Cationic lipids based on tes

Abstract

The present invention provides, in part, TES-based lipid compounds of Formula (Ia) and subformulae thereof, or pharmaceutically acceptable salts thereof. The compounds provided herein can be used, for example, as components of liposomal delivery vehicles for the delivery and expression of mRNA and encoded proteins, and thus can be used to treat various diseases, disorders, and conditions, such as those associated with a deficiency in one or more proteins.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 082,090, filed September 23, 2020, the entire disclosure of which is hereby incorporated by reference. Background Technology

[0003] Nucleic acid delivery has been extensively explored as a potential therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for treating a variety of diseases, including those related to deficiencies in one or more proteins.

[0004] Efficient delivery of nucleic acids encapsulated in liposomes remains an active area of ​​research. Cationic lipid components play a crucial role in promoting efficient encapsulation of nucleic acids during liposome loading. Furthermore, cationic lipids can play an important role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. Various cationic lipids suitable for in vivo use have been identified. However, there is still a need to identify lipids that can be synthesized efficiently and inexpensively without forming potentially toxic byproducts.

[0005] "Good" buffers (or Good's buffers) are buffering agents originally selected and described by Norman Good and his colleagues for use in biochemical and biological research (Good, NE et al. (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry 5(2), 467-477). Most biological reactions occur at near-neutral pH between 6 and 8. Therefore, Good deduced that the ideal buffer for biochemical or biological applications would have a pKa value in this region to provide maximum buffering capacity. Other selection criteria included high solubility, non-toxicity, limited interference with biochemical reactions, very low absorbance between 240 nm and 700 nm, enzyme and hydrolytic stability, minimal variation due to temperature and concentration, limited influence due to the ionic or salt composition of the solution, limited interaction with mineral cations, and limited permeability to biological membranes.

[0006] These characteristics make "Good" buffers a particularly good starting point for the synthesis of cationic lipids for use in the in vivo environment. Many "Good" buffers remain essential tools in modern biochemistry and biology laboratories and are therefore readily available at low cost. Summary of the Invention

[0007] This invention particularly provides a novel class of cationic lipid compounds for the in vivo delivery of therapeutic agents such as nucleic acids. These compounds are envisioned to be efficiently delivered in vivo while maintaining a favorable toxicity profile.

[0008] The cationic lipids of the present invention can be synthesized from readily available starting reagents, such as Good's buffer (see Table 1). The cationic lipids of the present invention also unexpectedly exhibit high encapsulation efficiency. The cationic lipids of the present invention also contain cleavable groups (e.g., esters and disulfide bonds), which are envisioned to improve biodegradability and thus contribute to their favorable toxicity profile.

[0009] In one respect, this article provides cationic lipids having a structure according to formula (Ia):

[0010]

[0011] Or its pharmaceutically acceptable salt, wherein

[0012] R 1 R 2 and R 3 Each is selected independently from:

[0013] Optional substituted alkyl, optional substituted alkenyl, optional substituted alkynyl, and optional substituted acyl groups;

[0014] R 4 yes

[0015] Each R 5 Independently selected from optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl groups;

[0016] Each R 6 It is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, and optionally substituted C2-C6 alkenyl;

[0017] A is -NR 9 -or -O-;

[0018] D is either O or S;

[0019] E and G are each independently selected from -NR 10 -、-O- and -S-;

[0020] R 8 R 9 and R 10 Each is independently selected from hydrogen, optionally substituted C1-C6 alkyl groups, and optionally substituted C2-C6 alkenyl groups;

[0021] Each b is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and

[0022] Each c is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0023] In one respect, this article provides cationic lipids, which are pharmaceutically acceptable salts of formula (Ia).

[0024] In one aspect, this document provides compositions comprising the cationic lipids of the present invention or pharmaceutically acceptable salts thereof, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids. In another aspect, the compositions are lipid nanoparticles, optionally liposomes.

[0025] In one respect, compositions containing the cationic lipids of the present invention can be used in therapy. Brief description of the attached diagram

[0027] Figure 1 illustrates in vivo protein production generated by delivery of mRNA (i.e., FFL mRNA) using lipid nanoparticles containing compounds V, XII, or I as described herein. As shown in the figure, the use of these compounds can lead to high levels of in vivo protein production (i.e., FFL protein) after administration. Detailed Implementation

[0028] definition

[0029] To facilitate understanding of the invention, certain terms are defined below. Further definitions of the following and other terms are stated throughout the specification. Publications and other references cited herein to describe the background of the invention and to provide further details on its practice are hereby incorporated by reference.

[0030] Amino acids: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, amino acids have the universal structure H₂N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids; in some embodiments, amino acids are d-amino acids; in some embodiments, amino acids are l-amino acids. "Standard amino acid" refers to any one of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids in a peptide (including carboxyl-terminal and / or amino-terminal amino acids) can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups, which may alter the cyclic half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl, acetate, acetyl, phosphate, formyl moiety, isoprene-like group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.). The terms "amino acid" and "amino acid residue" are used interchangeably and may refer to a free amino acid and / or an amino acid residue of a peptide. It will be clear from the context in which the term is used whether it refers to a free amino acid or a peptide residue.

[0031] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0032] Approximately or about: As used herein, the term “approximately” or “about” when applied to one or more intended values ​​refers to a value similar to the stated reference value. In some embodiments, unless otherwise stated or the context clearly indicates otherwise, the term “approximately” or “about” refers to a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value (except where this number would exceed 100% of the possible value).

[0033] Biologically active: As used herein, the term "biologically active" 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 applied to that organism is considered biologically active.

[0034] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, mRNA delivery includes situations where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also known as “local distribution” or “local delivery”); and situations where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient’s circulatory system (e.g., serum) and distributed systemically and absorbed by other tissues (also known as “systemic distribution” or “systemic delivery”).

[0035] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or post-translational modifications of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production” and their grammatical equivalents are used interchangeably.

[0036] Functionality: As used herein, a “functional” biomolecule is a biomolecule that exhibits its characteristic properties and / or activities in the form in which it is formed.

[0037] Half-life: As used herein, the term “half-life” is the time required for an amount of, such as the concentration or activity of, nucleic acid or protein to decrease to half the value of the amount measured at the beginning of a time period.

[0038] Supporting lipids: As used herein, the term “supporting lipid” refers to any neutral or zwitterionic lipid material (including cholesterol). Without being bound by any particular theory, supporting lipids can increase the stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles.

[0039] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A “control subject” is a subject suffering from the same form of disease as the treated subject and of approximately the same age.

[0040] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment (e.g., in test tubes or reactor dishes, in cell cultures, etc.) rather than in multicellular organisms.

[0041] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the case of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0042] Separated: As used herein, the term “separated” means a substance and / or entity that has been (1) separated from at least some of the components associated with its original production (whether in nature or in an experimental setting) and / or (2) artificially produced, prepared, and / or manufactured. Separated substances and / or entities may be separated from other components of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of their original association. In some embodiments, 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 about 99% pure. As used herein, a substance is considered “pure” if it is substantially free of other components. As used herein, the calculation of the purity percentage of the isolated substance and / or entity should not include excipients (e.g., buffer solutions, solvents, water, etc.).

[0043] Liposomes: As used herein, the term "liposome" refers to any layered, multilayered, or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids or by mixing one or more lipids with one or more polymers. In some embodiments, liposomes suitable for use in 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.

[0044] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The term “modified mRNA” refers to mRNA containing at least one chemically modified nucleotide. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, generated and optionally purified using a recombinant expression system, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs (such as analogs of chemically modified bases or sugars), backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' orientation. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); or a nucleoside analogue (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylur ... Cytidine, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose); and / or modified phosphate groups (e.g., thiophosphate and 5'-N-phosphoramide linked).

[0045] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance incorporated into or potentially incorporated into a polynucleotide chain. In some embodiments, nucleic acid is a compound and / or substance incorporated into or potentially incorporated into a polynucleotide chain via a phosphodiester linker. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to a polynucleotide chain containing a single nucleic acid residue. In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acid (RNA), including but not limited to interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), polynucleotide coding (MCNA), polymeric RNA coding (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, DNA can be in the following forms: antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a product of polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups.In the implementation scheme, RNA may be in the following forms: messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), small nucleus RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-native antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), transacting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some implementations, nucleic acids are mRNAs that encode proteins (such as enzymes).

[0046] Patient: As used herein, the term "patient" or "subject" means any organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include both prenatal and postnatal forms.

[0047] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means a substance that is suitable for contact with human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0048] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by reacting amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable alkalis include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates). Other pharmaceutically acceptable salts include quaternized alkylated amino salts formed using suitable electrophiles (e.g., haloalkanes), and salts formed from the quaternization of amines.

[0049] Whole-body distribution or delivery: As used herein, the terms “whole-body distribution” or “whole-body delivery” or their grammatical equivalents refer to a mechanism or method of delivery or distribution that affects the whole body or the entire organism. Generally, whole-body distribution or delivery is accomplished via the body’s circulatory system (e.g., blood flow). Compare this to the definition of “local distribution or delivery”.

[0050] Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a person presented to a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used interchangeably herein with “individual” or “patient.” A subject may suffer from or be susceptible to a disease or disorder, but may or may not exhibit symptoms of said disease or disorder.

[0051] Essentially: As used herein, the term “essentially” refers to a qualitative situation that exhibits the overall or near-overall range or extent of a intended characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if ever) complete and / or proceed to the point of achieving or avoiding absolute results. Therefore, the term “essentially” is used herein to capture the inherent lack of completeness in many biological and chemical phenomena.

[0052] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some implementations, target tissue includes those tissues that exhibit disease-related symptoms, signs, or characteristics.

[0053] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of a disease, disorder, and / or condition when administered to a subject who has or is susceptible to such a disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0054] Treatment: As used herein, the terms “treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease for the purpose of reducing the risk of developing symptoms associated with said disease.

[0055] Chemical definition

[0056] Acyl group: As used herein, the term "acyl group" refers to R Z -(C=O)-, where R Z It is, for example, any alkyl, alkenyl, ynyl, heteroalkyl, or heteroalkylene.

[0057] Aliphatic: As used in this article, the term aliphatic refers to C1-C 50 Hydrocarbons include both saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be straight-chain, branched, and / or cyclic. For example, C1-C 20 Aliphatic can include C1-C 20 Alkyl groups (e.g., straight-chain or branched C1-C) 20 saturated alkyl), C2-C 20 Alkenyl (e.g., straight-chain or branched C4-C) 20 Dieneyl, straight-chain or branched C6-C 20 Trienyl groups, etc., and C2-C 20 Alkyne groups (e.g., straight-chain or branched C2-C) 20 Alkyne group). C1-C 20 Aliphatic compounds can include C3-C 20 Cyclic aliphatic (e.g., C3-C) 20 cycloalkyl, C4-C 20 Cycloalkenyl or C8-C 20 (Cycloalkyne). In some embodiments, the aliphatic group may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur), and may optionally be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). The aliphatic group is either unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R" is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In the embodiments, R” is independently an unsubstituted alkyl group (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In embodiments, "R" is independently an unsubstituted C1-C3 alkyl. In embodiments, aliphatic is unsubstituted. In embodiments, aliphatic does not include any heteroatoms. Alkyl: As used herein, the term "alkyl" means acyclic straight-chain and branched hydrocarbon groups, such as "C1-C3 alkyl". 30"Alkyl" refers to an alkyl group having 1 to 30 carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl-tert-pentylhexyl, isohexyl, etc. The term "lower alkyl" means a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art for the benefit of this disclosure. Alkyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkyl group can be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents), wherein each example of R" is independently C1-C2. 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In the embodiments, R” is independently an unsubstituted alkyl group (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In embodiments, "R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkyl is substituted with a -OH group and may also be referred to herein as "hydroxyalkyl", wherein the prefix indicates the -OH group and "alkyl" is as described herein.

