CN115667207A

CN115667207A - Cationic lipids based on phenolic acid lipids

Info

Publication number: CN115667207A
Application number: CN202180039080.XA
Authority: CN
Inventors: S·卡夫; F·德罗萨; A·旺威拉翁; S·卡马卡
Original assignee: Translation Bio Co
Current assignee: Translation Bio Co
Priority date: 2020-04-01
Filing date: 2021-03-31
Publication date: 2023-01-31
Also published as: EP4126813A1; JP2023520047A; WO2021202694A1

Abstract

The present invention provides, in part, phenolic acid lipid compounds of formula (I) and subformulae thereof, or pharmaceutically acceptable salts thereof. Compounds provided hereinCan be used to deliver and express mRNA and encoded protein, e.g., as components of a liposomal delivery vehicle, and thus can be used to treat a variety of diseases, disorders, and conditions, such as those associated with a deficiency in one or more proteins.

Description

Cationic lipids based on phenolic acid lipids

Cross reference to related applications

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

Background

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

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

Phenolic acids have a number of advantageous properties that make them good starting points for the synthesis of cationic lipids for use in an in vivo environment. For example, phenolic acids show no toxicity, are available in large quantities, and are readily derivatized. Phenolic acids can be broadly divided into two groups: benzoic and cinnamic acids, and derivatives thereof.

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

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

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

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

disclosure of Invention

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

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

In one aspect, cationic lipids are provided having a structure according to formula (I):

wherein L is ₁ Is a bond, (C) ₁ -C ₆ ) Alkyl or (C) ₂ -C ₆ ) An alkenyl group;

wherein X is O or S;

wherein R is ¹ 、R ² 、R ³ 、R ⁴ And R ⁵ Each independently selected from H, OH, optionally substituted (C) ₁ -C ₆ ) Alkyl, optionally substituted (C) ₂ -C ₆ ) Alkenyl, optionally substituted (C) ₂ -C ₆ ) Alkynyl, optionally substituted (C) ₁ -C ₆ ) Alkoxy and-OC (O) R';

wherein R is ¹ 、R ² 、R ³ 、R ⁴ Or R ⁵ At least one of which is-OC (O) R';

wherein R' is

Wherein R is ⁶ Is composed of

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

wherein R is ⁷ Selected from H, optionally substituted (C) ₁ -C ₆ ) Alkyl, optionally substituted (C) ₂ -C ₆ ) Alkenyl, optionally substituted (C) ₂ -C ₆ ) Alkynyl, optionally substituted (C) ₁ -C ₆ ) Acyl, - (CH) ₂ ) _k R ^A Or- (CH) ₂ ) _k CH(OR ¹¹ )R ^A ；

Wherein R is ⁸ Selected from H, optionally substituted (C) ₁ -C ₆ ) Alkyl, optionally substituted (C) ₂ -C ₆ ) Alkenyl, optionally substituted (C) ₂ -C ₆ ) Alkynyl, optionally substituted (C) ₁ -C ₆ ) Acyl, - (CH) ₂ ) _n R ^B Or- (CH) ₂ ) _n CH(OR ¹² )R ^B ；

Wherein R is ⁹ Selected from H, optionally substituted (C) ₁ -C ₆ ) Alkyl, optionally substituted (C) ₂ -C ₆ ) Alkenyl, optionally substituted (C) ₂ -C ₆ ) Alkynyl, optionally substituted (C) ₁ -C ₆ ) Acyl, - (CH) ₂ ) _q R ^C Or- (CH) ₂ ) _q CH(OR ¹³ )R ^C ；

Wherein R is ¹⁰ Selected from H, optionally substituted (C) ₁ -C ₆ ) Alkyl, optionally substituted (C) ₂ -C ₆ ) Alkenyl, optionally substituted (C) ₂ -C ₆ ) Alkynyl, optionally substituted (C) ₁ -C ₆ ) Acyl, - (CH) ₂ ) _r R ^D Or- (CH) ₂ ) _r CH(OR ¹⁴ )R ^D ；

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

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

wherein R is ¹¹ 、R ¹² 、R ¹³ And R ¹⁴ Each independently selected from H, methyl, ethyl or propyl;

wherein R is ^A 、R ^B 、R ^C And R ^D Each independently selected from optionally substituted (C) ₆ -C ₂₀ ) Alkyl, optionally substituted (C) ₆ -C ₂₀ ) Alkenyl, optionally substituted (C) ₆ -C ₂₀ ) Alkynyl, optionally substituted (C) ₆ -C ₂₀ ) Acyl, optionally substituted-OC (O) alkyl, optionally substituted-OC (O) alkenyl, optionally substituted (C) ₁ -C ₆ ) Monoalkylamino, optionally substituted (C) ₁ -C ₆ ) Dialkylamino, optionally substituted (C) ₁ -C ₆ ) Alkoxy, -OH, -NH ₂ ；

Wherein R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ At least one of which respectively comprises R ^A 、R ^B 、R ^C Or R ^D Moiety wherein said R ^A 、R ^B 、R ^C Or R ^D Independently selected from optionally substituted (C) ₆ -C ₂₀ ) Alkyl, optionally substituted (C) ₆ -C ₂₀ ) Alkenyl, optionally substituted (C) ₆ -C ₂₀ ) Alkynyl, optionally substituted (C) ₆ -C ₂₀ ) Acyl, optionally substituted-OC (O) (C) ₆ -C ₂₀ ) Alkyl or optionally substituted-OC (O) (C) ₆ -C ₂₀ ) An alkenyl group;

or a pharmaceutically acceptable salt thereof.

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

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

In one aspect, the compositions comprising the cationic lipids of the present invention may be used for therapy.

Drawings

Figure 1 illustrates in vivo protein expression following intratracheal administration of lipid nanoparticles comprising one of the cationic lipid compounds 1-12. Based on positive luciferase activity, lipid nanoparticles comprising the cationic lipids described herein efficiently deliver FFL mRNA in vivo.

Detailed Description

Definition of

We have so made the present invention more understandable, first, certain 32681 is described below. Additional definitions for the following terms and other terms are set forth throughout this specification. The publications and other reference materials used herein to describe the background of the invention and to provide additional details respecting the practice are incorporated by reference.

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

Animals: as used herein, a "35486" refers to any member of the "lower" object. In some embodiments 2352626, "the" processes "refer to humans that perform the order on any machine. In some embodiments 23526, "" is a non-human machine fragment at any order culture. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and/or a pig). In some 23526embodiments, the virus includes, but is not limited to, a mammal, a 40165, a reptilis, a two, a fish and/or a worm. In some embodiments of 23526, the promoter may be a rotation promoter, a genetic engineering promoter, and/or a clone.

Large, 32004or\32004: as used herein, the term "about" or "approximately" when applied to one or more stated values refers to a value similar to the stated reference value. In certain embodiments, the term "about" or "approximately" refers to a series of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of any direction (greater than or less than) of the stated value, unless otherwise stated or otherwise apparent from the context (unless the number exceeds 100% of the possible values).

The biological activity is as follows: as used herein, the term "bioactive" refers to the characteristics 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 is considered to be biologically active when administered to that organism.

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

Expressing: 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 the post-translational modification of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms "expression" and "generation" and their grammatical equivalents are used interchangeably.

Functionality: as used herein, a "functional" biomolecule is a biomolecule that exhibits a form of property and/or activity that characterizes it.

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

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

Improving, increasing or decreasing: as used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents thereof, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. "biological blood" is suffering from the same form of disease, people who live their year 408011 who live their living being, who living being, live their living being 32004.

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

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

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

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

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

Nucleic acid (A): as used herein, the term "nucleic acid" in its broadest sense refers to any compound and/or substance that can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain from a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising a single nucleic acid residue. In some embodiments, "nucleic acid" encompasses RNA as well as single-and/or double-stranded DNA and/or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acids (RNAs), including, but not limited to, any one or more of interfering RNAs (RNAi), small interfering RNAs (siRNA), short hairpin RNAs (shRNA), antisense RNAs (aRNA), messenger RNAs (mRNA), modified messenger RNAs (mmRNA), long noncoding RNAs (lncRNA), micrornas (miRNA), polynuclear coding nucleic acids (MCNA), polymeric Coding Nucleic Acids (PCNA), guide RNAs (gRNA), and CRISPR RNAs (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, but not limited to, any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses RNA and DNA. In embodiments, the DNA may be in the form of antisense DNA, plasmid DNA, portions of plasmid DNA, pre-condensed DNA, products of Polymerase Chain Reaction (PCR), vectors (e.g., P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups. In embodiments, the RNA may be in the form of messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7 SL RNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), micronucleus RNA (snRNA), micronucleus RNA (snoRNA), smY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncrrna), microrna (miRNA), RNA that interacts with piwi (piRNA), small interfering RNA (siRNA), transactivating siRNA (tassirna), repeat-associated siRNA (rasiRNA), 73K RNA, reverse transcription, viral genome, viroid, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA encoding a protein, such as an enzyme.

The patients: as used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and/or humans). In some of the embodiments of 2352626. Humans include pre-pivot and post-pivot.

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

Pharmaceutically acceptable salts: pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in journal of pharmaceutical Sciences (j. Pharmaceutical Sciences), (1977) 66, by s.m. berge et al. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoate, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valeric salts, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N ⁺ (C _1-4 Alkyl radical) ₄ And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Where appropriate, additional pharmaceutically acceptable salts include those which employ, for example, halides, hydroxides, carboxylates, sulfates, phosphoric acidSalts, nitrates, sulfonates, and aryl sulfonates, and the like, form non-toxic ammonium, quaternary ammonium, and amine cations. Additional pharmaceutically acceptable salts include salts formed by quaternization of amines using a suitable electrophile (e.g., an alkyl halide) to form a quaternized alkylated amino salt.

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

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). People include pivot and post-pivot. In the kyo poly 23526, the living being is a human being. The patient can be a patient who is located at the disease affecting person who is lifted by the navigation provider, 2603964. TV (35486) "can be used herein either" living being "or" patient "each other. Tissue can be suffering from or susceptible to disease or barrier 31001, but can or cannot show the disease or barrier 31001.

Essentially: as used herein, "substantially" refers to a qualitative tip that exhibits all or nearly all levels of extent 22285or degree of a target feature or characteristic. A living being person of ordinary skill in the art of the field of living beings 23416. Thus, the term "substantially" is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena.

Target tissue: as used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some embodiments, the target tissues include those tissues exhibiting a disease-associated pathology, symptom, or feature.

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

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

Chemical definition

Acyl group: as used herein, the term "acyl" refers to R ^Z - (C = O) -, wherein R ^Z For example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene group.

