Auxiliary lipids for nucleic acid delivery

Abstract

Provided herein is a class of helper lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids.

Description

Related applications This application claims priority to European application EP 23306048.2, filed on 28 June 2023, the entire disclosure of which is hereby incorporated by reference. Background Technology

[0001] Nucleic acid delivery has been extensively explored as a potential treatment option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for the prevention and treatment of various diseases, such as in the use of vaccines.

[0002] Efficient delivery of liposome-encapsulated nucleic acids remains an active area of ​​research. Liposome-encapsulated nucleic acids can be administered intramuscularly (IM).

[0003] The auxiliary lipid components of liposomes play an important role in improving the efficiency of nucleic acid transfection. Various auxiliary lipids suitable for in vivo use have been identified. However, there is still a need to identify auxiliary lipids effective for intramuscular delivery of mRNA (e.g., in vaccines, such as those against influenza or respiratory syncytial virus (RSV)). It is also necessary to identify auxiliary lipids that can be synthesized efficiently and inexpensively without forming potentially toxic byproducts. Summary of the Invention

[0004] This invention provides, in particular, a novel class of auxiliary lipid compounds for improving the in vivo delivery of therapeutic agents such as nucleic acids. The auxiliary lipids can increase the stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles, and promote cell fusion and endosome escape. The inventors of this invention have unexpectedly discovered that lipid nanoparticles containing the auxiliary lipids of this invention are highly effective for intramuscular delivery of mRNA encapsulated within said lipid nanoparticles. In fact, lipid nanoparticles containing the auxiliary lipids of this invention have exhibited high levels of expression of said peptides or proteins when delivering mRNA encoding said peptides or proteins intramuscularly.

[0005] Lipid nanoparticles containing these auxiliary lipid compounds are considered to be capable of efficiently delivering therapeutic agents and vaccines (e.g., against influenza or respiratory syncytial virus (RSV)) intramuscularly in vivo.

[0006] On the one hand, this paper provides an accessory lipid having a structure according to formula (I): (I) Or its pharmaceutically acceptable salt, wherein: Y is selected from C 2-6 Alkylene or C 4-6 alkenyl; R a Does not exist, or R a C is arbitrarily replaced1- C 10 Alkyl, wherein, when R a When it exists, with R a The bonded nitrogen carries a positive charge; Each R1 is independently selected from hydrogen or optionally substituted C. 1- C 10 alkyl; Each R2 is selected independently from: (i) Optional substitution of C4-C 24 Alkyl, optionally substituted C4-C 24 alkenyl, and optionally substituted C4-C 24 alkynyl group; (ii) , where each R A Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z A Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 alkenyl; (iii) , where each R B Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z B Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0007] On the one hand, this article provides auxiliary lipids having formula (I), which are pharmaceutically acceptable salts.

[0008] On the one hand, this document provides compositions comprising one or more auxiliary lipids of the present invention or pharmaceutically acceptable salts thereof, and further comprising: (i) one or more cationic lipids; (ii) one or more sterol-based lipids; and (iii) One or more PEG-modified lipids.

[0009] On the one hand, the composition is lipid nanoparticles, optionally liposomes.

[0010] On the one hand, compositions comprising cationic lipids and one or more auxiliary lipids of the present invention can be used in therapies, such as for the treatment, prevention or improvement of influenza or respiratory syncytial virus (RSV). Detailed Implementation definition

[0011] To facilitate understanding of the invention, certain terms are defined below. Further definitions of the following and other terms are set forth throughout the specification. Publications and other references cited herein to describe the background of the invention and to provide further details about its implementation are hereby incorporated by reference.

[0012] Amino acids: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the common structure H₂N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is a l-amino acid. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutes. Amino acids (including carboxyl-terminal 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 cyclic half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids may contain one or more translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprene-like groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The terms "amino acid" and "amino acid residue" are used interchangeably and can refer to free amino acids and / or amino acid residues of peptides. It is obvious from the context in which the term is used whether it refers to free amino acids or peptide residues.

[0013] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, an animal includes, but is not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0014] Approximately or about: As used herein, the term “approximately” or “about” when applied to one or more target values ​​refers to a value similar to the stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in either direction, unless otherwise stated or otherwise apparent from the context (except where such a number would exceed 100% of the possible value).

[0015] Biologically active: As used herein, the term "biologically active" refers to the characteristic of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered biologically active.

[0016] Cationic lipids: As used herein, the term "cationic lipid" refers to a lipid substance that has a net positive charge at a selected pH (e.g., physiological pH). Several cationic lipids have been described in the literature, many of which are commercially available. Other cationic lipids suitable for use in compositions include those described in the literature.

[0017] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery encompasses the delivery of mRNA to a target tissue and the expression and retention of the encoded protein within that target tissue (also known as “local distribution” or “local delivery”), as well as the delivery of mRNA to a target tissue and the expression and secretion of the encoded protein into the patient’s circulatory system (e.g., serum) and its systemic distribution and uptake by other tissues (also known as “systemic distribution” or “systemic delivery”).

[0018] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production”, and their grammatical equivalents, are used interchangeably.

[0019] Functionality: As used herein, a “functional” biomolecule is a biomolecule that exhibits the properties and / or activities that characterize it.

[0020] Half-life: As used herein, the term “half-life” is the time required for the concentration or activity of a nucleic acid or protein to decrease to half of its value measured at the beginning of a time period.

[0021] Supporting lipids: As used herein, the term "supporting lipid" refers to any neutral or zwitterionic lipid material. Without being bound by any particular theory, supporting lipids can increase the stability, rigidity, and / or flowability within lipid bilayers / nanoparticles.

[0022] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to a value relative to a baseline measurement, which is, for example, a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A “control subject” is a subject with the same form of disease as the subject being treated, and who is approximately the same age as the subject being treated.

[0023] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment (e.g., in test tubes or reaction vessels, in cell cultures, etc.) rather than within a multicellular organism.

[0024] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells, as opposed to, for example, in vitro systems.

[0025] Liposomes: As used herein, the term "liposome" refers to any layered, multilayered, or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids or by mixing one or more lipids with one or more polymers. In some embodiments, liposomes suitable for use in the present invention contain one or more of the assisting lipids or cationic lipids of the present invention, and optionally further include: (i) one or more other cationic lipids; (ii) one or more non-cationic lipids; (iii) one or more sterol-based lipids; and / or (iv) One or more PEG-modified lipids.

[0026] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The term “modified mRNA” refers to mRNA containing at least one chemically modified nucleotide. mRNA may contain one or more coding and noncoding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, such as in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs of chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' orientation. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); or a nucleoside analogue (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine). Glycosides, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., thiophosphates and 5'-N-phosphoramide bonds).

[0027] Nucleic acid: As used herein, the term “nucleic acid” in its broadest sense refers to any compound and / or substance incorporated into or potentially incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance incorporated into a polynucleotide chain via or potentially incorporated via a phosphodiester bond. In some embodiments, “nucleic acid” refers to a single nucleic acid residue (e.g., a nucleotide and / or nucleoside). In some embodiments, “nucleic acid” refers to a polynucleotide chain containing a single nucleic acid residue. In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, “nucleic acid” encompasses ribonucleic acid (RNA), including but not limited to interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mmRNA), long non-coding RNA (lncRNA), microRNA (miRNA), multi-coding nucleic acid (MCNA), polymeric coding nucleic acid (PCNA), guide RNA (gRNA), and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, DNA may be in the following forms: antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a polymerase chain reaction (PCR) product, a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups.In embodiments, RNA may be in the following forms: messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SL RNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA component (TERC), splicing leader RNA (SL RNA), antisense RNA (aRNA or asRNA), cis-natural antisense transcript (cis-NAT), CRISPR RNA (crRNA), long noncoding RNA (lncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat-associated siRNA (rasiRNA), 73K RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some embodiments, nucleic acid is mRNA encoding a protein (e.g., an enzyme).

[0028] Patient: As used herein, the terms "patient" or "subject" refer to any biological organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include both prenatal and postnatal forms.

[0029] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means a substance that, to the extent of reasonable medical judgment, is suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0030] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts of an amino group formed by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, sulfonate, and arylsulfonate). Other pharmaceutically acceptable salts include those formed by quaternizing amines with suitable electrophilic agents (e.g., alkyl halides) to form quaternized alkylamino salts.

[0031] Whole-body distribution or delivery: As used herein, the terms “whole-body distribution,” “whole-body delivery,” or their grammatical equivalents refer to a mechanism or method of delivery or distribution that affects the whole body or the whole organism. Typically, whole-body distribution or delivery is accomplished via the body’s circulatory system (e.g., blood flow). Compare this to the definition of “local distribution or delivery.”

[0032] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes both prenatal and postnatal forms. In many embodiments, the subject is a human being. A subject can be a patient, referring to a person who goes to a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably with "individual" or "patient" herein. A subject may have or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0033] Essentially: As used herein, the term “essentially” refers to a qualitative condition that exhibits all or nearly all of the intended characteristics or properties, or to a degree or extent. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if at all) complete and / or proceed to completion or achieve or avoid an absolute result. Therefore, the term “essentially” is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0034] Target tissue: As used herein, the term “target tissue” refers to any tissue affected by the disease to be treated. In some embodiments, target tissue includes those tissues that exhibit pathology, symptoms, or features associated with the disease.

[0035] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of a disease, disorder, and / or condition when administered to a subject who has or is susceptible to such disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0036] Treatment: As used herein, the term "treatment" means any method used to partially or completely alleviate, relieve, reduce, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease for the purpose of reducing the risk of developing a disease-related pathology. Chemical definition

[0037] Acyl group: As used herein, the term "acyl group" refers to R Z -(C=O)-, where R Z It is, for example, any alkyl, alkenyl, ynyl, heteroalkyl, or heteroalkylene group.

[0038] Aliphatic: As used in this article, the term "aliphatic" refers to (C1-C1) 50Hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be straight-chain, branched, or cyclic. For example, (C1-C2) hydrocarbons... 20 Aliphatic ali ... 20 )alkyl (e.g., straight-chain or branched (C1-C1) 20 ) saturated alkyl), (C2-C 20 Alkenyl (e.g., straight-chain or branched (C4-C5)) 20 diene-based, straight-chain or branched (C6-C) 20 (trienyl, etc.), and (C2-C 20 ) alkynyl group (e.g., straight-chain or branched (C2-C) 20 )alkynyl group). (C1-C 20 Aliphatic ali ... 20 ) Cyclic aliphatic (e.g., (C3-C) 20 )cycloalkyl, (C4-C 20 )cycloalkenyl, or (C8-C 20 (Cycloalkyne). In some embodiments, the aliphatic group may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur), and may optionally be substituted with one or more substituents (e.g., alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). The aliphatic group is unsubstituted or substituted with one or more substituent groups as described herein. For example, the aliphatic group may be substituted with one or more of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R'' is independently (C1-C1). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl. In the examples, R'' is independently an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl). In the examples, R'' is independently an unsubstituted (C1-C3)alkyl. In the examples, the aliphatic group is unsubstituted. In the examples, the aliphatic group does not include any heteroatoms. Alkyl: As used herein, the term "alkyl" means an acyclic straight-chain and branched hydrocarbon group, such as "(C1-C3)alkyl". 30"alkyl" refers to an alkyl group having 1 to 30 carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, etc. The term "lower alkyl" means a straight-chain alkyl group or a branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art upon understanding this disclosure. Alkyl groups can be unsubstituted or... The alkyl group is substituted by one or more substituent groups as described herein. For example, the alkyl group may be substituted by one or more of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R'' is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl. In the examples, R'' is independently an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl). In the examples, R'' is independently an unsubstituted (C1-C3)alkyl. In the examples, the alkyl group is substituted (e.g., 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In the examples, the alkyl group is substituted with a -OH group and may also be referred to herein as a "hydroxyalkyl" group, wherein the prefix indicates a -OH group and "alkyl" is as described herein.

[0039] As used herein, "alkyl" also refers to a group having a straight-chain or branched saturated hydrocarbon group having 1 to 50 carbon atoms ("(C1-C50")). 50 Alkyl group ("(C1-C4")). In some embodiments, the alkyl group has 1 to 40 carbon atoms ("(C1-C4")). 40 Alkyl group ("(C1-C2")). In some embodiments, the alkyl group has 1 to 30 carbon atoms ("(C1-C2")). 30 Alkyl group ("(C1-C2")). In some embodiments, the alkyl group has 1 to 20 carbon atoms ("(C1-C2")). 20 Alkyl group ("(C1-C1")). In some embodiments, the alkyl group has 1 to 10 carbon atoms ("(C1-C1")). 10(C1-C9)alkyl. In some embodiments, the alkyl group has 1 to 9 carbon atoms ("(C1-C8)alkyl". In some embodiments, the alkyl group has 1 to 8 carbon atoms ("(C1-C7)alkyl". In some embodiments, the alkyl group has 1 to 7 carbon atoms ("(C1-C7)alkyl". In some embodiments, the alkyl group has 1 to 6 carbon atoms ("(C1-C6)alkyl". In some embodiments, the alkyl group has 1 to 5 carbon atoms ("(C1-C5)alkyl". In some embodiments, the alkyl group has 1 to 4 carbon atoms ("(C1-C4)alkyl". In some embodiments, the alkyl group has 1 to 3 carbon atoms ("(C1-C3)alkyl". In some embodiments, the alkyl group has 1 to 2 carbon atoms ("(C1-C2)alkyl". In some embodiments, the alkyl group has 1 to 2 carbon atoms ("(C1-C2)alkyl"). 2 to 6 carbon atoms (“C1 alkyl”). In some embodiments, the alkyl group has 2 to 6 carbon atoms (“(C2-C6)alkyl”). Examples of (C1-C6)alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). Other examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise stated, each example of an alkyl group is independently unsubstituted (“unsubstituted alkyl”) or substituted with one or more substituents (“substituted alkyl”). In some embodiments, the alkyl group is unsubstituted (C1-C6) 50 Alkyl groups. In some embodiments, the alkyl group is substituted (C1-C2). 50 )alkyl.

[0040] Adding the suffix "-ene" to a group indicates that the group is a divalent moiety. For example, arylene is the divalent moiety of aryl, while heteroarylene is the divalent moiety of heteroaryl.

[0041] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight-chain or branched hydrocarbon group, such as methylene, ethylene, isopropylene, etc. Similarly, the term "alkenylene" as used herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds, which may occur at any stable point on the chain; while the term "alkynylene" herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds, which may occur at any stable point on the chain. In some embodiments, the alkylene, alkenyl, or alkynyl group may comprise one or more cyclic aliphatic and / or one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur) and may optionally be substituted with one or more substituents (e.g., alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). For example, the alkylene, alkenylene, or ynylene group may be substituted with one or more of the following: halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R'' is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl. In the examples, R'' is independently an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl). In the embodiments, R'' is independently an unsubstituted (C1-C3)alkyl. In some embodiments, the alkylene, alkenyl, or ynylene is unsubstituted. In some embodiments, the alkylene, alkenyl, or ynylene does not include any heteroatoms. Alkenyl: As used herein, "alkenyl" means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds, which may be present at any stable point on the chain, such as (C2-C3)alkyl. 30"Alkenyl" refers to an alkenyl group having 2-30 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In embodiments, the alkenyl group comprises 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group comprises a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated. The alkenyl group may be unsubstituted or substituted as described herein. One or more substituent groups are described. For example, the alkenyl group may be substituted by one or more of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R'' is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl. In the examples, R'' is independently an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl). In the examples, R'' is independently an unsubstituted (C1-C3)alkyl. In the examples, the alkenyl group is unsubstituted. In the examples, the alkenyl group is substituted (e.g., 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In the examples, the alkenyl group is substituted with a -OH group and may also be referred to herein as a "hydroxyalkenyl" group, wherein the prefix indicates a -OH group and "alkenyl" is as described herein.

[0042] As used herein, "alkenyl" also refers to a straight-chain or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). 50 ()alkenyl". In some embodiments, the alkenyl group has 2 to 40 carbon atoms ("(C2-C") 40 ()alkenyl". In some embodiments, the alkenyl group has 2 to 30 carbon atoms ("(C2-C"). 30 ()alkenyl". In some embodiments, the alkenyl group has 2 to 20 carbon atoms ("(C2-C") 20 ()alkenyl". In some embodiments, the alkenyl group has 2 to 10 carbon atoms ("(C2-C") 10(C2-C9)alkenyl. In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("(C2-C8)alkenyl"). In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("(C2-C7)alkenyl"). In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("(C2-C7)alkenyl"). In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("(C2-C6)alkenyl"). In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("(C2-C5)alkenyl"). In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("(C2-C4)alkenyl"). In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("(C2-C3)alkenyl"). In some embodiments, the alkenyl group has 2 carbon atoms ("(C2)alkenyl"). The one or more The carbon-carbon double bond can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of (C2-C4) alkenyl groups include, but are not limited to, vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. Examples of (C2-C6) alkenyl groups include the above-described (C2-C4) alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Further examples of alkenyl groups include heptenyl (C7), octenyl (C8), octtrienyl (C8), etc. Unless otherwise stated, each example of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted by one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is unsubstituted (C2-C4). 50 Alkenyl group. In some embodiments, the alkenyl group is substituted (C2-C). 50 )alkenyl.