[0058] As used herein, "alkyl" also refers to a group having 1 to 50 carbon atoms, either a straight-chain or branched saturated hydrocarbon group ("C1-C50"). 50 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 40 carbon atoms (“C1-C4”). 40 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 30 carbon atoms (“C1-C3”). 30 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 20 carbon atoms (“C1-C2”). 20 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 10 carbon atoms (“C1-C1”). 10Alkyl group (“C1-C9 alkyl”). In some embodiments, the alkyl group has 1 to 9 carbon atoms (“C1-C8 alkyl”). In some embodiments, the alkyl group has 1 to 8 carbon atoms (“C1-C7 alkyl”). In some embodiments, the alkyl group has 1 to 7 carbon atoms (“C1-C7 alkyl”). In some embodiments, the alkyl group has 1 to 6 carbon atoms (“C1-C6 alkyl”). In some embodiments, the alkyl group has 1 to 5 carbon atoms (“C1-C5 alkyl”). In some embodiments, the alkyl group has 1 to 4 carbon atoms (“C1-C4 alkyl”). In some embodiments, the alkyl group has 1 to 3 carbon atoms (“C1-C3 alkyl”). In some embodiments, the alkyl group has 1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, the alkyl group has 1 carbon atom (“C1-C2 alkyl”). Alkyl group (“C1-C6 alkyl”). In some embodiments, the alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”). Examples of C1-C6 alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). Other examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise stated, each example of an alkyl group is independently unsubstituted (“unsubstituted alkyl”) or substituted with one or more substituents (“substituted alkyl”). In some embodiments, the alkyl group is an unsubstituted C1-C6 alkyl group. 50 Alkyl group. In some embodiments, the alkyl group is a substituted C1-C alkyl group. 50 alkyl.

[0059] The suffix "-ene" is added to a group to indicate that the group is a divalent moiety. For example, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0060] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight-chain or branched hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, etc. Similarly, the term "alkenylene" as used herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds (which may appear at any stable point along the chain), and the term "alkynylene" herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds (which may appear at any stable point along the chain). In some embodiments, the alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and may optionally be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). For example, the alkylene, alkenylene, or ynylene group may be substituted with one or more of the following (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents): halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R", wherein each example of R" is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In the embodiments, R” is independently an unsubstituted alkyl group (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In embodiments, "R" is independently an unsubstituted C1-C3 alkyl. In some embodiments, the alkylene, alkenyl, or ynylene is unsubstituted. In some embodiments, the alkylene, alkenyl, or ynylene 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 can appear at any stable point along the chain), for example, "C2-C 30"Alkenyl" refers to an alkenyl group having 2-30 carbon atoms. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In embodiments, the alkenyl group comprises 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group comprises a single carbon-carbon double bond. In embodiments, multiple double bonds are concatenated (e.g., 2 or 3). The alkenyl group may be unsubstituted. Or it may be substituted by one or more substituents as described herein. For example, the alkenyl group may be substituted by one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCO2R", -NH2, -NHR", -N(R")2, -SR" or -SO2R" (e.g., 1, 2, 3, 4, 5 or 6 independently chosen substituents), wherein each example of R" is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In the embodiments, R” is independently an unsubstituted alkyl group (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In embodiments, "R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl group is unsubstituted. In embodiments, the alkenyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkenyl group is substituted with a -OH group and may also be referred to herein as "hydroxyalkenyl," wherein the prefix indicates the -OH group and "alkenyl" is as described herein.

[0061] As used herein, “alkenyl” also refers to a straight-chain 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) (“C2-C”). 50 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 40 carbon atoms (“C2-C”). 40 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 30 carbon atoms (“C2-C”). 30 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 20 carbon atoms (“C2-C”). 20 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 10 carbon atoms (“C2-C”). 10The alkenyl group has 2 to 9 carbon atoms (“C2-C9 alkenyl”). In some embodiments, the alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, the alkenyl group has 2 to 7 carbon atoms (“C2-C7 alkenyl”). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”). In some embodiments, the alkenyl group has 2 to 4 carbon atoms (“C2-C4 alkenyl”). In some embodiments, the alkenyl group has 2 to 3 carbon atoms (“C2-C3 alkenyl”). In some embodiments, the alkenyl group has 2 carbon atoms (“C2 alkenyl”). One or more carbon-carbon double bonds may... It can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of C2-C4 alkenyl groups include, but are not limited to, vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. Examples of C2-C6 alkenyl groups include the C2-C4 alkenyl groups described above, as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Other examples of alkenyl groups include heptenyl (C7), octenyl (C8), octetrinyl (C8), etc. Unless otherwise stated, each example of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is an unsubstituted C2-C4 alkenyl group. 50 Alkenyl group. In some embodiments, the alkenyl group is a substituted C2-C group. 50 Alkenyl group.

[0062] Alkynyl: As used herein, “alkynyl” means any straight-chain or branched hydrocarbon chain having one or more carbon-carbon triple bonds (occurring at any stable point along the chain), e.g., “C2-C 30 "Alynyl" refers to an alkynyl group having 2-30 carbons. Examples of alkynyl groups include prop-2-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-2-alkynyl, 3-methylpent-4-alkynyl, hex-2-alkynyl, hex-5-alkynyl, etc. In embodiments, the alkynyl group comprises a carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group can be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R" is independently C1-C 20 Aliphatic (e.g., C1-C) 20 Alkyl, C1-C15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In the embodiments, R” is independently an unsubstituted alkyl group (e.g., unsubstituted C1-C3 alkyl). 20 Alkyl, C1-C 15 Alkyl, C1-C 10 Alkyl or C1-C3 alkyl). In embodiments, R” is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkynyl group is unsubstituted. In embodiments, the alkynyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5 or 6 substituents as described herein).

[0063] As used herein, “alkynyl” also refers to a straight-chain or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) (“C2-C”). 50 The alkynyl group ("alkynyl group"). An alkynyl group having one or more triple bonds and one or more double bonds is also called an "alkynyl group". In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("C2-C"). 40 The alkynyl group (“C2-C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 30 carbon atoms (“C2-C”). 30 The alkynyl group (“C2-C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 20 carbon atoms (“C2-C”). 20 The alkynyl group (“C2-C”) is present in some embodiments. In some embodiments, the alkynyl group has 2 to 10 carbon atoms (“C2-C”). 10The alkynyl group (“C2-C9 alkynyl”) is used in some embodiments. In some embodiments, the alkynyl group has 2 to 8 carbon atoms (“C2-C8 alkynyl”). In some embodiments, the alkynyl group has 2 to 7 carbon atoms (“C2-C7 alkynyl”). In some embodiments, the alkynyl group has 2 to 6 carbon atoms (“C2-C6 alkynyl”). In some embodiments, the alkynyl group has 2 to 5 carbon atoms (“C2-C5 alkynyl”). In some embodiments, the alkynyl group has 2 to 4 carbon atoms (“C2-C4 alkynyl”). In some embodiments, the alkynyl group has 2 to 3 carbon atoms (“C2-C3 alkynyl”). In some embodiments, the alkynyl group has 2 carbon atoms (“C2 alkynyl”). One or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of C2-C4 alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. Examples of C2-C6 alkenyl groups include the aforementioned C2-C4 alkynyl groups as well as pentynyl (C5), hexynyl (C6), etc. Further examples of alkynyl groups include heptyynyl (C7), octyynyl (C8), etc. Unless otherwise stated, each example of an alkynyl group is independently unsubstituted (“unsubstituted alkynyl”) or substituted with one or more substituents (“substituted alkynyl”). In some embodiments, the alkynyl group is an unsubstituted C2-C... 50 Alkyne group. In some embodiments, the alkynyl group is a substituted C2-C group. 50 Alkyne group.

[0064] Aryl: The term "aryl" used alone or as part of a larger portion (such as "araneyl") refers to a monocyclic, bicyclic, or tricyclic carbocyclic system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic, and wherein each ring in the system contains four to seven ring members. In embodiments, the aryl group has six ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl", for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl" (e.g., anthracene). "Aryl" also includes ring systems in which an aromatic ring, as defined above, is fused with one or more carbocyclic or heterocyclic groups, wherein the attached groups or dots are on the aryl ring, and in such cases, the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

[0065] As used herein, “aryl” also refers to a group having a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared π electrons in a cyclic arrangement) of 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C”). 14 Aryl group (“C6 aryl”). In some embodiments, the aryl group has 6 ring carbon atoms (“C6 aryl”; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms (“C6 aryl”). 10 Aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“C”). 14 "Aryl"; for example, anthracene. "Aryl" also includes ring systems in which an aromatic ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attached group or dot is on the aryl ring, and in such cases, the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Unless otherwise stated, each example of an aryl is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In some embodiments, the aryl is an unsubstituted C6-C 14 Aryl group. In some embodiments, the aryl group is a substituted C6-C group. 14 Aryl.

[0066] arylene: As used herein, the term "arylene" refers to a divalent aryl group (that is, having two sites that attach to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted or substituted phenylene).

[0067] Carbocyclic group: As used herein, "carbocyclic group" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms in a non-aromatic ring system ("C3-C"). 10 The carbocyclic group (“C3-C8 carbocyclic group”) is a carbonyl group. In some embodiments, the carbocyclic group has 3 to 8 ring carbon atoms (“C3-C7 carbocyclic group”). In some embodiments, the carbocyclic group has 3 to 7 ring carbon atoms (“C3-C7 carbocyclic group”). In some embodiments, the carbocyclic group has 3 to 6 ring carbon atoms (“C3-C6 carbocyclic group”). In some embodiments, the carbocyclic group has 4 to 6 ring carbon atoms (“C4-C6 carbocyclic group”). In some embodiments, the carbocyclic group has 5 to 6 ring carbon atoms (“C5-C6 carbocyclic group”). In some embodiments, the carbocyclic group has 5 to 10 ring carbon atoms (“C5-C6 carbocyclic group”). 10Carbocyclic groups (C3-C6) include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Exemplary C3-C8 carbocyclic groups include, but are not limited to, the above-mentioned C3-C6 carbocyclic groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptanetrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Exemplary C3-C 10 Carbocyclic groups include, but are not limited to, the C3-C8 carbocyclic groups mentioned above, as well as cyclononyl (C9), cyclononenyl (C9), and cyclodecyl (C9). 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C9) 10 ), spiro[4.5]decyl(C 10 As illustrated in the foregoing examples, in some embodiments, the carbocyclic group is monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., containing fused, bridged, or spirocyclic systems, such as bicyclic systems (“bicyclic carbocyclic”) or tricyclic systems (“tricyclic carbocyclic”)) and may be saturated or may contain one or more carbon-carbon double or triple bonds. “Carbocyclic” also includes ring systems in which the carbocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the attachment point is on the carbocyclic ring, and in such cases, the number of carbons continues to specify the number of carbons in the carbocyclic system. Unless otherwise stated, each example of a carbocyclic group is independently unsubstituted (“unsubstituted carbocyclic”) or substituted with one or more substituents (“substituted carbocyclic”). In some embodiments, the carbocyclic group is an unsubstituted C3-C 10 Carbocyclic group. In some embodiments, the carbocyclic group is a substituted C3-C group. 10 Carbon cyclic group.

[0068] In some embodiments, "carbocyclic group" or "carbocyclic" is referred to as "cycloalkyl," that is, a monocyclic saturated carbocyclic group ("C3-C") having 3 to 10 ring carbon atoms. 10 The cycloalkyl group has 3 to 8 cyclic carbon atoms (“C3-C8 cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 6 cyclic carbon atoms (“C3-C6 cycloalkyl”). In some embodiments, the cycloalkyl group has 4 to 6 cyclic carbon atoms (“C4-C6 cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 6 cyclic carbon atoms (“C5-C6 cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 10 cyclic carbon atoms (“C5-C6 cycloalkyl”). 10Cycloalkyl. Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the C5-C6 cycloalkyl groups described above, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the C3-C6 cycloalkyl groups described above, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise stated, each example of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In some embodiments, the cycloalkyl group is an unsubstituted C3-C6 cycloalkyl group. 10 Cycloalkyl. In some embodiments, the cycloalkyl group is a substituted C3-C. 10 Cycloalkyl.

[0069] Halogen: As used in this article, the term “halogen” means fluorine, chlorine, bromine or iodine.

[0070] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from N, O, S, and P. Heteroalkyl groups include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphate diesters, aminophosphates, sulfonamides, and disulfides. Heteroalkyl groups may optionally include monocyclic, bicyclic, or tricyclic rings, wherein each ring ideally has three to six members. Examples of heteroalkyl groups include polyethers such as methoxymethyl and ethoxyethyl.

[0071] Heteroalkyl: As used herein, the term “heteroalkyl” refers to the divalent form of heteroalkyl as described herein.

[0072] 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).

[0073] As used herein, “heteroaryl” also refers to a group having a 5-14 member monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared π electrons in a cyclic arrangement) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In heteroaryls containing one or more nitrogen atoms, the attachment point can be a carbon or nitrogen atom if the valence allows. Heteroaryl polycyclic systems may include one or more heteroatoms in one or two rings. “Heteroaryl” includes ring systems in which the heteroaryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment point is on the heteroaryl ring, and in such cases, the number of ring members continues to specify 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 site is on the aryl or heteroaryl ring, and in such cases, the number of ring members specifies the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. A polycyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) may have its attachment site on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

[0074] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system (“5-10 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system (“5-8 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system (“5-6 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the 5-6-membered heteroaryl group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise stated, each example of a heteroaryl group is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some embodiments, the heteroaryl group is an unsubstituted 5-14-membered heteroaryl group. In some embodiments, the heteroaryl group is a substituted 5-14-membered heteroaryl group.