Aliphatic: as used herein, the term aliphatic refers to C _1- C ₄₀ Hydrocarbons, and includes both saturated hydrocarbons and unsaturated hydrocarbons. The aliphatic may be linear, branched or cyclic. For example, C ₁ -C ₂₀ The aliphatic may include C ₁ -C ₂₀ Alkyl (e.g., straight or branched C) ₁ -C ₂₀ Saturated alkyl), C ₂ -C ₂₀ Alkenyl (e.g., straight or branched C) ₄ -C ₂₀ Dienyl, straight-chain or branched C ₆ -C ₂₀ Trienyl, etc.) and C ₂ -C ₂₀ Alkynyl (e.g., straight or branched C) ₂ -C ₂₀ Alkynyl). C ₁ -C ₂₀ The aliphatic group may include C ₃ -C ₂₀ Cyclic aliphatic (e.g. C) ₃ -C ₂₀ Cycloalkyl radical, C ₄ -C ₂₀ Cycloalkenyl or C ₈ -C ₂₀ Cycloalkynyl). In certain embodiments, the aliphatic may comprise one or more cyclic aliphatics and/or one or more heteroatoms such as oxygen, nitrogen or sulfur, andand may be optionally substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester or amide. An aliphatic radical is unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic can be substituted with one or more (e.g., 1,2,3,4,5, or 6 independently selected substituents) of: halogen, -COR " ₂ H、-CO ₂ R”、-CN、-OH、-OR”、-OCOR'、-OCO ₂ R”、-NH ₂ 、-NHR”、-N(R”) ₂ -SR "or-SO ₂ R 'wherein each instance of R' is independently C ₁ -C ₂₀ Aliphatic (e.g. C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted C ₁ -C ₃ An alkyl group. In embodiments, the aliphatic is unsubstituted. In embodiments, aliphatic does not include any heteroatoms. Alkyl groups: as used herein, the term "alkyl" means acyclic straight and branched chain hydrocarbon radicals, e.g., "C ₁ -C ₃₀ Alkyl "refers to an alkyl group having 1 to 30 carbons. The alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like. The term "lower alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art, given the benefit of this disclosure. An alkyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkyl group can be substituted with one or more (e.g., 1,2,3,4,5, or 6 independently selected substituents) of: halogen, -COR " ₂ H、-CO ₂ R”、-CN、-OH、-OR”、-OCOR'、-OCO ₂ R”、-NH ₂ 、-NHR”、-N(R”) ₂ -SR "or-SO ₂ R ', wherein each instance of R' is independently C ₁ -C ₂₀ Aliphatic (e.g. C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted C ₁ -C ₃ An alkyl group. In embodiments, alkyl is substituted (e.g., with 1,2,3,4,5, or 6 substituent groups as described herein). In embodiments, an alkyl group is substituted with an-OH group, and may also be referred to herein as "hydroxyalkyl," where the prefix represents an-OH group, and "alkyl" is as described herein.

The term "alkyl" as used herein also refers to a radical of a straight or branched chain saturated hydrocarbon group having from 1 to 50 carbon atoms ("C) ₁ -C ₅₀ Alkyl "). In some embodiments, the alkyl group has 1 to 40 carbon atoms ("C) ₁ -C ₄₀ Alkyl "). In some embodiments, the alkyl group has 1 to 30 carbon atoms ("C) ₁ -C ₃₀ Alkyl "). In some embodiments, the alkyl group has 1 to 20 carbon atoms ("C) ₁ -C ₂₀ Alkyl "). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("C) ₁ -C ₁₀ Alkyl "). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C) ₁ -C ₉ Alkyl "). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C) ₁ -C ₈ Alkyl "). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C) ₁ -C ₇ Alkyl "). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C) ₁ -C ₆ Alkyl "). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C) ₁ -C ₅ Alkyl "). In a 1In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C) ₁ -C ₄ Alkyl "). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C) ₁ -C ₃ Alkyl "). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C) ₁ -C ₂ Alkyl "). In some embodiments, the alkyl group has 1 carbon atom ("C) ₁ Alkyl "). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C) ₂ -C ₆ Alkyl "). C ₁ -C ₆ Examples of alkyl groups include, but are not limited to, methyl (C) ₁ ) Ethyl (C) ₂ ) N-propyl (C) ₃ ) Isopropyl (C) ₃ ) N-butyl (C) ₄ ) Tert-butyl (C) ₄ ) Sec-butyl (C) ₄ ) Isobutyl (C) ₄ ) N-pentyl group (C) ₅ ) 3-pentyl radical (C) ₅ ) Pentyl radical (C) ₅ ) Neopentyl (C) ₅ ) 3-methyl-2-butyl (C) ₅ ) Tert-amyl (C) ₅ ) And n-hexyl (C) ₆ ). Further examples of alkyl groups include n-heptyl (C) ₇ ) N-octyl (C) ₈ ) And the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents. In certain embodiments, alkyl is unsubstituted C ₁ -C ₅₀ An alkyl group. In certain embodiments, alkyl is substituted C ₁ -C ₅₀ An alkyl group.

The prefix "ene" for a group indicates that the group is a divalent moiety, e.g., arylene is a divalent moiety of aryl and heteroarylene is a divalent moiety of heteroaryl.

Alkylene group: as used herein, the term "alkylene" denotes a saturated divalent straight or branched chain hydrocarbon group, and is exemplified by methylene, ethylene, isopropylidene, and the like. Also, as used herein, the term "alkenylene" refers to an unsaturated divalent straight or branched chain hydrocarbon radical having one or more unsaturated carbon-carbon double bonds that may be present at any stable point along the chain, and the term "alkynylene" refers herein to an unsaturated divalent straight or branched chain hydrocarbon radical having one or more unsaturated carbon-carbon triple bondsA chain or branched hydrocarbon group, the unsaturated carbon-carbon triple bond may be present at any point of stability along the chain. In certain embodiments, the alkylene, alkenylene, or alkynylene group may contain one or more cycloaliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may be optionally substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. For example, an alkylene, alkenylene, or alkynylene group may be substituted with one or more (e.g., 1,2,3,4,5, or 6 independently selected substituents) of: halogen, -COR ", -CO ₂ H、-CO ₂ R”、-CN、-OH、-OR”、-OCOR”、-OCO ₂ R”、-NH ₂ 、-NHR”、-N(R”) ₂ -SR "or-SO ₂ R ', wherein each instance of R' is independently C ₁ -C ₂₀ Aliphatic (e.g. C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted C ₁ -C ₃ An alkyl group. In certain embodiments, the alkylene, alkenylene, or alkynylene group is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene group does not include any heteroatoms. Alkenyl: as used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may occur at any point of stability along the chain, for example: "C ₂ -C ₃₀ Alkenyl "means alkenyl having 2 to 30 carbons. For example, alkenyl includes prop-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-5-enyl, 2, 3-dimethylbut-2-enyl and the like. In embodiments, the alkenyl group contains 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 (e.g., 2or 3) are conjugated. Alkenyl may be unsubstitutedSubstituted or substituted with one or more substituents described herein. For example, an alkenyl group can be substituted with one or more (e.g., 1,2,3,4,5, or 6 independently selected substituents) of: halogen, -COR " ₂ H、-CO ₂ R”、-CN、-OH、-OR”、-OCOR”、-OCO ₂ R”、-NH ₂ 、-NHR”、-N(R”) ₂ -SR "or-SO ₂ R ', wherein each instance of R' is independently C ₁ -C ₂₀ Aliphatic (e.g. C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted C ₁ -C ₃ An alkyl group. In embodiments, the alkenyl group is unsubstituted. In embodiments, the alkenyl group is substituted (e.g., with 1,2,3,4,5, or 6 substituent groups as described herein). In embodiments, an alkenyl group is substituted with an-OH group, and may also be referred to herein as a "hydroxyalkenyl," where the prefix represents an-OH group, and "alkenyl" is as described herein.

As used herein, "alkenyl" also refers to a radical ("C") having a straight or branched chain hydrocarbyl group of 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1,2,3, or 4 double bonds) ₂ -C ₅₀ Alkenyl "). In some embodiments, alkenyl groups have 2 to 40 carbon atoms ("C) ₂ -C ₄₀ Alkenyl "). In some embodiments, alkenyl groups have 2 to 30 carbon atoms ("C) ₂ -C ₃₀ Alkenyl "). In some embodiments, alkenyl groups have 2 to 20 carbon atoms ("C) ₂ -C ₂₀ Alkenyl "). In some embodiments, alkenyl groups have 2 to 10 carbon atoms ("C) ₂ -C ₁₀ Alkenyl "). In some embodiments, alkenyl groups have 2 to 9 carbon atoms ("C) ₂ -C ₉ Alkenyl "). In some embodiments, the alkenyl group hasHaving 2 to 8 carbon atoms (' C) ₂ -C ₈ Alkenyl "). In some embodiments, alkenyl groups have 2 to 7 carbon atoms ("C) ₂ -C ₇ Alkenyl "). In some embodiments, alkenyl groups have 2 to 6 carbon atoms ("C) ₂ -C ₆ Alkenyl "). In some embodiments, alkenyl groups have 2 to 5 carbon atoms ("C) ₂ -C ₅ Alkenyl "). In some embodiments, alkenyl groups have 2 to 4 carbon atoms ("C) ₂ -C ₄ Alkenyl "). In some embodiments, alkenyl groups have 2 to 3 carbon atoms ("C) ₂ -C ₃ Alkenyl "). In some embodiments, alkenyl has 2 carbon atoms ("C) ₂ Alkenyl "). The carbon-carbon double bond(s) may be internal (e.g., in the 2-butenyl group) or terminal (e.g., in the 1-butenyl group). C ₂ -C ₄ Examples of alkenyl groups include, but are not limited to, vinyl (C) ₂ ) 1-propenyl group (C) ₃ ) 2-propenyl (C) ₃ ) 1-butenyl (C) ₄ ) 2-butenyl (C) ₄ ) Butadienyl radical (C) ₄ ) And so on. C ₂ -C ₆ Examples of the alkenyl group include the aforementioned C ₂ -C ₄ Alkenyl and pentenyl (C) ₅ ) Pentadienyl (C) ₅ ) Hexenyl (C6), and the like. Further examples of alkenyl groups include heptenyl (C) ₇ ) Octenyl (C) ₈ ) Octrienyl (C) ₈ ) And the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In certain embodiments, alkenyl is unsubstituted C ₂ -C ₅₀ An alkenyl group. In certain embodiments, alkenyl is substituted C ₂ -C ₅₀ An alkenyl group.

Alkynyl: as used herein, "alkynyl" means a hydrocarbon chain of any straight or branched configuration having one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "C ₂ -C ₃₀ Alkynyl "refers to alkynyl groups having 2-30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl and the like. In the examples, alkynyl groups includeContaining a carbon-carbon triple bond. An alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkynyl group can be substituted with one or more (e.g., 1,2,3,4,5, or 6 independently selected substituents) of: halogen, -COR " ₂ H、-CO ₂ R”、-CN、-OH、-OR”、-OCOR”、-OCO ₂ R”、-NH ₂ 、-NHR”、-N(R”) ₂ -SR "or-SO ₂ R 'wherein each instance of R' is independently C ₁ -C ₂₀ Aliphatic (e.g. C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl groups). In embodiments, R "is independently unsubstituted alkyl (e.g., unsubstituted C) ₁ -C ₂₀ Alkyl radical, C ₁ -C ₁₅ Alkyl radical, C ₁ -C ₁₀ Alkyl or C ₁ -C ₃ Alkyl). In embodiments, R "is independently unsubstituted C ₁ -C ₃ An alkyl group. In embodiments, the alkynyl group is unsubstituted. In embodiments, the alkynyl group is substituted (e.g., with 1,2,3,4,5, or 6 substituent groups as described herein).

