEG-2K. The addition of such 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) in the european society of biochemistry (FEBS Letters), 268 (1): 235-237), or the components can be selected to rapidly change out of the formulation in vivo (see U.S. patent No. 5,885,613).

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

Pharmaceutical formulation and therapeutic use

The compounds of the invention as described herein can be used to prepare compositions (e.g., to construct liposome compositions) that facilitate or enhance delivery and release of encapsulating material (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeation or fusion with the lipid membrane of such target cells).

For example, when a liposome composition (e.g., a lipid nanoparticle) comprises or is otherwise enriched for one or more of the compounds disclosed herein, a phase change in the lipid bilayer of one or more target cells can facilitate delivery of an encapsulating material (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) into the one or more target cells.

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

Thus, pharmaceutical formulations comprising the described compounds and the nucleic acids provided herein may be used for a variety of therapeutic purposes. To facilitate delivery of the nucleic acids in vivo, the compounds and nucleic acids described herein may be formulated in combination with one or more additional pharmaceutically acceptable carriers, targeting ligands, or stabilizers. In some embodiments, the compounds described herein may be formulated by pre-mixed lipid solutions. In other embodiments, the post-insertion techniques may be used to formulate compositions comprising the compounds described herein into lipid membranes of nanoparticles. Techniques for formulating and administering pharmaceuticals are found in "Remington' sPharmaceutical Sciences," Mack Publishing co., easton, pa., latest edition.

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

A common route of administration of the liposome composition of the present invention may be intravenous delivery, particularly when treating metabolic disorders, especially those affecting the liver (e.g., ornithine Transcarbamylase (OTC) deficiency). Alternatively, the liposome composition can be administered by pulmonary delivery (e.g., for treating cystic fibrosis), depending on the disease or disorder to be treated. For vaccination, the liposome compositions of the invention are typically administered intramuscularly. Diseases or conditions affecting the eye can be treated by intravitreal administration of the liposome compositions of the present invention.

Alternatively or in addition, the pharmaceutical formulation of the invention may be administered in a local rather than systemic manner, for example by direct injection of the pharmaceutical formulation into the targeted tissue, preferably in the form of a slow release formulation. Local delivery can be affected in various ways depending on the tissue to be targeted. Exemplary tissues in which the delivered mRNA may be delivered and/or expressed include, but are not limited to, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and/or cerebrospinal fluid. In an embodiment, the tissue to be targeted is in the liver. For example, aerosols containing the compositions of the invention may be inhaled (for nasal, tracheal or bronchial delivery), the compositions of the invention may be injected, for example, into the site of injury, disease manifestations or pain, the compositions may be provided in the form of lozenges for oral, tracheal or esophageal applications, may be supplied to the stomach or intestine in liquid, tablet or capsule form, may be supplied to rectal or vaginal applications in the form of suppositories, or may even be delivered to the eye by use of creams, drops or even injections.

The compositions described herein can include an mRNA encoding a peptide, including a peptide (e.g., a polypeptide, such as a protein) described herein.

In embodiments, the mRNA encodes a polypeptide.

In embodiments, the mRNA encodes a protein.

Described herein are exemplary peptides encoded by mRNA (e.g., exemplary proteins encoded by mRNA).

The present invention provides methods for delivering a composition having a full-length mRNA molecule encoding a peptide or protein of interest for use in treating a subject, such as a human subject or cells of a human subject, or cells that have been treated and delivered to a human subject.

Thus, in certain embodiments, the invention provides methods for preparing a therapeutic composition comprising full-length mRNA encoding a peptide or protein for delivery to or treatment of a lung or lung cell of a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding ATP-binding cassette subfamily a member 3 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding the motor protein axon middle chain 1 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding the motor protein axon heavy chain 5 (DNAH 5) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding alpha-1-antitrypsin protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding fork cassette P3 (FOXP 3) protein. In certain embodiments, the invention provides methods for producing therapeutic compositions having full-length mRNA encoding one or more surface-active proteins (e.g., one or more of surface-active protein a, surface-active protein B, surface-active protein C, and surface-active protein D).

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

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with urea cycle disorders. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding Ornithine Transcarbamylase (OTC) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding argininosuccinate synthetase 1 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding carbamoyl phosphate synthetase I protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding argininosuccinate lyase proteins. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding arginase proteins.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with a lysosomal storage disorder. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an alpha-galactosidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding glucocerebrosidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding isocyanate-2-sulfatase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding iduronidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding N-acetyl-alpha-D-glucosidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a heparan N-sulfatase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a galactosamine-6 sulfatase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a β -galactosidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a lysosomal lipase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding Transcription Factor EB (TFEB).

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with a glycogen storage disorder. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an acid alpha-glucosidase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a glucose-6-phosphatase (G6 PC) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a liver glycogen phosphorylase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a muscle phosphoglycerate mutein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a glycogenolytic branching enzyme.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with amino acid metabolism. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding phenylalanine hydroxylase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding glutaryl-CoA dehydrogenase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding propionyl-CoA carboxylase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an oxalate alanine-glyoxylate aminotransferase.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with a disorder of lipid metabolism or fibrosis. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an mTOR inhibitor. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding ATPase phospholipid transporter 8B1 (ATP 8B 1) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding one or more NF- κB inhibitors, such as one or more of I- κBα, interferon-related developmental regulator 1 (IFRD 1), and Sirtuin 1 (SIRT 1). In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding PPAR-gamma proteins or active variants.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein associated with methylmalonic acid. For example, in certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a methylmalonyl-coa mutant enzyme protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a methylmalonyl-coa epimerase protein.