[0075] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrroloyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridyl, quinolinyl, isoquinolinyl, cenolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridine, dibenzofuranyl, carbazoleyl, acridineyl, phenothiazinyl, phenothiazinyl, and phenothiazinyl.

[0076] As used herein, "heterocyclic group" or "heterocycle" refers to a group ("3-14-membered heterocyclic group") having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In heterocyclic groups containing one or more nitrogen atoms, the attachment point may be a carbon or nitrogen atom, if the valence allows. Heterocyclic groups may be monocyclic ("monocyclic heterocyclic group") or polycyclic (e.g., fused, bridged, or spirocyclic systems, such as bicyclic systems ("bicyclic heterocyclic group") or tricyclic systems ("tricyclic heterocyclic group")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclic polycyclic systems may include one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems in which a heterocyclic ring as defined above is fused with one or more carbocyclic groups, wherein the attachment point is on the carbocyclic or heterocyclic ring; or ring systems in which a heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the attachment point is on the heterocyclic ring, and in such cases, the number of ring members continues to specify the number of ring members in the heterocyclic ring system. Unless otherwise stated, each example of a heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In some embodiments, the heterocyclic group is an unsubstituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a substituted 3-14 membered heterocyclic group.

[0077] In some embodiments, the heterocyclic group is a 5-10 membered non-aromatic ring system (“5-10 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heterocyclic group is a 5-8 membered non-aromatic ring system (“5-8 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heterocyclic group is a 5-6 membered non-aromatic ring system (“5-6 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the 5-6 membered heterocyclic group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0078] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxathiolanyl, oxathiolanyl, and dithiorenyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazineyl, morpholinyl, dithiaalkyl, and dioxaneyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary bicyclic heterocyclic groups include, but are not limited to, indololinyl, isoindololinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthidyl, decahydro-1,8-naphthidyl, octahydropyrrolo[3,2-b]pyrrole, indololinyl, phthalimidyl, naphthalimidyl, chromealkyl, chromenyl, 1H-benzo[e][1,4]diazazolyl, 1, 4,5,7-Tetrahydropyrano[3,4-b]pyrrole, 5,6-dihydro-4H-furano[3,2-b]pyrrole, 6,7-dihydro-5H-furano[3,2-b]pyrrole, 5,7-dihydro-4H-thieno[2,3-c]pyrrole, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridyl, 2,3-dihydrofurano[2,3-b]pyridyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridyl, 4,5,6,7-tetrahydrofurano[3,2-c]pyridyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridyl, 1,2,3,4-tetrahydro-1,6-naphthidyl, etc.

[0079] Heterocyclic alkyl: As used herein, the term "heterocyclic alkyl" 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. The heterocyclic alkyl may be substituted or unsubstituted.

[0080] As understood from the foregoing, in some embodiments, alkyl, alkenyl, alkynyl, acyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups as defined herein are optionally substituted. Optionally substituted means that the group 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" carbocyclic, "substituted" or "unsubstituted" heterocyclic, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). Generally, the term "substituted" means a substituent on which at least one hydrogen atom is permitted (e.g., substitution that produces a stable compound (e.g., does not self-substitute)). Substituents (such as those in compounds that undergo transformation through rearrangement, cyclization, elimination, or other reactions) are substituted. Unless otherwise indicated, a “substituted” group has substituents at one or more substituted positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of an organic compound, and any substituents described herein that result in the formation of a stable compound. Any and all such combinations are contemplated for obtaining stable compounds. For the purposes of this invention, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any suitable substituents that satisfy the valence state of the heteroatom as described herein and result in the formation of a stable moiety.

[0081] Exemplary carbon atom substituents include, but are not limited to, halogens, -CN, -NO2, -N3, -SO2, -SO3H, -OH, and -OR. aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3+X - -N(OR) cc )R bb -SeH, -SeR aa -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc)2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3-OSi(R aa )3-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa)2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 14 Carbocyclic group, 3-14 membered heterocyclic group, C6-C 14 Aryl and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0082] Alternatively, the two hydrogen atoms on the carbon atom can be replaced by the following groups: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc ;

[0083] R aa Each example is independently selected from C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic group, 3-14 membered heterocyclic group, C6-C 14 aryl and 5-14 heteroaryl, or two R aaGroups are linked to form 3-14 membered heterocyclic rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0084] R bb Each example is independently selected from hydrogen, -OH, -OR aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic group, 3-14 membered heterocyclic group, C6-C 14 aryl and 5-14 heteroaryl, or two R bb The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0085] R cc Each example is independently selected from hydrogen, C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic group, 3-14 membered heterocyclic group, C6-C 14 aryl and 5-14 heteroaryl, or two Rcc The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0086] R dd Each example is independently selected from halogens, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2、-N(R ff )3+X - -N(OR) ee )R ff -SH, -SR ee -SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee -OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee -S(=O)R ee 、-Si(R ee3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee -P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee 2. C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic groups, 3-10 membered heterocyclic groups, C6-C 10 Aryl, 5-10-membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can connect to form =O or =S;

[0087] R ee Each example is independently selected from C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 carbonyl group, C6-C 10 Aryl, 3-10 membered heterocyclic and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl and heteroaryl group is independently bounded by 0, 1, 2, 3, 4 or 5 R groups. gg Group substitution;

[0088] R ff Each example is independently selected from hydrogen, C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic groups, 3-10 membered heterocyclic groups, C6-C 10 aryl and 5-10 heteroaryl, or two R ff The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution; and

[0089] R ggEach example independently is a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C 50 Alkyl, -ON (C1-C) 50 Alkyl)2, -N(C1-C 50 Alkyl)2, -N(C1-C 50 Alkyl)3+X - -NH(C1-C 50 Alkyl)2+X - -NH2(C1-C 50 alkyl)+X - -NH3+X - -N(OC1-C 50 Alkyl) (C1-C 50 Alkyl group, -N(OH) (C1-C 50 Alkyl), -NH(OH), -SH, -SC1-C 50 Alkyl, -SS (C1-C 50 Alkyl), -C(=O)(C1-C 50 Alkyl groups, -CO2H, -CO2 (C1-C) 50 Alkyl), -OC (=O) (C1-C) 50 Alkyl), -OCO2 (C1-C 50 Alkyl groups, -C(=O)NH2, -C(=O)N(C1-C 50 Alkyl)2、-OC(=O)NH(C1-C 50 Alkyl), -NHC(=O)(C1-C 50 Alkyl), -N(C1-C 50 Alkyl)C(=O)(C1-C 50 Alkyl), -NHCO2 (C1-C 50 Alkyl), -NHC(=O)N(C1-C 50 Alkyl)2、-NHC(=O)NH(C1-C 50 Alkyl groups), -NHC(=O)NH2, -C(=NH)O(C1-C 50 Alkyl), -OC (=NH)(C1-C 50 Alkyl), -OC (=NH)OC1-C 50 Alkyl group, -C(=NH)N(C1-C) 50 Alkyl)2、-C(=NH)NH(C1-C 50 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C1-C 50 Alkyl)2、-OC(NH)NH(C1-C 50Alkyl groups, -OC(NH)NH2, -NHC(NH)N(C1-C 50 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C1-C 50 Alkyl), -SO2N(C1-C 50 alkyl)2、-SO2NH(C1-C 50 Alkyl groups, -SO2NH2, -SO2 (C1-C 50 Alkyl), -SO2O(C1-C 50 Alkyl), -OSO2 (C1-C6 alkyl), -SO (C1-C6 alkyl), -Si (C1-C 50 Alkyl)3, -OSi (C1-C6 alkyl)3, -C(=S)N (C1-C 50 Alkyl)2, C(=S)NH(C1-C 50 Alkyl), C(=S)NH2, -C(=O)S(C1-C6 alkyl), -C(=S)S(C1-C6 alkyl), -SC(=S)S(C1-C6 alkyl), -P(=O)2(C1-C 50 Alkyl), -P (=O) (C1-C) 50 Alkyl)2、-OP(=O)(C1-C 50 Alkyl)2、-OP(=O)(OC1-C 50 Alkyl)2, C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 carbonyl group, C6-C 10 aryl, 3-10 heterocyclic, 5-10 heteroaryl; or two geminal Rs gg Substituents can be linked to form =O or =S; where X - It is an antiion.

[0090] As used herein, the term “halogen” or “halogen” refers to fluorine (fluorinated, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0091] As used herein, a "counterion" is a negatively charged group associated with a positively charged quaternary ammonium in order to maintain electronic 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-sulfonate-5-sulfonate, ethane-1-sulfonate-2-sulfonate, etc.) and carboxyl groups (e.g., acetate, ethanoate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0092] If the valence allows, the nitrogen atom can be substituted or unsubstituted, and includes primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen substituents include, but are not limited to, hydrogen, -OH, and -OR. aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc 2. C1-C 50 Alkyl, C2-C 50 alkenyl, C2-C 50 alkynyl group, C3-C 10 Carbocyclic group, 3-14 membered heterocyclic group, C6-C 14 aryl and 5-14 heteroaryl, or two R cc The alkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups together with the attached nitrogen atoms form 3-14 membered heterocyclic or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R atoms. dd Group substitution, and wherein R aa R bb Rcc and R dd It is as defined above.

[0093] In some embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also known as an amino protecting group). Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0094] For example, amide groups (e.g., -C(=O)R) aa The nitrogen protecting group of () includes, but is not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridinecarboxamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0095] Such as urethane groups (e.g., -C(=O)OR) aaThe nitrogen-protecting groups of ) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfonyl)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoylmethyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), and 1-(1-adamantyl)-1-methylethyl Carbamates (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylcarbamate)ethylcarbamate, tert-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), carbamic acid Ethylene ester (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamon ester carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolinyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, 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-anthraylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonyl Acyl ethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithiaalkyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphoethyl carbamate (Peoc), 2-triphenylphosphoisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolyl methyl carbamate, 2-(trifluoromethyl)-6-chromone methyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-Dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyylvinyl carbamate, o-(N,N-dimethylformamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodobenzyl Ethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicocarbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0096] Such as sulfonamide groups (e.g., -S(=O)2R) aa The nitrogen protecting groups of () 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), 2,2,5,7,8-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4',8'-dimethoxynaphthyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0097] 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-oxazoline-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldimethylsilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptanylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro... -9-fluorenylmethyleneamine, N-ferrocene methylamine (Fcm), N-2-pyridinemethylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidene diamine, N-p-nitrobenzylamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-oxo-l-cyclohexene) N-Borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentaacylchromium or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylaminophosphate, dibenzylaminophosphate, diphenylaminophosphate, benzylsulfonamide, o-nitrobenzenesulfonamide (Nps), 2,4-dinitrobenzenesulfonamide, pentachlorobenzenesulfonamide, 2-nitro-4-methoxybenzenesulfonamide, triphenylmethylsulfonamide, and 3-nitropyridinesulfonamide (Npys).

[0098] In some embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also known as a hydroxy protecting group). Oxyprotecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0099] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), and tetrahydropyranyl (THP). 3-Bromotetrahydropyranyl, Tetrahydrothiopyranyl, 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-bridged methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl- 1-Benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenyloxyselenoyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinemethyl, 4-pyridinemethyl, 3-methyl-2-pyridinemethyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)benzyl methyl, tris(p-methoxyphenyl)methyl, 4-(4'-bromobenzoylmethyloxyphenyl)diphenylmethyl, 4,4',4”-tris(4,5-dichlorobenzoiminophenyl)methyl, 4,4',4”-tris(acetylpropionyloxyphenyl)methyl, 4,4',4”-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4”-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xanthrayl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodisulfuran-2-yl, benzoisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenyl Methoxyacetic acid ester, phenoxyacetic acid ester, p-chlorophenoxyacetic acid ester, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (milkone ester), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonium)ethyl carbonate Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6 -Dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrate esters, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate esters, dimethylphosphinothionyl, alkyl 2,4-dinitrophenyl sulfenate, sulfate esters, methanesulfonate (methanesulfonate), benzyl sulfonate, and toluenesulfonate (Ts).