[0043] Alkynyl: As used herein, "alkynyl" means any straight-chain or branched hydrocarbon chain having one or more carbon-carbon triple bonds, which can be present at any stable point on the chain, such as "(C2-C2)". 30"Alynyl" refers to an alkynyl group having 2-30 carbons. Examples of alkynyl groups include prop-2-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-2-alkynyl, 3-methylpent-4-alkynyl, hex-2-alkynyl, hex-5-alkynyl, etc. In the examples, the alkynyl group contains a carbon-carbon triple bond. The alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, the alkynyl group may be substituted with one or more of halogen, -COR'', -CO2H, -CO2R'', -CN, -OH, -OR'', -OCOR'', -OCO2R'', -NH2, -NHR'', -N(R'')2, -SR'', or -SO2R'' (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R'' is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl. In the examples, R'' is independently an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 (C1-C3)alkyl). In the examples, R'' is independently an unsubstituted (C1-C3)alkyl. In the examples, the alkynyl group is unsubstituted. In the examples, the alkynyl group is substituted (e.g., 1, 2, 3, 4, 5, or 6 substituent groups as described herein).

[0044] As used herein, “alkynyl” also refers to a straight-chain or branched hydrocarbon group having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds). 50 The alkynyl group, having one or more triple bonds and one or more double bonds, is also called an "alkynyl group". In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("(C2-C"). 40 () ynyl group). In some embodiments, the ynyl group has 2 to 30 carbon atoms ("(C2-C"). 30 () ynyl group). In some embodiments, the ynyl group has 2 to 20 carbon atoms ("(C2-C"). 20 () ynyl group). In some embodiments, the ynyl group has 2 to 10 carbon atoms ("(C2-C") 10(C2-C9) ynyl group. In some embodiments, the ynyl group has 2 to 9 carbon atoms ("(C2-C8) ynyl group"). In some embodiments, the ynyl group has 2 to 8 carbon atoms ("(C2-C8) ynyl group"). In some embodiments, the ynyl group has 2 to 7 carbon atoms ("(C2-C7) ynyl group"). In some embodiments, the ynyl group has 2 to 6 carbon atoms ("(C2-C6) ynyl group"). In some embodiments, the ynyl group has 2 to 5 carbon atoms ("(C2-C5) ynyl group"). In some embodiments, the ynyl group has 2 to 4 carbon atoms ("(C2-C4) ynyl group"). In some embodiments, the ynyl group has 2 to 3 carbon atoms ("(C2-C3) ynyl group"). In some embodiments, the ynyl group has 2 carbon atoms ("(C2-C5) ynyl group"). 2) Alynyl group. The one or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of (C2-C4) alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), etc. Examples of (C2-C6) alynyl groups include the above-mentioned (C2-C4) alkynyl groups as well as pentynyl (C5), hexynyl (C6), etc. Other examples of alkynyl groups include heptyynyl (C7), octyynyl (C8), etc. Unless otherwise stated, each example of an alkynyl group is independently unsubstituted (“unsubstituted alkynyl”) or substituted by one or more substituents (“substituted alkynyl”). In some embodiments, the alkynyl group is unsubstituted (C2-C4). 50 ) alkynyl group. In some embodiments, the alkynyl group is substituted (C2-C) 50 ) yyn group.

[0045] Aryl: The term "aryl" used alone or as part of a larger portion (such as in "arylene") refers to a monocyclic, bicyclic, or tricyclic carbocyclic system having a total of six to fourteen ring members, wherein the ring system has a single attachment site with the remainder of the molecule, at least one ring in the system is aromatic, and each ring in the system contains four to seven ring members. In embodiments, the aryl group has six ring carbon atoms ("(C6)aryl", for example, phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("(C6)aryl", for example, phenyl). 10 aryl (e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“(C 14 "Aryl" also includes ring systems in which the aryl ring (as defined above) is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment group or attachment point is located on the aryl ring, and in such cases, the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

[0046] As used herein, "aryl" also refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic array) having 6–14 ring carbon atoms and no heteroatoms ("(C6-C")). 14 (C6)aryl. In some embodiments, the aryl group has 6 ring carbon atoms ("(C6)aryl"; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("(C6)aryl"). 10 aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“(C 14 "Aryl"; for example, anthracene. "Aryl" also includes ring systems in which the aryl ring (as defined above) is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment group or attachment point is located on the aryl ring, and in such cases, the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Unless otherwise stated, each example of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In some embodiments, the aryl group is unsubstituted (C6-C6). 14 aryl group. In some embodiments, the aryl group is substituted (C6-C6). 14 Aryl.

[0047] arylene: As used herein, the term "arylene" refers to a divalent aryl group (i.e., having two attachment sites with the molecule). Exemplary arylene groups include phenylene (e.g., unsubstituted or substituted phenylene).

[0048] Carbocyclic group: As used herein, "carbocyclic group" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms in its non-aromatic ring system ("(C3-C10")). 10 The carbocyclic group has 3 to 8 cyclic carbon atoms (“(C3-C8) carbocyclic”). In some embodiments, the carbocyclic group has 3 to 7 cyclic carbon atoms (“(C3-C7) carbocyclic”). In some embodiments, the carbocyclic group has 3 to 6 cyclic carbon atoms (“(C3-C6) carbocyclic”). In some embodiments, the carbocyclic group has 4 to 6 cyclic carbon atoms (“(C4-C6) carbocyclic”). In some embodiments, the carbocyclic group has 5 to 6 cyclic carbon atoms (“(C5-C6) carbocyclic”). In some embodiments, the carbocyclic group has 5 to 10 cyclic carbon atoms (“(C5-C6) carbocyclic”). 10(C3-C6) Carbocyclic groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), etc. Exemplary (C3-C8) carbocyclic groups include, but are not limited to, the above (C3-C6) carbocyclic groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptanetrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Exemplary (C3-C6) 10 The carbocyclic groups include, but are not limited to, the aforementioned (C3-C8) carbocyclic groups, as well as cyclononyl (C9), cyclononenyl (C9), and cyclodecyl (C9). 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C9) 10 ), spiro[4.5]decyl (C 10 As illustrated in the foregoing examples, in some embodiments, the carbocyclic group is monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., containing fused, bridged, or spirocyclic systems, such as bicyclic systems (“bicyclic carbocyclic”) or tricyclic systems (“tricyclic carbocyclic”)), and may be saturated or may contain one or more carbon-carbon double or triple bonds. “Carbocyclic” also includes ring systems in which the carbocyclic ring (as defined above) is fused with one or more aryl or heteroaryl groups, wherein the attachment point is located on the carbocyclic ring, and in such cases, the number of carbons continues to represent the number of carbons in the carbocyclic system. Unless otherwise stated, each example of a carbocyclic group is independently unsubstituted (“unsubstituted carbocyclic”) or substituted with one or more substituents (“substituted carbocyclic”). In some embodiments, the carbocyclic group is an unsubstituted C3-C… 10 Carbocyclic group. In some embodiments, the carbocyclic group is substituted (C3-C4). 10 ) carbon cyclic group.

[0049] In some embodiments, "carbocyclic" or "carbocyclic" refers to "cycloalkyl", that is, a monocyclic saturated carbocyclic group having 3 to 10 ring carbon atoms ("(C3-C10")). 10(C3-C8)cycloalkyl. In some embodiments, the cycloalkyl group has 3 to 8 cyclic carbon atoms ("(C3-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 6 cyclic carbon atoms ("(C3-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 4 to 6 cyclic carbon atoms ("(C4-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 6 cyclic carbon atoms ("(C5-C6)cycloalkyl"). In some embodiments, the cycloalkyl group has 5 to 10 cyclic carbon atoms ("(C5-C6)cycloalkyl"). 10 (C5-C6) cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). (C3-C6) cycloalkyl groups include the above-described (C5-C6) cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). (C3-C8) cycloalkyl groups include the above-described (C3-C6) cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise stated, each example of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In some embodiments, the cycloalkyl group is unsubstituted (C3-C6). 10 )cycloalkyl. In some embodiments, the cycloalkyl group is substituted (C3-C4). 10 )cycloalkyl.

[0050] Halogen: As used in this article, the term “halogen” means fluorine, chlorine, bromine or iodine.

[0051] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to having 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, phosphate diesters, phosphoramides, sulfonamides, and disulfides. Heteroalkyl groups may optionally include monocyclic, bicyclic, or tricyclic rings, wherein each ring is preferably a three- to six-membered ring. Examples of heteroalkyl groups include polyethers, such as methoxymethyl and ethoxyethyl.

[0052] Heteroalkylene: As used herein, the term “heteroalkylene” refers to the divalent form of a heteroalkyl group as described herein.

[0053] Heteroaryl: As used herein, the term “heteroaryl” is a fully unsaturated ring containing heteroatoms, wherein at least one ring atom is a heteroatom, such as, but not limited to, nitrogen and oxygen.

[0054] As used herein, “heteroaryl” also refers to a group of a 5-14 member monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic array), having a cyclic carbon atom and one or more cyclic heteroatoms (e.g., 1, 2, 3, or 4 cyclic heteroatoms), wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-14 member heteroaryl”). In heteroaryl groups containing one or more nitrogen atoms, the attachment point can be a carbon atom or a nitrogen atom, where the valence allows. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or two rings. “Heteroaryl” includes ring systems in which a heteroaryl ring (as defined above) is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment point is located on the heteroaryl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring (as defined above) is fused with one or more aryl groups, wherein the attachment site is located on the aryl ring or the 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. A polycyclic heteroaryl group, in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazoyl, etc.), may have the attachment site on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

[0055] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heteroaryl”). In some embodiments, the 5-6-membered heteroaryl group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise stated, each example of a heteroaryl group is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some embodiments, the heteroaryl group is an unsubstituted 5-14-membered heteroaryl. In some embodiments, the heteroaryl group is a substituted 5-14-membered heteroaryl.

[0056] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrroleyl, furanyl, and phenylthioyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, aziryl, oxazinyl, and thioazinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indole, isoindole, indazole, benzotriazolyl, benzobenzylthio, isobenzobenzylthio, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indoleazinyl, and purinel. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphridinyl, pteridinyl, quinolinyl, isoquinolinyl, cenolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinel, dibenzofuranyl, carbazoleyl, acridinel, phenothiazinyl, phenotoxazinyl, and phenothiazinyl.

[0057] As used herein, "heterocyclic group" or "heterocycle" refers to a group having a 3- to 14-membered non-aromatic ring system with a ring carbon atom and one 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 heterocyclic groups"). In heterocyclic groups containing one or more nitrogen atoms, the attachment point can be a carbon atom or a nitrogen atom, where the valence allows. Heterocyclic groups can be monocyclic ("monocyclic heterocyclic group") or polycyclic (e.g., fused, bridged, or spirocyclic systems, such as bicyclic systems ("bicyclic heterocyclic group") or tricyclic systems ("tricyclic heterocyclic group")), and can be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclic polycyclic ring systems may contain one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems in which a heterocyclic ring (as defined above) is fused with one or more carbocyclic groups (where the attachment point is located on the carbocyclic or heterocyclic ring), or ring systems in which a heterocyclic ring (as defined above) is fused with one or more aryl or heteroaryl groups (where the attachment point is located on the heterocyclic ring), and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Unless otherwise stated, each example of a heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In some embodiments, the heterocyclic group is an unsubstituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a substituted 3-14 membered heterocyclic group.

[0058] In some embodiments, the heterocyclic group is a 5-10 membered nonaromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-10 membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5-8 membered nonaromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-8 membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5-6 membered nonaromatic ring system having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms, wherein each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 membered heterocyclic group”). In some embodiments, the 5-6 membered heterocyclic group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0059] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, ethylene oxide, and thioalkyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirrobutyl, oxadiazolinyl, and thiobutyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrophenylthio, dihydrophenylthio, pyrrolyl, dihydropyrrolyl, and pyrrolyl-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxopentyl, oxothiopentyl, and dithiopentyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiocyclohexyl, and dioxacyclohexyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazineyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocycloheptyl, oxecanyl, and thiocycloheptyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary bicyclic heterocyclic groups include, but are not limited to, indololinyl, isoindololinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthidyl, decahydro-1,8-naphthidyl, octahydropyrrolo[3,2-b]pyrrole, indololinyl, benzo[o]dicarboximide, naphthimide, benzo[dihydropyranyl]chromenyl, 1H-benzo[e][1,4]diazazonyl, 1,4,5,7-tetrahydropyranolo[3,4-b]pyrroleyl. 5,6-Dihydro-4H-furano[3,2-b]pyrrolithyl, 6,7-Dihydro-5H-furano[3,2-b]pyrrolithyl, 5,7-Dihydro-4H-thieno[2,3-c]pyrrolithyl, 2,3-Dihydro-1H-pyrroli[2,3-b]pyridyl, 2,3-Dihydrofurano[2,3-b]pyridyl, 4,5,6,7-Tetrahydro-1H-pyrroli-[2,3-b]pyridyl, 4,5,6,7-Tetrahydrofurano[3,2-c]pyridyl, 4,5,6,7-Tetrahydrothieno[3,2-b]pyridyl, 1,2,3,4-Tetrahydro-1,6-naphthidyl, etc.

[0060] Heterocyclic alkyl: As used herein, the term "heterocyclic alkyl" is a non-aromatic ring in which at least one atom is a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocyclic alkyl groups may be substituted or unsubstituted.

[0061] As can be understood from the foregoing, in some embodiments, alkyl, alkenyl, alkynyl, acyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups as defined herein are optionally substituted. "Optionally substituted" means that the group may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclic, "substituted" or "unsubstituted" heterocyclic, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Generally, the term "substituted" means that at least one hydrogen atom present on the group is replaced by a permitted substituent, which, for example, results in a stable compound (e.g., does not spontaneously regenerate). Substituents in a compound that has undergone transformation (e.g., by rearrangement, cyclization, elimination, or other reactions). Unless otherwise indicated, a “substituted” group has substituents at one or more substituted positions of the group, and when more than one position is substituted in any given structure, the substituents at each position may be the same or different. The term “substitution” is considered to include substitution with all permissible substituents of an organic compound (any substituents described herein that result in the formation of a stable compound). The invention contemplates any and all such combinations to obtain a stable compound. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents as described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0062] Exemplary carbon atom substituents include, but are not limited to, halogens, -CN, -NO2, -N3, -SO2, -SO3H, -OH, and -OR. aa -ON(R) bb )2、-N(R bb )2、-N(R bb )3+X - -N(OR) cc )R bb -SeH, -SeR aa -SH, -SR aa -SSR cc -C(=O)R aa -CO2H, -CHO, -C(OR) cc )2、-CO2R aa、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、- OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、- C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3 -OSi(R aa )3 -C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、- SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、-OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa)2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、- P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、- P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa 2. -B(OR) cc )2、-BR aa (OR cc (C1-C) 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 14 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C 14 ) aryl, and 5-14 heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl is independently bounded by 0, 1, 2, 3, 4, or 5 R dd Group substitution; Or two hydrogen atom on a carbon atom with =O, =S, =NN(R) groups bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb 、or =NOR cc replace;

[0063] R aa Each example is independently selected from (C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C 14 ) aryl, and 5-14 heteroaryl, or two R aaGroups are linked to form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0064] R bb Each example is independently selected from hydrogen, -OH, -OR aa - N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) cc OR aa - C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc - C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2、(C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C 14 ) aryl, and 5-14 heteroaryl, or two R bb The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0065] R cc Each example is independently selected from hydrogen, (C1-C) 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C14 ) aryl, and 5-14 heteroaryl, or two R cc The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0066] R dd Each example is independently selected from halogens, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee -ON(R) ff )2、-N(R ff )2、-N(R ff )3+X - -N(OR) ee )R ff -SH, -SR ee - SSR ee -C(=O)R ee -CO2H, -CO2R ee -OC(=O)R ee -OCO2R ee -C(=O)N(R) ff )2、- OC(=O)N(R ff )2、-NR ff C(=O)R ee -NR ff CO2R ee -NR ff C(=O)N(R ff )2、-C(=NR ff OR ee - OC(=NR) ff )R ee -OC(=NR) ff OR ee -C(=NR) ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee -SO2N(R) ff )2、-SO2R ee -SO2OR ee -OSO2R ee-S(=O)R ee 、-Si(R ee 3. -OSi(R) ee 3. -C(=S)N(R) ff )2、-C(=O)SR ee -C(=S)SR ee -SC(=S)SR ee -P(=O)2R ee - P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2、(C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-10 membered heterocyclic group, (C6-C 10 ) aryl, 5-10 heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal R groups dd Substituents can be linked to form =O or =S;