In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mRNA for which delivery to or treatment of the liver may provide therapeutic benefits. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding ATP7B protein (also known as Wilson's disease protein). In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding porphobilinogen deaminase. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding one or more clotting enzymes, such as factor VIII, factor IX, factor VII, and factor X. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding human Hemochromatosis (HFE) proteins.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of cardiovascular system or cardiovascular cells in a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding vascular endothelial growth factor a protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a relaxin protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding bone morphogenic protein 9 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a bone morphogenic protein 2 receptor protein.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of a muscle or muscle cell of a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a dystrophin protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of a myocardium or cardiomyocyte in a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding proteins that modulate one or both of potassium and sodium channels in muscle tissue or muscle cells. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein that modulates kv7.1 channels in muscle tissue or muscle cells. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a protein that modulates the nav1.5 channel in muscle tissue or muscle cells.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of a nervous system or nervous system cells of a subject. For example, in certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a survivin motor neuron 1 protein. For example, in certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a survivin motor neuron 2 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a human mitochondrial protein (frataxin). In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding ATP-binding cassette subfamily D member 1 (ABCD 1) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding CLN3 protein.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of blood or bone marrow or blood cells or bone marrow cells of a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding beta globulin. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a bruton's tyrosine kinase protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding one or more clotting enzymes, such as factor VIII, factor IX, factor VII, and factor X.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of a kidney or kidney cell of a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding type IV collagen α5 chain (COL 4 A5) protein.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivery to or treatment of an eye or eye cell of a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding ATP-binding cassette subfamily a member 4 (ABCA 4) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a retinol chitins protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a retinal pigment epithelium-specific 65kDa (RPE 65) protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding the 290kDa centrosome protein (CEP 290).

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding a peptide or protein for delivering a vaccine to a subject or cells of a subject or for treatment with a vaccine. For example, in certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from an infectious source such as a virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from an influenza virus. In certain embodiments, the invention provides methods of producing therapeutic compositions having full-length mRNA encoding an antigen from respiratory syncytial virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from rabies virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the invention provides methods of producing therapeutic compositions having full-length mRNA encoding an antigen from a rotavirus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a hepatitis virus, such as hepatitis a virus, hepatitis b virus, or hepatitis c virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from human papillomavirus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a human interstitial pneumovirus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a malaria virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from a zika virus. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen from chikungunya virus.

In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding an antigen associated with cancer in a subject or an antigen identified from cancer cells in a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen determined from cancer cells of the subject itself, i.e., providing a personalized cancer vaccine. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antigen expressed from a mutated KRAS gene.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antibody to OX 40. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to VEGF. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding antibodies to tissue necrosis factor α. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antibody to CD 3. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an antibody to CD 19.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an immunomodulatory agent. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding interleukin 12. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding interleukin 23. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding interleukin 36 γ. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding constitutively active variants of one or more interferon gene (STING) protein stimulators.

In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding an endonuclease. In certain embodiments, the invention provides methods for preparing a therapeutic composition having full-length mRNA encoding an RNA-guided DNA endonuclease protein, such as a Cas 9 protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a meganuclease protein. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full-length mRNA encoding a transcription activator-like effector nuclease protein. In certain embodiments, the invention provides a method of preparing a therapeutic composition having full-length mRNA encoding a zinc finger nuclease protein.

Delivery method

The delivery route used in the methods of the invention allows for non-invasive self-administration of the compounds of the invention. In some embodiments, the methods involve intratracheal or pulmonary administration by nebulization, gasification, or instillation of a composition comprising mRNA encoding the therapeutic protein in a suitable transfection or lipid carrier vehicle, as described above. In some embodiments, the protein is encapsulated by a liposome. In some embodiments, the liposome comprises a lipid, which is a compound of the invention. As used hereinafter, the administration of the compounds of the present invention comprises the administration of a composition comprising the compounds of the present invention.

Although local cells and tissues of the lung represent potential targets for biological reservoirs or reservoirs for production and secretion of proteins encoded by mRNA, applicants have found that administration of the compounds of the invention to the lung by nebulization, vaporization or instillation results in even non-secreted proteins being distributed outside the lung cells. Without wishing to be bound by any particular theory, it is contemplated that nanoparticle compositions of the present invention achieve complete nanoparticle transfer to non-lung cells and tissues, e.g., heart, liver, spleen, through the lung airway-blood barrier, which results in the production of encoded proteins in these non-lung tissues. Thus, the compounds of the invention and methods of the invention find use beyond the production of therapeutic proteins in lung cells and lung tissue, and may be used for delivery to non-lung target cells and/or tissues. They are useful in the management and treatment of a wide variety of diseases, and in particular peripheral diseases caused by secreted and non-secreted proteins and/or enzyme deficiency (e.g., one or more lysosomal storage disorders). In certain embodiments, the compounds of the invention used in the methods of the invention achieve distribution of mRNA encapsulated nanoparticles in liver, spleen, heart and/or other non-lung cells and production of encoded proteins in liver, spleen, heart and/or other non-lung cells. For example, administration of a compound of the invention to the lung by nebulization, seven-component or instillation will result in the composition itself and its protein products (e.g., functional β -galactosidase protein) being detectable in local cells and tissues of the lung as well as peripheral target cells, tissues and organs due to translocation of mRNA and delivery vehicle to non-lung cells.