As used herein, "alkynyl" also refers to a radical ("C") of a straight or branched chain hydrocarbyl 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) ₂ -C ₅₀ Alkynyl "). Alkynyl groups having one or more triple bonds and one or more double bonds are also referred to as "ene-ynes". In some embodiments, alkynyl has 2 to 40 carbon atoms ("C) ₂ -C ₄₀ Alkynyl "). In some embodiments, alkynyl has 2 to 30 carbon atoms ("C) ₂ -C ₃₀ Alkynyl "). In some embodiments, alkynyl has 2 to 20 carbon atoms ("C) ₂ -C ₂₀ Alkynyl "). In some embodiments, alkynyl has 2 to 10 carbon atoms ("C) ₂ -C ₁₀ Alkynyl "). In some embodiments, alkynyl has 2 to 9 carbon atoms ("C) ₂ -C ₉ Alkynyl radical"). In some embodiments, alkynyl has 2 to 8 carbon atoms ("C) ₂ -C ₈ Alkynyl "). In some embodiments, alkynyl has 2 to 7 carbon atoms ("C) ₂ -C ₇ Alkynyl "). In some embodiments, alkynyl has 2 to 6 carbon atoms ("C) ₂ -C ₆ Alkynyl "). In some embodiments, alkynyl has 2 to 5 carbon atoms ("C) ₂ -C ₅ Alkynyl "). In some embodiments, alkynyl has 2 to 4 carbon atoms ("C) ₂ -C ₄ Alkynyl "). In some embodiments, alkynyl has 2 to 3 carbon atoms ("C) ₂ -C ₃ Alkynyl "). In some embodiments, alkynyl has 2 carbon atoms ("C) ₂ Alkynyl "). The carbon-triple bond or bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). C ₂ -C ₄ Examples of alkynyl groups include, but are not limited to, ethynyl (C) ₂ ) 1-propynyl (C) ₃ ) 2-propynyl (C) ₃ ) 1-butynyl (C) ₄ ) 2-butynyl (C) ₄ ) And the like. C ₂ -C ₆ Examples of the alkenyl group include the aforementioned C ₂ -C ₄ Alkynyl and pentynyl (C) ₅ ) Hexynyl (C) ₆ ) And so on. Further examples of alkynyl groups include heptynyl (C) ₇ ) Octynyl (C) ₈ ) And the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In certain embodiments, alkynyl is unsubstituted C ₂ -C ₅₀ Alkynyl. In certain embodiments, alkynyl is substituted C ₂ -C ₅₀ Alkynyl.

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

As used herein, "aryl" also refers to a radical ("C") of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n +2 aromatic ring system (e.g., having 6, 10, or 14 π electrons in common in the ring array) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ₆ -C ₁₄ Aryl "). In some embodiments, an aryl group has 6 ring carbon atoms ("C) ₆ Aryl "; for example, phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C) ₁₀ Aryl "; for example, naphthyl groups such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C) ₁₄ Aryl "; for example, an anthracene group). "aryl" also includes ring systems in which an aromatic ring as defined above is fused to one or more carbocyclic or heterocyclic groups in which the linking radical or point of attachment is on the aromatic ring, and in which case the number of carbon atoms continues to indicate the number of carbon atoms in the aromatic ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, aryl is unsubstituted C ₆ -C ₁₄ And (3) an aryl group. In certain embodiments, aryl is substituted C ₆ -C ₁₄ And (3) an aryl group.

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

A carbocyclic group: as used herein, "carbocyclyl" or "carbocycle" refers to a ring having from 3 to 10 ring carbon atoms ("C") in a non-aromatic ring system ₃ -C ₁₀ Carbocyclyl ") and zero heteroatom non-aromatic cyclic hydrocarbyl groups. In some embodiments, carbocyclyl has 3 to 8 ring carbon atoms ("C) ₃ -C ₈ Carbocyclyl "). In some embodiments, carbocyclyl has 3 to 7 ring carbon atoms ("C) ₃ -C ₇ Carbocyclyl "). In some embodiments, carbocyclyl has 3 to 6 ring carbon atoms ("C) ₃ -C ₆ Carbocyclyl "). In some embodiments, carbocyclyl has 4 to 6 ring carbon atoms ("C) ₄ -C ₆ Carbocyclyl "). In some embodiments, carbocyclyl has 5 to 6 ring carbon atoms ("C) ₅ -C ₆ Carbocyclyl "). In some embodiments, carbocyclyl has 5 to 10 ring carbon atoms ("C) ₅ -C ₁₀ Carbocyclyl "). Exemplary C ₃ -C ₆ Carbocyclyl includes, but is not limited to, cyclopropyl (C) ₃ ) Cyclopropenyl group (C) ₃ ) Cyclobutyl (C) ₄ ) Cyclobutenyl (C4), cyclopentyl (C ₅ ) Cyclopentenyl group (C) ₅ ) Cyclohexyl (C) ₆ ) Cyclohexenyl (C) ₆ ) Cyclohexadienyl (C) ₆ ) And so on. Exemplary C ₃ -C ₈ Carbocyclyl groups including, but not limited to, the foregoing C ₃ -C ₆ Carbocyclyl and cycloheptyl (C) ₇ ) Cycloheptenyl (C) ₇ ) Cycloheptadienyl (C) ₇ ) Cycloheptatrienyl (C) ₇ ) Cyclooctyl (C) ₈ ) Cyclooctenyl (C) ₈ ) Bicyclo [2.2.1]Heptyl (C) ₇ ) Bicyclo [2.2.2 ] s]Octyl radical (C) ₈ ) And the like. Exemplary C ₃ -C ₁₀ Carbocyclyl includes, but is not limited to, C as previously described ₃ -C ₈ Carbocyclyl and cyclononyl (C) ₉ ) Cyclononenyl (C) ₉ ) Cyclodecyl (C) ₁₀ ) Cyclodecenyl (C) ₁₀ ) octahydro-1H-indenyl (C) ₉ ) Decahydronaphthyl (C) ₁₀ ) Spiro [4.5 ]]Decyl (C) ₁₀ ) And the like. As shown in the foregoing examples, in certain embodiments, a carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring)A ring system, such as a bicyclic system ("bicyclic carbocyclyl") or tricyclic system) ("tricyclic carbocyclyl")), and may be saturated or may contain one or more carbon-carbon double or triple bonds. "carbocyclyl" also includes ring systems in which a carbocyclic ring, as defined above, is fused to one or more aryl or heteroaryl groups, with the point of attachment being on the carbocyclic ring, and in such cases the number of carbons always represents the number of carbons in the carbocyclic system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In certain embodiments, carbocyclyl is unsubstituted C ₃ -C ₁₀ A carbocyclic group. In certain embodiments, carbocyclyl is substituted C ₃ -C ₁₀ A carbocyclic group.

In some embodiments, "carbocyclyl" or "carbocycle" refers to "cycloalkyl," i.e., a monocyclic saturated carbocyclyl ("C") having 3 to 10 ring carbon atoms ₃ -C ₁₀ Cycloalkyl "). In some embodiments, cycloalkyl groups have 3 to 8 ring carbon atoms ("C) ₃ -C ₈ Cycloalkyl "). In some embodiments, cycloalkyl groups have 3 to 6 ring carbon atoms ("C) ₃ -C ₆ Cycloalkyl "). In some embodiments, cycloalkyl groups have 4 to 6 ring carbon atoms ("C) ₄ -C ₆ Cycloalkyl "). In some embodiments, cycloalkyl groups have 5 to 6 ring carbon atoms ("C) ₅ -C ₆ Cycloalkyl "). In some embodiments, cycloalkyl groups have 5 to 10 ring carbon atoms ("C) ₅ -C ₁₀ Cycloalkyl "). C ₅ -C ₆ Examples of cycloalkyl groups include cyclopentyl (C) ₅ ) And cyclohexyl (C) ₅ )。C3-C ₆ Examples of cycloalkyl groups include the foregoing C ₅ -C ₆ Cycloalkyl cyclopropyl (C) ₃ ) And cyclobutyl (C) ₄ )。C ₃ -C ₈ Examples of cycloalkyl groups include the foregoing C ₃ -C ₆ Cycloalkyl and cycloheptyl (C) ₇ ) And cyclooctyl (C) ₈ ). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted with one or more substituentsSubstituted ("substituted cycloalkyl"). In certain embodiments, cycloalkyl is unsubstituted C ₃ -C ₁₀ A cycloalkyl group. In certain embodiments, cycloalkyl is substituted C ₃ -C ₁₀ A cycloalkyl group.

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

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

A heteroalkylene group: as used herein, the term "heteroalkylene" refers to a divalent form of a heteroalkyl group as described herein.

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

As used herein, "heteroaryl" also refers to a radical of a 5-to 14-membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n +2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons in common in the ring array) having ring carbon atoms and 1 or more (e.g., 1,2,3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from the group consisting of oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-to 14-membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in such cases the number of ring members always represents the number of ring members in the heteroaryl ring system. "heteroaryl" also encompasses ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases the number of ring members represents the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups do not contain a heteroatom in one ring (e.g., indolyl, quinolinyl, carbazolyl, etc.), and the point of attachment can be on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

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

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

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

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

Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, thioalkenyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiocyclopentyl. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thiacyclohexanyl. Exemplary 6 membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepinyl, and thiacycloheptyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azacyclooctyl, oxocyclooctyl, and thiepinyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphtyridyl, decahydro-1, 8-naphthyridinyl, decahydropyrrolo [3,2-b ] pyrrole, indolyl, phthalimidyl, naphthoylimino, chromanyl, benzothienyl, 1H-benzo [ e ] [1,4] diazepinyl, 1,4,5, 7-tetrahydropyrano [3,4-b ] pyrrolyl, 5, 6-dihydro-4H-furo [3,2-b ] pyrrolyl, 6, 7-dihydro-5H-furo [3,2-b ] pyranyl, 5, 7-dihydro-4H-thieno [2,3-c ] pyranyl, 2, 3-1H-thieno [2, 2-b ] pyridinyl, 5, 7-dihydro-4H-thieno [2,3-c ] pyridinyl, 2, 3-dihydro-1, 2-tetrahydropyranyl, 2-b ] pyridinyl, 1, 5, 4H-thieno [2, 2-b ] pyridinyl, 5, 7-dihydro-4H-thieno [2,3-b ] pyridinyl, 2,3-b ] pyridinyl, 4H-tetrahydropyranyl, and the like.