[0067] R ee Each example is independently selected from (C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, (C6-C 10 aryl, 3-10 membered heterocyclic, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution;

[0068] R ff Each example is independently selected from hydrogen, (C1-C) 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-10 membered heterocyclic group, (C6-C 10 ) aryl and 5-10 heteroaryl, or two R ffThe groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution; and

[0069] R gg Each example independently is a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -O(Cl-C) 50 )alkyl, -ON((C1-C 50 )alkyl)2、-N((C1-C 50 )alkyl)2、-N((C1-C 50 )alkyl)3+X - -NH((C1-C 50 )alkyl)2+X - -NH2((C1-C 50 )alkyl) +X - -NH3+X - -N(O(C1-C) 50 )alkyl)((C1-C 50 )alkyl), -N(OH)((C1-C 50 )alkyl), -NH(OH), -SH, -S(C1-C 50 )alkyl, -SS((C1-C 50 )alkyl), -C(=O)((C1-C 50 )alkyl), -CO2H, -CO2((C1-C 50 )alkyl), -OC(=O)((C1-C 50 )alkyl), -OCO2((C1-C 50 )alkyl), -C(=O)NH2, -C(=O)N((C1-C 50 )alkyl)2、-OC(=O)NH((C1-C 50 )alkyl), -NHC(=O)((C1-C 50 )alkyl), -N((C1-C 50 )alkyl)C(=O)((C1-C 50 )alkyl), -NHCO2((C1-C 50 )alkyl), -NHC(=O)N((C1-C 50 )alkyl)2、-NHC(=O)NH((C1-C 50 )alkyl), -NHC(=O)NH2, -C(=NH)O((C1-C 50 )alkyl), -OC(=NH)((C1-C50 )alkyl), -OC(=NH)O(C1-C 50 )alkyl, -C(=NH)N((C1-C 50 )alkyl)2、-C(=NH)NH((C1-C 50 )alkyl), -C(=NH)NH2, -OC(=NH)N((C1-C 50 )alkyl)2、-OC(NH)NH((C1-C 50 )alkyl), -OC(NH)NH2, -NHC(NH)N((C1-C 50 )alkyl)2、-NHC(=NH)NH2、-NHSO2((C1-C 50 )alkyl), -SO2N((C1-C 50 )alkyl)2、-SO2NH((C1-C 50 )alkyl), -SO2NH2, -SO2((C1-C 50 )alkyl), -SO2O((C1-C 50 -alkyl), -OSO2((C1-C6)alkyl), -SO((C1-C6)alkyl), -Si ... 50 )alkyl)3, -OSi((C1-C6)alkyl)3, -C(=S)N((C1-C 50 )alkyl)2、C(=S)NH((C1-C 50 )alkyl), C(=S)NH2, -C(=O)S((C1-C6)alkyl), -C(=S)S((C1-C6)alkyl), -SC(=S)S((C1-C6)alkyl), -P(=O)2((C1-C 50 )alkyl), -P(=O)((C1-C 50 )alkyl)2、-OP(=O)((C1-C 50 )alkyl)2、-OP(=O)(O(C1-C 50 )alkyl)2、(C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, (C6-C 10 ) aryl, 3-10 heterocyclic, 5-10 heteroaryl; or two geminal R gg Substituents can be linked to form =O or =S; where X - It is an antiion.

[0070] As used herein, the term “halo” or “halogen” refers to fluorine (fluorinated, -F), chlorine (chlorinated, -Cl), bromine (brominated, -Br), or iodine (iodinated, -I).

[0071] As used herein, a "counterion" is a negatively charged group that binds to a positively charged quaternary ammonium to maintain electroneutrality. 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-5-sulfonate, ethane-1-sulfonic-2-sulfonate, etc.) and carboxylate ions (e.g., acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0072] Where valence permits, the nitrogen atom can be substituted or unsubstituted, and includes primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, and -OR. aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa - C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc)2、(C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C 14 ) aryl, and 5-14 heteroaryl, or two R cc The groups, together with the nitrogen atoms to which they are attached, form 3-14 membered heterocyclic groups or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R atoms. dd Group substitution, and wherein R aa R bb R cc and R dd As defined above.

[0073] In some embodiments, the substituents present on the nitrogen atom are nitrogen protecting groups (also known as amino protecting groups). Nitrogen protecting groups are well known in the art and include those described in detail in: Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0074] For example, nitrogen-protecting groups (such as amide groups, e.g., -C(=O)R) aa This includes, but is not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridine amide, 3-pyridylformamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N'-dithiobenzyloxyamido)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0075] Nitrogen protecting groups, such as urethane groups (e.g., -C(=O)OR), aaThis includes, but is not limited to, methyl carbamates, ethyl carbamates, 9-fluorenyl methyl carbamate (Fmoc), 9-(2-sulfonyl)fluorenyl methyl carbamate, 9-(2,7-dibromo)fluoroenyl methyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilyl ethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), and 1-(1-adamantyl)-1-methylethyl carbamate. (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylamido)ethylcarbamate, tert-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), vinylcarbamate (Voc), Allyl Carbamate (Alloc), 1-Isopropyl Allyl Carbamate (Ipaoc), Cinnamyl Carbamate (Coc), 4-Nitrocinnamyl Carbamate (Noc), 8-Quinolinyl Carbamate, N-Hydroxypiperidinyl Carbamate, Alkyl Dithiocarbamate, Benzyl Carbamate (Cbz), p-Methoxybenzyl Carbamate (Moz), p-Nitrobenzyl Carbamate, p-Bromobenzyl Carbamate, p-Chlorobenzyl Carbamate, 2,4-Dichlorobenzyl Carbamate, 4-Methylsulfinylbenzyl Carbamate (Msz), 9-Anthrylmethyl Carbamate, Diphenylmethyl Carbamate, 2-Methylthioethyl Carbamate, 2-Methylsulfonyl 2-(p-Toluenesulfonyl)ethylcarbamate, [2-(1,3-dithionyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphoethylcarbamate (Peoc), 2-triphenylphosphoisopropylcarbamate (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonelmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-Dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl The following are listed as carbamates: 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-l-cyclopropylmethylcarbamate, 1-methyl-1(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-l-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-tert-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, and 2,4,6-trimethylbenzylcarbamate.

[0076] Nitrogen protecting groups, such as sulfonamide groups (e.g., -S(=O)2R), aa This includes, but is not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilyl ethyl sulfonamide (SES), 9-anthracitesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzyl sulfonamide, trifluoromethyl sulfonamide, and benzoylmethyl sulfonamide.

[0077] Other nitrogen-protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldimethylsilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridinone, and N-methylamine. N-Allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptanamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl] ]amine (MMTr), N-9-phenylfluoreneamine (PhF), N-2,7-dichloro-9-fluorenemethyleneamine, N-ferroceneylmethylamino (Fcm), N-2-picoylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N' N,N'-Dimethylaminomethylene)amine, N,N'-isopropylidene diamine, N-p-nitrobenzylamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylimine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentaacylchromium or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N -Nitroamines, N-nitrosamines, N-oxides of amines, diphenylphosphamides (Dpp), dimethylthiophosphamides (Mpt), diphenylthiophosphamides (Ppt), dialkylphosphamides, dibenzylphosphamides, diphenylphosphamides, benzenesulfinamides, o-nitrobenzenesulfinamides (Nps), 2,4-dinitrobenzenesulfinamides, pentachlorobenzenesulfinamides, 2-nitro-4-methoxybenzenesulfinamides, triphenylmethylsulfinamides, and 3-nitropyridinesulfinamides (Npys).

[0078] In some embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also known 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, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0079] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyl dimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl ( THP), 3-bromotetrahydropyranyl, tetrahydrothiaranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiaranyl, 4-methoxytetrahydrothiaranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl 1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenyloxyselenoethyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picoyl, 4-picoyl, 3-methyl-2-picoyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl Di(p-methoxyphenyl)phenylmethyl, Tris(p-methoxyphenyl)methyl, 4-(4'-bromobenzoylmethyloxyphenyl)diphenylmethyl, 4,4',4”-tris(4,5-dichlorophthalimidephenyl)methyl, 4,4',4”-tris(acetylpropionyloxyphenyl)methyl, 4,4',4”-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4”-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenemethyl, 9-anthrayl, 9-(9-phenyl)xanthrayl, 9-(9-phenyl-10-oxo)anthrayl, 1,3-benzodithiofuran-2-yl, benzoisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate Triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (methyl benzoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (P sec), 2-(triphenylphosphino)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-( (Methiomethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetic acid, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid, dichlorophenylacetic acid, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyyl)benzoate, α-naphthylcarbamate, nitrates, alkyl N,N,N',N'-tetramethylphosphinodiacamide, alkyl N-phenylcarbamate, borates, dimethylphosphinothio, alkyl 2,4-dinitrophenylsulfinate, sulfates, methanesulfonates (methanesulfonates), benzylsulfonates, and toluenesulfonates (Ts).

[0080] In some embodiments, the substituents present on the sulfur atom are sulfur protecting groups (also known as thiol protecting groups). Sulfur protecting groups are well known in the art and include those nitrogen protecting groups described in detail in: Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0081] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picoyl, 2-quinolinylmethyl, 2-picoyl N-oxide, 9-anthraylmethyl, 9-fluorenylmethyl, xanthyl, ferroceneylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzocycloheptanyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, tert-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolyl, acetaminomethyl, trimethylacetaminomethyl, benzamide methyl, allyloxycarbonylaminomethyl , phenylacetamidomethyl, phthaliminomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(ethoxycarbonyl)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2 -Methylpropyl-2-yl, Acetyl, Benzoyl, Trifluoroacetyl, N-[[(p-Biphenyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, Tert-butoxycarbonyl, Benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, Sulfonate, Sulfoylthiocarbonate, 3-Nitro-2-pyridinesulfinyl sulfide, Oxazothione. The compounds of the present invention

[0082] Liposome-based mediators are considered attractive carriers for therapeutics and continue to be developed. While liposome-based mediators containing certain lipid components have shown good results in terms of encapsulation, stability, and site localization, liposome-based delivery systems still require significant improvement. For example, significant drawbacks of liposome delivery systems involve the construction of liposomes that are stable enough in cell culture or in vivo to reach the desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release their encapsulating material into such target cells.

[0083] In particular, there remains a need for improved cofactor lipids to efficiently deliver mRNA intramuscularly (e.g., for the treatment of influenza or respiratory syncytial virus (RSV)). There is also a need for improved cofactor lipid compounds that exhibit improved pharmacokinetic properties and are capable of enhancing the delivery efficiency of macromolecules (e.g., nucleic acids) to a variety of cell types and tissues. Novel cofactor lipid compounds characterized by improved safety and the ability to enhance the delivery efficiency of encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs are also in particular needed.

[0084] This article describes a novel class of cofactor lipid compounds for improving the in vivo delivery of therapeutic agents such as nucleic acids (e.g., against influenza or respiratory syncytial virus (RSV)). Specifically, the cofactor lipids described herein can be used in combination with cationic lipids and optionally other lipids to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic purposes such as disease treatment and prevention (vaccines, e.g., against influenza or respiratory syncytial virus (RSV)).

[0085] In the embodiments, the compounds of the present invention as described herein may provide one or more desired features or properties. That is, in some embodiments, the compounds of the present invention as described herein may be characterized as having one or more properties that give such compounds an advantage over other similarly classified lipids. For example, the compounds disclosed herein can be used to control and modulate the properties of liposome compositions (e.g., lipid nanoparticles) in which they are components. In particular, the compounds disclosed herein may be characterized by enhanced transfection efficiency and their ability to elicit specific biological outcomes. Such outcomes may include, for example, enhanced cellular uptake, endosome / lysosome disruption, and / or promotion of the release of encapsulating materials (e.g., polynucleotides) into cells. Lipid nanoparticles comprising one or more of the assistive lipids of the present invention may also be characterized by achieving high levels of expression of said peptides or proteins when delivered via intravenous, intrathecal, intramuscular, intranasal, sublingual, or pulmonary delivery (optionally via nebulization) of mRNA encoding said peptides or proteins. Furthermore, the compounds disclosed herein have favorable pharmacokinetic properties, biodistribution, and efficiency.

[0086] This application demonstrates that the assisting lipids of the present invention can not only be synthesized from readily available starting materials, but also that lipid nanoparticles containing one or more of the assisting lipids of the present invention have certain other advantages.

[0087] Furthermore, the assist lipids of the present invention have cleavable groups, such as ester groups. These cleavable groups (e.g., esters, thioesters, disulfides, carbonates, carbamates, and thiocarbamates) are considered to improve biodegradability, thereby contributing to the good safety of the lipids. Lipid nanoparticles comprising one or more of the assist lipids of the present invention are contemplated for efficient intramuscular delivery of therapeutic agents and vaccines (e.g., against influenza or respiratory syncytial virus (RSV)). Lipid nanoparticles comprising the assist lipids of the present invention are also contemplated for efficient escape from the body. Lipid nanoparticles comprising the assist lipids of the present invention are also contemplated for efficient in vivo delivery while maintaining good safety. Lipid nanoparticles comprising the assist lipids of the present invention are also contemplated for exhibiting beneficial degradation in vivo. The novel assist lipids of the present invention are also contemplated for providing improved stability to the compositions disclosed herein.

[0088] This document provides compounds as assistive lipids. In the embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (I): (I) Or its pharmaceutically acceptable salt, wherein: Y is selected from C, which can be substituted at will. 2- C6 alkylene or optionally substituted C 4-C6-olefinic; R a Does not exist, or R a C is arbitrarily replaced 1- C 10 Alkyl, wherein, when R a When it exists, with R a The bonded nitrogen carries a positive charge; Each R1 is independently selected from hydrogen or optionally substituted C. 1- C 10 alkyl; Each R2 is selected independently from: (i) Optional substitution of C4-C 24 Alkyl, optionally substituted C4-C 24 alkenyl, and optionally substituted C4-C 24 alkynyl group; (ii) , where each R A Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z A Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 alkenyl; (iii) , where each R B Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z B Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0089] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (II): (II) Or its pharmaceutically acceptable salt.

[0090] In embodiments, the assistive lipids of the present invention comprise compounds having the structure according to formula (IIa): (IIa) Or its pharmaceutically acceptable salt.

[0091] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (IIb): (IIb) Or its pharmaceutically acceptable salt.

[0092] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (IIc): (IIc) Or its pharmaceutically acceptable salt.

[0093] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (IId): (IId) Or its pharmaceutically acceptable salt.

[0094] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (III): (III) Or its pharmaceutically acceptable salt.

[0095] In the embodiments, Y is selected from C, which is optionally replaced. 2-6 Alkylene. In the examples, Y is an optionally substituted C2 alkylene. In the examples, Y is an optionally substituted C3 alkylene. In the examples, Y is an optionally substituted C4 alkylene. In the examples, Y is an optionally substituted C5 alkylene. In the examples, Y is an optionally substituted C6 alkylene.

[0096] In a preferred embodiment, Y is -CH2CH2-.

[0097] In the embodiments, Y is selected from C, which is optionally replaced. 4-6 Alkenyl group. In the examples, Y is an optionally substituted C4 alkenyl group. In the examples, Y is an optionally substituted C5 alkenyl group. In the examples, Y is an optionally substituted C6 alkenyl group.

[0098] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (IV): (IV) Or its pharmaceutically acceptable salt.

[0099] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (V): (V) Or its pharmaceutically acceptable salt.

[0100] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (Va): (Va) Or its pharmaceutically acceptable salt.

[0101] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (Vb): (Vb) Or its pharmaceutically acceptable salt.

[0102] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (Vc): (Vc) Or its pharmaceutically acceptable salt.

[0103] In embodiments, the assistive lipids of the present invention comprise compounds having a structure according to formula (Vd): (Vd) Or its pharmaceutically acceptable salt.

[0104] In the embodiments, the stereochemistry of the carbon atom located between R2C(=O)O-CH2- and -CH2-OP(=O)(OH)-O- is as shown in the following structure: .

[0105] In the embodiments, the stereochemistry of the carbon atom located between R2C(=O)O-CH2- and -CH2-OP(=O)(OH)-O- is as shown in the following structure: .

[0106] In one embodiment, each R2 is the same. In another embodiment, each R2 is different.