In certain embodiments, the compounds of the invention may be used in the methods of the invention to specifically target peripheral cells or tissues. After pulmonary delivery, it is contemplated that the compounds of the invention cross the pulmonary airway-blood barrier and are distributed into cells other than local lung cells. Thus, the compounds disclosed herein can be administered to a subject by pulmonary administration using a variety of methods known to those of skill in the art (e.g., by inhalation), and distributed to localized target cells and tissues of the lung, as well as cells in peripheral non-lung cells and tissues (e.g., liver, spleen, kidney, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid and plasma). Thus, both local cells and peripheral non-lung cells of the lung may act as a biological reservoir or reservoir capable of producing and/or secreting a translation product encoded by one or more polynucleotides. Thus, the invention is not limited to the treatment of lung diseases or conditions, but may be used as a non-invasive means of facilitating delivery of polynucleotides or production of enzymes and proteins encoded thereby in peripheral organs, tissues and cells (e.g., hepatocytes) that would otherwise be achievable only by systemic administration. Exemplary peripheral non-lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

After administration of the composition to the subject, a protein product (e.g., a functional protein or enzyme) encoded by the mRNA can be detected in the peripheral target tissue at least about one to seven days or more after administration of the compound to the subject. The amount of protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the protein encoded, and the condition of the patient. For example, a protein product may be detected in a peripheral target tissue at a concentration (e.g., therapeutic 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, at least 1.3 μg/ml, at least 1.4 μg/ml, or at least 1.5 μg/ml) for at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45 days or more after administration of the compound to a subject.

It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of an aerosol mist generated by a liquid nebulizer or using a dry powder device such as the device described in U.S. patent 5,780,014, incorporated herein by reference.

In certain embodiments, the compounds of the present invention may be formulated such that they may be aerosolized or otherwise delivered as a particulate liquid or solid prior to or after administration to a subject. Such compounds may be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., atomized aqueous solutions or suspensions) to produce particles that are readily 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 administration of a predetermined mass, volume, or dose of the composition (e.g., about 0.5mg/kg of mRNA per dose) to a subject. For example, in certain embodiments, a compound of the present invention is 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 may be formulated as a particulate powder (e.g., respirable dry particles) for inhalation. In certain embodiments, the compositions of the invention formulated as inhalable particles are of a suitable size such that they can be inhaled by a subject or delivered using a suitable device (e.g., an average D50 or D90 particle size of less than about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or less). In still other embodiments, the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, a compound of the invention is administered to a subject such that the concentration administered in a single dose is at least 0.05mg/kg, at least 0.1mg/kg, at least 0.5mg/kg, at least 1.0mg/kg, at least 2.0mg/kg, at least 3.0mg/kg, at least 4.0mg/kg, at least 5.0mg/kg, at least 6.0mg/kg, at least 7.0mg/kg, at least 8.0mg/kg, at least 9.0mg/kg, at least 10mg/kg, at least 15mg/kg, at least 20mg/kg, at least 25mg/kg, At least 30mg/kg, at least 35mg/kg, at least 40mg/kg, at least 45mg/kg, at least 50mg/kg, at least 55mg/kg, at least 60mg/kg, at least 65mg/kg, at least 70mg/kg, at least 75mg/kg, at least 80mg/kg, at least 85mg/kg, at least 90mg/kg, at least 95mg/kg, or at least 100mg/kg body weight. in some embodiments, a compound of the invention is administered to a subject such that the total amount administered in one or more doses is at least 0.1mg, at least 0.5mg, at least 1.0mg, at least 2.0mg, at least 3.0mg, at least 4.0mg, at least 5.0mg, at least 6.0mg, at least 7.0mg, at least 8.0mg, at least 9.0mg, at least 10mg, at least 15mg, at least 20mg, at least 25mg, at least 30mg, at least 35mg, at least 40mg, at least 45mg, at least 50mg, at least 55mg, at least 60mg, At least 65mg, at least 70mg, at least 75mg, at least 80mg, at least 85mg, at least 90mg, at least 95mg, or at least 100mg of mRNA.

Examples

While certain compounds, compositions, and methods of the present invention have been described in detail in terms of certain examples, the following examples are illustrative of the compounds of the present invention and are not intended to be limiting thereof.

Synthetic scheme of vanillic acid lipid

Synthesis of 3- (dimethylamino) propyl 4-hydroxy-3-methoxybenzoate (3)

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

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

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

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

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

All other lipids were prepared in similar yields following representative procedures.