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

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

Exemplary carbon atom substituents include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2, -SO3H, -OH, -ORaa, -ON (Rbb) 2, -N (Rbb) 3+ X-, -N (ORcc) Rbb, -SeH, -SeRaa, -SH, -SRaa, -SSRc, -C (= O) Raa, -CO2H, -CHO, -C (ORcc) 2, -CO2Raa, -OC (= O) Raa, -OCO2Raa, -C (= O) N (Rbb) 2, -OC (= O) N (Rbb) 2, -NRbbC (= O) Raa, -NRbbCO2Raa, -NRC (= O) N (Rbb) 2, -C (= NRbb) Raa, -C (= O) NRbb) ORaa, -NRbb (= O) Raa, -NRbb (= C (= O) NRbb) N (Rbb) 2, -C (= O) Raa, -NRbb) NRbb 2, -NRbb (= C (= O) Rab) Raa, -NRbb) NRbb 2, -NRO 2 NRbb, -C (= S) SRaa, -SC (= O) SRaa, -OC (= O) SRaa, -SC (= O) ora, -SC (= O) Raa, -P (= O) 2Raa, -OP (= O) 2Raa, -P (= O) (Raa) 2, -OP (= O) (ORcc) 2, -P (= O) 2N (Rbb) 2, -OP (= O) 2N (Rbb) 2, -P (= O) (NRbb) 2, -OP (= O) (nrcc) 2, -OP (= O) (NRbb) 2-NRbbP (= O) (ORcc) 2, -NRbbP (= O) (NRbb) 2, -P (Rcc) 3, -OP (Rcc) 2, -OP (Rcc) 3, -B (Raa) 2, -B (ORcc) 2, -BRaa (ORcc), C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C14 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0,1, 2,3,4 or 5 Rdd groups;

or two twinned hydrogens on a carbon atom are replaced by a group = O, = S, = NN (Rbb) 2, = NNRbbC (= O) Raa, = NNRbbC (= O) ORaa, = NNRbbS (= O) 2Raa, = NRbb or = NORcc;

each instance of Raa is independently selected from C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl and 5-14 membered heteroaryl, or two Raa groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0,1, 2,3,4 or 5 Rdd groups;

each instance of Rbb is independently selected from the group consisting of hydrogen, -OH, -ORaa, -N (Rcc) 2, -CN, -C (= O) Raa, -C (= O) N (Rcc) 2, -CO2Raa, -SO2Raa, -C (= NRcc) ORaa, -C (= NRcc) N (Rcc) 2, -SO2Rcc, -SO2ORcc, -SORaa, -C (= S) N (Rcc) 2, -C (= O) SRcc, -C (= S) SRcc, -P (= O) 2Raa, -P (= O) (Raa) 2, -P (= O) 2N (Rcc) 2, -P (= O) (NRcc) 2, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered carbocyclyl, C6-C14 membered heteroaryl, and heteroaryl, or two Rbb groups and the heteroatom to which they are attached form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3,4, or 5 Rdd groups;

each instance of Rcc is independently selected from hydrogen, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-14 membered heterocyclyl, C6-C14 aryl, and 5-14 membered heteroaryl, or two Rcc groups together with the heteroatom to which they are attached form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3,4, or 5 Rdd groups;

<xnotran> Rdd , -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -ORee, -ON (Rff) 2, -N (Rff) 2, -N (Rff) 3+X-, -N (ORee) Rff, -SH, -SRee, -SSRee, -C (= O) Ree, -CO2H, -CO2Ree, -OC (= O) Ree, -OCO2Ree, -C (= O) N (Rff) 2, -OC (= O) N (Rff) 2, -NRffC (= O) Ree, -NRffCO2Ree, -NRffC (= O) N (Rff) 2, -C (= NRff) ORee, -OC (= NRff) Ree, -OC (= NRff) ORee, -C (= NRff) N (Rff) 2, -OC (= NRff) N (Rff) 2, -NRffC (= NRff) N (Rff) 2, -NRffSO2Ree, -SO2N (Rff) 2, -SO2Ree, -SO2ORee, -OSO2Ree, -S (= O) Ree, -Si (Ree) 3, -OSi (Ree) 3, -C (= S) N (Rff) 2, -C (= O) SRee, -C (= S) SRee, -SC (= S) SRee, -P (= O) 2Ree, -P (= O) (Ree) 2, </xnotran> -OP (= O) (Ree) 2, -OP (= O) (ore) 2, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0,1, 2,3,4 or 5 Rgg groups or two Rdd substituents may be attached to form = O or = S;

each instance of Ree is independently selected from C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3,4, or 5 Rgg groups;

each instance of Rff is independently selected from hydrogen, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, 3-10 membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl, or two Rff groups together with the heteroatom to which they are attached form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2,3,4, or 5 Rgg groups; and is

<xnotran> Rgg , -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C50 , -ON (C1-C50 ) 2, -N (C1-C50 ) 2, -N (C1-C50 ) 3+X-, -NH (C1-C50 ) 2+X-, -NH2 (C1-C50 ) + X-, -NH3+ X-, -N (OC 1-C50 ) (C1-C50 ), -N (OH) (C1-C50 ), -NH (OH), -SH, -SC1-C50 , -SS (C1-C50 ), -C (= O) (C1-C50 ), -CO2H, -CO2 (C1-C50 ), -OC (= O) (C1-C50 ), -OCO2 (C1-C50 ), -C (= O) NH2, -C (= O) N (C1-C50 ) 2, -OC (= O) NH (C1-C50 ), -NHC (= O) (C1-C50 ), -N (C1-C50 ) C (= O) (C1-C50 ), -NHCO2 (C1-C50 ), -NHC (= O) N (C1-C50 ) 2, </xnotran> -NHC (= O) NH (C1-C50 alkyl), -NHC (= O) NH2, -C (= NH) O (C1-C50 alkyl), -OC (= NH) (C1-C50 alkyl), -OC (= NH) OC1-C50 alkyl, -C (= NH) N (C1-C50 alkyl) 2, -C (= NH) NH (C1-C50 alkyl), -C (= NH) NH2, -OC (= NH) N (C1-C50 alkyl) 2, -OC (NH) NH (C1-C50 alkyl), -OC (NH) NH2, -NHC (NH) N (C1-C50 alkyl) 2, -NHC (= NH) NH2, -NHSO2 (C1-C50 alkyl), -SO2N (C1-C50 alkyl) 2, -SO2NH (C1-C50 alkyl), -SO2NH2, -SO2 (C1-C50 alkyl), -SO2O (C1-C50 alkyl), -OSO2 (C1-C6 alkyl), -SO (C1-C6 alkyl), -Si (C1-C50 alkyl) 3, -OSi (C1-C6 alkyl) 3, -C (= S) N (C1-C50 alkyl) 2, C (= S) NH (C1-C50 alkyl), C (= S) NH2, -C (= O) S (C1-C6 alkyl), -C (= S) S (C1-C6 alkyl), -SC (= S) S (C1-C6 alkyl), -P (= O) 2 (C1-C50 alkyl), -P (= O) (C1-C50 alkyl) 2, -OP (= O) (OC 1-C50 alkyl) 2, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C6-C10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal Rgg substituents may be linked to form = O or = S; wherein X-is a counter ion.

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

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

The nitrogen atoms may be substituted or unsubstituted, as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -ORaa, -N (Rcc) 2, -CN, -C (= O) Raa, -C (= O) N (Rcc) 2, -CO2Raa, -SO2Raa, -C (= NRbb) Raa, -C (= NRcc) ORaa, -C (= NRcc) N (Rcc) 2, -SO2Rcc, -SO2ORcc, -sora, -C (= S) N (Rcc) 2, -C (= O) SRcc, -C (= S) SRcc, -P (= O) 2Raa, -P (= O) (Raa) 2, -P (= O) 2N (Rcc) 2, -P (= O) (NRcc) 2, C1-C50alkyl, C2-C50 alkenyl, C2-C50 alkynyl, C3-C10 carbocyclyl, C3-C10-aryl, C5-C50 heterocyclyl, 14-membered heterocyclyl, 14-aryl, 14-membered heterocyclyl, 14-membered heteroaryl, or 14 membered heteroaryl, wherein each of the substituents are independently substituted as defined above, and each of which form a heteroaryl group, and a heteroaryl group, wherein each of the group is substituted.

In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen Protecting Groups are well known in the art and include those described in detail in Organic Synthesis Protecting Groups in Organic Synthesis, t.w.greene and p.g.m.wuts, 3 rd edition, john Wiley father, 1999, which references are incorporated herein by reference.

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

Nitrogen protecting groups such as urethane groups (e.g., -C (= O) ORaa) include, but are not limited to: methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), 9- (2-sulfo) fluorenylmethylcarbamate, 9- (2, 7-dibromo) fluoroalkenylmethylcarbamate, 2, 7-di-t-butyl- [9- (10, 10-dioxo-10, 10-tetrahydrothioxanth) ] methylcarbamate (DBD-Tmoc), 4-methoxybenzocarbamate (Phenoc), 2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (hZ) 1- (1-adamantyl) -1-methylethylcarbamate (Adpoc), 1-dimethyl-2-ethylcarbamate, 1-dimethyl-2, 2-dibromoethylcarbamate (DB-t-BOC), 1-dimethyl-2, 2-Trichloroethylcarbamate (TCBOC) 1-methyl-1- (4-biphenylyl) carbamic acid ethyl ester (Bpoc), 1- (3, 5-di-tert-butylphenyl) -1-methylethyl carbamate (t-Bumeoc), 2- (2 '-and 4' -pyridyl) carbamic acid ethyl ester (Pyoc), 2- (N, N-dicyclohexylcarboxamido) carbamic acid ethyl ester, <xnotran> (BOC), 1- (Adoc), (Voc), (Alloc), 1- (Ipaoc), (Coc), 4- (Noc), 8- , N- , , (Cbz), (Moz), , , ,2,4- ,4- (Msz), 9- , ,2- ,2- ,2- ( ) , [2- (1,3- ) ] (Dmoc), 4- (Mtpc), 2,4- (Bmpc), 2- (Peoc), 2- (Ppoc), 1,1- -2- , - - , </xnotran> <xnotran> ( ) ,5- ,2- ( ) -6- (Tcroc), ,3,5- , ,3,4- -6- , ( ) , , S- , , , , , , ,2,2- , (N, N- ) ,1,1- -3- (N, N- ) ,1,1- , (2- ) ,2- ,2- , , , , p- (p' - ) ,1- ,1- , 1-1- -1- ,1- -1 (3,5- ) , </xnotran> 1-methyl-1- (p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, ethyl 1-methyl-1- (4-pyridyl) carbamate, phenyl carbamate, benzyl p- (phenylazo) carbamate, 2,4, 6-tri-tert-butylphenyl carbohydrate, 4- (trimethylammonium) benzyl carbamate and 2,4, 6-trimethylbenzyl carbamate.

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

Other nitrogen protecting groups include, but are not limited to: phenothiazinyl- (10) -acyl derivatives, N '-p-toluenesulfonylaminoyl derivatives, N' -phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4, 5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithienylimide (Dts), N-2, 3-diphenylmaleimide, N-2, 5-dimethylpyrrole, N-1, 4-tetramethyldisilylcyclopentane adduct (STABASE), 5-substituted 1, 3-dimethyl-1, 3, 5-triazacyclohexan-2-one, phenothiazinyl- (10) -acyl derivatives, N '-p-toluenesulfonylaminoyl derivatives, N' -phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, N-2-diphenyl-3-oxazolin-2-one, N-dithiopheneylimide, N-2-imide (Dts), N-2, N-diphenylmaleimide, N-2-dimethylpyrrole, N-1, 4-tetramethyldisilylcyclopentane adduct (STABASE), 5-substituted 1,3, 5-triazacyclohexan-2-one, and mixtures thereof 5-substituted 1, 3-dibenzyl-1, 3, 5-triazacyclohexan-2-ones, 1-substituted 3, 5-dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2- (trimethylsilyl) ethoxy ] methylamine (SEM), N-3-acetoxypropylamine, N- (1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl) amine, quaternary ammonium salts, N-benzylamine, N-bis (4-methoxyphenyl) methylamine, N-5-dibenzofuran amine, N-tritylamine (Tr), N- [ (4-methoxyphenyl) benzhydryl ] amine (MMTr), N-9-phenylfluorofluorenamine (PhF), <xnotran> N-2,7- -9- , N- (Fcm), N-2- N '- , N-1,1- , N- , np- , N- , N- [ (2- ) ] , N- (N', N '- ) , N, N' - , np- , N- , N-5- , N- (5- -2- ) , N- , N- (5,5- -3- -1- ) , N- , N- , N- [ ( - ) ] , N- , N- , N- , N- , N- , (Dpp), (Mpt), (Ppt), , , , , (Nps), 2,4- , ,2- -4- , </xnotran> Triphenylmethylsulfinamide and 3-nitropyridine sulfinamide (Npys).