[0107] In the embodiment, R a No. In the embodiments, each R1 is independently selected from the optionally substituted C. 1-C 10 Alkyl. In the examples, each R1 is methyl. In the examples, each R1 is ethyl. In the examples, each R1 is propyl. In the examples, each R1 is butyl. In the examples, each R1 is pentyl. In the examples, R a It does not exist, and each R1 is independently selected from the optionally substituted C. 1- C 10 Alkyl group. In the examples, R a It does not exist, and each R1 is a methyl group. In the examples, R a It does not exist, and each R1 is ethyl. In the examples, R a It does not exist, and each R1 is propyl. In the embodiments, R a It does not exist, and each R1 is butyl. In the embodiment, R a It does not exist, and each R1 is pentyl.

[0108] In an embodiment, one R1 is independently selected from the optionally substituted C. 1- C 10 Alkyl group, and another R1 is independently selected from C1 groups substituted with hydroxyl groups. 1- C 10 Alkyl group. In the examples, one R1 is methyl, and the other R1 is a C1 alkyl group with a terminal hydroxyl group substituted. 1- C 10 Alkyl group. In an example, one R1 is methyl and the other R1 is hydroxymethyl. In an example, one R1 is methyl and the other R1 is hydroxyethyl, such as 2-hydroxyethyl. In an example, one R1 is methyl and the other R1 is hydroxypropyl, such as 3-hydroxypropyl. In an example, R... a No, an R1 is independently selected from the optionally substituted C. 1- C 10 Alkyl group, and another R1 is independently selected from C1 groups substituted with hydroxyl groups. 1- C 10 Alkyl group. In the examples, R a No, one R1 is a methyl group, and the other R1 is a C-terminus substituted with a hydroxyl group. 1- C 10 Alkyl group. In the examples, R a No, one R1 is methyl and the other R1 is hydroxymethyl. In the examples, R a No, one R1 is methyl and the other R1 is hydroxyethyl, such as 2-hydroxyethyl. In the examples, R a No, one R1 is methyl and the other R1 is hydroxypropyl, such as 3-hydroxypropyl.

[0109] In the embodiment, R a It exists. In the embodiment, R... a There exist, and each R1 is independently selected from the optionally substituted C. 1- C 10 Alkyl group. In the examples, R a It exists, and each R1 is a methyl group. In the examples, R... a It exists, and each R1 is ethyl. In the examples, R... a It exists, and each R1 is propyl. In the embodiments, R... a It exists, and each R1 is butyl. In the embodiment, R a It exists, and each R1 is pentyl.

[0110] In the embodiments, each R2 is independently selected from optionally substituted C4-C. 24 Alkyl, optionally substituted C4-C 24 alkenyl, and optionally substituted C4-C 24 Alkyne group. In the examples, each R2 is independently selected from optionally substituted C5-C groups. 20 Alkyl, optionally substituted C5-C 20 alkenyl groups, and optionally substituted C5-C groups 20 Alkyne group. In the examples, each R2 is independently selected from optionally substituted C6-C groups. 18 Alkyl, optionally substituted C6-C 18 alkenyl, and optionally substituted C6-C 18 Alkyne group. In the examples, each R2 is independently selected from optionally substituted C7-C groups. 15 Alkyl, optionally substituted C7-C 15 alkenyl groups, and optionally substituted C7-C groups 15 Alkyne group.

[0111] In the embodiments, each R2 is independently selected from optionally substituted C4-C. 24 Alkyl groups, such as branched C4-C 24 Alkyl group. In the examples, each R2 is independently selected from optionally substituted C5-C alkyl groups. 20 Alkyl groups, such as branched C5-C 20 Alkyl groups. In the examples, each R2 is independently selected from optionally substituted C6-C alkyl groups. 18 Alkyl groups, such as branched C6-C 18 Alkyl groups. In the examples, each R2 is independently selected from optionally substituted C7-C alkyl groups. 15 Alkyl groups, such as branched C7-C 15 alkyl.

[0112] In the embodiment, each R2 is independently selected (i) Each n is independently selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23. (ii) , (iii) , (iv) , (v) , (vi) , (vii) , (viii) , (ix) , (x) ,or (xi) .

[0113] In this embodiment, each R2 is independently selected from: (i) , (ii) ,or (iii) .

[0114] In the embodiment, each R2 is independently selected Each n is independently selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23. In an embodiment, each n is 3. In an embodiment, each n is 4. In an embodiment, each n is 5. In an embodiment, each n is 6. In an embodiment, each n is 7. In an embodiment, each n is 8. In an embodiment, each n is 9. In an embodiment, each n is 10. In an embodiment, each n is 11. In an embodiment, each n is 12. In an embodiment, each n is 13. In an embodiment, each n is 14. In an embodiment, each n is 15. In an embodiment, each n is 16. In an embodiment, each n is 17. In an embodiment, each n is 18. In an embodiment, each n is 19. In an embodiment, each n is 20. In an embodiment, each n is 21. In an embodiment, each n is 22. In the embodiment, each n is 23.

[0115] In the embodiment, each R2 is independently selected Each n is independently selected from 3 to 23. In the embodiment, each R2 is independently selected from... Each n is independently selected from 5-21. In the embodiment, each R2 is independently selected from... Each n is independently selected from 7-19. In the embodiment, each R2 is independently selected from... Each n is independently selected from 9-17. In the embodiment, each R2 is independently selected from... , where each n is independently selected from 11-15.

[0116] In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is .

[0117] In the embodiments, each R2 is independently selected from optionally substituted C4-C. 24 Alkenyl group.

[0118] In this embodiment, R2 is independently selected from: (i) (ii) , (iii) , (iv) ,or (v) .

[0119] In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is In the embodiment, each R2 is .

[0120] In the embodiment, each R2 is independently selected , where each R A Independently selected from optionally substituted C1-C 31Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and each Z in it A Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0121] In the embodiments, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, such as optionally substituted C4-C... 24 Alkyl groups, such as branched C4-C 24 Alkyl group. In some embodiments, each R A Independently selected from optionally substituted C5-C 20 Alkyl, each R A Independently selected from optionally substituted C8-C 15 alkyl.

[0122] In the embodiments, each R A Independently selected from branch C 17 Alkyl group. In the examples, each R A yes In the embodiment, each R A Independently selected from branch C 15 Alkyl group. In the examples, each R A yes In the embodiment, each R A Independently selected from branch C 13 Alkyl group. In the examples, each R A yes .

[0123] In the embodiments, each R A Selected independently from: (i) Each n is independently selected from 0, 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, and 30. (ii) , (iii) , (iv) , (v) , (vi) , (vii) , (viii) , (ix) , (x) ,or (xi) .

[0124] In the embodiments, each R A Selected independently Each n is independently selected from 0, 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, and 30. In an embodiment, each n is 0. In an embodiment, each n is 1. In an embodiment, each n is 2. In an embodiment, each n is 3. In an embodiment, each n is 4. In an embodiment, each n is 5. In an embodiment, each n is 6. In an embodiment, each n is 7. In an embodiment, each n is 8. In an embodiment, each n is 9. In an embodiment, each n is 10. In an embodiment, each n is 11. In an embodiment, each n is 12. In an embodiment, each n is 13. In an embodiment, each n is 14. In an embodiment, each n is 15. In an embodiment, each n is 16. In an embodiment, each n is 17. In this embodiment, each n is 18. In this embodiment, each n is 19. In this embodiment, each n is 20. In this embodiment, each n is 21. In this embodiment, each n is 22. In this embodiment, each n is 23. In this embodiment, each n is 24. In this embodiment, each n is 25. In this embodiment, each n is 26. In this embodiment, each n is 27. In this embodiment, each n is 28. In this embodiment, each n is 29. In this embodiment, each n is 30.

[0125] In the embodiments, each R A Selected independently Each n is independently selected from 1 to 29. In the embodiment, each R A Selected independently Each n is independently selected from 3 to 25. In the embodiment, each R A Selected independently Each n is independently selected from 5-21. In the embodiment, each R A Selected independently , where each n is independently selected from 7-19.

[0126] In the embodiments, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes .

[0127] In the embodiments, each R A Independently selected from optionally substituted C2-C 31 Alkenyl groups, such as optionally substituted C4-C groups 24 Alkenyl group.

[0128] In the embodiments, each R A Selected independently from: (i) (ii) , (iii) , (iv) ,or (v) .

[0129] In the embodiments, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes In the embodiment, each R A yes .

[0130] In the embodiment, each Z A Independently selected from optionally substituted C1-C 10 Alkylenes, such as optionally substituted C2-C7 alkylenes.

[0131] In the embodiment, each Z A Independently selected from optionally substituted C6 alkylene groups. In the examples, each Z... A Independently selected from unsubstituted C6 alkylene groups. In the examples, each Z... A Independently selected from unsubstituted straight-chain C6 alkylene groups.

[0132] In the embodiment, each Z A It is an optionally substituted C1 alkylene group. In the examples, each Z... A It is an optionally substituted C2 alkylene group. In the examples, each Z... A It is an optionally substituted C3 alkylene group. In the examples, each Z... A It is an optionally substituted C4 alkylene group. In the examples, each Z... A It is an optionally substituted C5 alkylene group. In the examples, each Z... A It is an optionally substituted C6 alkylene group. In the examples, each Z... A It is an optionally substituted C7 alkylene group. In the examples, each Z A It is an optionally substituted C8 alkylene group. In the examples, each Z... A It is an optionally substituted C9 alkylene group. In the examples, each Z... A C is arbitrarily replaced 10 Alkylene.

[0133] In the embodiment, each Z A Independently selected from optionally substituted C2-C 10 Alkenyl groups, such as optionally substituted C2-C7 alkenyl groups.

[0134] In the embodiment, each Z A It is an optionally substituted C2-olefin. In the examples, each Z... A It is an optionally substituted C3-olefin. In the examples, each Z... A It is an optionally substituted C4 alkenyl group. In the examples, each Z A It is an optionally substituted C5-olefin. In the examples, each Z A It is an optionally substituted C6-olefin. In the examples, each Z... A It is an optionally substituted C7 alkenyl group. In the examples, each Z A It is an optionally substituted C8 alkenyl group. In the examples, each Z A It is an optionally substituted C9-olefin. In the examples, each Z... A C is arbitrarily replaced 10 Alkenyl group.

[0135] In the embodiment, Y is -CH2CH2- when present, and each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A Independently selected from optionally substituted C1-C 10 Alkylene.

[0136] In the embodiments, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C1 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C2 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C3 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C4 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C5 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C6 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C7 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C8 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C9 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is C 10 Alkylene.

[0137] In the embodiments, each R AIndependently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A Independently selected from optionally substituted C2-C7 alkylene groups.

[0138] In the embodiments, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C2 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C3 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C4 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C5 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C6 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C7 alkylene group.

[0139] In the embodiments, each R A yes And each Z A It is a C7 alkylene group.

[0140] In the embodiments, each R A yes And each Z A It is a C6 alkylene group.

[0141] In the embodiments, each R A yes And each Z A It is a C2 alkylene group.

[0142] In the embodiments, each R A yes And each Z A Independently selected from C5-C7 alkylene groups, for example, each Z A It is a C6 alkylene group.

[0143] In the embodiments, each R A yes And each Z A It is a C6 alkylene group.

[0144] In the embodiment, Y is -CH2CH2- when present, and each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A Independently selected from optionally substituted C2-C 10 Alkenyl group.

[0145] In the embodiments, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C2-olefinic group. In the examples, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C3-olefinic group. In the examples, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C4 alkenyl group. In the examples, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is C5-olefinic. In the examples, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is C6-olefinic. In the examples, each R A Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z A It is a C7 subene group.

[0146] In the embodiments, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A Independently selected from optionally substituted C2-C7 alkenyl groups.

[0147] In the embodiments, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C2-olefinic group. In the examples, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C3-olefinic group. In the examples, each R AIndependently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C4 alkenyl group. In the examples, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is C5-olefinic. In the examples, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is C6-olefinic. In the examples, each R A Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z A It is a C7 subene group.

[0148] In the embodiments, each R A yes And each Z A It is a C6 subene group.

[0149] In the embodiment, Y is -CH2CH2- when present, and each R A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A Independently selected from optionally substituted C1-C 10 Alkylene.

[0150] In the embodiments, each R A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C1 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C2 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C3 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C4 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C5 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each ZA It is a C6 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C7 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C8 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is a C9 alkylene group. In the examples, each R... A Independently selected from optionally substituted C1-C 31 alkenyl, and each Z A It is C 10 Alkylene.

[0151] In the embodiments, each R A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A Independently selected from optionally substituted C2-C7 alkylene groups.

[0152] In the embodiments, each R A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C2 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C3 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C4 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C5 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C6 alkylene group. In the examples, each R... A Independently selected from optionally substituted C4-C 24 alkenyl, and each Z A It is a C7 alkylene group.

[0153] In the embodiments, each R Ayes And each Z A It is a C6 alkylene group.

[0154] In the embodiments, each R A yes And each Z A It is a C6 alkylene group.

[0155] In the embodiment, each R2 is independently selected , where each R B Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C1-C 31 alkenyl, and optionally substituted C1-C 31 alkynyl group; and each Z in it B Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0156] In the embodiments, each R B Independently selected from optionally substituted C1-C 31 Alkyl groups, such as optionally substituted C4-C... 24 Alkyl groups, such as branched C4-C 24 alkyl.

[0157] In the embodiments, each R B Independently selected from branch C 17 Alkyl group. In the examples, each R B yes In the embodiment, each R B Independently selected from branch C 15 Alkyl group. In the examples, each R B yes In the embodiment, each R B Independently selected from branch C 13 Alkyl group. In the examples, each R B yes .

[0158] In the embodiments, each R B Selected independently from: (i) Each n is independently selected from 0, 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, and 30. (ii) , (iii) , (iv) , (v) , (vi) , (vii) , (viii) , (ix) , (x) ,or (xi) .

[0159] In the embodiments, each R B Selected independently Each n is independently selected from 0, 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, and 30. In an embodiment, each n is 0. In an embodiment, each n is 1. In an embodiment, each n is 2. In an embodiment, each n is 3. In an embodiment, each n is 4. In an embodiment, each n is 5. In an embodiment, each n is 6. In an embodiment, each n is 7. In an embodiment, each n is 8. In an embodiment, each n is 9. In an embodiment, each n is 10. In an embodiment, each n is 11. In an embodiment, each n is 12. In an embodiment, each n is 13. In an embodiment, each n is 14. In an embodiment, each n is 15. In an embodiment, each n is 16. In an embodiment, each n is 17. In this embodiment, each n is 18. In this embodiment, each n is 19. In this embodiment, each n is 20. In this embodiment, each n is 21. In this embodiment, each n is 22. In this embodiment, each n is 23. In this embodiment, each n is 24. In this embodiment, each n is 25. In this embodiment, each n is 26. In this embodiment, each n is 27. In this embodiment, each n is 28. In this embodiment, each n is 29. In this embodiment, each n is 30.

[0160] In the embodiments, each R B Selected independently Each n is independently selected from 1 to 29. In the embodiment, each R B Selected independently Each n is independently selected from 3 to 25. In the embodiment, each R B Selected independently Each n is independently selected from 5-21. In the embodiment, each R B Selected independently Each n is independently selected from 7-19

[0161] In the embodiments, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes .

[0162] In the embodiments, each R B Independently selected from optionally substituted C2-C 31 Alkenyl groups, such as optionally substituted C4-C groups 24 Alkenyl group.

[0163] In the embodiments, each R B Selected independently from: (i) (ii) , (iii) , (iv) ,or (v) .

[0164] In the embodiments, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes In the embodiment, each R B yes .

[0165] In the embodiment, each Z B Independently selected from optionally substituted C1-C 10 Alkylenes, such as optionally substituted C2-C7 alkylenes.

[0166] In the embodiment, each Z B Independently selected from optionally substituted C6 alkylene groups. In the examples, each Z... B Independently selected from unsubstituted C6 alkylene groups. In the examples, each Z... B Independently selected from unsubstituted straight-chain C6 alkylene groups. In the examples, each Z... B It is an optionally substituted C1 alkylene group. In the examples, each Z... B It is an optionally substituted C2 alkylene group. In the examples, each Z... B It is an optionally substituted C3 alkylene group. In the examples, each Z... B It is an optionally substituted C4 alkylene group. In the examples, each Z... B It is an optionally substituted C5 alkylene group. In the examples, each Z... B It is an optionally substituted C6 alkylene group. In the examples, each Z... B It is an optionally substituted C7 alkylene group. In the examples, each Z B It is an optionally substituted C8 alkylene group. In the examples, each Z... B It is an optionally substituted C9 alkylene group. In the examples, each Z... B C is arbitrarily replaced 10 Alkylene.

[0167] In the embodiment, each Z B Independently selected from optionally substituted C2-C 10 Alkenyl groups, such as optionally substituted C2-C7 alkenyl groups.