[0100] In some embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0101] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-pyridinemethyl, 2-quinolinylmethyl, 2-pyridinemethyl N-oxide, 9-anthraylmethyl, 9-fluorenylmethyl, xanthyl, ferroceneylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzocycloheptanyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, tert-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamoxymethyl, trimethylacetamoxymethyl, benzamidemethyl, allyloxycarbonylamino Methyl, phenylacetamidomethyl, phthaliminomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carbonylethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylprop-2- Acetyl, benzoyl, trifluoroacetyl, N-[[(p-biphenyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, sulfenyl thiocarbonate, 3-nitro-2-pyridine sulfenyl sulfide, oxathiolone.

[0102] The compounds of the present invention

[0103] Liposome-based mediators are considered attractive carriers for therapeutics and require continued development efforts. While liposome-based mediators containing certain lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a significant need to improve liposome-based delivery systems. For example, significant drawbacks of liposome delivery systems involve the construction of liposomes with sufficient cell culture or in vivo stability to achieve desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release their encapsulated materials into such target cells.

[0104] In particular, there remains a need for improved lipid compounds that exhibit improved pharmacokinetic properties and can deliver macromolecules (such as nucleic acids) to a variety of cell types and tissues with enhanced efficiency. Importantly, there is still a particular need for novel lipid compounds characterized by reduced toxicity and the ability to efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0105] This article describes a novel class of cationic lipid compounds for improving the in vivo delivery of therapeutic agents, such as nucleic acids. In particular, the cationic lipids described herein can 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, microRNA) for therapeutic purposes.

[0106] In embodiments, the compounds of the present invention as described herein may provide one or more desired features or properties. That is, in some embodiments, the compounds of the present invention as described herein may be characterized by having one or more properties that provide an advantage over other similarly classified lipids. For example, the compounds disclosed herein may allow for the control and customization of the properties of liposome compositions (e.g., lipid nanoparticles) in which they are components. In particular, the compounds disclosed herein may be characterized by enhanced transfection efficiency and their ability to elicit specific biological outcomes. Such outcomes may include, for example, enhanced cellular uptake, endosome / lysosome disruption, and / or promotion of the release of encapsulated materials (e.g., polynucleotides) into cells. Additionally, the compounds disclosed herein have favorable pharmacokinetic properties, biodistribution, and efficiency (e.g., due to the different dissociation rates of the polymer groups used).

[0107] This application demonstrates that the cationic lipids of the present invention can not only be easily synthesized and processed from readily available starting materials, but also unexpectedly possess high encapsulation efficiency.

[0108] Furthermore, the cationic lipids of the present invention have cleavable groups, such as ester groups and disulfide bonds. It is envisioned that these cleavable groups (e.g., esters and disulfide bonds) can enhance biodegradability and thus contribute to their favorable toxicity profile.

[0109] This document provides compounds that are cationic lipids. For example, the cationic lipids of the present invention include compounds having a structure according to formula (Ia):

[0110]

[0111] Or its pharmaceutically acceptable salt, wherein

[0112] R 1 R 2 and R 3 Each is selected independently from:

[0113] Optional substituted alkyl, optional substituted alkenyl, optional substituted alkynyl, and optional substituted acyl groups;

[0114] R 4 yes

[0115] Each R 5 Independently selected from optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl groups;

[0116] Each R 6 It is independently selected from hydrogen, halogen, optionally substituted C1-C6 alkyl, and optionally substituted C2-C6 alkenyl;

[0117] A is -NR 9 -or -O-;

[0118] D is either O or S;

[0119] E and G are each independently selected from -NR 10 -、-O- and -S-;

[0120] R 8 R 9 and R 10 Each is independently selected from hydrogen, optionally substituted C1-C6 alkyl groups, and optionally substituted C2-C6 alkenyl groups;

[0121] Each b is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and

[0122] Each c is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0123] In embodiments, the cationic lipids of the present invention comprise compounds having the structure according to formula (Ib):

[0124]

[0125] Or a pharmaceutically acceptable salt thereof, wherein R 5 A and R 4 It is as defined in equation (Ia).

[0126] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ic):

[0127]

[0128] Or a pharmaceutically acceptable salt thereof, wherein R 5 A, c, G and R 8 It is as defined in equation (Ia).

[0129] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Id):

[0130]

[0131] Or a pharmaceutically acceptable salt thereof, wherein R 5 c, G and R 8 It is as defined in equation (Ia).

[0132] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ie):

[0133]

[0134] Or a pharmaceutically acceptable salt thereof, wherein R 5 c, G and R 8 It is as defined in equation (Ia).

[0135] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (If):

[0136]

[0137] Or a pharmaceutically acceptable salt thereof, wherein R 5 A and A are defined as in equation (Ia).

[0138] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ig):

[0139]

[0140] Or a pharmaceutically acceptable salt thereof, wherein R 5 It is as defined in equation (Ia).

[0141] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ih):

[0142]

[0143] Or a pharmaceutically acceptable salt thereof, wherein R 5 It is as defined in equation (Ia).

[0144] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ii):

[0145] Or a pharmaceutically acceptable salt thereof, wherein R 5 A and A are defined as in equation (Ia).

[0146] In embodiments, the cationic lipids of the present invention comprise compounds having the structure according to formula (Ij):

[0147]

[0148] Or a pharmaceutically acceptable salt thereof, wherein R 5 It is as defined in equation (Ia).

[0149] In embodiments, the cationic lipids of the present invention comprise compounds having a structure according to formula (Ik):

[0150]

[0151] Or a pharmaceutically acceptable salt thereof, wherein R 5 It is as defined in equation (Ia).

[0152] In the implementation scheme (e.g., a compound of formula (Ia)), R 1 R 2 and R 3 Each independently is

[0153] In the implementation scheme (e.g., a compound of formula (Ia)), R 1 R 2 and R 3 Each independently is And D and E are each O.

[0154] In the implementation scheme (e.g., a compound of formula (Ia)), R 1 R 2 and R3 Each independently is

[0155] In the implementation scheme (e.g., a compound of formula (Ia)), R 1 R 2 and R 3 Each independently is And D and E are each O.

[0156] In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 Selected from In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 4 yes

[0157] In the implementation plan, each R 5 Independently selected from arbitrarily substituted C1-C 50 Alkyl groups, optionally substituted C2-C 50 Alkenyl and optionally substituted C2-C 50 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C1-C 40 Alkyl groups, optionally substituted C2-C 40 Alkenyl and optionally substituted C2-C 40 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C1-C 30 Alkyl groups, optionally substituted C2-C 30 Alkenyl and optionally substituted C2-C 30 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C1-C 20Alkyl groups, optionally substituted C2-C 20 Alkenyl and optionally substituted C2-C 20 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C1-C 10 Alkyl groups, optionally substituted C2-C 10 Alkenyl and optionally substituted C2-C 10 Alkyne group.

[0158] In the implementation plan, each R 5 Independently selected from arbitrarily substituted C5-C 50 Alkyl groups, optionally substituted C5-C 50 Alkenyl and optionally substituted C5-C 50 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C5-C 40 Alkyl groups, optionally substituted C5-C 40 Alkenyl and optionally substituted C5-C 40 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C5-C 30 Alkyl groups, optionally substituted C5-C 30 Alkenyl and optionally substituted C5-C 30 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C5-C 20 Alkyl groups, optionally substituted C5-C 20 Alkenyl and optionally substituted C5-C 20 Alkyne group. In the implementation scheme, each R 5 Independently selected from arbitrarily substituted C5-C 10 Alkyl groups, optionally substituted C5-C 10 Alkenyl and optionally substituted C5-C 10 Alkyne group.

[0159] In the implementation plan, each R 5 It is an optional substituted alkyl group. In the embodiments, each R 5 It is an optional, replaced C5-C 50 Alkyl group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 40 Alkyl group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 30 Alkyl group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 25 Alkyl group. In the implementation scheme, each R 5It is an optional, replaced C5-C 20 Alkyl group. In the implementation scheme, each R 5 It is an optional, substituted C8-C 16 alkyl.

[0160] In the implementation plan, each R 5 It is an optionally substituted alkenyl group. In the embodiments, each R 5 It is an optional, replaced C5-C 50 Alkenyl. In the implementation scheme, each R... 5 It is an optional, replaced C5-C 40 Alkenyl. In the implementation scheme, each R... 5 It is an optional, replaced C5-C 30 Alkenyl. In the implementation scheme, each R... 5 It is an optional, replaced C5-C 25 Alkenyl. In the implementation scheme, each R... 5 It is an optional, replaced C5-C 20 Alkenyl. In the implementation scheme, each R... 5 It is an optional substituted C 8-16 Alkenyl group.

[0161] In the implementation plan, each R 5 It is an optionally substituted alkynyl group. In the embodiments, each R 5 It is an optional, replaced C5-C 50 Alkyne group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 40 Alkyne group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 30 Alkyne group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 25 Alkyne group. In the implementation scheme, each R 5 It is an optional, replaced C5-C 20 Alkyne group. In the implementation scheme, each R 5 It is an optional, substituted C8-C 16 Alkyne group.

[0162] In the implementation plan, each R 5 Independently selected from optionally substituted alkyl groups and optionally substituted alkenyl groups.

[0163] In the implementation plan, each R 5 Selected independently from:

[0164]

[0165] In the implementation plan, R1 R 2 and R 3 Each is selected independently from: (For example, ), optionally where D and E are each O, and each R 5 Selected independently from:

[0166]

[0167] In the implementation scheme (e.g., compounds of formula (Ia) or (Ib), R 6 It is hydrogen;

[0168] In the embodiments (e.g., compounds of formula (Ia), (Ib), (Ic), (If), or (Ii), A is -NH- or -O-. In the embodiments (e.g., compounds of formula (Ia), (Ib), (Ic), (If), or (Ii), A is -NH-. In the embodiments (e.g., compounds of formula (Ia), (Ib), (Ic), (If), or (Ii),) A is -O-.

[0169] In the implementation scheme, D is O. In the implementation scheme, D is S.

[0170] In the implementation plan, E is -NR 10 - In the implementation, E is -O-. In the implementation, E is -S-. In the implementation, G is -NR. 10 - In the implementation, G is -O-. In the implementation, G is -S-.

[0171] In the implementation plan, R 8 It is H. In the implementation plan, R 8 It is an optionally substituted C1-C6 alkyl group. In the embodiments, R 8 It is an optionally substituted C1-C5 alkyl group. In the embodiments, R 8 It is an optionally substituted C1-C4 alkyl group. In the embodiments, R 8 It is an optionally substituted C1-C3 alkyl group. In the embodiments, R 8 It is an optionally substituted C1-C2 alkyl group. In the embodiments, R 8 It is an optionally substituted C1 alkyl group. In the embodiments, R 8 It is a methyl group.

[0172] In the implementation plan, R 8 It is an optionally substituted C2-C6 alkenyl group. In the embodiments, R 8 It is an optionally substituted C2-C5 alkenyl group. In the embodiments, R 8It is an optionally substituted C2-C4 alkenyl group. In the embodiments, R 8 It is an optionally substituted C2-C3 alkenyl group. In the embodiments, R 8 It is an optional substituted C2 alkenyl group.

[0173] In the implementation plan, R 9 It is hydrogen.

[0174] In the implementation plan, R 9 It is an optionally substituted C1-C6 alkyl group. In the embodiments, R 9 It is an optionally substituted C1-C5 alkyl group. In the embodiments, R 9 It is an optionally substituted C1-C4 alkyl group. In the embodiments, R 9 It is an optionally substituted C1-C3 alkyl group. In the embodiments, R 9 It is an optionally substituted C1-C2 alkyl group. In the embodiments, R... 9 It is a methyl group.

[0175] In the implementation plan, R 9 It is an optionally substituted C2-C6 alkenyl group. In the embodiments, R 9 It is an optionally substituted C2-C5 alkenyl group. In the embodiments, R 9 It is an optionally substituted C2-C4 alkenyl group. In the embodiments, R 9 It is an optionally substituted C2-C3 alkenyl group. In the embodiments, R 9 It is an optional substituted C2 alkenyl group.

[0176] In the implementation plan, each R 10 Independently selected from hydrogen and optionally substituted C1-C6 alkyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C1-C5 alkyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C1-C4 alkyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C1-C3 alkyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C1-C2 alkyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C1 alkyl groups. In the embodiments, each R 10 It is independently selected from hydrogen and methyl.