Synthetic scheme of syringic acid lipid

Synthesis of 3- (dimethylamino) propyl 4-hydroxy-3, 5-dimethoxy benzoate (6)

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

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

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

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

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

Scheme for synthesizing sinapic acid lipid

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

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

Synthesis of (E) -4- (3- (3- (dimethylamino) propoxy) -3-oxoprop-1-en-1-yl) -2, 6-dimethoxyphenyl 4- (bis (2- ((tert-butyldimethylsilyloxy) dodecylamino) butyrate (10)

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

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

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

Synthesis of phenolic acid lipid scheme A

Synthesis of intermediate (3 a) in synthesis scheme a:

To a solution of (1) (2.00 g,2.86 mmol) in anhydrous CH ₂Cl₂ (10 mL) at 0deg.C was added dropwise oxalyl chloride (0.98 mL,4.0 eq.) followed by slow warming of the reaction mixture to room temperature and stirring for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was then redissolved in anhydrous CH ₂Cl₂ (30 mL). To the stirred acid chloride solution was added (2 a) (55mg, 1.0 eq), DMAP (349 mg,1.0 eq) and then triethylamine (3.18 ml,8.0 eq) at 0 ℃. The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature for 16 hours. After 16 hours, the reaction mixture was diluted with CH ₂Cl₂ and washed with saturated NaHCO _{3( Solution )} solution. The separated organic layer was washed with brine, dried over Na ₂SO₄, and concentrated under reduced pressure to give crude material. The crude material was first purified using 50% EtOAc in hexanes and then repurified using 15% EtOAc in CH ₂Cl₂ to give (3 a) as a viscous oil (623 mg, 25%). C ₅₀H₉₃NO₇Si₂,[M+H]⁺ = 876.65 calculated for MS (esi+), observed = 876.6.

Synthesis of intermediate (3 b) in synthesis scheme a:

The procedure of (3 a) was followed using (2 b) to provide (3 b) as a viscous oil (557 mg, 23%). C ₄₉H₉₁NO₆Si₂,[M+H]⁺ = 846.64 calculated for MS (esi+), observed = 846.6.

Synthesis of intermediate (5 a) in synthesis scheme a:

To a solution of (3 a) (308 mg,0.351 mmol) in anhydrous CH ₂Cl₂ (3 mL) at room temperature was added oxalyl chloride (0.15 mL,5.0 eq.) and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was then redissolved in anhydrous CH ₂Cl₂ (3 mL). To the stirred acid chloride solution was added 3-dimethylaminopropanol (4) (109 mg,3 eq.) followed by triethylamine (0.10 ml,2.0 eq.) at 0 ℃. The reaction mixture was slowly warmed to room temperature and then stirred at the same temperature for 16 hours. After completion of the reaction monitored by MS, the reaction mixture was concentrated to dryness under reduced pressure and purified using CH ₂Cl₂ containing 0-10% MeOH to give (5 a) as a viscous oil (204 mg, 60%). C ₅₅H₁₀₄N₂O₇Si₂,[M+H]⁺ = 961.74 calculated for MS (esi+), observed = 961.7.

Synthesis of intermediate (5 b) in synthesis scheme a:

The procedure of (5 a) was followed using (3 b) to provide (5 b) as a viscous oil (248 mg, 90%). C ₅₄H₁₀₂N₂O₆Si₂,[M+H]⁺ = 931.73 calculated for MS (esi+), observed = 931.7.

Synthesis of TBL-0731 Compound 355 (6 a) in scheme A:

To a stirred solution of (5 a) (204 mg,0.212 mmol) in anhydrous THF (3 mL) at 0 ℃ was added pyridine containing 70% hydrogen fluoride (1.09 mL,197 equivalents) dropwise, followed by slow warming to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction monitored by MS, the reaction mixture was cooled to 0 ℃ and quenched by addition of solid NaHCO ₃ in portions. After gas formation was minimized, the resulting mixture was diluted with EtOAc and neutralized with saturated NaHCO _{3( aqueous solution )} solution. The separated organic layer was washed with brine, dried over Na ₂SO₄, and concentrated under reduced pressure to give crude material. The crude material was purified using CH ₂Cl₂ with 0-20% MeOH to give TBL-0731 (6 a) (132 mg, 85%) as a viscous oil. C ₄₃H₇₆N₂O₇,[M+H]⁺ = 733.57 calculated for MS (esi+), observed value ＝733.5.¹H NMR(500MHz,CDCl₃)δ7.64(d,J＝15.9Hz,1H),7.14–7.08(m,2H),7.05(d,J＝8.1,3.5Hz,1H),6.37(d,J＝16.0Hz,1H),4.27(t,J＝6.4Hz,2H),3.86(s,3H),3.72–3.63(m,2H),2.78–2.70(m,2H),2.67–2.47(m,6H),2.46–2.40(m,2H),2.36(s,6H),2.02–1.90(m,4H),1.49–1.35(m,4H),1.34–1.15(m,32H),0.87(t,J＝6.9Hz,6H).

Synthesis of TBL-0750 Compound 467 (6 b) in scheme A:

The procedure of (6 a) was followed using (5 b) to provide TBL-0750 (6 b) (153 mg, 82%) as a viscous oil. C ₄₂H₇₄N₂O₆,[M+H]⁺ = 703.55 calculated for MS (esi+), observed value ＝703.6.¹HNMR(400MHz,CDCl₃)δ7.66(d,J＝16.0Hz,1H),7.53(d,J＝8.7,1.3Hz,2H),7.12(d,J＝8.6,1.5Hz,2H),6.38(d,J＝16.0,0.9Hz,1H),4.26(t,J＝6.4Hz,2H),3.70–3.63(m,2H),2.73–2.64(m,2H),2.64–2.46(m,6H),2.45–2.40(m,2H),2.34(s,6H),1.99–1.88(m,4H),1.43–1.34(m,4H),1.31–1.21(m,32H),0.87(t,J＝6.8Hz,6H).