[0168] In the embodiment, each Z B It is an optionally substituted C2-olefin. In the examples, each Z... B It is an optionally substituted C3-olefin. In the examples, each Z... B It is an optionally substituted C4 alkenyl group. In the examples, each Z B It is an optionally substituted C5-olefin. In the examples, each Z B It is an optionally substituted C6-olefin. In the examples, each Z... B It is an optionally substituted C7 alkenyl group. In the examples, each Z B It is an optionally substituted C8 alkenyl group. In the examples, each Z BIt is an optionally substituted C9-olefin. In the examples, each Z... B C is arbitrarily replaced 10 Alkenyl group.

[0169] In the embodiment, Y is -CH2CH2- when present, and each R B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B Independently selected from optionally substituted C1-C 10 Alkylene.

[0170] In the embodiments, each R B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C1 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C2 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C3 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C4 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C5 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C6 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C7 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C8 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each Z B It is a C9 alkylene group. In the examples, each R... B Independently selected from optionally substituted C1-C 31 Alkyl groups, and each ZB It is C 10 Alkylene.

[0171] In the embodiments, each R B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B Independently selected from optionally substituted C2-C7 alkylene groups.

[0172] In the embodiments, each R B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C2 alkylene group. In the examples, each R... B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C3 alkylene group. In the examples, each R... B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C4 alkylene group. In the examples, each R... B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C5 alkylene group. In the examples, each R... B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C6 alkylene group. In the examples, each R... B Independently selected from optionally substituted C4-C 24 Alkyl groups, and each Z B It is a C7 alkylene group.

[0173] In the embodiments, each R B Selected independently from: (i) , (ii) , (iii) , (iv) , (v) ,or (vi) .

[0174] In the embodiments, each R B yes And each Z B It is a C5 alkylene group.

[0175] In the embodiments, each R B yes And each Z B It is a C6 alkylene group.

[0176] In an embodiment, one of R2 is optionally replaced by C4-C. 24 Alkyl, optionally substituted C4-C 24 alkenyl, or optionally substituted C4-C 24 Alkyne group, and the other R2 is , where each R A Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and each Z in it A Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0177] In an embodiment, one of R2 is optionally replaced by C4-C. 24 Alkyl, optionally substituted C4-C 24 alkenyl, or optionally substituted C4-C 24 Alkyne group, and the other R2 is , where each R B Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C1-C 31 alkenyl, and optionally substituted C1-C 31 alkynyl group; and each Z in it B Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

[0178] In the embodiments, each R A They are identical when they exist. In the embodiment, each R A They are different when they exist.

[0179] In the embodiment, each Z A They are identical when they exist. In the embodiment, each Z A They are different when they exist.

[0180] In the embodiments, each R B They are identical when they exist. In the embodiment, each R B They are different when they exist.

[0181] In the embodiment, each Z B They are identical when they exist. In the embodiment, each Z B They are different when they exist.

[0182] In the embodiments, each R A When present, it is independently selected from straight-chain C1-C. 31 Alkyl group. In the examples, each R A When present, it is independently selected from branches C1-C. 31 Alkyl group. In the examples, each R A When present, it is independently selected from linear C4-C. 24 Alkyl group. In the examples, each R A When present, it is independently selected from branch C4-C. 24 Alkyl group. In the examples, an R A It is a straight-chain C1-C 31 alkyl, and another R A It is a branch C1-C 31 Alkyl group. In the examples, an R A It is a straight-chain C4-C 24 alkyl, and another R A It is a C4-C branch. 24 alkyl.

[0183] In an embodiment, an R A yes And another R A It is C1-C 31 Alkyl group. In the examples, an R A yes And another R A It is C4-C 24 Alkyl group. In the examples, an R A yes And another R A It is a C8 alkyl group.

[0184] In the embodiments, each R B When present, it is independently selected from straight-chain C1-C. 31 Alkyl group. In the examples, each R B When present, it is independently selected from branches C1-C. 31 Alkyl group. In the examples, each R B When present, it is independently selected from linear C4-C. 24 Alkyl group. In the examples, each R B When present, it is independently selected from branch C4-C. 24 alkyl.

[0185] In an embodiment, an R A It is a straight-chain C1-C 31 alkyl, and another R A It is a branch C1-C 31 Alkyl group. In the examples, an R A It is a straight-chain C4-C 24 alkyl, and another R A It is a C4-C branch. 24 alkyl.

[0186] In the embodiments, the assistive lipids of the present invention have any of the structures in Table A, or pharmaceutically acceptable salts thereof.

[0187] In the embodiments, the assisting lipid is .

[0188] In the embodiments, the assisting lipid is .

[0189] In the embodiments, the assisting lipid is .

[0190] In the embodiments, the assisting lipid is .

[0191] In the embodiments, the assisting lipid is .

[0192] In the embodiments, the assisting lipid is .

[0193] In the embodiments, the assisting lipid is .

[0194] In the embodiments, the assisting lipid is .

[0195] In the embodiments, the assisting lipid is .

[0196] In the embodiments, this document provides compositions comprising one or more auxiliary lipids of the present invention or pharmaceutically acceptable salts thereof, and further comprising: (i) One or more cationic lipids, (ii) one or more sterol-based lipids, and (iii) One or more PEG-modified lipids.

[0197] In the embodiments, the one or more sterol-based lipids are cholesterol-based lipids, such as cholesterol.

[0198] In the embodiments, the composition is lipid nanoparticles, optionally liposomes. In the embodiments, the one or more cationic lipids account for about 20 mol% to about 60 mol% of the lipid nanoparticles. In the embodiments, the one or more lipids of the present invention account for about 10 mol% to about 50 mol% of the lipid nanoparticles. In the embodiments, the one or more PEG-modified lipids account for about 1 mol% to about 4 mol% of the lipid nanoparticles. In the embodiments, the one or more sterol-based lipids account for about 10 mol% to about 50 mol% of the lipid nanoparticles.

[0199] In embodiments, lipid nanoparticles encapsulate nucleic acids, optionally encoding mRNA of peptides or proteins. In embodiments, lipid nanoparticles encapsulate mRNA encoding peptides or proteins, optionally for use in vaccines. In embodiments, the peptide is an antigen. As used herein, the phrase "encapsulation percentage" refers to the portion of a therapeutic agent (e.g., mRNA) effectively encapsulated within a liposome-based medium (e.g., lipid nanoparticles) relative to the initial portion of the therapeutic agent present in the lipid phase. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 50%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 55%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 60%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 65%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 70%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 75%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 80%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 85%. In one embodiment, the encapsulation percentage of the lipid nanoparticles on the mRNA was at least 90%. In another embodiment, the encapsulation percentage of the lipid nanoparticles on the mRNA was at least 95%. In yet another embodiment, the encapsulation percentage was calculated by Ribogreen assay (Invitrogen) with and without 0.1% Triton-X 100.

[0200] In the embodiments, the compositions of the present invention are used in therapeutic applications.

[0201] In the embodiments, the compositions of the present invention are used in a method of treating or preventing a disease that can be treated or prevented by a peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen, and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0202] In an embodiment, a method for treating or preventing a disease is provided, wherein the method comprises administering the composition of the invention to a subject in need, and wherein the disease is treatable or preventable by a peptide or protein encoded by mRNA, optionally wherein the mRNA encodes an antigen, and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0203] In the embodiments, the composition is administered intravenously, intrathecally, or intramuscularly, or via pulmonary delivery (optionally via nebulization). In the embodiments, the composition is administered intramuscularly. Exemplary compounds

[0204] In the embodiments, the auxiliary lipids of the present invention comprise compounds selected from those shown in Table A or pharmaceutically acceptable salts thereof.

[0205] Exemplary compounds include those described in Table A, or pharmaceutically acceptable salts thereof. Table A

[0206] Any of the compounds (1-65) identified in Table A above may be provided in the form of pharmaceutically acceptable salts, and such salts are intended to be covered in this invention.

[0207] The compounds of the present invention as described herein can be prepared according to methods known in the art, including exemplary synthesis of the examples provided herein. Nucleic acid

[0208] The compounds of the present invention, as described herein, can be used to prepare compositions for the delivery of nucleic acids. Nucleic acid synthesis

[0209] The nucleic acids according to the invention can be synthesized according to any known method. For example, the mRNA according to the invention can be synthesized via in vitro transcription (IVT). In short, IVT is typically performed using: a linear or circular DNA template containing a promoter, a ribonucleotide triphosphate pool, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, mutant T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0210] In some embodiments, to prepare the mRNA according to the invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription (e.g., T3, T7, mutated T7, or SP6 promoter), followed by the desired nucleotide sequence for the desired mRNA and a termination signal.

[0211] The desired mRNA sequence according to the invention can be determined using standard methods and incorporated into a DNA template. For example, virtual reverse translation can be performed based on a degenerate genetic code, starting with the desired amino acid sequence (e.g., an enzyme sequence). An optimization algorithm can then be used to select appropriate codons. Typically, the G / C ratio can be optimized to achieve the highest possible G / C ratio, while also considering the frequency of tRNA based on codon usage as much as possible. The optimized RNA sequence can be constructed and displayed, for example, using a suitable display device and compared with the original (wild-type) sequence. Secondary structures can also be analyzed to calculate stable and unstable properties, or to calculate RNA regions separately. Modified mRNA

[0212] In some embodiments, the mRNA according to the invention can be synthesized as unmodified or modified mRNA. Modified mRNA includes nucleotide modifications in the RNA. Therefore, modified mRNA according to the invention can include nucleotide modifications, such as backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotides, purine and pyrimidine analogs or derivatives, such as, for example, 1-methyladenine, 2-methyladenine, 2-methylthio-N-6-isopentene. N-6-methyladenine, N-6-isopentenyladenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil), dihydrouracil, 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-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio- Uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, β-D-mannosyl-queuosine, wybutoxosine, and phosphoramide, thiophosphate, peptide nucleotide, methylphosphonate, 7-deazoguanosine, 5-methylcytosine, and inosine. Those skilled in the art will know the preparation of such analogues, for example, from the following documents: U.S. Patent Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the disclosures of which are incorporated herein by reference in their entirety. Pharmaceutical formulations comprising the compounds of the present invention

[0213] In some embodiments, pharmaceutical compositions and liposome compositions comprising compounds of the present invention as described herein may be used in formulations to facilitate the delivery of encapsulating materials (e.g., one or more polynucleotides, such as mRNA) to one or more target cells, and subsequently to transfect the one or more target cells.

[0214] According to the present invention, nucleic acids, such as mRNA encoding proteins as described herein (e.g., full-length, fragment, or portion of a protein), can be delivered via a delivery medium comprising compounds of the present invention as described herein.

[0215] As used herein, the terms “delivery medium,” “transfer medium,” “nanoparticle,” or their grammatical equivalents are used interchangeably.

[0216] For example, the present invention provides compositions comprising one or more compounds described herein and one or more polynucleotides (e.g., pharmaceutical compositions). The compositions (e.g., pharmaceutical compositions) may further comprise... (i) one or more cationic lipids, (ii) one or more non-cationic lipids, (iii) one or more sterol-based lipids, and / or (iv) One or more PEG-modified lipids.

[0217] In some embodiments, the composition exhibits an enhanced (e.g., increased) ability to transfect one or more target cells. Therefore, methods for transfecting one or more target cells are also provided herein. Such methods generally include the steps of contacting the one or more target cells with a pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising a compound described herein (encapsulating one or more polynucleotides), such that the one or more target cells are transfected with a material therein encapsulated (e.g., one or more polynucleotides). As used herein, the term “transfect” refers to the introduction of one or more encapsulating materials (e.g., nucleic acids and / or polynucleotides) into cells (e.g., into target cells). The introduced polynucleotides may be stably or temporarily maintained in the target cells. The term “transfection efficiency” refers to the relative amount of such encapsulating material (e.g., polynucleotides) that is absorbed, introduced, and / or expressed by the transfected target cells. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by the target cells after transfection. In some embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby increasing the possibility of delivering an appropriate dose of encapsulating material (e.g., one or more polynucleotides) to a pathological site and subsequently expressing it, while minimizing potential systemic adverse reactions or toxicities associated with the compound or its encapsulated contents.

[0218] For example, transfection of one or more target cells with a polynucleotide encapsulated in one or more lipid nanoparticles containing a pharmaceutical or liposome composition disclosed herein can stimulate the production of a product (e.g., a polypeptide or protein) encoded by such a polynucleotide and enhance the ability of such target cells to express the polynucleotide and produce, for example, the target polypeptide or protein. For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating mRNA will enhance (i.e. increase) the production of proteins or enzymes encoded by such mRNA.

[0219] Furthermore, delivery mediators described herein (e.g., liposome delivery mediators) can be prepared for preferential distribution to other target tissues, cells, or organs, such as the heart, lungs, kidneys, and spleen. In embodiments, delivery mediators described herein (e.g., liposome delivery mediators) can be prepared for preferential distribution to the lungs. In embodiments, lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, polynucleotides (e.g., mRNA) encapsulated in one or more pharmaceutical compositions and liposome compositions described herein can be delivered to and / or transfected into target cells or tissues. In some embodiments, the encapsulated polynucleotides (e.g., mRNA) are capable of being expressed by target cells and producing (and in some cases excreted) functional polypeptide products, thereby conferring, for example, beneficial properties to the target cells or tissues. Such encapsulated polynucleotides (e.g., mRNA) may encode, for example, antigens, hormones, enzymes, receptors, polypeptides, peptides, or other target proteins. Liposome delivery mediators

[0220] In some embodiments, the composition is a suitable delivery medium. In examples, the composition is a liposome delivery medium, such as lipid nanoparticles.

[0221] The terms “liposome delivery medium” and “liposome composition” are used interchangeably.

[0222] Enriching liposome compositions using one or more of the auxiliary lipids disclosed herein can be used as a means to improve safety or otherwise confer one or more desired properties upon such enriched liposome compositions (e.g., improved stability, improved endosome escape, improved delivery of encapsulated polynucleotides to one or more target cells, and / or reduced in vivo toxicity of the liposome composition). Therefore, pharmaceutical compositions comprising one or more of the lipids disclosed herein, particularly liposome compositions, are also contemplated.

[0223] Therefore, in some embodiments, the compounds of the present invention as described herein can be used as components of liposome compositions to facilitate or enhance the delivery and release of encapsulating materials (e.g., one or more therapeutic agents) into one or more target cells (e.g., by permeation or fusion with the lipid membrane of such target cells).

[0224] As used herein, liposome delivery mediators (e.g., lipid nanoparticles) are typically characterized as microvesicles with an internal aqueous space isolated from an external medium by a membrane having one or more bilayers. The bilayer membrane of a liposome is typically formed from amphiphilic molecules, such as synthetic or naturally derived lipids containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol. 16: 307-321, 1998). The bilayer membrane of a liposome can also be formed from amphiphilic polymers and surfactants (e.g., polymers, nonionic surfactant vesicles, etc.). In the context of this invention, liposome delivery mediators are typically used to transport desired mRNA to target cells or tissues.

[0225] In some embodiments, such compositions (e.g., liposome compositions) are loaded with or otherwise encapsulated with materials such as one or more bioactive polynucleotides (e.g., mRNA).

[0226] In examples, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a peptide or protein encapsulated within liposomes. In examples, the liposomes comprise: (i) one or more cationic lipids, (ii) one or more non-cationic lipids, (iii) one or more sterol-based lipids, and (iv) One or more PEG-modified lipids, At least one of the lipids is a compound of the present invention as described herein.

[0227] In some embodiments, the composition comprises mRNA encoding a peptide or protein (e.g., any peptide or protein described herein). In some embodiments, the composition comprises mRNA encoding a peptide (e.g., any peptide described herein). In some embodiments, the composition comprises mRNA encoding a protein (e.g., any protein described herein).

[0228] In the embodiments, the composition (e.g., a pharmaceutical composition) comprises nucleic acids encapsulated in liposomes, wherein the liposomes contain compounds described herein.

[0229] In some embodiments, the nucleic acid is mRNA encoding a peptide or protein. In some embodiments, the mRNA encodes a peptide or protein for use in the lungs or lung cells of a subject, delivered to or treating the subject. In some embodiments, the mRNA encodes a peptide or protein for use in the liver or hepatocytes of a subject, delivered to or treating the subject. In some embodiments, the mRNA encodes a peptide or protein for use in muscle cells, delivered to or treating the subject. In some embodiments, the mRNA encodes a peptide or protein for use in immune cells, delivered to or treating the subject. Other exemplary mRNAs are also described herein.

[0230] In the embodiments, the liposome delivery medium (e.g., lipid nanoparticles) may have a net positive charge.

[0231] In the embodiments, the liposome delivery medium (e.g., lipid nanoparticles) may have a net negative charge.