[0177] In the implementation plan, each R 10 Independently selected from hydrogen and optionally substituted C2-C6 alkenyl groups. In the embodiments, each R 10Independently selected from hydrogen and optionally substituted C2-C5 alkenyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C2-C4 alkenyl groups. In the embodiments, each R 10 Independently selected from hydrogen and optionally substituted C2-C3 alkenyl groups. In the embodiments, each R 10 It is independently selected from hydrogen and optionally substituted C2 alkenyl groups.

[0178] In the implementation plan, G is NR 10 And R 8 and R 10 The two are the same. In some implementations, R 8 and R 10 Both are selected from H and optionally substituted C1-C6 alkyl groups. In some embodiments, R 8 and R 10 Each is a methyl group.

[0179] In the implementation scheme, for any of the aforementioned implementation schemes having an integer b, b is 1, 2, 3, 4, or 5. In the implementation scheme, for any of the aforementioned implementation schemes having an integer b, b is 1, 2, 3, or 4. In the implementation scheme, for any of the aforementioned implementation schemes having an integer b, b is 1, 2, or 3. In the implementation scheme, for any of the aforementioned implementation schemes having an integer b, b is 1 or 2. In the implementation scheme, for any of the aforementioned implementation schemes having an integer b, b is 1.

[0180] In the implementation scheme, for any of the aforementioned implementation schemes having an integer c, c is 1, 2, 3, 4, or 5. In the implementation scheme, for any of the aforementioned implementation schemes having an integer c, c is 1, 2, 3, or 4. In the implementation scheme, for any of the aforementioned implementation schemes having an integer c, c is 1, 2, or 3. In the implementation scheme, for any of the aforementioned implementation schemes having an integer c, c is 2. In the implementation scheme, for any of the aforementioned implementation schemes having an integer c, c is 3.

[0181] In the implementation scheme, the substituent is not optionally substituted.

[0182] In the embodiments, the cationic lipids of the present invention have any of the structures in Table A, or a pharmaceutically acceptable salt thereof.

[0183] In one embodiment, a composition is provided comprising any one of the cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids as described in the preceding embodiments. In another embodiment, the composition is lipid nanoparticles. In another embodiment, the one or more cationic lipids constitute about 30 mol%-60 mol% of the lipid nanoparticles. In another embodiment, the one or more non-cationic lipids constitute 10 mol%-50 mol% of the lipid nanoparticles. In another embodiment, the one or more PEG-modified lipids constitute 1 mol%-10 mol% of the lipid nanoparticles. In another embodiment, the cholesterol-based lipids constitute 10 mol%-50 mol% of the lipid nanoparticles. In another embodiment, the lipid nanoparticles encapsulate nucleic acids, optionally encapsulating mRNA encoding peptides or proteins. In another embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 70%. In another embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 75%. In another embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 80%. In another embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 85%. In one embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 90%. In another embodiment, the encapsulation percentage of mRNA in the lipid nanoparticles is at least 95%.

[0184] In the embodiments, the composition described in any one of the foregoing embodiments is used in a therapeutic application.

[0185] In the embodiments, the composition of any one of the foregoing embodiments is used in a method of treating or preventing a disease suitable for treatment or prevention by a peptide or protein encoded by the mRNA, optionally wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0186] In the embodiments, the composition is administered intravenously, intrathecally, or intramuscularly, or delivered via the lungs, optionally via nebulization.

[0187] Exemplary compounds

[0188] In embodiments, the cationic lipids of the present invention comprise compounds selected from those depicted in Table A, or pharmaceutically acceptable salts thereof.

[0189] Exemplary compounds include those described in Table A, or pharmaceutically acceptable salts thereof.

[0190]

[0191]

[0192]

[0193] Any of the compounds identified in Table A above may be provided in the form of pharmaceutically acceptable salts and such salts are intended to be covered by this invention.

[0194] The compounds of the present invention as described herein can be prepared according to methods known in the art, including exemplary synthesis of the embodiments provided herein.

[0195] Nucleic acid

[0196] The compounds of the present invention, as described herein, can be used to prepare compositions that can be used to deliver nucleic acids.

[0197] Nucleic acid synthesis

[0198] The nucleic acids according to the invention can be synthesized according to any known method. For example, the mRNA according to the invention can be synthesized via in vitro transcription (IVT). In short, IVT is generally performed using: a straight or circular DNA template containing a promoter, a library of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, mutant T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0199] In some embodiments, for the preparation of the mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription (e.g., T3, T7, mutated T7, or SP6 promoter), followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0200] One or more mRNA sequences required according to the present invention can be determined using standard methods and incorporated into a DNA template. For example, virtual reverse translation is performed based on a degenerate genetic code, starting from the desired amino acid sequence (e.g., an enzyme sequence). An optimization algorithm can then be used to select suitable codons. Typically, the G / C ratio can be optimized to achieve the highest possible G / C ratio on the one hand, and the frequency of tRNA can be considered as much as possible based on codon usage on the other. For example, with the aid of a suitable display device, the optimized RNA sequence can be constructed and displayed, and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate stable and unstable properties or corresponding RNA regions.

[0201] Modified mRNA

[0202] In some embodiments, the mRNA according to the invention can be synthesized as unmodified or modified mRNA. Modified mRNA includes nucleotide modifications in the RNA. Therefore, the modified mRNA according to the invention can include nucleotide modifications such as backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), said naturally occurring nucleotides and / or nucleotide analogs including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil ( 5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxy Aminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, piracetamidine, β-D-mannosyl-piracetamidine, wybutoxosine and aminophosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-dezoguanosine, 5-methylcytosine and inosine. The preparation of such analogues is known to those skilled in the art, for example, from U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety.

[0203] Pharmaceutical formulations containing cationic lipids and nucleic acids

[0204] In some embodiments, the compounds of the present invention as described herein, as well as pharmaceutical and liposome compositions comprising such lipids, can be used in formulations to facilitate the delivery of encapsulated material (e.g., one or more polynucleotides, such as mRNA) to one or more target cells, and subsequently transfection of said one or more target cells. For example, in some embodiments, the cationic lipids (and compositions, such as liposome compositions comprising such lipids) described herein are characterized by one or more of the following: receptor-mediated endocytosis, clathrin-mediated and pituitary-mediated endocytosis, phagocytosis and macropinocytosis, fusogenicity, endosome or lysosomal disruption, and / or releasability, which provides advantages of such compounds over other similarly classified lipids.

[0205] According to the present invention, nucleic acids (e.g., mRNA encoding proteins (e.g., full-length, fragments, or portions of proteins) as described herein can be delivered via a delivery medium comprising the compounds of the present invention as described herein.

[0206] As used herein, the terms “delivery medium,” “transfer medium,” “nanoparticle,” or their grammatical equivalents are used interchangeably.

[0207] For example, the present invention provides a composition comprising the compounds described herein and one or more polynucleotides (e.g., a pharmaceutical composition). The composition (e.g., a pharmaceutical composition) may further comprise one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and / or one or more PEG-modified lipids.

[0208] In some embodiments, the composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally involve contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a compound encapsulating one or more polynucleotides as described herein), such that the one or more target cells are transfected with the encapsulated material (e.g., one or more polynucleotides). As used herein, the term “transfect” or “transfection” refers to the introduction of one or more encapsulated materials (e.g., nucleic acids and / or polynucleotides) into the cells (e.g., into target cells). The introduced polynucleotides may be stably or transiently maintained in the target cells. The term “transfection efficiency” refers to the relative amount of such encapsulated material (e.g., polynucleotides) absorbed, introduced into, and / or expressed by the transfected target cells. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide products produced by the target cells after transfection. In some embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the likelihood of delivering an appropriate dose of the encapsulated material (e.g., one or more polynucleotides) to the pathological site and subsequently expressing it, while minimizing potential systemic adverse reactions or toxicities associated with the compounds or their encapsulation contents.

[0209] Following transfection of one or more target cells with polynucleotides, for example, encapsulated in one or more lipid nanoparticles (containing pharmaceutical compositions or liposome compositions disclosed herein), the production of products (e.g., peptides or proteins) encoded by such polynucleotides can be stimulated, and the ability of such target cells to express said polynucleotides and produce, for example, the target peptide or target protein can be enhanced. For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e., increase) the production of proteins or enzymes encoded by such mRNA.

[0210] Furthermore, delivery mediators described herein (e.g., liposome delivery mediators) can be prepared for preferential distribution to other target tissues, cells, or organs, such as the heart, lungs, kidneys, and spleen. In embodiments, lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical compositions and liposome compositions described herein can be delivered to and / or transfected into target cells or tissues. In some embodiments, the encapsulated polynucleotides (e.g., mRNA) are capable of being expressed by target cells and producing (and in some cases secreted) functional polypeptide products, thereby conferring beneficial properties to, for example, the target cells or tissues. Such encapsulated polynucleotides (e.g., mRNA) may encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other target proteins.

[0211] Liposome delivery mediators

[0212] In some embodiments, the composition is a suitable delivery medium. In one embodiment, the composition is a liposome delivery medium, such as lipid nanoparticles.

[0213] The terms “liposome delivery medium” and “liposome composition” are used interchangeably.

[0214] Liposome compositions enriched with one or more 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 the delivery of encapsulated polynucleotides to one or more target cells and / or reduce the in vivo toxicity of the liposome composition). Therefore, pharmaceutical compositions comprising one or more cationic lipids disclosed herein, and in particular liposome compositions, are also contemplated.

[0215] Therefore, in some embodiments, the compounds of the present invention as described herein can be used as components of liposome compositions to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells).

[0216] As used herein, liposome delivery mediators (e.g., lipid nanoparticles) are typically characterized as microvesicles having an internal water space isolated from an external medium by one or more bilayer membranes. The bilayer membranes of liposomes are typically formed of amphiphilic molecules, such as synthetic or naturally derived lipids, which contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membranes of liposomes can also be formed of amphiphilic polymers and surfactants (e.g., polymerosomes, nonionic surfactant vesicles, etc.). In the context of this invention, liposome delivery mediators are typically used to transport desired mRNA to target cells or tissues.

[0217] In some embodiments, such compositions (e.g., liposome compositions) are loaded with or otherwise encapsulated with materials, such as one or more bioactive polynucleotides (e.g., mRNA).

[0218] In embodiments, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a protein encapsulated in liposomes. In embodiments, the liposomes 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. In embodiments, the composition comprises mRNA encoding a protein (e.g., any protein described herein). In embodiments, the composition comprises mRNA encoding a cystic fibrosis transmembrane conduction regulation (CFTR) protein. In embodiments, the composition comprises mRNA encoding an ornithine transcarbamylase (OTC) protein.

[0219] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises nucleic acids encapsulated in liposomes, wherein the liposomes comprise compounds described herein.

[0220] In embodiments, the nucleic acid is mRNA encoding a peptide or protein. In embodiments, the mRNA encodes a peptide or protein for use in or to treat the lungs or lung cells of a subject (e.g., mRNA encoding a cystic fibrosis transmembrane conduction regulation (CFTR) protein). In embodiments, the mRNA encodes a peptide or protein for use in or to treat the liver or hepatocytes of a subject (e.g., mRNA encoding an ornithine transcarbamate (OTC) protein). Other exemplary mRNAs are described herein.

[0221] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net positive charge.

[0222] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net negative charge.

[0223] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net neutral charge.

[0224] In some embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein.

[0225] For example, the amount of the compound of the present invention as described herein in a composition can be described as a percentage (“wt%”) of the combined dry weight of all lipids in the composition (e.g., the combined dry weight of all lipids present in the liposome composition).

[0226] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in an amount of about 0.5 wt% to about 30 wt% (e.g., about 0.5 wt% to about 20 wt%) of the combined dry weight of all lipids present in the composition (e.g., a liposome composition).

[0227] In embodiments, the compounds of the present invention as described herein are present in amounts of about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in the composition (such as a liposome composition). In embodiments, the compounds of the present invention as described herein are present in amounts of about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in the composition (such as a liposome delivery medium).

[0228] In embodiments, the amount of the compounds of the invention as described herein is present in an amount of at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0229] In embodiments, the amount of the compounds of the invention as described herein is present in an amount not exceeding about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of the total lipids in the composition (e.g., a liposome composition).

[0230] In some embodiments, the composition (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises about 0.1 wt% to about 20 wt% (e.g., about 0.1 wt% to about 15 wt%) of the compounds described herein. In some embodiments, the delivery medium (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of the compounds described herein. In some embodiments, the delivery medium (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises as much as about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of the compounds described herein. In some embodiments, the percentages result in improved beneficial effects (e.g., improved delivery to target tissues, such as the liver or lungs).