In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen Protecting Groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, t.w.greene and p.g.m.wuts, 3 rd edition, john wiley parent, 1999, which references are incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to: <xnotran> , (MOM), (MTM), , ( ) (SMOM), (BOM), (PMBM), (4- ) (p-AOM), (GUM), ,4- (POM), ,2- (MEM), 2,2,2- , (2- ) ,2- ( ) (SEMOR), (THP), 3- , ,1- ,4- (MTHP), 4- ,4- S, S- ,1- [ (2- -4- ) ] -4- -4- (CTMP), 1,4- -2- , , ,2,3,3a,4,5,6,7,7 α - -7,8,8- -4,7- -2- ,1- ,1- (2- ) ,1- -1- , </xnotran> 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl) ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2, 4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3, 4-dimethoxybenzyl, o-nitrobenzyl, p-halobenzyl, 2, 6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxyanion group, diphenylmethyl, p, p ' -dinitrobenzhydryl, 5-dibenzosuberyl, trityl, α -naphthylbenzhydryl, p-methoxyphenyldiphenylmethyl, bis (p-methoxyphenyl) phenylmethyl, tris (p-methoxyphenyl) methyl, 4- (4 ' -bromobenzoylphenylmethylphenoxy) diphenylmethyl, 4',4 "-tris (4, 5-dichlorophthalimidophenyl) methyl, 4',4" -tris (acetylpropionyloxyphenyl) methyl, 4',4 "-tris (benzyloxy) methyl, 3- (imidazol-1-yl) bis (4 ',4" -dimethoxyphenyl) methyl, 1-bis (4-methoxyphenyl) -1' -pyrenylmethyl, 9-anthracenyl, 9- (9-phenyl) xanthenyl, <xnotran> 9- (9- -10- ) ,1,3- -2- , S, S- , (TMS), (TES), (TIPS), (IPDMS), (DEIPS), , (TBDMS), (TBDPS), , - , , (DPMS), (TBMPS), , , , , , , , , , , ,3- ,4- ( ), 4,4- ( ) ( ), , , ,4- , , ,2,4,6- (2,4,6-trimethylbenzoate mesitoate), , 9- (Fmoc), , 2,2,2- (Troc), 2- ( ) (TMSEC), 2- ( ) (Psec), 2- ( ) (Peoc), </xnotran> Isobutyl alkyl carbonate, vinyl alkyl carbonate, allyl alkyl carbonate, p-nitrophenyl alkyl carbonate, benzyl alkyl carbonate, p-methoxybenzyl alkyl carbonate, dimethoxybenzyl alkyl 3, 4-carbonate, o-nitrobenzyl alkyl carbonate, p-nitrobenzyl alkyl carbonate, benzyl alkyl S-thiocarbonate, 4-ethoxy-1-carbonate, methyl dithiocarbonate, 2-iodobenzoic acid, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2- (methylthiomethoxymethyl) benzoate, 2, 6-dichloro-4-methylphenoxyacetate, 2, 6-dichloro-4- (1, 3-tetramethylbutyl) phenoxyacetate, 2, 4-bis (1, 1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monomalate, (E) -2-methyl-2-butenoate, o- (methoxy) benzoate, alpha-naphthalene, nitrate, alkyl N, N, N ', N' -tetramethylphosphorodiamidite, alkyl N-phenylcarbamate, borate, dimethylthiophosphine, alkyl 2, 4-dinitrophenylsulfinate, sulfate, methanesulfonate (methanesulfonate or mesylate), benzylsulfonate, and tosylate (Ts).

In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in organic synthesis, t.w. greene and p.g. m.wuts, 3 rd edition, john wiley father, 1999, which references are incorporated herein by reference.

Exemplary sulfur protecting groups include, but are not limited to: <xnotran> , , ,2,4,6- ,2,4,6- , , , , , ,4- ,2- ,2- N- , 9- , 9- , , , , (4- ) ,5- , , -4- , ,2,4- , ,1- , (MOM), , ,2- , , , , , , , , , , , , , (2- -1- ) ,2- (2,4- ) ,2- ,2- ( ) ,2,2- () , (1- -2- ) ,2- ,2- (4- ) -2- -2- , , , , </xnotran> N- [ [ (p-biphenylyl) isopropoxy ] carbonyl ] -N-methyl ] -gamma-aminothiobutanoate, 2-trichloroethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, thiocarbonate, 3-nitro-2-pyridylthiosulfide, oxythiophene.

Compounds of the invention

Liposome-based vehicles are considered attractive carriers for therapeutic agents and continued development efforts are needed. Although liposome-based vehicles comprising certain lipid components show promising results in terms of encapsulation, stability and site localization, there is still a great need for improved liposome-based delivery systems. For example, significant drawbacks of liposome delivery systems relate to the construction of liposomes with sufficient cell culture or in vivo stability to achieve the desired target cells and/or intracellular compartments, and the ability of such liposome delivery systems to effectively release their encapsulated substances to such target cells.

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

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

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

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

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

Compounds of the invention

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

wherein X is O or S;

wherein R' is

Wherein R is ⁶ Is composed of

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

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

Wherein R is ⁷ 、R ⁸ 、R ⁹ 、R ¹⁰ Respectively include R ^A 、R ^B 、R ^C Or R ^D Moiety wherein said R ^A 、R ^B 、R ^C Or R ^D Independently selected from optionally substituted (C) ₆ -C ₂₀ ) Alkyl, optionally substituted (C) ₆ -C ₂₀ ) Alkenyl, optionally substituted (C) ₆ -C ₂₀ ) Alkynyl, optionally substituted (C) ₆ -C ₂₀ ) Acyl, optionally substituted-OC (O) (C) ₆ -C ₂₀ ) Alkyl or optionally substituted-OC (O) (C) ₆ -C ₂₀ ) An alkenyl group;

or a pharmaceutically acceptable salt thereof.

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

In an embodiment, L1 is independently a bond.

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

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

In embodiments, L1 is C2 alkenyl.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In embodiments, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are each independently selected from:

in embodiments, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are the same and are selected from:

in embodiments, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C8H17.

In the examples, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C10H21.

In embodiments, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both C12H25.

In the examples, R7= - (CH 2) kCH (OR 11) RA, R8 is- (CH 2) nCH (OR 12) RB, and RA and RB are both

In embodiments, X is O.

In an embodiment, X is S.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In embodiments, R7 and R8 are each optionally substituted (C1-C6) alkyl, e.g., -CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In the examples, R ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa C ₁ -C ₄₀ An alkyl group. In the examples, R ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa C ₁ -C ₃₀ An alkyl group. In the examples, R ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa C ₁ -C ₂₀ An alkyl group.

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

In the examples, R7 and R8 are each

In the examples, R7 and R8 are each

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In an embodiment, R' is:

in the examples, R' is

And k and n =1, and m = 2or 3.

In the examples, R' is

And k and n =1, and m =2.

In the examples, R' is

And k and n =1, and m =3.

In the examples, R' is

And R is ¹¹ And R ¹² Is H.

In the examples, R' is

And k and n =1, m = 2or 3, and R ¹¹ And R ¹² Is H.

In the examples, R' is

And k and n =1,m =2, and R ¹¹ And R ¹² Is H.

In the examples, R' is

And k and n =1,m =3, and R ¹¹ And R ¹² Is H.

In an embodiment, R' is:

wherein R' has the structure

In any of the preceding embodiments, R ^A And R ^B Can be as paragraph [0104 ]]To [0129]As defined in any of the paragraphs above.

In the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Is composed of

q and r =1, and p =2.

In the examples, R ⁶ Is composed of

And R is ¹³ And R ¹⁴ Is H.

In the examples, R ⁶ Is composed of

q and R =1, p =2, and R ¹³ And R ¹⁴ Is H.

In the examples, R ⁶ Selected from the group consisting of:

in the examples, R ⁶ Selected from the group consisting of:

in the examples, R ⁶ Selected from the group consisting of:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Selected from the group consisting of:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ Comprises the following steps:

in the examples, R ⁶ And R' are the same.

In the examples, R ⁶ Is composed of

R' is

m is 2 and p is 2.

In the examples, R ⁶ Is composed of

R' is

m is 3 and p is 2.

In the examples, L ₁ Bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

And R' is

In the examples, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

And R' is

In embodiments, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

And R' is

In embodiments, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ¹ And R ⁵ Is H, R ⁶ Is composed of

And R' is

And R' is

And R' is

In the examples, L ₁ Is a bond, R ³ is-OC (O) R', R ² Is OMe, R ⁶ Is composed of

And R' is

In embodiments, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² Is OMe, R ⁶ Is composed of

And R' is

In embodiments, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² Is OMe, R ¹ 、R ⁴ And R ⁵ Is H, R ⁶ Is composed of

And R' is

And R' is

And R' is

And R' is

In the examples, L ₁ Is C ₂ Alkenyl radical, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

And R' is

And R'Is composed of

In embodiments, X = O, L ₁ Is C ₂ Alkenyl radical, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

And R' is

In the examples, X = O, L ₁ Is C ₂ Alkenyl radical, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ¹ And R ⁵ Is H, R ⁶ Is composed of

And R' is

And R' is

And R' is

In the examples, R ⁶ Is composed of

R' is

R ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group.

In the examples, R ⁶ Is composed of

R' is

R ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, m is 2, and p is 2. In some embodiments, R ⁷ And R ⁸ The same is true. In some embodiments, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe.

R' is

And R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In some embodiments, R ⁷ And R ⁸ The same is true.

In the examples, L ₁ Is a bond, R ³ is-OC(O)R'，R ² And R ⁴ Is OMe, R ⁶ Is composed of

R' is

And R is ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, and m is 2. In some embodiments, R ⁷ And R ⁸ The same is true.

R' is

And R is ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In some embodiments, R ⁷ And R ⁸ The same is true. In some embodiments, m is 2.

R' is

And R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In some embodiments, R ⁷ And R ⁸ The same is true. In some embodiments of the present invention, the,m is 2.

In the examples, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

R' is

In the examples, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ¹ And R ⁵ Is H, R ⁶ Is composed of

R' is

R ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, and m is 2. In some embodiments, R ⁷ And R ⁸ The same is true.

R' is

And R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In some embodiments, R ⁷ And R ⁸ The same is true. In some embodiments, m is 2.

In the examples, X = O, L ₁ Is a bond, R ³ is-OC (O) R', R ² Is OMe, R ⁶ Is composed of

R' is

R' is

R' is

And R is ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) An alkyl group, a carboxyl group,wherein R is ^aa Is C ₁ -C ₅₀ Alkyl, and m is 2. In some embodiments, R ⁷ And R ⁸ The same is true.

R' is

R ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, and m is 2. In some embodiments, R ⁷ And R ⁸ The same is true.

R' is

R' is

And R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, and m is 2. In some embodiments, R ⁷ And R ⁸ The same is true.

In the examples, X = O, L ₁ Is C ₂ Alkenyl radical, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ⁶ Is composed of

R' is

And R is ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ An alkyl group. In some embodiments, R ⁷ And R ⁸ The same is true.