[0232] In the embodiments, the liposome delivery medium (e.g., lipid nanoparticles) may have a net neutral charge.

[0233] In the embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein.

[0234] The amount of the compounds of the present invention in the composition as described herein can also be described as a percentage (“mol%”) of the combined molar amount of total lipids in the composition (e.g., the combined molar amount of all lipids present in a liposome delivery medium (e.g., lipid nanoparticles).

[0235] In the embodiments of the pharmaceutical compositions described herein, the amount of compounds of the invention as described herein is about 5 mol% to about 60 mol% of the combined molar amount of all lipids present in the composition (e.g., liposome delivery medium, such as lipid nanoparticles), for example about 10 mol% to about 50 mol%.

[0236] In the embodiments, the compounds of the present invention as described herein are present in amounts of about 5 mol% to about 15 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol%, or about 45 mol% to about 60 mol%.

[0237] In the embodiments, the compounds of the present invention as described herein are present in amounts of about 5 mol% to about 60 mol%, 5 mol% to about 50 mol%, 5 mol% to about 40 mol%, 5 mol% to about 30 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol%, or about 5 mol% to about 25 mol%.

[0238] In some embodiments, the compounds of the present invention as described herein may comprise about 10 mol% to about 50 mol% of the total lipids in the composition (e.g., a liposome delivery medium, such as lipid nanoparticles), or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol%, or from about 35 mol% to about 50 mol%, or from about 40 mol% to about 50 mol%, or from about 45 mol% to about 50 mol%.

[0239] In some embodiments, the compounds of the present invention as described herein may constitute more than about 5 mol%, or more than about 10 mol%, or more than about 15 mol%, or more than about 20 mol%, or more than about 25 mol%, or more than about 30 mol%, or more than about 35 mol%, or more than about 40 mol% of the total lipids in the lipid nanoparticles.

[0240] In some embodiments, the described compound may comprise less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, or less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol% of the total lipids in the composition (e.g., a liposome delivery medium, such as lipid nanoparticles).

[0241] In the embodiments, the compounds of the present invention as described herein are present in amounts of about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the combined molar amount of total lipids in the composition (e.g., liposome composition, such as lipid nanoparticles).

[0242] In the embodiments, the compounds of the present invention as described herein are present in amounts not exceeding about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol of the total molar amount of lipids in the composition (e.g., liposome composition, such as lipid nanoparticles).

[0243] In the embodiments, this percentage results in improved beneficial effects (e.g., improved delivery to target tissues such as the liver, lungs, or muscles).

[0244] In typical embodiments, the compositions of the present invention (e.g., liposome compositions) comprise: (i) one or more cationic lipids, (ii) one or more non-cationic lipids, (iii) one or more sterol-based lipids, and (iv) One or more PEG-modified lipids, At least one of the lipids is a compound of the present invention as described herein.

[0245] For example, compositions suitable for practicing the present invention have four lipid components, comprising compounds of the present invention as described herein, and further comprising: (i) cationic lipids; (ii) Sterol-based lipids; and (iii) PEG-modified lipids.

[0246] Non-cationic lipids can be the auxiliary lipids of the present invention. Cationic lipids suitable for the compositions of the present invention are well known in the art. For example, the cationic lipid can be OF-02 ((3R,6S)-3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecyl-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione) (Fenton et al., doi: 10.1002 / adma.201505822). For example, the cationic lipid can be cKK-E10 ((3R,6S)-3,6-bis(4-(bis(2-hydroxydecyl)amino)butyl)piperazine-2,5-dione) (Dong et al., doi: 10.1073 / pnas.1322937111). For example, the cationic lipid can be 2-(4-(2-((4-(bis(2-hydroxydecyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butyrate). For example, the cationic lipid can be SM-102. For example, the cationic lipid can be ALC-0315. For example, the cationic lipid can be D-Lin-MC3-DMA. For example, the cationic lipid can be DOTMA (dioleoyl-3-trimethylammonium propane) or DOTAP (1,2-dioleoyl-3-trimethylammonium propane).

[0247] Sterol-based lipids can be cholesterol. PEG-modified lipids can be DMG-PEG2K.

[0248] In another embodiment, the pharmaceutical (e.g., liposome) composition comprises one or more of cationic lipids, PEG-modified lipids, non-cationic lipids, and sterol lipids, wherein at least one lipid present in the composition is an auxiliary lipid of the present invention.

[0249] In other embodiments, such pharmaceutical (e.g., liposome) compositions comprise: one or more cationic lipids; one or more PEG-modified lipids; one or more non-cationic lipids; and one or more sterol lipids, wherein at least one lipid present in the composition is an auxiliary lipid of the present invention.

[0250] In embodiments, compositions (e.g., lipid nanoparticles) encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein, one or more cationic lipids, and one or more lipids selected from the group consisting of non-cationic lipids and PEGylated lipids.

[0251] In embodiments, compositions encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) (e.g., lipid nanoparticles) comprise one or more compounds of the invention as described herein; one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids; and further comprise sterol-based lipids. Typically, such compositions have four lipid components comprising compounds of the invention as described herein, cationic lipids, and further comprising: (i) Sterol-based lipids (e.g., cholesterol); and (ii) PEG-modified lipids (e.g., DMG-PEG2K).

[0252] In embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein, and one or more lipids selected from the group consisting of: (i) cationic lipids, (ii) Non-cationic lipids, (iii) PEGylated lipids, and (iv) Sterol-based lipids.

[0253] In the examples, the cationic lipid is any cationic lipid disclosed in the literature. In the examples, the cationic lipid is (3R,6S)-3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecyl-9,12-dien-1-yl)amino)piperazine-2,5-dione. In the examples, the cationic lipid is (3R,6S)-3,6-bis(4-(bis(2-hydroxydecyl)amino)butyl)piperazine-2,5-dione. In the examples, the cationic lipid is 2-(4-(2-((4-(bis(2-hydroxydecyl)amino)butyl)dithioalkyl)ethyl)piperazine-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butyrate).

[0254] According to several embodiments, the following are selected based on the characteristics of the selected one or more lipids, the nature of the intended target cells, the characteristics of the mRNA to be delivered, the lipids of the present invention, cationic lipids and / or PEG-modified lipids (constituting lipid nanoparticles), and the relative molar ratios of such lipids to each other. Other considerations include, for example, the saturation of the alkyl chain and the size, charge, pH, pKa, fusion properties, and toxicity of the selected one or more lipids. Therefore, the molar ratios can be adjusted accordingly. cationic lipids

[0255] The compositions described herein, such as lipid nanoparticles, may contain one or more cationic lipids.

[0256] In some embodiments, liposomes may comprise one or more cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a variety of lipid substances that have a net positive charge at a selected pH (such as physiological pH). Several cationic lipids have been described in the literature, many of which are commercially available.

[0257] Cationic lipids suitable for the composition include those described in the literature. For example, the cationic lipid may be (3R,6S)-3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadecyl-9,12-dien-1-yl)amino)piperazine-2,5-dione. For example, the cationic lipid may be (3R,6S)-3,6-bis(4-(bis(2-hydroxydecyl)amino)butyl)piperazine-2,5-dione. For example, the cationic lipid may be 2-(4-(2-((4-(bis(2-hydroxydecyl)amino)butyl)dithioalkyl)ethyl)piperazine-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butyrate). For example, the cationic lipid may be SM-102. For example, the cationic lipid may be ALC-0315. For example, the cationic lipid may be D-Lin-MC3-DMA. For example, cationic lipids can be DOTMA (dioleoyl-3-trimethylammonium propane) or DOTAP (1,2-dioleoyl-3-trimethylammonium propane). non-cationic lipids

[0258] Compositions (e.g., liposome compositions) may also comprise one or more noncationic lipids. As used herein, the phrase "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid substances that carry a net negative charge at a selected pH (e.g., physiological pH). Noncationic lipids include, but are not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine. The amines include POPE, dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof. The non-cationic lipid or co-lipid suitable for practicing the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-disorcinyl-sn-glycerol-3-phosphoethanolamine (DEPE) may be used as a non-cationic lipid or co-lipid.

[0259] In some embodiments, the non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge under the conditions of formulation and / or application of the composition.

[0260] In some embodiments, non-cationic lipids may be present in the following molar ratios (mol%): about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, total non-cationic lipids may be present in the following molar ratios (mol%): about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40%. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipids in liposomes may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in liposomes may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. Sterol-based lipids

[0261] In some embodiments, the composition comprising the auxiliary lipid of the present invention (e.g., a liposome composition) further comprises one or more sterol-based lipids. Suitable sterol-based lipids are known in the literature, for example. Sterol-based lipids may be cholesterol-based lipids, such as cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleoaminopropyl)piperazine (Gao et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent No. 5,744,335), β-sitosterol, or imidazole cholesterol ester (ICE) having the following structure: ("ICE").

[0262] In some embodiments, sterol-based lipids may be present in the following molar ratios (mol%): about 1% to about 30% of the total lipids present in the liposomes, or about 5% to about 20%. In some embodiments, sterol-based lipids may be present in the following molar ratios (mol%): about 10% to about 50% of the total lipids present in the liposomes, or about 40% to about 50%. In some embodiments, the percentage of sterol-based lipids in the lipid nanoparticles may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of sterol-based lipids in the lipid nanoparticles may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. PEGylated lipids

[0263] In some embodiments, the composition (e.g., a liposome composition) comprises one or more additional PEGylated lipids. A suitable PEG-modified lipid or PEGylated lipid for practicing the present invention is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0264] For example, the present invention also contemplates the use of polyethylene glycol (PEG) modified phospholipids and derived lipids (e.g., derived ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide)) in combination with one or more of the compounds of the present invention as described herein, and in some embodiments, in combination with other lipids that co-constitute the liposome. In some embodiments, particularly useful exchangeable lipids are those having shorter acyl chains (e.g., (C8 PEG-2000 ceramide)). 14 ) or (C 18 PEG-ceramide.

[0265] Other PEG-modified lipids under consideration (also referred to herein as PEGylated lipids, the term being interchangeable with PEG-modified lipids) include, but are not limited to, those having (C6-C) 20The lipid is a polyethylene glycol chain up to 5 kDa covalently attached to one or more alkyl chains of length 5 kDa. In some embodiments, the PEG-modified lipid or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components can prevent complex aggregation and can also provide a means of increasing cycle life and enhancing the delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters [European Federation of Biochemical Societies Letters], 268 (1): 235-237), or they can be selected for rapid in vivo exchange of formulations (see U.S. Patent No. 5,885,613).

[0266] The additional PEG-modified phospholipids and derived lipids of the present invention may be present in the following molar ratios (mol%): about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, about 3% to about 5%, about 1% to about 5%, or about 1.5% to about 3% of the total lipids present in the composition (e.g., liposome composition). Pharmaceutical preparations and therapeutic uses

[0267] The compounds of the present invention as described herein can be used to prepare compositions (e.g., to construct liposome compositions) that facilitate or enhance the delivery and release of encapsulating materials (e.g., one or more therapeutic polynucleotides) into one or more target cells (e.g., by permeation or fusion with the lipid membrane of such target cells).

[0268] For example, when a liposome composition (e.g., lipid nanoparticles) contains or otherwise enriches one or more compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate the delivery of encapsulating materials (e.g., one or more therapeutic polynucleotides encapsulated in lipid nanoparticles) to the one or more target cells.

[0269] Similarly, in some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by their reduced in vivo toxicity. In some embodiments, the reduced toxicity is a function of the high transfection efficiency associated with the compositions disclosed herein, making it possible to administer reduced amounts of such compositions to subjects to achieve the desired therapeutic response or outcome.

[0270] In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by effective intranasal delivery of mRNA. In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by effective pulmonary delivery of mRNA. In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by achieving high levels of expression of said peptide or protein when delivered via intravenous, intrathecal, intramuscular, intranasal, sublingual, or pulmonary delivery (optionally via nebulization). In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomal mediators characterized by achieving high levels of expression of said peptide or protein when delivered via intramuscular delivery of mRNA.

[0271] Therefore, pharmaceutical formulations comprising the compounds described herein and the provided nucleic acids can be used for a variety of therapeutic and / or preventative purposes. To facilitate the delivery of nucleic acids in vivo, the compounds and nucleic acids described herein can be formulated in combination with one or more additional drug carriers, targeting ligands, or stabilizers. In some embodiments, the compounds described herein can be formulated via a premixed lipid solution. In other embodiments, a post-insertion technique can be used to formulate compositions comprising the compounds described herein into lipid membranes of nanoparticles. Pharmaceutical formulation and administration techniques can be found in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania.

[0272] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary (including tracheal or inhalation), or enteral administration; parenteral delivery, including intradermal, transdermal (local), intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intravenous, intravenous, intraperitoneal, or intranasal administration. In embodiments, the route of administration is selected from intravenous, intrathecal, intramuscular, intranasal, sublingual, or pulmonary delivery (optionally via nebulization). In embodiments, the route of administration is intramuscular. In certain embodiments, intramuscular administration is directed to muscles selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, administration results in the delivery of nucleic acids to muscle cells. In some embodiments, administration results in the delivery of nucleic acids to hepatocytes (i.e., liver cells).

[0273] A common route of administration for the liposome compositions of the present invention is intravenous delivery, particularly in the treatment of metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamate (OTC) deficiency). Alternatively, depending on the disease or disorder to be treated, the liposome compositions may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposome compositions of the present invention are typically administered intramuscularly. Alternatively, vaccination may be performed via intranasal administration. Diseases or disorders affecting the eyes may be treated by intravitreal administration of the liposome compositions of the present invention.

[0274] Alternatively or additionally, the pharmaceutical formulations of the present invention can be administered locally rather than systemically, for example, by injecting the pharmaceutical formulation directly into a target tissue (e.g., in a sustained-release formulation). Depending on the tissue to be targeted, local delivery can be achieved in various ways. Exemplary tissues that can be delivered and / or express mRNA include, but are not limited to, the liver, kidneys, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In an example, the tissue to be targeted is the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); for example, the composition of the present invention can be injected into the site of injury, disease manifestation, or pain; the composition can be provided in the form of lozenges for oral, tracheal, or esophageal application; can be provided in the form of liquids, tablets, or capsules for gastric or intestinal administration; can be provided in the form of suppositories for rectal or vaginal application; or can even be delivered to the eyes by using creams, drops, or even injections.

[0275] Alternatively or additionally, the pharmaceutical formulations of the present invention can be administered intranasally. For example, the pharmaceutical formulations of the present invention can be administered via nasal spray. Exemplary tissues that can deliver and / or express mRNA include, but are not limited to, the lungs, heart, liver, spleen, and muscle. In one embodiment, the tissue to be targeted is in the lungs. In another embodiment, the tissue to be targeted is in muscle.

[0276] Alternatively or additionally, the pharmaceutical formulations of the present invention can be administered via pulmonary delivery (optionally via nebulization or dry powder inhalation). In embodiments, the pharmaceutical formulations of the present invention are administered via pulmonary delivery (via nebulization). In embodiments, the pharmaceutical formulations of the present invention are administered via pulmonary delivery (via dry powder inhalation). Exemplary tissues that can deliver and / or express mRNA include, but are not limited to, the lungs, heart, liver, spleen, and muscle. In embodiments, the tissue to be targeted is in the lungs. In embodiments, the tissue to be targeted is in muscle.

[0277] The compositions described herein may contain mRNA encoding peptides, including peptides (e.g., polypeptides, such as proteins) described herein.

[0278] In this embodiment, the mRNA encodes a polypeptide. In this embodiment, the mRNA encodes a peptide. In this embodiment, the peptide is an antigen. In this embodiment, the mRNA encodes a peptide for treating influenza. In this embodiment, the mRNA encodes a peptide for treating respiratory syncytial virus (RSV).

[0279] In this embodiment, the mRNA encodes a protein. In this embodiment, the mRNA encodes a protein used to treat influenza. In this embodiment, the mRNA encodes a protein used to treat respiratory syncytial virus (RSV).

[0280] The present invention provides a method for delivering a composition having a full-length mRNA molecule encoding a target peptide or protein for use in a treatment subject, such as a human subject, or in the cells of a human subject, or in the cells of a human subject that have been treated and delivered thereto. delivery method

[0281] The delivery routes used in the methods of this invention allow for non-invasive self-administration of the compounds of this invention. In some embodiments, the method involves administering a composition containing mRNA encoding a therapeutic peptide or protein (in a suitable transfection or lipocarrier medium as described above) via aerosolization, nebulization, or instillation into the nose, trachea, or lungs. In some embodiments, the method involves administering a composition containing mRNA encoding a therapeutic peptide or protein (in a suitable transfection or lipocarrier medium as described above) via intravenous, intrathecal, intramuscular, intranasal, sublingual, or pulmonary delivery (optionally via nebulization). In some embodiments, the peptide or protein is encapsulated in liposomes. In some embodiments, the liposomes contain lipids that are compounds of this invention. As used below, administration of the compounds of this invention includes administration of a composition containing the compounds of this invention.