[0231] The amount of the compounds of the present invention as described herein in the composition may also be described as a percentage (“mol%”) of the combined molar amount of the total lipids in the composition (e.g., the combined molar amount of all lipids present in a liposome delivery medium).

[0232] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in the following amounts: a combined molar amount of all lipids present in the composition (such as a liposome delivery medium) of about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%).

[0233] In embodiments, the compounds of the present invention as described herein are present in the following amounts as a combined molar amount of all lipids present in the composition (e.g., a liposome delivery medium): about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol%, or about 45 mol% to about 60 mol%. In embodiments, the compounds of the present invention as described herein are present in the following amounts as a combined molar amount of all lipids present in the composition (e.g., a liposome delivery medium): about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol%, or about 5 mol% to about 25 mol%.

[0234] In some embodiments, the compounds of the present invention as described herein may comprise a total amount of lipids in the composition (e.g., a liposome delivery medium) ranging from about 0.1 mol% to about 50 mol%, or from 0.5 mol% to about 50 mol%, or from about 1 mol% to about 50 mol%, or from about 5 mol% to about 50 mol%, or from about 10 mol% to about 50 mol%, or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol%.

[0235] In some embodiments, the compounds of the present invention as described herein may comprise a total amount of lipids in the lipid nanoparticles greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, or greater than about 5 mol%, or greater than about 10 mol%, or greater than about 20 mol%, or greater than about 30 mol%, or greater than about 40 mol%.

[0236] In some embodiments, the compound as described may comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, or less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total amount of lipids in the composition (e.g., a liposome delivery medium).

[0237] In embodiments, the amount of the compounds of the invention as described herein is present in the following amounts as a combined molar amount of total lipids in the composition (e.g., a liposome composition): at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol%.

[0238] In embodiments, the amount of the compounds of the invention as described herein is present in the following amounts as a combined molar amount of total lipids in the composition (e.g., a liposome composition): not more than about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol%.

[0239] In the implementation scheme, the percentage results in improved beneficial effects (e.g., improved delivery to target tissues, such as the liver or lungs).

[0240] In typical embodiments, the compositions of the present invention (e.g., liposome compositions) 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 comprising a compound of the present invention as described herein, a non-cationic lipid, a cholesterol-based lipid, and a PEG-modified lipid as a cationic lipid component. The non-cationic lipid may be DOPE or DEPE. The cholesterol-based lipid may be cholesterol. The PEG-modified lipid may be DMG-PEG2K.

[0241] In a further embodiment, the pharmaceutical (e.g., liposome) composition comprises one or more of PEG-modified lipids, non-cationic lipids, and cholesterol lipids. In other embodiments, such pharmaceutical (e.g., liposome) compositions comprise: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids. In yet another embodiment, such pharmaceutical (e.g., liposome) compositions comprise: one or more PEG-modified lipids and one or more cholesterol lipids.

[0242] In embodiments, the composition (e.g., lipid nanoparticles) encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprises one or more compounds of the present invention as described herein and one or more lipids selected from cationic lipids, non-cationic lipids, and PEGylated lipids.

[0243] In embodiments, the composition encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) (e.g., lipid nanoparticles) comprises one or more compounds of the present invention as described herein; one or more lipids selected from cationic lipids, non-cationic lipids, and PEGylated lipids; and further comprises cholesterol-based lipids. Typically, such a composition has four lipid components comprising, as a cationic lipid component, a compound of the present invention as described herein, a non-cationic lipid (e.g., DOPE), a cholesterol-based lipid (e.g., cholesterol), and a PEG-modified lipid (e.g., DMG-PEG2K).

[0244] In the embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein, and one or more lipids selected from cationic lipids, non-cationic lipids, PEGylated lipids, and cholesterol-based lipids.

[0245] According to various embodiments, the selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, and the relative molar ratios of such lipids to each other, are based on the characteristics of one or more selected lipids, the properties of the intended target cells, and the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, and the size, charge, pH, pKa, fusogenicity, and toxicity of one or more selected lipids. Therefore, the molar ratios can be adjusted accordingly.

[0246] cationic lipids

[0247] In addition to any of the compounds of the present invention as described herein, the composition may contain one or more other cationic lipids.

[0248] In some embodiments, liposomes may comprise one or more additional cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a number of lipid species that have 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.

[0249] Suitable additional cationic lipids for use in the composition include cationic lipids as described in the literature.

[0250] assist lipids

[0251] Compositions (e.g., liposome compositions) may also contain one or more accessory lipids. Such accessory lipids include noncationic lipids. As used herein, the phrase "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid types that carry a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include, but are not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (…). Dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof. A suitable non-cationic lipid or co-lipid for practicing this invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE) can be used as a non-cationic lipid or co-lipid.

[0252] In some embodiments, the non-cationic lipids are neutral lipids, i.e., lipids that do not carry a net charge under the conditions of formulation and / or application of the composition.

[0253] In some embodiments, non-cationic lipids may be present in the composition at the following molar ratios (mol%) of the total lipids present in the composition: 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%. In some embodiments, the total non-cationic lipids may be present in the composition at the following molar ratios (mol%) of the total lipids present in the composition: 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%. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipids in the liposomes may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0254] In some embodiments, non-cationic lipids may be present in the composition at the following weight percentages (wt%) of the total lipids present in the composition: 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%. In some embodiments, the percentage of non-cationic lipids in the liposomes may 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 may 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 may not exceed about 5 wt%, not exceed about 10 wt%, not exceed about 20 wt%, not exceed about 30 wt%, or not exceed about 40 wt%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may not exceed about 5 wt%, not exceed about 10 wt%, not exceed about 20 wt%, not exceed about 30 wt%, or not exceed about 40 wt%.

[0255] Cholesterol-based lipids

[0256] In some embodiments, the composition (e.g., a liposome composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleenylaminopropyl)piperazine (Gao et al. Biochem. Biophys. Res. Comm. 179,280 (1991); Wolf et al. BioTechniques 23,139 (1997); U.S. Patent No. 5,744,335), or imidazole cholesterol ester (ICE) having the following structure.

[0257]

[0258] In some embodiments, cholesterol-based lipids may be present in the liposomes at the following molar ratios (mol%) of the total lipids present in the liposomes: about 1% to about 30% or about 5% to about 20%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[0259] In some embodiments, cholesterol-based lipids may be present in the liposomes at the following weight percentages (wt%) of the total lipids present in the liposomes: about 1% to about 30% or about 5% to about 20%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may 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%.

[0260] PEGylated lipids

[0261] In some embodiments, the composition (e.g., a liposome composition) comprises one or more additional PEGylated lipids. A suitable PEG-modified or PEGylated lipid for practicing the present invention is 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2K).

[0262] For example, the present invention also contemplates the use of polyethylene glycol (PEG) modified phospholipids and derived lipids (such as derived ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8PEG-2000 ceramide)) in combination with one or more compounds of the present invention as described herein, and (in some embodiments) other lipids constituting liposomes. In some embodiments, particularly useful exchangeable lipids are those having shorter acyl chains (e.g., C... 14 Or C 18 PEG-ceramide.

[0263] Other envisioned PEG-modified lipids (also referred to herein as PEGylated lipids, the term being interchangeable with PEG-modified lipids) include, but are not limited to, those having one or more C6-C bonds. 20The lipid covalently attached alkyl chain is a polyethylene glycol chain of up to 5 kDa in 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 of increasing the cycle life of the lipid-nucleic acid composition and increasing the delivery of the lipid-nucleic acid composition to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected for rapid exchange of formulations in vivo (see U.S. Patent No. 5,885,613).

[0264] The additional PEG-modified phospholipids and derived lipids of the present invention may be present in the following molar ratios (mol%) of the total lipids present in the composition (e.g., liposome composition): from about 0% to about 10%, from about 0.5% to about 10%, from about 1% to about 10%, from about 2% to about 10%, or from about 3% to about 5%.

[0265] Pharmaceutical preparations and therapeutic uses

[0266] The compounds of the present invention as described herein can be used to prepare compositions (e.g., for constructing liposome compositions) that promote or enhance the delivery and release of encapsulating materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells).

[0267] For example, when a liposome composition (e.g., lipid nanoparticles) contains or is otherwise enriched with one or more of the compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate the delivery of encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in lipid nanoparticles) into said one or more target cells.

[0268] Similarly, in some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by their reduced in vivo toxicity. In some embodiments, the reduced toxicity is associated with high transfection efficiency of the compositions disclosed herein, making it possible to administer a reduced amount of this composition to the subject to achieve the desired therapeutic response or outcome.

[0269] Therefore, pharmaceutical formulations comprising the compounds described herein and the nucleic acids provided by the present invention can be used for a variety of therapeutic purposes. To facilitate in vivo delivery of nucleic acids, the compounds described herein and the nucleic acids can be formulated in combination with one or more additional drug carriers, targeting ligands, or stabilizing agents. In some embodiments, the compounds described herein can be formulated via a premixed lipid solution. In other embodiments, compositions comprising the compounds described herein can be formulated using a post-insertion technique into a lipid membrane of nanoparticles. Techniques for drug formulation and administration can be found in the following literature: “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0270] 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 intravenous, intravenous, intraperitoneal, or intranasal administration. In certain embodiments, intramuscular administration is administration to muscles selected from skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the administration results in the delivery of nucleic acids to muscle cells. In some embodiments, the administration results in the delivery of nucleic acids to hepatocytes (i.e., liver cells).

[0271] A common route of administration for the liposome compositions of the present invention is intravenous delivery, particularly when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamate (OTC) deficiency). Alternatively, depending on the disease or disorder to be treated, the liposome compositions may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposome compositions of the present invention are typically administered intramuscularly. Diseases or disorders affecting the eye can be treated by intravitreal administration of the liposome compositions of the present invention.

[0272] Alternatively or additionally, the pharmaceutical formulations of the present invention can be administered locally rather than systemically, for example, by direct injection of the pharmaceutical formulation into the targeted tissue (e.g., in a sustained-release formulation). Local delivery can be affected in a variety of ways, depending on the tissue to be targeted. Exemplary tissues in which the delivered mRNA can be delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In an embodiment, the tissue to be targeted is in the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention can be injected into, for example, sites of injury, disease manifestation, or pain; the composition can be provided as a lozenge for oral, tracheal, or esophageal application; it can be supplied in liquid, tablet, or capsule form for administration to the stomach or intestine; it can be supplied in suppository form for rectal or vaginal application; or it can even be delivered to the eye by using a cream, drops, or even injection.

[0273] The compositions described herein may contain mRNA encoding peptides (including those described herein, such as polypeptides, like proteins).

[0274] In the implementation scheme, mRNA encodes a polypeptide.

[0275] In the implementation plan, mRNA encodes a protein.

[0276] This article describes exemplary peptides (e.g., exemplary proteins encoded by mRNA) that are encoded by mRNA.

[0277] The present invention provides a method for delivering a composition having a full-length mRNA molecule encoding a target peptide or protein for treating a subject (e.g., a human subject) or cells of a human subject, or for treating and delivering cells of a human subject.

[0278] delivery method

[0279] The delivery routes used in the methods of the present invention allow for non-invasive self-administration of the compounds of the present invention. In some embodiments, the methods involve intratracheal or pulmonary administration of a composition containing mRNA encoding a therapeutic protein via aerosolization, nebulization, or instillation in a suitable transfection or lipid carrier medium as described above. In some embodiments, the protein is encapsulated in liposomes. In some embodiments, the liposomes contain lipids, which are compounds of the present invention. As used below, administering the compounds of the present invention includes administering a composition containing the compounds of the present invention.

[0280] While local lung cells and tissues represent potential targets for the production and secretion of mRNA-encoded proteins, the applicant has found that administration of the compounds of the present invention to the lungs via aerosolization, nebulization, or infusion results in the distribution of even non-secreting proteins outside lung cells. Without wishing to be bound by any particular theory, it is envisioned that the nanoparticle compositions of the present invention cross the lung-airway-blood barrier, resulting in the translocation of intact nanoparticles to non-lung cells and tissues (e.g., heart, liver, spleen), leading to the production of the encoded proteins in these non-lung tissues. Therefore, the utility of the compounds and methods of the present invention extends beyond the production of therapeutic proteins in lung cells and lung tissues and can be used for delivery to non-lung target cells and / or tissues. They can be used to manage and treat a wide range of diseases, particularly peripheral diseases caused by deficiencies in secreted and non-secreting proteins and / or enzymes (e.g., one or more lysosomal storage disorders). In some embodiments, the compounds of the present invention used in the methods of the present invention result in the distribution of mRNA-encapsulated nanoparticles and the production of the encoded proteins in liver, spleen, heart, and / or other non-lung cells. For example, as a result of the translocation of mRNA and delivery mediators to non-lung cells, the application of the compounds of the present invention to the lungs via aerosolization, nebulization, or infusion will result in the detection of the composition itself and its protein products (e.g., functional β-galactosidase protein) in local lung cells and tissues as well as in peripheral target cells, tissues, and organs.