R' is

R' is

In embodiments, X = O, L ₁ Is C ₂ Alkenyl radical, R ³ is-OC (O) R', R ² And R ⁴ Is OMe, R ¹ And R ⁵ Is H, R ⁶ Is composed of

R' is

In which R is ⁷ And R ⁸ Is a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ In any of the above embodiments of alkyl, R ^aa May alternatively be C ₁ -C ₄₀ An alkyl group.

In which R is ⁷ And R ⁸ Is a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ In any of the above embodiments of alkyl, R ^aa May alternatively be C ₁ -C ₃₀ An alkyl group.

In which R is ⁷ And R ⁸ Is a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ In any of the above embodiments of alkyl, R ^aa May alternatively be C ₁ -C ₂₀ An alkyl group.

In which R is ⁷ And R ⁸ is-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ In any of the examples for alkyl, R ⁷ And R ⁸ Can be respectively

In which R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ In any of the examples for alkyl, R ⁷ And R ⁸ Can each be

In embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (II):

or a pharmaceutically acceptable salt thereof, wherein R ¹ -R ⁶ And X is as already defined herein.

In embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (IIA):

or a pharmaceutically acceptable salt thereof, wherein R', R ⁶ And X is as already defined herein.

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

or a pharmaceutically acceptable salt thereof.

In embodiments, the cationic lipids of the present invention have the structure:

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

In embodiments, the cationic lipid of the present invention comprises a compound having a structure according to formula (IIF):

In embodiments, the cationic lipid of the present invention comprises a compound having a structure according to formula (IIG):

In embodiments, the cationic lipid of the present invention comprises a compound having a structure according to formula (IIH):

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

In embodiments, the cationic lipid of the present invention comprises a compound having a structure according to formula (III):

In embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (IIIA):

In embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (IIIB):

or a pharmaceutically acceptable salt thereof, wherein R ^A 、R ^B And p is as already defined herein. In embodiments, the cationic lipids of the present invention have the structure:

or a pharmaceutically acceptable salt thereof, wherein R ^A And R ^B As already defined herein.

or a pharmaceutically acceptable salt thereof.

or a pharmaceutically acceptable salt thereof.

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

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

or a pharmaceutically acceptable salt thereof.

in embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (IIIL):

In embodiments, the cationic lipid of the invention comprises a compound having a structure according to formula (IV):

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

or a pharmaceutically acceptable salt thereof, wherein R ^A 、R ^B M and p are as already defined herein.

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

or a pharmaceutically acceptable salt thereof,

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

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

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

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

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

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

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

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

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

Exemplary Compounds

Exemplary compounds include exemplary compounds described in tables 1-8

TABLE 1

TABLE 2

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

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

Unless otherwise stated, R = C ₁₆ H ₂₉ Has the structure:

unless otherwise stated, R = C ₁₆ H ₃₁ Has the structure:

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

Nucleic acids

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

Synthesis of nucleic acids

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

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

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

Modified mRNA

In some embodiments, mRNA according to the present invention may be synthesized as unmodified mRNA or modified mRNA. Modified mRNA contains nucleotide modifications in RNA. Thus, a modified mRNA according to the invention may include nucleotide modifications, e.g., backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (a), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotide analogs or derivatives of purines and pyrimidines, for example, 1-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-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl) -uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxygen acetic acid methyl ester, 5-methyl amino methyl uracil, 5-methoxy amino methyl-2-methyl uracil, 5' -methoxy carbonyl methyl uracil, 5-methoxy uracil, uracil-5-oxygen acetic acid methyl ester, uracil-5-oxygen acetic acid (V), 1-methyl-false uracil, braid glycoside, beta-D-mannose-braided glycoside, wye oxygen glycoside and phosphoramidate, thiophosphate, peptide nucleotides, methyl phosphonate, 7-nitrogen guanosine, 5-methyl cytosine and inosine. The preparation of such analogs is known to those skilled in the art, for example, from U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

Pharmaceutical formulations of cationic lipids and nucleic acids

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

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

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

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

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

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

In addition, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to preferentially distribute to other target tissues, cells, or organs, such as the heart, lungs, kidneys, spleen. In embodiments, the lipid nanoparticles of the present invention may be prepared to achieve enhanced delivery to target cells and tissues. For example, a polynucleotide (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 a target cell or target tissue. In some embodiments, the encapsulated polynucleotide (e.g., mRNA) is capable of being expressed by and produced (and in some cases secreted) by a target cell to confer, for example, a beneficial property to the target cell or target tissue. Such encapsulated polynucleotides (e.g., mrnas) may encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest.

Liposomal delivery vehicles

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

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

Enrichment of liposome compositions using one or more of the cationic lipids disclosed herein can be used as a means to improve (e.g., reduce) toxicity or otherwise impart one or more desirable properties to such enriched liposome compositions (e.g., improve delivery of the encapsulated polynucleotide to one or more target cells and/or reduce in vivo toxicity of the liposome compositions). Accordingly, pharmaceutical compositions, particularly liposomal compositions, comprising one or more of the cationic lipids disclosed herein are also contemplated.

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

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

In certain embodiments, such compositions (e.g., liposome compositions) load or otherwise encapsulate a material, such as one or more biologically active polynucleotides (e.g., mRNA).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In embodiments, the percentage results in an improvement in beneficial effect (e.g., improved delivery to a target tissue, such as the liver or lung).

In a typical embodiment, a composition of the invention (e.g., a liposome composition) comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the invention as described herein. For example, compositions suitable for practicing the invention have four lipid components, including the compounds of the invention described herein as cationic lipid components, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids. The non-cationic lipid may be DOPE or DEPE. The cholesterol-based lipid may be cholesterol. The PEG-modified lipid may be DMG-PEG2K.

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

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

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

In embodiments, a composition (e.g., a lipid nanoparticle) encapsulating a nucleic acid (e.g., mRNA encoding a peptide or protein) comprises one or more of the compounds of the invention described herein, one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and pegylated lipids; and further comprising a cholesterol-based lipid. Typically, such compositions have four lipid components, including as a cationic lipid component a compound of the 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-PEG 2K).

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

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

In some embodiments, the ratio of cationic lipid to non-cationic lipid to cholesterol-based lipid to PEG-modified lipid may be between about 30-60. In some embodiments, the ratio of the one or more cationic lipids to the one or more non-cationic lipids to the one or more cholesterol-based lipids to the one or more PEG-modified lipids is about 40. In some embodiments, the ratio of the one or more cationic lipids to the one or more non-cationic lipids to the one or more cholesterol-based lipids to the one or more PEG-modified lipids is about 40. In some embodiments, the ratio of the one or more cationic lipids to the one or more non-cationic lipids to the one or more cholesterol-based lipids to the one or more PEG-modified lipids is about 40. In some embodiments, the ratio of the one or more cationic lipids to the one or more non-cationic lipids to the one or more cholesterol-based lipids to the one or more PEG-modified lipids is about 50.

Cationic lipids

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

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

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

Helper lipids

The compositions (e.g., liposome compositions) can also include one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid substances that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1, 2-diethylene glycol-sn-glycerol-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylpE, 18-1-trans PE, L-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof. A non-cationic or helper lipid suitable for use in the practice of the present invention is dioleoyl phosphatidylethanolamine (DOPE). Alternatively, 1, 2-diethylene glycol-sn-glycerol-3-phosphoethanolamine (DEPE) can be used as the non-cationic or helper lipid.

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

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

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

Cholesterol-based lipids

In some embodiments, the composition (e.g., liposome composition) comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N, N-dimethyl-N-ethylcarboxyamide cholesterol), 1, 4-bis (3-N-oleylamino-propyl) piperazine (Gao et al, "communication of biochemistry and biophysical studies (biochem. Biophysis. Res. Comm.) -179, 280 (1991); wolf et al," biotechnology (BioTechniques)' 23,139 (1997); U.S. Pat. No. 5,744,335), or the Imidazole Cholesterol Ester (ICE) having the structure:

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

In some embodiments, the cholesterol-based lipids may be present in a weight ratio (% by weight) of about 1% to about 30% or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticle 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 nanoparticle 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.%.

Pegylated lipids

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

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

Contemplated additional PEG-modified lipids (also referred to herein asPegylated lipids, which term is interchangeable with PEG-modified lipids), include, but are not limited to, covalently linked to a lipid having a C ₆ -C ₂₀ The length of the lipid of the alkyl chain is at most 5 kDa. In some embodiments, the PEG-modified lipid or pegylated lipid is pegylated cholesterol or PEG-2K. Addition of such components can prevent complex aggregation and can also provide a means for increasing cycle life and delivery of lipid-nucleic acid compositions to target cells (Klibanov et al (1990) Federation of European Biochemical Association (FEBS Letters), 268 (1): 235-237), or can be selected to allow rapid in vivo replacement of formulations (see U.S. Pat. No. 5,885,613).

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

Pharmaceutical formulations and therapeutic uses

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

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

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

Thus, pharmaceutical formulations comprising the described compounds and the nucleic acids provided by the invention may be used for various therapeutic purposes. To facilitate delivery of nucleic acids in vivo, the compounds and nucleic acids described herein may be formulated in combination with one or more additional pharmaceutically acceptable carriers, targeting ligands, or stabilizers. In some embodiments, the compounds described herein may be formulated by pre-mixing lipid solutions. In other embodiments, compositions comprising the compounds described herein can be formulated into the lipid membrane of nanoparticles using post-intercalation techniques. Techniques for formulating and administering drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing co., easton, pa., latest edition.

Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary, including intratracheal or inhalation, or enteral administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections; and intrathecally, directly intraventricularly, intravenously, intraperitoneally, or intranasally. In particular embodiments, the intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the administration results in delivery of the nucleic acid to a muscle cell. In some embodiments, administration results in delivery of the nucleic acid to a hepatocyte (i.e., a liver cell).

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

Alternatively or in addition, the pharmaceutical formulation of the present invention may be administered locally rather than systemically, e.g., by direct injection of the pharmaceutical formulation into the targeted tissue, preferably in the form of a sustained release formulation. Local delivery can be affected in various 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, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph node, skin, and/or cerebrospinal fluid. In embodiments, the tissue to be targeted is in the liver. For example, an aerosol containing a composition of the invention (for nasal, tracheal, or bronchial delivery) may be inhaled; for example, the compositions of the invention may be injected into the site of injury, disease manifestation, or pain; the composition may be provided in the form of a lozenge for oral, tracheal, or esophageal use; may be supplied to the stomach or intestine in the form of liquids, tablets or capsules, or may be supplied in the form of suppositories for rectal or vaginal application; or may even be delivered to the eye by use of creams, drops or even injections.

The compositions described herein can include mRNA-encoded peptides, including peptides (e.g., polypeptides, such as proteins) described herein.

In embodiments, the mRNA encodes a polypeptide.

In embodiments, the mRNA encodes a protein.

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

The present invention provides methods for delivering compositions having full-length mRNA molecules encoding peptides or proteins of interest for treating a subject, e.g., a human subject or cells of a human subject or cells processed and delivered to a human subject.

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

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

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

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

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

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

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

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

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

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

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

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

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

In certain embodiments, the invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding a peptide or protein for delivery to or treatment of a kidney or kidney cells in a subject. In certain embodiments, the invention provides methods for preparing therapeutic compositions having full length mRNA encoding collagen type IV alpha 5 chain (COL 4 A5) protein.