[0282] Although local cells and tissues of the lung represent potential targets that can be used as biological reservoirs or repositories for the production and secretion of mRNA-encoded proteins, the applicant has found that administering the compounds of the present invention to the lungs via aerosolization, nebulization, or infusion results in the distribution of even non-secreting proteins outside the lung cells. Without wishing to be bound by any particular theory, the nanoparticle compositions of the present invention are contemplated to cross the lung airway-blood barrier, resulting in the translocation of intact nanoparticles to non-lung cells and tissues, such as the heart, liver, and spleen, in which encoded peptides or proteins are generated. Therefore, the uses of the compounds and methods of the present invention are not limited to the generation of therapeutic proteins in lung cells and lung tissues, but can also be used for delivery to non-lung target cells and / or tissues. They are useful for the management and treatment of a variety of diseases. In some embodiments, the compounds of the present invention used in the methods of the present invention result in the distribution of mRNA-encapsulated nanoparticles in the liver, spleen, heart, and / or other non-lung cells, and the generation of encoded peptides or proteins. For example, administering the compounds of the present invention to the lungs via aerosolization, nebulization, or infusion will result in the composition itself and its peptide or protein products (e.g., antigens or functional proteins) being detectable in local cells and tissues of the lungs, as well as in peripheral target cells, tissues, and organs (due to the translocation of mRNA and delivery mediators to non-lung cells).

[0283] In some embodiments, the compounds of the present invention can be used in the methods of the present invention to specifically target peripheral cells or tissues. Following pulmonary delivery, the compounds of the present invention are contemplated to cross the pulmonary airway-blood barrier and distribute to cells other than local lung cells. Therefore, the compounds disclosed herein can be administered to a subject via pulmonary administration (using various methods known to those skilled in the art, e.g., by inhalation) and distributed to local target cells and tissues of the lungs, as well as peripheral non-lung cells and tissues (e.g., cells of the liver, spleen, kidneys, heart, skeletal muscle, lymph nodes, brain, cerebrospinal fluid, and plasma). Thus, both local lung cells and peripheral non-lung cells can serve as bioreservoir or reservoir capable of producing and / or secreting translational products encoded by one or more polynucleotides. Therefore, the present invention is not limited to treating lung diseases or conditions, but can be used as a non-invasive means to facilitate the delivery of polynucleotides or the production of peptides or proteins encoded therefrom in peripheral organs, tissues, and cells (e.g., hepatocytes) that would otherwise only be achieved through systemic administration. Exemplary peripheral non-lung cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, osteocytes, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, β cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0284] Following administration of the composition to a subject, at least one to seven days or longer after administration of the compound, a peptide or protein product (e.g., a functional protein or enzyme) encoded by mRNA can be detected in the peripheral target tissue. The amount of peptide or protein product required to achieve a therapeutic effect will vary depending on the condition being treated, the encoded peptide or protein, and the patient's condition. For example, 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 longer after administration of the compound to the subject, a peptide or protein product can be detected in peripheral target tissue at a concentration (e.g., therapeutic concentration) of at least 0.025-1.5 µg / ml (e.g., at least 0.050 µg / ml, at least 0.075 µg / ml, at least 0.1 µg / ml, at least 0.2 µg / ml, at least 0.3 µg / ml, at least 0.4 µg / ml, at least 0.5 µg / ml, at least 0.6 µg / ml, at least 0.7 µg / ml, at least 0.8 µg / ml, at least 0.9 µg / ml). µg / ml, at least 1.0 µg / ml, at least 1.1 µg / ml, at least 1.2 µg / ml, at least 1.3 µg / ml, at least 1.4 µg / ml, or at least 1.5 µg / ml).

[0285] It has been demonstrated that nucleic acids can be delivered to the lungs via intratracheal administration of a liquid suspension of the compound, as well as via inhaled liquid nebulizers or aerosol mists generated using dry powder devices (such as those described in U.S. Patent 5,780,014, which is incorporated herein by reference).

[0286] In some embodiments, the compounds of the present invention may be formulated such that they can be atomized or otherwise delivered as particulate liquids or solids before or during administration to a subject. Such compounds may be administered by means of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., atomized aqueous solutions or suspensions) to produce particles readily inhaled or inhaled by the subject. In some embodiments, such devices (e.g., metered-dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers, or blowpipes) facilitate the administration of a predetermined mass, volume, or dose of the composition to a subject (e.g., about 0.5 mg / kg mRNA per dose). For example, in some embodiments, the compounds of the present invention are administered to a subject using a metered-dose inhaler containing a suspension or solution comprising the compound and a suitable propellant. In some embodiments, the compounds of the present invention may be formulated as particulate powders for inhalation (e.g., inhalable dry particles). In some embodiments, the compositions of the present invention formulated into inhalable particles have 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 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or smaller).

[0287] In other embodiments, the compositions of the present invention are formulated to contain one or more lung surfactants (e.g., lamellar bodies). In some embodiments, the compositions of the present invention are administered to a subject such that the concentration administered in a single dose is at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg. mg / kg body weight. In some embodiments, the compositions of the present invention are administered to a subject such that the total amount administered in one or more doses is at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg mRNA. Example