Synthesis of phenolic acid lipid scheme B

Synthesis of intermediate (3) in synthesis scheme B:

To a solution of acid intermediate (1) (4.58 g) in CH ₂Cl₂ (30 mL, anhydrous) was added oxalyl chloride (1.04 mL,2 eq.) and stirred at room temperature for 2 hours. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CH ₂Cl₂ (30 mL, anhydrous). To this solution was then added syringic acid (2) (1.32 g,1.1 eq.) followed by pyridine (2.93 ml,6 eq.) and the resulting mixture was stirred at room temperature overnight. After stirring overnight, the solvent was removed under reduced pressure and the remaining residue was dissolved with a minimum of CH ₂Cl₂, then filtered through a short plug of silica gel with 100% EtOAc as eluent. The clear filtrate was concentrated to dryness to provide a crude product material that was purified over MPLC using a 10-100% EtOAc/hexanes gradient over 10CV to provide the acid product (3) (3.70 g, 78%). C ₅₃H₁₀₁NO₈Si₂,[M+H]⁺ = 936.7 calculated for MS (esi+), observed = 936.7.

Synthesis of intermediate (5 a) in synthesis scheme B:

To a solution of benzoic acid (3) (400 mg) in CH ₂Cl₂ (4 mL, anhydrous) was added oxalyl chloride (0.18 mL,5 eq.) and stirred at room temperature for 2 hours. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CH ₂Cl₂ (3 mL, anhydrous). The resulting solution was cooled with an ice bath, and then a solution of 2-aminoethanol (4 a) (76 mg) in CH ₂Cl₂ (1 mL) was added. The reaction mixture was warmed to room temperature and stirred at the same temperature for 16 hours. After complete consumption of starting material was monitored by LC-MS, the reaction mixture was concentrated to dryness and the crude material was purified over MPLC over 12CV using a 0-20% meoh/CH ₂Cl₂ gradient to afford (5 a) as a viscous oil (225 mg, 52%). C ₅₇H₁₁₀N₂O₈Si₂,[M+H]⁺ = 1007.8 calculated for MS (esi+), observed = 1007.6.

Synthesis of intermediate (5B) in synthesis scheme B:

The procedure of synthesis (5 a) was followed using 4-aminobutanol (4 b) to provide (5 b) (360 mg, 81%) as a viscous oil. C ₅₉H₁₁₄N₂O₈Si₂,[M+H]⁺ = 1035.8 calculated for MS (esi+), observed = 1035.6.

Synthesis of intermediate (5 c) in Synthesis scheme B:

The synthetic procedure of (5 a) was followed using intermediate (3) (500 mg) and 3-morpholinopropanol (4 c) to afford product (5 c) (350 mg, 62%). C ₆₀H₁₁₄N₂O₉Si₂,[M+H]⁺ = 1063.8 calculated for MS (esi+), observed = 1063.6.

Synthesis of intermediate (5 d) in Synthesis scheme B:

the synthetic procedure of (5 a) was followed using intermediate (3) (500 mg) and 2-pyridinemethanol (4 d) to afford product (5 d) (237 mg, 43%). C ₆₀H₁₀₈N₂O₈Si₂,[M+H]⁺ = 1041.8 calculated for MS (esi+), observed = 1041.6.

Synthesis of intermediate (5 e) in Synthesis scheme B:

The synthetic procedure of 5a was followed using intermediate (3) (843 mg) and 4-methylpiperazine ethanol (4 e) to afford product (5 e) (348 mg, 36%). C ₆₀H₁₁₅N₃O₈Si₂,[M+H]⁺ = 1062.8 calculated for MS (esi+), observed = 1062.7.

Synthesis of TBL-0507 Compound 48 (6 a) in Synthesis scheme B:

To a stirred solution of TBS protected intermediate (5 a) (225 mg) in THF (3 mL, anhydrous) in a plastic polymer scintillation vial (non-glass) was added triethylamine (0.16 mL,5 eq.) dropwise followed by triethylamine-3 HF (0.36 mL,10 eq.) dropwise. The reaction mixture was stirred at 50 ℃ overnight. The reaction was monitored by suspending two drops of the reaction mixture between EtOAc and NaHCO _{3( aqueous solution )} layers and analyzing the organic layer (TLC or LC-MS). Once the starting material was consumed, excess HF and volatiles were removed by blowing off with N ₂ gas in a fume hood, and the remaining material was diluted with EtOAc and neutralized with saturated aqueous NaHCO ₃ (checked with pH paper). The separated organic layer was washed with brine, dried over Na ₂SO₄, and concentrated under reduced pressure to afford crude product material. The crude material was purified over MPLC with a gradient of 0-40% MeOH/CH ₂Cl₂ over 10CV to afford TBL-0507 (6 a) (90 mg, 52%). C ₄₅H₈₂N₂O₈,[M+H]⁺ = 779.6 calculated for MS (esi+), observed = 779.5.