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

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

In certain embodiments, the invention provides methods for preparing a therapeutic composition having a full-length mRNA encoding an antigen associated with a cancer in a subject or an antigen identified from a cancer cell in a subject. In certain embodiments, the present invention provides methods for preparing therapeutic compositions having full-length mrnas that encode antigens determined from a subject's own cancer cells, i.e., providing personalized cancer vaccines. In certain embodiments, the present invention provides methods for preparing a therapeutic composition having full-length mRNA encoding an antigen expressed from a mutated KRAS gene.

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

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

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

Delivery method

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

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

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

After administration of the composition to the subject, a protein product (e.g., a functional protein or enzyme) encoded by the mRNA can be detected in the peripheral target tissue at least about one to seven days or more after administration of the compound to the subject. The amount of protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the protein encoded and the condition of the patient. For example, a concentration of protein in a target tissue of at least about 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45 days or more after administration of a compound to a subject can be detected in a concentration of the protein of at least 0.025 to 1.5 μ g/ml (e.g., at least 0.050 μ g/ml, at least 0.075 μ g/ml, at least 0.1 μ g/ml, at least 0.2 μ g/ml, at least 0.3 μ g/ml, at least 0.4 μ g/ml, at least 0.5 μ g/ml, at least 0.6 μ g/ml, at least 0.7 μ g/ml, at least 0.8 μ g/ml, at least 0.9 μ g/ml, at least 1.0 μ g/ml, at least 1.1.1 μ g/ml, at least 1.2 μ g/ml, at least 1.5 μ g/ml, at least 0.4 μ g/ml, or more).

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

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

Examples of the invention

While certain compounds, compositions, and methods of this invention have been described with specificity in accordance with certain examples, the following examples are intended only to illustrate the compounds of this invention and are not intended to be limiting thereof.

Synthetic scheme for vanillic acid lipid

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

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

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

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

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

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

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

Synthetic scheme of syringic acid lipid

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

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

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

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

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

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

Synthetic scheme for sinapinic acid lipid

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

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

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

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

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

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

Synthesis of phenolic acid lipids scheme A

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

to (1) (2.00g, 2.86mmol) in anhydrous CH at 0 deg.C ₂ Cl ₂ To a solution in (10 mL) was added oxalyl chloride (0.98mL, 4.0 equiv.) dropwise, and the reaction mixture was slowly warmed to room temperature and stirred for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure and the remaining residue was redissolved in anhydrous CH ₂ Cl ₂ (30 mL). To the stirred acid chloride solution was added (2 a) (555mg, 1.0 equiv.), DMAP (349mg, 1.0 equiv.) and then triethylamine (3.18mL, 8.0 equiv.) at 0 ℃. The reaction mixture was slowly warmed to room temperature, and then stirred at the same temperature for 16 hours. After 16 hours, the reaction mixture was washed with CH ₂ Cl ₂ Diluted and saturated NaHCO _{3 (solution)} And (4) washing the solution. The separated organic layer was washed with brine, over Na ₂ SO ₄ Dried and concentrated under reduced pressure to give the crude material. The crude material was first purified using 50% EtOAc in hexane and then 15% EtOAc in CH ₂ Cl ₂ And further purified to obtain (3 a) (623mg, 25%) as a viscous oil. C calculated by MS (ESI +) ₅₀ H ₉₃ NO ₇ Si ₂ ，[M+H] ⁺ =876.65, observed value =876.6.

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

the procedure of (3 a) was followed using (2 b) to provide (3 b) as a viscous oil (557mg, 23%). C calculated by MS (ESI +) ₄₉ H ₉₁ NO ₆ Si ₂ ，[M+H] ⁺ =846.64, observed value =846.6.

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

(3 a) (308mg, 0.351mmol) in anhydrous CH at room temperature ₂ Cl ₂ To the solution in (3 mL) was added oxalyl chloride (0.15ml, 5.0 eq) and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was redissolved in anhydrous CH ₂ Cl ₂ (3 mL). To the stirred acid chloride solution was added 3-dimethylaminopropanol (4) (109mg, 3 equiv.) followed by triethylamine (0.10 mL,2.0 equiv.) at 0 deg.C. The reaction mixture was slowly warmed to room temperature, and then stirred at the same temperature for 16 hours. After monitoring the completion of the reaction by MS, the reaction mixture was concentrated to dryness under reduced pressure and CH with 0-10% MeOH was used ₂ Cl ₂ Purification to give (5 a) (204mg, 60%) as a viscous oil. C calculated by MS (ESI +) ₅₅ H ₁₀₄ N ₂ O ₇ Si ₂ ，[M+H] ⁺ =961.74, observed value =961.7.

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

use of (3b) The procedure of (5 a) was followed to provide (5 b) (248mg, 90%) as a viscous oil. C calculated by MS (ESI +) ₅₄ H ₁₀₂ N ₂ O ₆ Si ₂ ，[M+H] ⁺ =931.73, observed value =931.7.

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

to a stirred solution of (5 a) (204mg, 0.212mmol) in anhydrous THF (3 mL) was added dropwise pyridine containing 70% hydrogen fluoride (1.09mL, 197 equiv.) at 0 deg.C, then slowly warmed to room temperature. The reaction mixture was stirred at room temperature for 16 hours. After monitoring the completion of the reaction by MS, the reaction mixture was cooled to 0 ℃ and purified by the addition of solid NaHCO in portions ₃ And (4) quenching. After gas formation was minimized, the resulting mixture was diluted with EtOAc and saturated NaHCO _{3 (aqueous solution)} Neutralizing the solution. The separated organic layer was washed with brine, over Na ₂ SO ₄ Dried and concentrated under reduced pressure to give the crude material. The crude material was used with CH containing 0-20% MeOH ₂ Cl ₂ Purification to obtain TBL-0731 (6 a) (132mg, 85%) as a viscous oil. C calculated by MS (ESI +) ₄₃ H ₇₆ N ₂ O ₇ ，[M+H] ⁺ =733.57, observed value =733.5. ¹ H NMR(500MHz,CDCl ₃ )δ7.64(d,J＝15.9Hz,1H),7.14–7.08(m,2H),7.05(d,J＝8.1,3.5Hz,1H),6.37(d,J＝16.0Hz,1H),4.27(t,J＝6.4Hz,2H),3.86(s,3H),3.72–3.63(m,2H),2.78–2.70(m,2H),2.67–2.47(m,6H),2.46–2.40(m,2H),2.36(s,6H),2.02–1.90(m,4H),1.49–1.35(m,4H),1.34–1.15(m,32H),0.87(t,J＝6.9Hz,6H).

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

using the procedure of (5 b) following (6 a) to provide a viscous oilTBL-0750 (6 b) (153mg, 82%). C calculated by MS (ESI +) ₄₂ H ₇₄ N ₂ O ₆ ，[M+H] ⁺ =703.55, observed value =703.6. ¹ H NMR(400MHz,CDCl ₃ )δ7.66(d,J＝16.0Hz,1H),7.53(d,J＝8.7,1.3Hz,2H),7.12(d,J＝8.6,1.5Hz,2H),6.38(d,J＝16.0,0.9Hz,1H),4.26(t,J＝6.4Hz,2H),3.70–3.63(m,2H),2.73–2.64(m,2H),2.64–2.46(m,6H),2.45–2.40(m,2H),2.34(s,6H),1.99–1.88(m,4H),1.43–1.34(m,4H),1.31–1.21(m,32H),0.87(t,J＝6.8Hz,6H).

Synthesis scheme B of phenolic acid lipids

Synthesis of intermediate (3) in synthesis scheme B:

to acid intermediate (1) (4.58 g) in CH ₂ Cl ₂ To a solution (30 mL, anhydrous) was added oxalyl chloride (1.04ml, 2 equiv.) and stirred at room temperature for 2 hours. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CH ₂ Cl ₂ (30 mL, anhydrous). Syringic acid (2) (1.32g, 1.1 equiv) was then added to this solution followed by pyridine (2.93ml, 6 equiv) and the resulting mixture was stirred at room temperature overnight. After stirring overnight, the solvent was removed under reduced pressure and the remaining residue was treated with minimal CH ₂ Cl ₂ Dissolve and then filter through a short plug of silica gel with 100% EtOAc as eluent. The clear filtrate was concentrated to dryness to provide the crude product material, which was purified on MPLC over 10CV using a 10-100% EtOAc/hexanes gradient to provide acid product (3) (3.70g, 78%). C calculated by MS (ESI +) ₅₃ H ₁₀₁ NO ₈ Si ₂ ，[M+H] ⁺ =936.7, observed value =936.7.

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

to benzoic acid (3) (400 mg) in CH ₂ Cl ₂ To the solution (4 mL, anhydrous) was added oxalyl chloride (0.18mL, 5 eq) and stirred at room temperature for 2 h. All volatiles were removed under reduced pressure and the remaining residue was redissolved in CH ₂ Cl ₂ (3 mL, anhydrous). The resulting solution was cooled with an ice bath, and then 2-aminoethanol (4 a) (76 mg) was added to CH ₂ Cl ₂ (1 mL). The reaction mixture was warmed to room temperature and stirred at the same temperature for 16 hours. After monitoring complete consumption of starting material by LC-MS, the reaction mixture was concentrated to dryness and 0-20% MeOH/CH was used ₂ Cl ₂ The crude material was purified by gradient over MPLC over 12CV to afford (5 a) (225mg, 52%) as a viscous oil. C calculated by MS (ESI +) ₅₇ H ₁₁₀ N ₂ O ₈ Si ₂ ，[M+H] ⁺ =1007.8, observed value =1007.6.

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

the procedure for synthesis (5 a) was followed using 4-aminobutanol (4 b) to provide the product as a viscous oil (5 b) (360mg, 81%). C calculated by MS (ESI +) ₅₉ H ₁₁₄ N ₂ O ₈ Si ₂ ，[M+H] ⁺ =1035.8, observed value =1035.6.

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

the synthetic procedure of (5 a) was followed using intermediate (3) (500 mg) and 3-morpholinopropanol (4 c) to provide product (5 c) (350mg, 62%). MS (ESI +) calculationC of (A) ₆₀ H ₁₁₄ N ₂ O ₉ Si ₂ ，[M+H] ⁺ =1063.8, observed value =1063.6.

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

the synthetic procedure for (5 a) was followed using intermediate (3) (500 mg) and 2-pyridinemethanol (4 d) to provide product (5 d) (237mg, 43%). C calculated by MS (ESI +) ₆₀ H ₁₀₈ N ₂ O ₈ Si ₂ ，[M+H] ⁺ =1041.8, observed value =1041.6.

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

the synthetic procedure for 5a was followed using intermediate (3) (843 mg) and 4-methylpiperazineethanol (4 e) to provide product (5 e) (346mg, 36%). C calculated by MS (ESI +) ₆₀ H ₁₁₅ N ₃ O ₈ Si ₂ ，[M+H] ⁺ =1062.8, observed value =1062.7.

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

to a stirred solution of TBS-protected intermediate (5 a) (225 mg) in THF (3 mL, anhydrous) in a plastic polymer scintillation vial (non-glass) was added triethylamine (0.16ml, 5 equivalents) dropwise, followed by triethylamine-3 HF (0.36ml, 10 equivalents) dropwise. The reaction mixture was stirred at 50 ℃ overnight. By reacting EtOAc with NaHCO _{3 (aqueous solution)} Two drops of the reaction mixture were suspended between the layers to monitor the reaction and the organic layer was analyzed (TLC or LC-MS). Once the starting material was consumed, excess HF and volatiles were passed through N in a fume hood ₂ Gas purged to remove and the remaining material was diluted with EtOAc and saturated NaHCO ₃ Aqueous solution neutralization (checked with pH paper). The separated organic layer was washed with brine and Na ₂ SO ₄ Dried and concentrated under reduced pressure to provide the crude material. The crude material was taken up in 0-40% MeOH/CH ₂ Cl ₂ The gradient was purified over 10CV on MPLC to afford TBL-0507 (6 a) (90mg, 52%). C calculated by MS (ESI +) ₄₅ H ₈₂ N ₂ O ₈ ，[M+H] ⁺ =779.6, observed value =779.5.