[0281] In some embodiments, the compounds of the present invention can be used in the methods of the present invention to specifically target peripheral cells or tissues. Following pulmonary delivery, it is contemplated that the compounds of the present invention cross the pulmonary airway-blood barrier and distribute to cells other than local lung cells. Therefore, the compounds disclosed herein can be administered to a subject via the pulmonary administration route using various methods known to those skilled in the art (e.g., by inhalation) and distributed to both local target cells and tissues of the lungs and peripheral non-pulmonary cells and tissues (e.g., cells of the liver, spleen, kidney, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both local lung cells and peripheral non-pulmonary cells can act as biological reservoirs or repositories capable of producing and / or secreting translational products encoded by one or more polynucleotides. Therefore, the present invention is not limited to treating lung diseases or conditions, but can also be used as a non-invasive means to promote the delivery of polynucleotides or the production of enzymes and proteins encoded therein in peripheral organs, tissues, and cells (e.g., hepatocytes) (which would otherwise only be possible through systemic administration). Exemplary peripheral non-lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiomyocytes, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0282] Following administration of the composition to a subject, for at least one to seven days or longer after administration of the compound to the subject, a protein product (e.g., a functional protein or enzyme) encoded by mRNA may be detected in peripheral target tissues. The amount of protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the encoded protein, and the patient's condition. For example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer after administration of the compound to the subject, the following concentrations (e.g., therapeutic concentrations) of protein product may be detected in peripheral target tissues: 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.

[0283] 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 aerosol generated by a liquid nebulizer or by using a dry dispersant device (such as the device described in U.S. Patent 5,780,014, which is incorporated herein by reference).

[0284] In some embodiments, the compounds of the present invention can be formulated such that they can be aerosolized or otherwise delivered as particulate liquids or solids before or after administration to a subject. Such compounds can be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions) to produce particles that are easily respirable or inhalable by the subject. In some embodiments, such devices (e.g., metered-dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers, or blowpipes) assist in administering a predetermined mass, volume, or dose of the composition (e.g., about 0.5 mg / kg mRNA per dose). For example, in some embodiments, the compounds of the present invention are administered to a subject using a metered-dose inhaler containing a suspension or solution comprising the compound and a suitable propellant. In some embodiments, the compounds of the present invention can be formulated as particulate powders for inhalation (e.g., inhalable dry particles). In some embodiments, the compositions of the invention, formulated as inhalable particles, have an appropriate size (e.g., an average D50 or D90 particle size less than about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm, or smaller). In yet other embodiments, the compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, the compounds of the present invention are administered to a subject in a single dose at concentrations of at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, and so on. Less than 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight.In some embodiments, the compound of the invention is administered to a subject such that the total amount of mRNA administered in one or more doses is: at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg.

[0285] Synthesis of the compounds of the present invention

[0286] Cationic lipid MC3 is currently the gold standard for in vivo delivery of, for example, siRNA (see WO2010 / 144740). However, the synthesis of this lipid involves a six-step process and requires treatment with Grignard reagents. In contrast, the present invention provides cationic lipids that can be prepared from readily available starting reagents such as TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid, see Table 1 below)). These starting reagents can be coupled to a cationic head group and a lipid tail (see, for example, Table 2 below) using coupling reactions such as sulfonation, acetylation, and alkylation.

[0287] Table 1

[0288]

[0289] Table 2: Examples of lipid chains suitable for the present invention:

[0290]

[0291]

[0292] In the implementation, the cationic lipids described herein can be prepared by conjugating a lipid (e.g., a carboxylic acid of a lipid) with a “Good’s” buffer under suitable conditions. Exemplary lipid chains are described in Table 2. Therefore, suitable cationic lipids include those generated from any combination of precursors described in Tables 1 and 2.

[0293] In some embodiments, the sulfonic acid group of a compound (such as "Good's" buffer) can be derived by forming a sulfonyl chloride using a reagent (such as oxalyl chloride). The resulting sulfonyl chloride can undergo a variety of reactions, including but not limited to reduction with Zn / HCl to form the corresponding thiol, and coupling with a nucleophile (such as an amine or alcohol) to form the corresponding sulfonamide and sulfonate (see, for example, Scheme 1 below):

[0294]

[0295] Using the chemical reactions outlined in Scheme 1, a range of suitable cationic lipid head groups and lipid chain-derived sulfonic acid starting agents can be used.

[0296] For example, the cationic lipid (compound 3) according to the invention can be synthesized by reacting TES (compound 1) with acyl chloride (compound 2) in a first step. Then, in a second step, using oxalyl chloride, a cationic head group (such as compound 4) can be added to form lipid 5 (see, for example, scheme 2).

[0297]

[0298] The compounds of the present invention as described herein can be prepared according to methods known in the art, including exemplary synthesis of the embodiments provided herein.

[0299] Example

[0300] Although certain compounds, compositions and methods of the present invention have been specifically described according to certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the invention.

[0301] Example 1: A general synthetic scheme for cationic lipids based on TES

[0302] For example, the compounds of the present invention can be prepared according to Scheme 3:

[0303]

[0304] Linoleic acid is treated with a chlorinating agent (such as oxaloyl chloride) to provide acyl chloride compound 2. The reaction of compound 2 with a nucleophilic compound (such as buffer compound 1) provides compound 3.

[0305]

[0306]

[0307] Compound 3 is treated with a chlorinating agent (such as oxalyl chloride) to provide the electrophilic compound 3-Cl. Then, the reaction of 3-Cl with a nucleophile (such as compound 4a or 4b) provides compounds I and II, respectively (see Scheme 4 above).

[0308] Lipids according to schemes 3 and 4 were prepared using the following reaction conditions:

[0309]

[0310] Example 2: Specific Synthesis Scheme for TES-based Cationic Lipids

[0311] Scheme 5 was used to prepare compound I, as shown below:

[0312]

[0313] Specifically, compound I was prepared according to scheme 5 using the following synthetic steps:

[0314] Synthesis of (9Z,12Z)-octadec-9,12-dienoyl chloride (2)

[0315]

[0316] N,N-dimethylformamide (0.1 mL) and oxaloyl chloride (1.2 mL, 14.3 mmol) were added to a solution of linolenic acid (1.0 g, 3.6 mmol) in 10 mL of dichloromethane at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The solvent was removed under reduced pressure, and the crude product was used for the next step without further purification.

[0317] 2-((1,3-bis(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadec-9, Synthesis of 12-dienoyl)oxy)methyl)prop-2-yl)amino)ethane-1-sulfonic acid (3)

[0318]

[0319] To a solution of (9Z,12Z)-octadecano-9,12-dienoyl chloride 2 (1.1 g, 3.6 mmol) in anhydrous N,N-dimethylacetamide (5.0 mL) and N-methylmorpholine (3.0 mL), 2-((1,3-dihydroxy-2-(hydroxymethyl)propyl-2-yl)amino)ethane-1-sulfonic acid (1,TES) (200 mg, 0.87 mmol) was added. The reaction mixture was heated to 55 °C for 3 h. MS analysis showed the formation of the desired product. The reaction mixture was cooled to room temperature, diluted with water (100 mL), and extracted with dichloromethane (2 x 100 mL). The combined organic layers were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the residue was purified by column chromatography (0 to 10% methanol gradient in 40 g SiO2: dichloromethane) to obtain 2-((1,3-bis(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl-2-yl)amino)ethane-1-sulfonic acid (562 mg, 47% yield) as a colorless solid.

[0320] 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl Synthesis of propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)(3-Cl)

[0321]

[0322] N,N-dimethylformamide (0.05 mL) and oxaloyl chloride (0.08 mL, 2.1 mmol) were added to a solution of 2-((1,3-bis(((9Z,12Z)-octadec-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propyl-2-yl)amino)ethane-1-sulfonic acid 3 (210 mg, 0.82 mmol) in anhydrous dichloromethane (5.0 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h. The solvent was removed to dryness under reduced pressure to give 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate), which was used in the next step without further purification.

[0323] 2-((2-( N -(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadecyl) C-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecano-9,12-dienoic acid) Synthesis of ester (compound I)

[0324]

[0325] N2 was added to a solution of 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate)3-Cl (210 mg, 0.82 mmol) in anhydrous dichloromethane (5.0 mL) at 0 °C. 1 N 1 -Dimethylethane-1,2-diamine (182 mg, 2.1 mmol). The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched by adding water, and the mixture was extracted with dichloromethane (2 x 100 mL). The combined organic layers were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The solvent was removed, and the crude product was purified by column chromatography (0 to 15% methanol gradient in 40 g SiO2: dichloromethane) to obtain 2-((2-(N-(2-(dimethylamino)ethyl)amino)ethyl)amino)-2-((((9Z,12Z)-octadec-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate) (139 mg, 62% yield).

[0326] 1H NMR (300MHz, chloroform-d) δ5.26-5.44(m,12H),4.09(s,6H),3.06-3.18(m,6H),2.75(t,6H),2.47(t,2H), 2.32(t,6H),2.24(s,6H),2.00-2.10(m,12H),1.52-1.65(m,4H),1.20-1.40(m,44H),0.88(t,9H).

[0327] APCI-MS analysis: C64H115N3O8S, [M+H] calculated value = 1186.7, observed value = 1186.8.

[0328] All other cationic lipids were prepared in similar yields following the representative procedure described above.

[0329] 2-((2-(N-(3-(dimethylamino)propyl)aminosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadecyl) C-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9 Z ,9' Z ,12 Z ,12' Z )-bis(octadec-9,12-dienoic acid) Analytical data of ester (compound II)

[0330]

[0331] 1H NMR (300MHz, chloroform-d) δ5.24-5.42(m,12H),4.08(s,6H),3.17(t,2H),3.06(bs,4H),2.75(t,6H),2.43(t,2H),2.31 (t,6H),2.23(s,6H),1.98-2.08(m,12H),1.70(quint,2H),1.52-1.63(m,4H),1.17-1.45(m,44H),0.87(t,9H).

[0332] APCI-MS analysis: C65H117N3O8S, [M+H] calculated value = 1100.7, observed value = 1100.8.

[0333] 2-((2-( N -(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino)-2-((octanoyloxy)methyl) Analytical data for propane-1,3-dimethyldioctanoate (compound V)

[0334]

[0335] 1H NMR (300MHz, chloroform-d) δ4.09(s,6H),3.05-3.18(m,6H),2.44(t,2H),2.32(t,6H),2.22(s,6H),1.54-1.65(m,6H),1.20-1.40(m,24H),0.86(t,9H).

[0336] APCI-MS analysis: C34H67N3O8S, [M+H] calculated value = 678.4, observed value = 678.5.

[0337] 2-((decanoyloxy)methyl)-2-((2-( N -(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino) Analytical data for propane-1,3-dimethylbis(decanoate) (compound VI)

[0338]

[0339] 1H NMR (300MHz, chloroform-d) δ4.08(s,6H),3.04-3.16(m,6H),2.45(t,2H),2.31(t,6H),2.23(s,6H),1.52-1.65(m,6H),1.20-1.40(m,38H),0.86(t,9H).

[0340] APCI-MS analysis: C40H79N3O8S, [M+H] calculated value = 762.4, observed value = 762.5.

[0341] 2-((2-(N-(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino)-2-((dodecanoyloxy)methyl Analytical data for propane-1,3-dimethyldi-dodecanoate (compound VII)

[0342]

[0343] 1H NMR (300MHz, chloroform-d) δ4.08(s,6H),3.04-3.19(m,6H),2.46(t,2H),2.32(t,6H),2.23(s,6H),1.52-1.66(m,6H),1.16-1.34(m,50H),0.86(t,9H).

[0344] APCI-MS analysis: C46H91N3O8S, [M+H] calculated value = 846.6, observed value = 846.7.

[0345] 2-((2-(N-(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino)-2-((tetradecyloxy)methyl Analytical data for propane-1,3-dimethylditetradecanoate (compound VIII)

[0346]

[0347] 1H NMR (300MHz, chloroform-d) δ4.07(s,6H),3.15(t,2H),2.98-3.12(m,4H),2.46(t,2H), 2.30(t,6H),2.23(s,6H),1.50-1.64(m,6H),1.16-1.34(m,62H),0.86(t,9H).