Synthesis of TBL-0508 Compound 49 (6B) in Synthesis scheme B:

The procedure of (6 a) was followed using TBS protected intermediate (5 b) (360 mg) to afford TBL-0508 (6 b) (71 mg, 25%). C ₄₇H₈₆N₂O₈,[M+H]⁺ = 807.6 calculated for MS (esi+), observed = 807.5.

Synthesis of TBL-0517 Compound 562 (6 c) in scheme B:

The procedure of (6 a) was followed using TBS protected intermediate (5 c) (350 mg) and purified with a gradient of 0-10% MeOH/CH ₂Cl₂ to afford TBL-0517 (6 c) (164 mg, 60%). C ₄₈H₈₆N₂O₉,[M+H]⁺ = 835.6 calculated for MS (esi+), observed = 835.5.

Synthesis of TBL-0518 Compound 563 (6 d) in Synthesis scheme B:

The procedure of (6 a) was followed using TBS protected intermediate (5 d) (237 mg) and purified with a gradient of 0-10% MeOH/CH ₂Cl₂ to afford TBL-0518 (6 d) (111 mg, 60%). C ₄₈H₈₀N₂O₈,[M+H]⁺ = 813.6 calculated for MS (esi+), observed = 813.5.

Synthesis of TBL-0535 Compound 564 (6 e) in scheme B:

The procedure of (6 a) was followed using TBS protected intermediate (5 e) (346 mg) and purification with a 0-20% MeOH/CH ₂Cl₂ gradient provided TBL-0535 (6 e) (88 mg, 32%). C ₄₈H₈₇N₃O₈,[M+H]⁺ = 834.6 calculated for MS (esi+), observed = 834.6.

Synthesis of phenolic acid lipid scheme C

To a flask containing amino acid (7) (500 mg) and dodecyl acrylate (8 a) (2.91 g,2.5 eq) was added isopropanol (5 mL) and triethylamine (1.35 mL,2 eq). The resulting mixture was heated at 90 ℃ for 3 hours. After completion of the reaction monitored by MS, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The remaining material was purified on MPLC using 0-12% MeOH/CH ₂Cl₂ to afford product (9 a) (987 mg, 35%). C ₃₄H₆₅NO₆,[M+H]⁺ = 584.5 calculated for MS (esi+), observed = 584.5.

Synthesis of intermediate (9 b) in Synthesis scheme C:

The procedure of (9 a) was followed using amino acid (7) (1.00 g), tetradecyl acrylate (8 b) (6.51 g,2.5 eq), isopropanol (10 mL) and triethylamine (2.70 mL,2 eq) to provide product (9 b) (1.80 g, 29%). C ₃₈H₇₃NO₆,[M+H]⁺ = 640.5 calculated for MS (esi+), observed = 640.5.

Synthesis of TBL-0484 compound 565 (11 a) in FIG. C:

Oxalyl chloride (1.0 ml,23 eq) was added dropwise to a solution of intermediate (9 a) (300 mg) in anhydrous CH ₂Cl₂ (3 mL) at room temperature and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was then redissolved in anhydrous CH ₂Cl₂ (3 mL). Phenolic acid (10) (146 mg,1.0 eq) and pyridine (0.21 ml,5 eq) were added to the stirred acid chloride solution at room temperature, and then stirred at the same temperature for 16 hours. After completion of the reaction monitored by MS, the reaction mixture was concentrated under reduced pressure. The remaining crude material was purified on MPLC using 0-10% meoh/CH ₂Cl₂ to afford product TBL-0484 (11 a) (90 mg, 21%). C ₄₈H₈₄N₂O₁₀,[M+H]⁺ = 849.6 calculated for MS (esi+), observed = 849.5.

Synthesis of TBL-0485 Compound 566 (11 b) in scheme C:

The procedure of (11 a) was followed using intermediate (9 b) (300 mg) to afford TBL-0485 (11 b) (80 mg, 19%). C ₅₂H₉₂N₂O₁₀,[M+H]⁺ = 905.7 calculated for MS (esi+), observed = 905.6.

Example 1 lipid nanoparticle formulation

The cationic lipids described herein can be used to prepare lipid nanoparticles according to methods known in the art. For example, suitable methods include those described in International publication No. WO 2018/089801, which is incorporated herein by reference in its entirety.

One exemplary method of lipid nanoparticle formulation is method a of WO 2018/089801 (see, e.g., example 1 and fig. 1 of WO 2018/089801). Method a ("a") involves a conventional method of encapsulating mRNA by mixing the mRNA with a lipid mixture without first preforming the lipid into lipid nanoparticles. In an exemplary method, an ethanol lipid solution and an aqueous buffer of mRNA were prepared separately. Solutions of lipid mixtures (cationic lipids, helper lipids, zwitterionic lipids, PEG lipids, etc.) were prepared by dissolving the lipids in ethanol. mRNA solutions were prepared by dissolving mRNA in citrate buffer. The mixture was then heated to 65 ℃ before mixing. The two solutions were then mixed using a pump system. In some cases, a gear pump system is used to mix the two solutions. In certain embodiments, the two solutions are mixed using a 'T' confluence (or "Y" confluence). The mixture was then purified by diafiltration by TFF method. The resulting formulation was concentrated and stored at 2-8 ℃ until further use.