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

intermediate (5 b) (360 mg) protected with TBS followed the procedure of (6 a) to afford TBL-0508 (6 b) (71mg, 25%). C calculated by MS (ESI +) ₄₇ H ₈₆ N ₂ O ₈ ，[M+H] ⁺ =807.6, observed value =807.5.

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

intermediate (5 c) (350 mg) protected with TBS following the procedure of (6 a) and using 0-10% MeOH/CH ₂ Cl ₂ Gradient purification to afford TBL-0517 (6 c) (164mg, 60%). C calculated by MS (ESI +) ₄₈ H ₈₆ N ₂ O ₉ ，[M+H] ⁺ =835.6, observed value =835.5.

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

intermediate (5 d) (237 mg) protected with TBS the procedure of (6 a) was followed, and MeOH/CH was 0-10% ₂ Cl ₂ Gradient purification to afford TBL-0518 (6 d) (111mg, 60%). C calculated by MS (ESI +) ₄₈ H ₈₀ N ₂ O ₈ ，[M+H] ⁺ =813.6, observed value =813.5.

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

intermediate (5 e) (346 mg) protected with TBS following the procedure of (6 a) and using 0-20% MeOH/CH ₂ Cl ₂ Gradient purification to afford TBL-0535 (6 e) (88mg, 32%). C calculated by MS (ESI +) ₄₈ H ₈₇ N ₃ O ₈ ，[M+H] ⁺ =834.6, observed value =834.6.

Synthesis of phenolic acid lipids scheme C

Synthesis of intermediate (9 a) in synthesis scheme C:

to a flask containing amino acid (7) (500 mg) and dodecyl acrylate (8 a) (2.91g, 2.5 equivalents) were added isopropanol (5 mL) and triethylamine (1.35mL, 2 equivalents). The resulting mixture was heated at 90 ℃ for 3 hours. After monitoring the completion of the reaction by MS, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The remaining material was used 0-12% MeOH/CH ₂ Cl ₂ Purification on MPLC afforded product (9 a) (987 mg, 35%). C calculated by MS (ESI +) ₃₄ H ₆₅ NO ₆ ，[M+H] ⁺ =584.5, observed value =584.5.

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

the procedure of (9 a) was followed using amino acid (7) (1.00 g), tetradecyl acrylate (8 b) (6.51g, 2.5 equivalents), isopropanol (10 mL), and triethylamine (2.70ml, 2 equivalents) to provide product (9 b) (1.80g, 29%). C calculated by MS (ESI +) ₃₈ H ₇₃ NO ₆ ，[M+H] ⁺ =640.5, observed value =640.5.

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

intermediate (9 a) (300 mg) in anhydrous CH at room temperature ₂ Cl ₂ (3 mL) was added oxalyl chloride (1.0 mL,23 equiv.) dropwise, and stirred at the same temperature for 2 hours. Excess solvent and oxalyl chloride were removed under reduced pressure, and the remaining residue was redissolved in anhydrous CH ₂ Cl ₂ (3 mL). Phenolic acid (10) (146mg, 1.0 equiv.) and pyridine (0.21ml, 5 equiv.) were added to the stirred acid chloride solution at room temperature, followed by stirring at the same temperature for 16 hours. After monitoring the completion of the reaction by MS, the reaction mixture was concentrated under reduced pressure. The remaining crude material was used 0-10% MeOH/CH ₂ Cl ₂ Purification on MPLC provided the product TBL-0484 (11 a) (90mg, 21%). C calculated by MS (ESI +) ₄₈ H ₈₄ N ₂ O ₁₀ ，[M+H] ⁺ =849.6, observed value =849.5.

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

the procedure of (11 a) was followed using intermediate (9 b) (300 mg) to provide TBL-0485 (11 b) (80mg, 19%). C calculated by MS (ESI +) ₅₂ H ₉₂ N ₂ O ₁₀ ，[M+H] ⁺ =905.7, observed value =905.6.

Example 1: lipid nanoparticle formulations

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

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

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

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

TABLE 5 exemplary lipid nanoparticle formulations for intratracheal administration

Delivering FFLs by intratracheal administration mRNA

By means of

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

Numbered examples

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

wherein X is O or S;

wherein R' is

Wherein R is ⁶ Is composed of

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

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

wherein R is ^A 、R ^B 、R ^C And R ^D Each independently selected from optionally substituted (C) ₆ -C ₂₀ ) Alkyl, optionally substituted (C) ₆ -C ₂₀ ) Alkenyl, optionally substituted (C) ₆ -C ₂₀ ) Alkynyl, optionally substituted (C) ₆ -C ₂₀ ) Acyl, optionally substituted-OC (O) alkyl, optionally substituted-OC (O) alkenyl, optionally substituted (C) ₁ -C ₆ ) Monoalkylamino, optionally substituted (C) ₁ -C ₆ ) Dialkylamino, optionally substituted (C) ₁ -C ₆ ) Alkane (I) and its preparation methodOxy, -OH, -NH ₂ ；

or a pharmaceutically acceptable salt thereof.

2. The cationic lipid according to numbered example 1, or a pharmaceutically acceptable salt thereof, wherein any alkyl, alkenyl, alkynyl, acyl, alkoxy, monoalkylamino, dialkylamino, heterocycloalkyl, or heteroaryl is optionally substituted with one or more substituents selected from the group consisting of: (C) ₁ -C ₆ ) Alkyl, (C) ₂ -C ₆ ) Alkenyl, (C) ₂ -C ₆ ) Alkynyl, (C) ₁ -C ₆ ) Acyl group, (C) ₁ -C ₆ ) Alkoxy, halogen, -COR, -CO ₂ H、-CO ₂ R、-CN、-OH、-OR、-OCOR、-OCO ₂ R、-NH ₂ 、-NHR、-N(R) ₂ -SR or-SO ₂ R, or two twin hydrogens on a carbon atom are substituted with a group = NH, wherein each instance of R is independently C ₁ -C ₁₀ An aliphatic alkyl group.

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

i)R ^A and R ^B The same; and/or

ii)R ^C And R ^D The same is true.

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

i)R ^A and R ^B Different; and/or

ii)R ^C And R ^D Different.

5. The cationic lipid according to any one of numbered embodiments 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R ^A 、R ^B 、R ^C And R ^D The same is true.

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

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

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

9. The cationic lipid of any one of the preceding numbered embodiments, or pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁴ And R ⁵ Only one of which is-OC (O) R'.

10. The cationic lipid according to numbered embodiment 9, or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁴ Or R ⁵ Are not OH.

11. The cationic lipid according to any one of numbered embodiments 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁴ And R ⁵ Two of which are-OC (O) R'.

12. The cationic lipid according to numbered embodiment 11, or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁴ Or R ⁵ Are not OH.

13. The cationic lipid according to any one of numbered embodiments 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁴ And R ⁵ Three of (1)OC(O)R'。

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

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

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

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

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

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

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

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

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

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

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

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

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

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

22. the cationic lipid according to numbered embodiment 21, or a pharmaceutically acceptable salt thereof, wherein:

i) k, m and n =1; or

ii) k, m and n =1, and R ¹¹ And R ¹² = H; or

iii) k and n =1, and m =2; or alternatively

iv) k and n =1,m =2, and R ¹¹ And R ¹² = H; or alternatively

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

vi) k and n =1,m =3, and R ¹¹ And R ¹² ＝H。

23. The cationic lipid of any one of the preceding numbered embodiments, or pharmaceutically acceptable salt thereof, wherein R ⁶ Comprises the following steps:

24. the cationic lipid according to numbered embodiment 23, or a pharmaceutically acceptable salt thereof, wherein:

i) p, q and r =1; or

ii) p, q and R =1, and R ¹³ And R ¹⁴ Is H; or alternatively

iii) q and r =1, and p =2; or alternatively

iv) q and R =1, p =2, and R ¹³ And R ¹⁴ Is H.

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

26. the cationic lipid according to numbered embodiment 25, or a pharmaceutically acceptable salt thereof, wherein R ⁶ Comprises the following steps:

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

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

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

or a pharmaceutically acceptable salt thereof.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

61. The composition of any one of numbered embodiments 54 through 60 for use in therapy.

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

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

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

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

Claims

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

wherein X is O or S;

wherein R' is

Wherein R is ⁶ Is composed of

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

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

or a pharmaceutically acceptable salt thereof.

2. The cationic lipid according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is O.

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

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

5. The cationic lipid of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R' is:

6. the cationic lipid according to claim 5, or a pharmaceutically acceptable salt thereof, wherein:

vii) k, m and n =1; or

viii) k, m and n =1, and R ¹¹ And R ¹² = H; or

ix) k and n =1, and m =2; or

x) k and n =1, m =2, and R ¹¹ And R ¹² = H; or

xi) k and n =1, and m =3; or

xii) k and n =1,m =3, and R ¹¹ And R ¹² ＝H。

7. The cationic lipid according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein R' is

And R is ⁷ And R ⁸ Each is by-CO ₂ R ^aa Substituted optionally substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa Is C ₁ -C ₅₀ Alkyl, preferably wherein R ⁷ And R ⁸ Each being a quilt-CO ₂ R ^aa Substituted (C) ₁ -C ₆ ) Alkyl radical, wherein R ^aa C ₁ -C ₂₀ Alkyl, more preferably wherein R ⁷ And R ⁸ Each is as follows:

(i)

or

(ii)

8. The cationic lipid according to any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein R ⁶ Comprises the following steps:

9. the cationic lipid according to any one of the preceding claims, having a structure according to:

(a) Formula (II):

or a pharmaceutically acceptable salt thereof,

(b) Formula (IIA):

or a pharmaceutically acceptable salt thereof,

(c) Formula (IIF):

or a pharmaceutically acceptable salt thereof,

(d) Formula (IIG):

or a pharmaceutically acceptable salt thereof,

(e) Formula (IIH):

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

(f) Formula (III):

or a pharmaceutically acceptable salt thereof,

(g) Formula (IIIA):

or a pharmaceutically acceptable salt thereof,

(h) Formula (IIIB):

or a pharmaceutically acceptable salt thereof,

(i) Formula (IIID):

or a pharmaceutically acceptable salt thereof,

(j) Formula (IIIL):

or a pharmaceutically acceptable salt thereof, or

(k) Formula (IV):

10. The cationic lipid according to any one of claims 1 to 8, having a structure according to formula (VI), (VII), (VIII), (IX) or (X):

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

12. A composition comprising the cationic lipid of any one of the preceding claims, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids, optionally wherein the composition is a lipid nanoparticle.

13. The composition of claim 12, wherein the composition is a lipid nanoparticle and the lipid nanoparticle encapsulates a nucleic acid, optionally an mRNA encoding a peptide or protein.

14. The composition of claim 13, wherein the lipid nanoparticle has a percent encapsulation of mRNA of at least 70%.

15. A composition according to any one of claims 12 to 14 for use in therapy.