[0348] APCI-MS analysis: C52H103N3O8S, [M+H] calculated value = 930.7, observed value = 930.6.

[0349] 2-((2-(N-(2-(dimethylamino)ethyl)aminosulfonyl)ethyl)amino)-2-((palmitoyloxy)methyl Analytical data for propane-1,3-dimethyldipalmitate (compound IX)

[0350]

[0351] 1H NMR (300MHz, chloroform-d) δ4.09(s,6H),3.15(t,2H),2.98-3.12(m,4H),2.48(t,2H), 2.32(t,6H),2.25(s,6H),1.53-1.64(m,6H),1.16-1.34(m,74H),0.86(t,9H).

[0352] APCI-MS analysis: C58H115N3O8S, [M+H] calculated value = 1014.8, observed value = 1014.7.

[0353] 2-((2-(N-(3-(dimethylamino)propyl)aminosulfonyl)ethyl)amino)-2-((octanoyloxy)methyl) Analytical data for propane-1,3-dimethyldioctanoate (compound X)

[0354]

[0355] 1H NMR (300MHz, chloroform-d) δ4.08(s,6H),3.18(t,2H),3.02-3.12(m,4H),2.48(t,2H),2.31(t, 6H),2.27(s,6H),1.73(quint,2H),1.52-1.65(m,6H),1.20-1.40(m,24H),0.86(t,9H).

[0356] APCI-MS analysis: C35H69N3O8S, [M+H] calculated value = 692.4, observed value = 692.5.

[0357] 2-((decanoyloxy)methyl)-2-((2-(N-(3-(dimethylamino)propyl)aminosulfonyl)ethyl)amino) Analytical data for propane-1,3-dimethylbis(decanoate) (compound XI)

[0358]

[0359] 1H NMR (300MHz, chloroform-d) δ4.09(s,6H),3.18(t,2H),3.04-3.14(m,4H),2.48(t,2H),2.32(t, 6H),2.26(s,6H),1.73(quint,2H),1.52-1.65(m,6H),1.16-1.38(m,38H),0.86(t,9H).

[0360] APCI-MS analysis: C41H81N3O8S, [M+H] calculated value = 776.4, observed value = 776.5.

[0361] 2-((2-(N-(3-(dimethylamino)propyl)aminosulfonyl)ethyl)amino)-2-((tetradecyloxy)methyl Analytical data for propane-1,3-dimethylditetradecanoate (compound XII)

[0362]

[0363] 1H NMR (300MHz, chloroform-d) δ4.06(s,6H),3.15(t,2H),3.04-3.12(m,4H),2.47(t,2H),2.29(t, 6H),2.25(s,6H),1.71(quint,2H),1.50-1.64(m,6H),1.16-1.38(m,62H),0.84(t,9H).

[0364] APCI-MS analysis: C53H105N3O8S, [M+H] calculated value = 944.7, observed value = 944.7.

[0365] 2-((2-(N-(3-(dimethylamino)propyl)aminosulfonyl)ethyl)amino)-2-((palmitoyloxy)methyl Analytical data for propane-1,3-dimethyldipalmitate (compound XIII)

[0366]

[0367] 1H NMR (300MHz, chloroform-d) δ4.09(s,6H),3.22(t,2H),3.04-3.14(m,4H),2.66(t,2H),2.41(s, 6H),2.32(t,6H),1.82(quint,2H),1.54-1.66(m,6H),1.16-1.38(m,74H),0.86(t,9H).

[0368] APCI-MS analysis: C59H117N3O8S, [M+H] calculated value = 1028.8, observed value = 1028.7.

[0369] Example 3: Lipid Nanoparticle Formulation

[0370] The cationic lipids described herein can be used to prepare lipid nanoparticles according to methods known in the art. Suitable methods include, for example, those described in International Publication No. WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[0371] An exemplary method for the formulation of lipid nanoparticles is Method A of WO 2018 / 089801 (see, for example, Example 1 and Figure 1 of WO 2018 / 089801). Method A (“A”) relates to a conventional method of encapsulating mRNA by mixing a mixture of mRNA and lipids without first pre-forming the lipids into lipid nanoparticles. In the exemplary method, an ethanol lipid solution and an aqueous buffer solution of mRNA are prepared separately. A solution of a mixture of lipids (cationic lipids, cofactor lipids, zwitterionic lipids, PEG lipids, etc.) is prepared by dissolving the lipids in ethanol. An mRNA solution is prepared by dissolving the mRNA in citrate buffer. The two solutions are then mixed using a pump system. In some cases, a gear pump system is used to mix the two solutions. In some embodiments, a 'T' connector (or a 'Y' connector) is used to mix the two solutions. The mixture is then purified by percolation using a TFF method. The resulting formulation is concentrated and stored at 2°C–8°C until further use.

[0372] A second exemplary method for the formulation of lipid nanoparticles is Method B of WO 2018 / 089801 (see, for example, Example 2 and Figure 2 of WO2018 / 089801). Method B (“B”) refers to a method of encapsulating messenger RNA (mRNA) by mixing pre-formed lipid nanoparticles with mRNA. A range of different conditions can be employed in Method B, such as different temperatures (i.e., heating or not heating the mixture), buffers, and concentrations. In the exemplary method, lipids dissolved in ethanol and citrate buffer are mixed using a pump system. The instantaneous mixing of the two streams results in the formation of empty lipid nanoparticles, which is a self-assembly process. The resulting formulation mixture is empty lipid nanoparticles in citrate buffer containing alcohol. The formulation is then subjected to a TFF purification method, in which buffer exchange is performed. The resulting suspension of pre-formed empty lipid nanoparticles is then mixed with mRNA using a pump system. For some cationic lipids, heating the mixed solution results in a higher percentage of lipid nanoparticles containing mRNA and a higher total mRNA yield.

[0373] The lipid nanoparticle formulations listed in Table 3 were prepared using method A. All lipid nanoparticle formulations contained hEPO mRNA and different lipids in the following mol% ratio: cationic lipid: DMG-PEG2000: cholesterol: DOPE = 40:5:25:30.

[0374] Table 3. Exemplary lipid nanoparticle formulations

[0375]

[0376] Example 4: Delivery of firefly luciferase (FFL) mRNA via intratracheal administration

[0377] During anesthesia (50 μL / animal) Male CD1 mice (6–8 weeks old) were administered the lipid nanoparticle formulation containing FFL mRNA, cationic lipids, DMG-PEG2000, cholesterol, and DOPE as listed in Table 3 via intratracheal aerosol administration. Approximately 24 hours after administration, animals were given 150 mg / kg (60 mg / ml) of luciferin via intraperitoneal injection at 2.5 ml / kg. All animals were imaged using an IVIS imaging system 5–15 minutes later to measure luciferase production in the lungs. Figure 1 illustrates the efficient in vivo delivery of FFL mRNA by the lipid nanoparticles containing the cationic lipids described herein, based on positive luciferase activity.

[0378] Based on the above description, those skilled in the art can readily determine the basic features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention.

[0379] All U.S. or foreign references, patents or applications cited in this application are incorporated herein by reference as if they were written entirely herein. In the event of any inconsistency, the material disclosed herein in its literal sense shall prevail.

Claims

1. A compound having a structure according to formula (Ia): (him) Or its pharmaceutically acceptable salt, wherein R 1 R 2 and R 3 Each independently is: ； R 4 yes ; Each R 6 It is independently selected from hydrogen, halogen, C1-C6 alkyl, and C2-C6 alkenyl; D is O; E and G are each independently selected from -NR 10 -、-O- and -S-; R 8 R 9 and R 10 Each is independently selected from hydrogen, C1-C6 alkyl, and C2-C6 alkenyl; Each b is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and in: (a) A is -O-; each R 5 Independently selected from C1-C 20 Alkyl, C2-C 20 alkenyl and C2-C 20 Alkyne group; and each C is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; (b) A is -NR 9 -;R 9 Selected from C1-C6 alkyl and C2-C6 alkenyl; each R 5 Independently selected from C1-C 20 Alkyl, C2-C 20 alkenyl and C2-C 20 Alkyne group; and each C is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; (c) A is -NH-; each R 5 Independently selected from C1-C 20 Alkyl and C2-C 20 Alkyne group; and each carbon group is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or (d) A is -NH-; each R 5 Independently selected from C1-C 20 Alkyl, C2-C 20 alkenyl and C2-C 20 Alkyne group; and each c is independently selected from 1, 3, 4, 5, 6, 7, 8, 9 and 10.

2. The compound according to claim 1, wherein the compound has a structure according to formula (Ib): (One) Or its pharmaceutically acceptable salt.

3. The compound according to claim 1, wherein the compound has a structure according to formula (Ic): (Ic) Or its pharmaceutically acceptable salt.

4. The compound according to claim 1, wherein the compound has a structure according to formula (Id): (Id) Or its pharmaceutically acceptable salt.

5. The compound according to claim 1, wherein the compound has a structure according to formula (Ie): (Ie) Or its pharmaceutically acceptable salt.

6. The compound according to claim 1, wherein the compound has a structure according to formula (If): (If) Or its pharmaceutically acceptable salt.

7. The compound according to claim 6, wherein the compound has a structure according to formula (Ig): (Ig) Or its pharmaceutically acceptable salt.

8. The compound according to claim 6, wherein the compound has a structure according to formula (Ih): (Them) Or its pharmaceutically acceptable salt.

9. The compound according to claim 1, wherein the compound has a structure according to formula (Ii): (Ii) Or its pharmaceutically acceptable salt.

10. The compound according to claim 9, wherein the compound has a structure according to formula (Ij): (Ij) Or its pharmaceutically acceptable salt.

11. The compound according to claim 9, wherein the compound has a structure according to formula (Ik): (I) Or its pharmaceutically acceptable salt.

12. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, Where R 1 R 2 and R 3 Each independently is .

13. The compound of claim 12 or a pharmaceutically acceptable salt thereof, wherein E is O.

14. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein each R 5 Independently selected from C1-C 20 Alkyl and C2-C 20 Alkenyl group.

15. The compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, wherein each R 5 Selected independently from: 、 、 、 、 、 、 、 ,and 。 16. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 4 yes .

17. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 4 yes .

18. The compound of claim 17 or a pharmaceutically acceptable salt thereof, wherein R 4 Selected from and .

19. The compound of claim 18 or a pharmaceutically acceptable salt thereof, wherein R 4 yes .

20. The compound of claim 18 or a pharmaceutically acceptable salt thereof, wherein R 4 yes .

21. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein b is 1.

22. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein c is 2 or 3.

23. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein A is NR. 9 Or NH.

24. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein A is O.

25. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from the group consisting of compounds II-XIII: 。 26. A compound I: Pharmaceutically acceptable salts.

27. A composition comprising any one of the cationic lipids of claims 1 to 26, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

28. The composition of claim 27, wherein the composition is a lipid nanoparticle, optionally a liposome.

29. The composition of claim 28, wherein the one or more cationic lipids constitute about 30 mol%-60 mol% of the lipid nanoparticles.

30. The composition of claim 28, wherein the one or more non-cationic lipids constitute 10 mol%-50 mol% of the lipid nanoparticles.

31. The composition according to claim 28, wherein one or more PEG-modified lipids constitute 1 mol%-10 mol% of the lipid nanoparticles.

32. The composition of claim 28, wherein the cholesterol-based lipid constitutes 10 mol%-50 mol% of the lipid nanoparticles.

33. The composition according to any one of claims 28-32, wherein the lipid nanoparticles encapsulate nucleic acids, optionally encoding the mRNA of a peptide or protein.

34. The composition according to any one of claims 28-32, wherein the lipid nanoparticles encapsulate mRNA encoding peptides or proteins.

35. The composition of claim 34, wherein the encapsulation percentage of mRNA in the lipid nanoparticles is: (i) at least 70%; (ii) at least 75%; (iii) At least 80%; (iv) at least 85%; (v) at least 90%; or (vi) At least 95%.

36. Use of the composition according to claim 34 in the preparation of a medicament.

37. Use of the composition according to claim 34 in the preparation of a medicament for treating or preventing a disease suitable for treatment or prevention by means of a peptide or protein encoded by said mRNA.

38. The use according to claim 37, wherein the disease is (a) a protein deficiency, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

39. The use according to claim 38, wherein the protein deficiency affects the liver, lungs, brain, or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

40. The use according to claim 36 or 37, wherein the composition is formulated for intravenous, intrathecal, or intramuscular administration, or for delivery via the lungs.

41. The use according to claim 40, wherein, The composition is formulated for application by atomization.

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