A second exemplary method for lipid nanoparticle formulation is method B of WO 2018/089801 (see e.g., example 2 and fig. 2 of WO 2018/089801). Method B ("B") refers to the process of encapsulating messenger RNA (mRNA) by mixing preformed lipid nanoparticles with mRNA. A range of different conditions may be used in method B, such as varying temperatures (i.e., with or without heating the mixture), buffers, and concentrations. In an exemplary method, lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process. The resulting formulation mixture was empty lipid nanoparticles in citrate buffer containing alcohol. The formulation is then subjected to a TFF purification process, wherein buffer exchange occurs. The resulting suspension of preformed hollow lipid nanoparticles is then mixed with mRNA using a pump system. For certain cationic lipids, heating the solution after mixing results in a higher percentage of mRNA-containing lipid nanoparticles and a higher total mRNA yield.

Lipid nanoparticle formulations of table 5 were prepared by methods a or B. Each formulation included mRNA (FFL mRNA) and lipids (cationic lipid: DMG-PEG2000; cholesterol: DOPE) encoding firefly luciferase proteins in the mole% ratios listed in Table 5.

Table 5 exemplary lipid nanoparticle formulations for intratracheal administration

Delivery of FFL mRNA by intratracheal administration

By means ofA single intratracheal aerosol administration was performed to administer the lipid nanoparticle formulation (50 ul/animal) including FFL mRNA in table 5 to male CD1 mice (6-8 weeks old) under anesthesia. About 24 hours after administration, 150mg/kg (60 mg/ml) of fluorescein was administered to the animals by intraperitoneal injection at 2.5 ml/kg. After 5-15 minutes, all animals were imaged using an IVIS imaging system to measure luciferase production in the lungs. Figure 1 shows that lipid nanoparticles comprising the cationic lipids described herein are effective in delivering FFL mRNA in vivo based on positive luciferase activity.

Numbered embodiments

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

Wherein L ₁ is a bond, (C ₁-C₆) alkyl or (C ₂-C₆) alkenyl;

Wherein X is O or S;

wherein at least one of R ¹、R²、R³、R⁴ or R ⁵ is-OC (O) R';

wherein R' is

Wherein R ⁶ is

Wherein m and p are each independently 0, 1, 2, 3, 4 or 5;

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

Or a pharmaceutically acceptable salt thereof.

2. The cationic lipid of numbered embodiment 1, or a pharmaceutically acceptable salt thereof, wherein any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of (C ₁-C₆) alkyl, (C ₂-C₆) alkenyl, (C ₂-C₆) alkynyl, (C ₁-C₆) acyl, (C ₁-C₆) alkoxy, halogen 、-COR、-CO₂H、-CO₂R、-CN、-OH、-OR、-OCOR、-OCO₂R、-NH₂、-NHR、-N(R)₂、-SR, or-SO ₂ R, or two geminal hydrogens on a carbon atom are substituted with a group = NH, wherein each instance of R is independently C ₁-C₁₀ aliphatic alkyl.

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

i) R ^A and R ^B are identical and/or

Ii) R ^C and R ^D are identical.

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

i) R ^A and R ^B are different and/or

Ii) R ^C and R ^D are different.

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

6. The cationic lipid or pharmaceutically acceptable salt thereof of any one of numbered embodiments 1-4, wherein one or more of R ^A、R^B、R^C and R ^D are different.

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

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

9. The cationic lipid or pharmaceutically acceptable salt thereof of any of the preceding numbered embodiments, wherein only one of R ¹、R²、R³、R⁴ and R ⁵ is-OC (O) R'.

10. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered example 9, wherein neither R ¹、R²、R³、R⁴ nor R ⁵ is OH.

11. The cationic lipid or pharmaceutically acceptable salt thereof of any one of numbered embodiments 1-8, wherein both of R ¹、R²、R³、R⁴ and R ⁵ are-OC (O) R'.

12. The cationic lipid of numbered example 11, or a pharmaceutically acceptable salt thereof, wherein neither R ¹、R²、R³、R⁴ nor R ⁵ is OH.

13. The cationic lipid or pharmaceutically acceptable salt thereof of any one of numbered embodiments 1-8, wherein three of R ¹、R²、R³、R⁴ and R ⁵ are-OC (O) R'.

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

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

16. The cationic lipid or pharmaceutically acceptable salt thereof of any of the preceding numbered embodiments, wherein R ³ is-OC (O) R'.

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

i) p, q and r are the same, or

Ii) one or more of p, q and r are different, or

Iii) q and r are the same and p is different.

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

i) k, m and n are the same, or

Ii) one or more of k, m and n are different, or

Iii) k and n are the same and m is different.

19. The cationic lipid or pharmaceutically acceptable salt thereof according to any one of the preceding numbered embodiments, wherein m is 1, 2, or 3.

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

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

22. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered example 21, wherein:

i) k, m and n=1, or

Ii) k, m and n=1, and R ¹¹ and R ¹² =h, or

Iii) k and n=1 and m=2, or

Iv) k and n=1, m=2, and R ¹¹ and R ¹² =h, or

V) k and n=1 and m=3, or

Vi) k and n=1, m=3, and R ¹¹ and R ¹² =h.

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

24. The cationic lipid or pharmaceutically acceptable salt thereof according to numbered example 23, wherein:

i) p, q and r=1, or

Ii) p, q and r=1, and R ¹³ and R ¹⁴ are H, or

Iii) q and r=1 and p=2, or

Iv) q and r=1, p=2, and R ¹³ and R ¹⁴ are H.

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

26. The cationic lipid of numbered embodiment 25, or a pharmaceutically acceptable salt thereof, wherein R ⁶ is:

27. The cationic lipid of any one of the preceding numbered embodiments, having a structure according to formula (II):

Or a pharmaceutically acceptable salt thereof.

28. The cationic lipid according to numbered example 27, having a structure according to formula (IIA):

Or a pharmaceutically acceptable salt thereof.

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

Or a pharmaceutically acceptable salt thereof.

30. The cationic lipid according to numbered example 27, having a structure according to formula (IIF):

Or a pharmaceutically acceptable salt thereof.

31. The cationic lipid according to numbered example 27, having a structure according to formula (IIG):

Or a pharmaceutically acceptable salt thereof.

32. The cationic lipid according to numbered example 27, having a structure according to formula (IIH):

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

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

Or a pharmaceutically acceptable salt thereof.

34. The cationic lipid according to numbered example 33, having a structure according to formula (IIIA):

Or a pharmaceutically acceptable salt thereof.

35. The cationic lipid of numbered example 33 or numbered example 34 having a structure according to formula (IIIB):

Or a pharmaceutically acceptable salt thereof.

36. The cationic lipid according to numbered example 35, having a structure according to formula (IIIC):

Or a pharmaceutically acceptable salt thereof.

37. The cationic lipid according to numbered example 33, having a structure according to formula (IIID):

Or a pharmaceutically acceptable salt thereof.

38. The cationic lipid of numbered example 37 having a structure selected from formulas (IIIE), (IIIF), (IIIG), (IIIH), (IIII), (IIIJ), or (IIIK):

Or a pharmaceutically acceptable salt thereof.

39. The cationic lipid according to numbered example 33, having a structure according to formula (IIIL):

Or a pharmaceutically acceptable salt thereof.

40. The cationic lipid according to numbered example 33, having a structure according to formula (IV):

Wherein M is selected from H, OH, OMe or Me, or a pharmaceutically acceptable salt thereof.

41. The cationic lipid according to numbered example 33, having a structure according to formula (VI), (VII), (VIII), (IX) or (X):

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

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

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

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

46. A compound selected from the compounds listed in tables 1 to 8, or a pharmaceutically acceptable salt thereof.

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

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

49. The composition of example 48, wherein the one or more cationic lipids comprise about 30mol% to 60mol% of the lipid nanoparticle.

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

51. The composition of any one of numbered embodiments 48-50, wherein the one or more PEG-modified lipids comprise 1mol% to 10mol% of the lipid nanoparticle.

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

53. The composition of any one of numbered embodiments 48-52, the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.

54. The composition of any one of numbered embodiments 48-52, wherein the lipid nanoparticle encapsulates an mRNA encoding a peptide or protein.

55. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 70%.

56. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 75%.

57. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 80%.

58. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 85%.

59. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 90%.

60. The composition of numbered embodiment 54, wherein the percent encapsulation of mRNA by the lipid nanoparticle is at least 95%.

61. The composition of any one of numbered embodiments 54-60 for use in performing a treatment.

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

63. The composition for use according to numbered examples 61 or 62, wherein the composition is administered intravenously, intrathecally, or intramuscularly, or by pulmonary delivery, optionally by nebulization.

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

65. The method of numbered embodiment 64, wherein the composition is administered intravenously, intrathecally, or intramuscularly, or by pulmonary delivery, optionally by nebulization.

Claims

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

wherein L ₁ is a bond, C ₁ -C ₆ alkyl or C ₂ -C ₆ alkenyl;

Where X is O;

wherein R ¹ , R ² , R ⁴ and R ⁵ are each independently selected from H and C ₁ -C ₆ alkoxy;

wherein R ³ is -OC(O)R';

Where R' is

Where ^R6 is

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

wherein R ⁹ is an optionally substituted C ₁ -C ₆ alkyl group;

wherein R ¹⁰ is an optionally substituted C ₁ -C ₆ alkyl group;

wherein k and n are each independently 1;

wherein R ¹¹ and R ¹² are each H;

wherein ^RA and ^RB are each independently selected from optionally substituted C ₆ -C ₂₀ alkyl and optionally substituted C ₆ -C ₂₀ alkenyl;

Wherein, "optionally substituted" refers to a substituent selected from -CO ₂ R", OH and -OCOR", wherein R" is C ₁ -C ₂₀ alkyl;

or a pharmaceutically acceptable salt thereof.

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

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

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

5. The cationic lipid of claim 1 , having a structure according to:

(a) Formula (IIIA):

or a pharmaceutically acceptable salt thereof,

(c) Formula (IIID):

or a pharmaceutically acceptable salt thereof,

(d) Formula (IIA):

or a pharmaceutically acceptable salt thereof,

(e) Formula (IIF):

or a pharmaceutically acceptable salt thereof, or (f) Formula (IIG):

or a pharmaceutically acceptable salt thereof.

6. The cationic lipid according to claim 1, having a structure selected from compounds 1-12:

or a pharmaceutically acceptable salt thereof.

7. The cationic lipid according to claim 6, having the structure of compound 5:

or a pharmaceutically acceptable salt thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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