[0288] While certain compounds, compositions, and methods of the present invention have been specifically described according to certain embodiments, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention. List of abbreviations: DMAP: 4-Dimethylaminopyridine DMF: Dimethylformamide EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: Ethyl acetate LDA: Lithium diisopropylamino THF: Tetrahydrofuran MS: Mass Spectrometry APCI-MS: Atmospheric Pressure Chemical Ionization Mass Spectrometry ESI-MS: Electrospray ionization mass spectrometry TLC: Thin-layer chromatography Example 1: Synthesis of the compound of the present invention Option 1: Synthesis of intermediates Synthesis of 9-(octyloxy)-9-oxonanoic acid (3) A solution of azelaic acid 1 (25 g, 0.133 mol), octane-1-ol 2 (21 g, 0.133 mol), EDCI (25.5 g, 0.133 mol), and DMAP (3.25 g, 26.5 mmol) in 300 mL of dichloromethane was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with dichloromethane (2 × 500 mL), and the combined organic layers were washed with brine. After drying with sodium sulfate, the solvent was evaporated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 10% methanol in dichloromethane) to give 9-(octyloxy)-9-oxonanoic acid (15 g, 37%) as a colorless oil. Synthesis of 8-((5-methylhexyl)oxy)-8-oxooctanoic acid (7) Step 1: A solution of 5-methylhexanoic acid 4 (21.8 g, 0.167 mol) in 90 mL THF was slowly added to a cold suspension of LiAlH4 (21.4 g, 0.564 mol) in 200 mL THF. After addition, the resulting mixture was stirred overnight at room temperature. The reaction was carefully quenched by adding 21 mL of water, 21 mL of 15% NaOH, and 60 mL of water. The resulting mixture was diluted with 400 mL of diethyl ether and stirred for 1 h. After filtration through celite, the filtrate was dried over magnesium sulfate, and the solvent was removed under vacuum to give 5-methyl-1-hexanol (19 g, 98%) as a colorless oil. Step 2: A solution of 6-octanoic acid (30.3 g, 0.174 mol), 5-methyl-1-hexanol 5 (19 g, 0.163 mol), EDCI (33.25 g, 0.174 mol), and DMAP (4.25 g, 34.8 mmol) in 300 mL of dichloromethane was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with dichloromethane (2 × 500 mL), and the combined organic layers were washed with brine. After drying with sodium sulfate, the solvent was evaporated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 10% methanol in dichloromethane) to give 9-(octyloxy)-9-oxononanoic acid (14 g, 29%) as a colorless oil. Synthesis of 2-nonylundecanoic acid (9) A solution of nonanoic acid 8 (10 g, 52.5 mmol) in 30 mL THF was added dropwise to a suspension of NaH (60%, 2.32 g, 58 mmol) in 50 mL THF. After the addition, the reaction mixture was cooled to 0°C, and a solution of LDA (2.0 M in THF / heptane / ethylbenzene, 31.7 mL, 63.5 mmol) was slowly added, followed by stirring of the mixture at room temperature for 30 min. Octyl iodide (33 g, 63.5 mmol) was added, and the reaction was heated at 45°C for 16 h. After cooling to room temperature, the reaction was quenched to pH 2 with 1N HCl. After separation, the organic layer was dried over magnesium sulfate and concentrated, and the residue was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give 2-nonyl undecanoic acid (8 g, 50%) as a white solid. Synthesis of 4-oxo-4-((2-pentyldecyl)oxy)butyric acid (13) Step 1: A solution of decanoic acid 10 (20 g, 0.116 mol) in 20 mL THF was added dropwise to a suspension of NaH (60%, 5.11 g, 0.128 mol) in 30 mL THF. After the addition, the reaction mixture was cooled to 0°C, and a solution of LDA (2.0 M in THF / heptane / ethylbenzene, 69.8 mL, 0.14 mol) was slowly added, followed by stirring of the mixture at room temperature for 30 min. Pentyl iodide (26.6 g, 0.14 mol) was added, and the reaction was heated at 45°C for 16 h. After cooling to room temperature, the reaction was quenched to pH 2 with 1 N HCl. After separation, the organic layer was dried over magnesium sulfate and concentrated, and the residue was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give 2-pentyldecanoic acid (14 g, 50%) as a white solid. Step 2: A solution of 2-pentyldecanoic acid 11 (14 g, 57.9 mmol) in 60 mL THF was slowly added to a cold suspension of LiAlH4 (7.4 g, 0.197 mol) in 100 mL THF. After addition, the resulting mixture was stirred overnight at room temperature. The reaction was carefully quenched by adding 7.4 mL of water, 7.4 mL of 15% NaOH, and 24 mL of water. The resulting mixture was then diluted with 200 mL of diethyl ether and stirred for 1 h. After filtration through celite, the filtrate was dried over magnesium sulfate, and the solvent was removed under vacuum to give 2-pentyldecane-1-ol (13 g, 98%) as a colorless oil. Step 3: A mixture of 2-pentyldecane-1-ol 12 (13 g, 57 mmol), succinic anhydride (11.4 g, 0.114 mol), and DMAP (17.4 g, 0.142 mol) in 200 mL of dichloromethane was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with dichloromethane (2 × 500 mL), and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the crude product was purified by rapid column chromatography (SiO2: 0% to 10% methanol in dichloromethane) to give 4-oxo-4-((2-pentyldecyl)oxy)butyric acid (15 g, 80%) as a colorless oil. Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (15) A mixture of octanediic acid 6 (19 g, 0.109 mol), 1-octylnonanol 14 (20 g, 78.1 mmol), EDCI (14.9 g, 78.1 mmol), and DMAP (1.9 g, 15.6 mmol) in 300 mL of dichloromethane was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with dichloromethane (2 × 500 mL), and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the residue was purified by rapid column chromatography (SiO2: 0% to 10% methanol in dichloromethane) to give 8-(heptadecan-9-yloxy)-8-oxooctanoic acid (13 g, 26%) as a colorless oil. Scheme 2: Synthesis of compounds having formula (Vb) Synthesis procedure for compound 7: Step 1: O ’1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)9-dioctyl di(azelate) (17) A solution of 9-(octyloxy)-9-oxononanoic acid 3 (9.43 g, 31.4 mmol), 3-(benzyloxy)propane-1,2-diol 16 (2.86 g, 15.7 mmol), EDCI (6.6 g, 34.5 mmol), and DMAP (0.38 g, 3.1 mmol) in 60 mL of dichloromethane was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with dichloromethane (2 × 150 mL), and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give an O2 solution as a colorless oil. ’1 O 1 -(3-(benzyloxy)propane-1,2-diyl)9-dioctyl di(azelate) (6.44 g, 55%). Step 2: O ’1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)9-dioctyl di(azelate) (18) O ’1 O 1A mixture of 3-(benzyloxy)propane-1,2-diyl)9-dioctyl di(azelate)17 (4 g, 5.35 mmol) and 5% Pd / C (200 mg) in 30 mL of ethyl acetate was purged three times each with nitrogen and hydrogen, and then the reaction was stirred for 16 h at room temperature under a hydrogen gas chamber. After filtration and concentration, O was obtained as a colorless oil. ’1 O 1 -(3-hydroxypropane-1,2-diyl)9-dioctyl di(azelate) (3.2 g, 91%). Step 3: 9-Dioctyl O ’1 O 1 Synthesis of -(3-((2-O-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(azelate) (20) O ’1 O 1 A solution of 18-(3-hydroxypropane-1,2-diyl)9-dioctyl di(azelate) 18 (1.5 g, 2.28 mmol) and triethylamine (0.23 g, 2.28 mmol) in 10 mL THF was cooled to 0°C, and then a solution of 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane 19 (0.32 g, 2.28 mol) in 4 mL THF was added dropwise over 30 min. The reaction mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. Step 4: O ’1 O 1 Synthesis of -(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)9-dioctyl di(azelate) (compound 7) In a sealed tube, 9-dioctyl O ’1 O 1 A solution of 3-(((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)di(azelate)20 (0.5 g, 0.66 mmol) in 10 mL of acetonitrile was cooled in a dry ice bath, and a solution of dimethylamine (2 M in THF, 1.65 mL, 3.3 mmol) was added. The flask was sealed and heated at 65°C for 16 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (gold column: CHCl3 / MeOH / H2O 65:25:4) to give the desired product (0.11 g, 20%) as a white solid. 1 H NMR (300 MHz, CDCl3) δ 5.26-5.19 (m, 1H), 4.37 (dd, 1H), 4.27-4.21(m, 2H), 4.14 (dd, 1H), 4.02 (m, 6H), 3.17 (m, 2H), 2.86 (s, 6H), 2.31-2.25 (m, 8H), 1.65-1.51 (m, 12H), 1.29 (m, 32H), 0.86 (t, 6H). ESI-MS analysis: C41H78NO12P [M+H] calculated value = 808.5, observed value = 808.7. O' 1 O 1 -(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)-oxy)propane-1,2-diyl)8-bis(5-methylhexyl)di(octanoate) (compound 8) The title compound was prepared in a manner similar to that of compound 7. 1 H NMR (300 MHz, CDCl3) δ 5.23-5.13 (1H), 4.35 (dd, 1H), 4.23 (m, 2H), 4.12 (dd, 1H), 4.04-3.97 (m, 6H), 3.17 (m, 2H), 2.85 (s, 6H), 2.26 (t, 8H), 1.65-1.45 (m, 10H), 1.26 (m, 28H), 0.85 (t, 6H). ESI-MS analysis: C37H70NO12P [M+H] calculated value = 752.5, observed value = 752.6. O,O'-(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)-oxy)propane-1,2-diyl)bis(2-pentyldecyl)succinate) (Compound 10) The title compound was prepared in a manner similar to that of compound 7. 1H NMR (300 MHz, CDCl3) δ 5.30-5.22 (m, 1H), 4.38 (dd, 1H), 4.26-4.18 (m, 4H), 4.10-4.04 (m, 2H), 3.98-3.93(m, 4H), 3.21-3.16 (m, 2H), 2.85 (s, 6H), 2.64-2.62 (m, 8H), 1.70-1.55 (m,4H), 1.26 (m, 40H), 0.87 (t, 12H). ESI-MS analysis: C45H86NO12P [M+H] calculated value = 864.5, observed value = 864.6. 1-(1-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-3-((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)9-octyl azelate (compound 11) The title compound was prepared in a manner similar to that of compound 7. 1 ¹H NMR (300 MHz, CDCl₃) δ 5.28–5.20 (m, 1H), 4.84 (quintet, 1H), 4.37 (dd, 1H), 4.26–4.19 (m, 2H), 4.15 (dd, 1H), 4.04 (t, 4H), 3.20–3.15 (m, 2H), 2.85 (s, 6H), 2.32–2.24 (m, 8H), 1.66–1.44 (m, 12H), 1.36–1.20 (m, 48H), 0.87 (t, 9H). ESI-MS analysis: C49H94NO12P [M+H] calculated value = 920.6, observed value = 920.1. O' 1 O 1 -(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)-oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (compound 12) The title compound was prepared in a manner similar to that of compound 7. 1¹H NMR (300 MHz, CDCl₃) δ 5.28–5.21 (m, 1H), 4.85 (quintet, 2H), 4.39 (dd, 1H), 4.27–4.20 (m, 2H), 4.16 (dd, 1H), 4.08–4.02 (m, 2H), 3.22–3.17 (m, 2H), 2.86 (s, 6H), 2.34–2.24 (m, 8H), 1.68–1.42 (m, 12H), 1.25 (m, 60H), 0.88 (t, 12H). ESI-MS analysis: C57H110NO12P [M+H] calculated value = 1032.8, observed value = 1032.9. O'1,O1-(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)7-di(heptadecane-9-yl)di(heptanediate) (Compound 15) The title compound was prepared in a manner similar to that of compound 7. 1 ¹H NMR (300 MHz, CDCl₃) δ 5.20 (m, 1H), 4.82 (quintet, 2H), 4.38 (dd, 1H), 4.21 (m, 2H), 4.12 (dd, 1H), 3.97 (t, 2H), 3.21 (m, 2H), 2.85 (s, 6H), 2.32–2.21 (m, 8H), 1.64–1.42 (m, 12H), 1.22 (m, 56H), 0.84 (t, 12H). APCI-MS analysis: C55H106NO12P [M+H] calculated value = 1004.7, observed value = 1004.8. O'1,O1-(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)9-di(heptadecane-9-yl)di(azelate) (compound 16) The title compound was prepared in a manner similar to that of compound 7. 1¹H NMR (300 MHz, CDCl₃) δ 5.21 (m, 1H), 4.84 (quintet, 2H), 4.38 (dd, 1H), 4.23 (m, 2H), 4.12 (dd, 1H), 3.99 (t, 2H), 3.24 (m, 2H), 2.85 (s, 6H), 2.30–2.21 (m, 8H), 1.64–1.42 (m, 12H), 1.23 (m, 64H), 0.86 (t, 12H). APCI-MS analysis: C59H114NO12P [M+H] calculated value = 1060.5, observed value = 1060.8. ((3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (Compound 17) The title compound was prepared in a manner similar to that of compound 7. 1 H NMR (300 MHz, CDCl3) δ 5.23 (m,1H), 4.38 (dd, 1H), 4.22 (m, 2H), 4.13 (dd, 1H), 4.04 (t, 4H), 4.00-3.93 (m,2H), 3.23 (m, 2H), 2.87 (s, 6H), 2.36-2.24 (m, 6H), 1.68-1.52 (m, 8H), 1.45-1.34 (m, 4H), 1.24 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: C55H106NO12P [M+H] calculated value = 1004.7, observed value = 1004.7. ((3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (Compound 18) The title compound was prepared in a manner similar to that of compound 7. 1H NMR (300 MHz, CDCl3) δ 5.26 (m,1H), 4.38 (dd, 1H), 4.22 (m, 2H), 4.17 (dd, 1H), 4.04 (t, 4H), 4.00-3.93 (m,2H), 3.22 (m, 2H), 2.86 (s, 6H), 2.36-2.24 (m, 6H), 1.68-1.52 (m, 8H), 1.45-1.34 (m, 4H), 1.24 (m, 60H), 0.86 (t, 12H). APCI-MS analysis: C57H110NO12P [M+H] calculated value = 1032.7, observed value = 1032.7. O'1,O1-(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-bis(eicosano-11-yl)bis(octanoate) (compound 19) The title compound was prepared in a manner similar to that of compound 7. 1 ¹H NMR (300 MHz, CDCl₃) δ 5.23 (m, 1H), 4.84 (quintet, 2H), 4.38 (dd, 1H), 4.23 (m, 2H), 4.15 (dd, 1H), 4.01 (t, 2H), 3.24 (m, 2H), 2.86 (s, 6H), 2.34–2.21 (m, 8H), 1.64–1.42 (m, 16H), 1.24 (m, 72H), 0.86 (t, 12H). APCI-MS analysis: C65H126NO12P [M+H] calculated value = 1144.9, observed value = 1144.1. Synthesis procedure for compound 9: Step 1: Synthesis of 3-(benzyloxy)propane-1,2-dimethylbis(2-nonylundecanoate) (17c) A mixture of 2-nonylundecanoic acid 9 (6.27 g, 20 mmol) and 1,1'-carbonyldiimidazole (3.57 g, 22 mmol) in 40 mL THF was stirred for 40 min. A solution of 3-(benzyloxy)propane-1,2-diol 16 (911 mg, 5 mmol) in 50 mL DMF was cooled to 0°C, sodium hydride (60% in mineral oil, 880 mg, 22 mmol) was added, and the resulting mixture was stirred at this temperature for 15 min. An aliquot of 1-(1H-imidazol-1-yl)-2-nonylundecane-1-one was slowly added, and the mixture was stirred at room temperature for 2 h. TLC showed that the reaction was complete. The reaction mixture was decanted into 1 N HCl and extracted with ethyl acetate, and the combined organic layers were washed with water and brine. After drying with sodium sulfate, the solvent was concentrated and the crude product was purified by column chromatography (SiO2: EtOAc / hexane 0% to 10%) to obtain a mixture, which was further purified by column chromatography (SiO2: diethyl ether / hexane 0% to 20%) to obtain a pure product in a waxy state (2.27 g, 57%). Step 2: Synthesis of 3-hydroxypropane-1,2-dimethylbis(2-nonylundecanoate) (18c) A mixture of 3-(benzyloxy)propane-1,2-dimethylbis(2-nonylundecanoate) 17c (2.27 g, 2.94 mmol) and 5% Pd / C (300 mg) in 30 mL of ethyl acetate was purged three times each with nitrogen and hydrogen, and the reaction was then carried out at room temperature in a Parr hydrogenator for 16 h. After filtration and concentration, 3-hydroxypropane-1,2-dimethylbis(2-nonylundecanoate) (1.94 g, 97%) was obtained as a colorless oil. Step 3: Synthesis of 3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diylbis(2-nonyldecanoate) (20c) A solution of 3-hydroxypropane-1,2-dimethylbis(2-nonylundecanoate)18c (1.0 g, 1.47 mmol) and triethylamine (0.20 mL, 1.47 mmol) in 10 mL THF was cooled to 0°C, and then a solution of 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane 19 (0.21 g, 1.47 mol) in 4 mL THF was added dropwise over 30 min. The reaction mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. Step 4: 3-(((2(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-dimethylbis(2-nonylundecanoate) (Compound 9) In a sealed tube, a solution of 3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diylbis(2-nonylundecanoate)20c (1.26 g, 1.47 mmol) in 10 mL of acetonitrile was cooled in a dry ice bath, and a solution of dimethylamine (2 M in THF, 3.65 mL, 7.3 mmol) was added. The flask was sealed and heated at 65°C for 16 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (gold column: CHCl3 / MeOH / H2O 65:25:4) to give the desired product (110 mg, 9%) as a white solid. 1 H NMR (300 MHz, CDCl3) δ 5.28-5.16 (1H), 4.50 (dd, 1H), 4.24 (m, 2H), 4.10 (dd, 1H), 4.05-3.90 (m, 4H), 3.20 (m, 2H), 2.86 (s, 6H), 2.35-2.21 (m,2H), 1.65-1.36 (m, 6H), 1.24 (m, 56H), 0.87 (t, 12H). APCI-MS analysis: C47H94NO8P [M+H] calculated value = 832.6, observed value = 832.2. Scheme 2: Synthesis of compounds having formula (III) Synthesis of 2-(((2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propoxy)(hydroxy)-phosphoryl)oxy)-N,N,N-trimethylethane-1-ammonium (compound 24) In a sealed tube, 9-dioctyl O ’1 O 1A solution of 3-(((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)di(azelate)20 (900 mg, 1.0 mmol) in 10 mL of acetonitrile was cooled in a dry ice bath, and a solution of trimethylamine (2 M in THF, 2.5 mL, 5 mmol) was added. The flask was sealed and heated at 65°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (gold column: CHCl3 / MeOH / H2O 65:25:4) to give the desired product (560 mg, 54%) as a white solid. 1 ¹H NMR (300 MHz, CDCl₃) δ 5.17 (m, 1H), 4.83 (quintet, 2H), 4.38 (dd, 1H), 4.30 (m, 2H), 4.09 (dd, 1H), 3.91 (m, 2H), 3.78 (m, 2H), 3.35 (s, 9H), 2.32–2.21 (m, 8H), 1.64–1.42 (m, 16H), 1.24 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: C58H113NO12P [M+] calculated value = 1046.8, observed value = 1046.8. Scheme 2: Synthesis of compounds having formula (Va) Synthesis procedure for compound 1: Step 1: O' 1 O 1 Synthesis of -(3-(((2-(((benzyloxy)carbonyl)amino)ethoxy)(hydroxy)-phosphoryl)oxy)propane-1,2-diyl)9-dioctyl di(azelate) (23) A mixture of POCl3 (266 mg, 1.7 mmol) in 20 mL of dichloromethane and 20 mL of diethyl ether was cooled to 0°C, and then triethylamine (193 mg, 1.9 mmol) was added under a N2 atmosphere, followed by the addition of O' 1 O 1A solution of 1-(3-hydroxypropane-1,2-diyl)8-bis(5-methylhexyl)di(octanoate)18 (1.0 g, 1.59 mmol) in 5 mL of dichloromethane was added, and the resulting mixture was stirred at this temperature for 2 h. TLC showed the disappearance of ethanol. The mixture was cooled to 0°C, and then a solution of benzyl(2-hydroxyethyl)carbamate (341 mg, 1.75 mmol) and triethylamine (241 mg, 2.4 mmol) in 10 mL of dichloromethane was added, and the mixture was stirred overnight at room temperature. 2 mL of water was added, and the mixture was stirred for 2 h. After concentration, the crude product was purified by column chromatography (SiO2: 0%–10% methanol in dichloromethane) to give the desired product (200 mg, 14%) as a pale yellow oil. Step 2: O' 1 O 1 Synthesis of -(3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-bis(5-methylhexyl)di(octanoate) (Compound 1) To O' 1 O 1 Three drops of acetic acid were added to a mixture of -(3-(((2-((((benzyloxy)carbonyl)amino)ethoxy)(hydroxy)-phosphoryl)oxy)propane-1,2-diyl)9-dioctyl di(azelate)23 (200 mg, 0.23 mmol) and 5% Pd / C (70 mg) in 50 mL of CH2Cl2. The mixture was then placed in a Pal reactor and incubated overnight at 38 psi under hydrogen. The reaction mixture was filtered through a Celite filter and concentrated. The residue was purified by column chromatography, eluting with 0%–30% methanol in dichloromethane to give the desired product (50 mg, 30%) as a white waxy substance. 1 H NMR (300 MHz, CD3OD) δ 5.28 (m, 1H), 4.42 (dd, 1H), 4.16 (dd, 1H), 4.12-3.94 (m, 8H), 3.17 (m, 2H), 2.40-2.27 (m, 8H), 1.70-1.53 ​​(m, 10H), 1.33(m, 36H), 0.97-0.86(m, 6H). APCI-MS analysis: C39H74NO12P [M+H] calculated value = 779.5, observed value = 779.5. O' 1 O 1-(3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-bis(5-methylhexyl)di(octanoate) (Compound 2) The title compound was prepared in a manner similar to that of compound 1. 1 H NMR (300 MHz, DMSO-d6) δ 5.05(m, 1H), 4.28 (dd, 1H), 4.08 (dd, 1H), 3.97 (t, 4H), 3.85-3.73 (m, 4H), 2.95(m, 2H), 2.47 (t, 8H), 1.56-1.44 (m, 14H), 1.32-1.09 (m, 16H), 0.82 (d, 12H). ESI-MS analysis: C35H66NO12P [M+H] calculated value = 724.4, observed value = 724.5. Synthesis procedure for compound 6: Step 1: O' 1 O 1 Synthesis of -(3-(((2-((tert-butoxycarbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (22) A mixture of POCl3 (191 mg, 1.25 mmol) and triethylamine (0.24 mL, 1.7 mmol) in 10 mL THF was cooled to 0°C, and then 8-di(heptadecane-9-yl)O' was slowly added. 1 O 1 A solution of 18-(3-hydroxypropane-1,2-diyl)bis(octanoate)18 (1.0 g, 1.13 mmol) in 5 mL THF was added, and the resulting mixture was stirred at this temperature for 2 h. TLC showed the disappearance of ethanol. The volatiles were evaporated under vacuum, and the residue was redissolved in 10 mL dichloromethane. After cooling to 0°C, a solution of tert-butyl(2-hydroxyethyl)carbamate (183 mg, 1.13 mmol) and triethylamine (0.24 mL, 1.7 mmol) in 5 mL dichloromethane was added, and the mixture was stirred overnight at room temperature. 2 mL of water was added, and the mixture was stirred for 2 h. After concentration, the crude product was purified by column chromatography (SiO2: 0%–10% methanol in dichloromethane) to give the desired product containing triethylamine, which was partitioned between water and dichloromethane to give a pure product as a wax (400 mg, 35%). Step 2: Synthesis of 2-ammonium ethyl (2,3-bis((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)propyl) phosphate (compound 6) To O' 1 O 1 -(3-(((2-((tert-butoxycarbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)22 (400 mg, 0.46 mmol) was prepared by adding 2 mL of trifluoroacetic acid to a solution in 5 mL of CH2Cl2, and then stirring the mixture overnight. After concentration, the residue was purified by column chromatography, eluting with chloroform / methanol / water 35:13:2 to give the desired product (250 mg, 68%) as a white waxy substance. 1 ¹H NMR (300 MHz, CDCl₃) δ 8.45 (s, 3H), 5.21 (m, 1H), 4.84 (quintet, 2H), 4.36 (dd, 1H), 4.18–4.04 (m, 3H), 3.93 (m, 2H), 3.17 (m, 2H), 2.36–2.23 (m, 8H), 1.66–1.53 (m, 8H), 1.51–1.43 (m, 8H), 1.24 (m, 56H), 0.86 (t, 12H). APCI-MS analysis: C55H106NO12P [M+H] calculated value = 1004.7, observed value = 1004.1. 3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-dimethylbis(2-nonylundecanoate) (Compound 3) The title compound was prepared in a manner similar to that of compound 6. 1 H NMR (300 MHz, CDCl3) δ 8.44 (m,3H), 5.21 (m, 1H), 4.50-4.44 (m, 1H), 4.35-3.85 (m, 5H), 3.17 (m, 2H), 2.44-2.02 (m, 6H), 1.63-1.33 (m, 4H), 1.24 (m, 56H), 0.87 (t, 12H). APCI-MS analysis: C45H90NO8P [M+H] calculated value = 804.2, observed value = 804.1. O,O'-(3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)bis(2-pentyldecyl)succinate (compound 4) The title compound was prepared in a manner similar to that of compound 6. 1 H NMR (300 MHz, CDCl3) δ 8.47 (s,3H), 5.21 (m, 1H), 4.36 (dd, 1H), 4.20 (dd, 1H), 4.10 (m, 2H), 3.96 (d, 6H),3.17 (m, 2H), 2.61 (s, 8H), 1.61 (m, 2H), 1.25 (m, 44H), 0.87 (t, 12H). ESI-MS analysis: C43H82NO12P [M+H] calculated value = 836.5, observed value = 836.0. O,O'-(3-(((2-aminoethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)bis(2-pentyldecyl)succinate (compound 5) The title compound was prepared in a manner similar to that of compound 6. 1 H NMR (300 MHz, CDCl3) δ 8.47 (s,3H), 5.21 (m, 1H), 4.36 (dd, 1H), 4.20 (dd, 1H), 4.10 (m, 2H), 3.96 (d, 6H),3.17 (m, 2H), 2.61 (s, 8H), 1.61 (m, 2H), 1.25 (m, 44H), 0.87 (t, 12H). ESI-MS analysis: C43H82NO12P [M+H] calculated value = 836.5, observed value = 836.0. Synthesis scheme of compound 34 Synthesis procedure for compound 34: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) A mixture of octanediic acid 2 (22.0 g, 0.126 mol), 1-octylnonanol 1 (16.2 g, 63.1 mmol), EDCI (24.0 g, 0.126 mol), and DMAP (771 mg, 6.3 mmol) in 400 mL of dichloromethane was stirred overnight at room temperature. TLC showed the presence of ethanol. The reaction mixture was diluted with water, extracted with dichloromethane, and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the residue was purified by rapid column chromatography (SiO2: 0% to 40% EtOAc in hexane) to give 8-(heptadecan-9-yloxy)-8-oxooctanoic acid (13.2 g, 50%) as a colorless oil. Step 2: O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A solution of 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid 3 (5.1 g, 12 mmol), 3-(benzyloxy)propane-1,2-diol 4 (1.0 g, 5.5 mmol), EDCI (5.5 g, 29 mmol), and DMAP (1.5 g, 12 mmol) in 50 mL of dichloromethane was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was ground three times with hexane. The solution was concentrated, and the residue was purified by rapid column chromatography (SiO2: 0% to 30% ethyl acetate in hexane) to give O2 as a colorless oil. '1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5.1 g, 97%). Step 3: 8-Di(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) O '1 O 1 A mixture of 5-(3-(benzyloxy)propane-1,2-diyl)8-di(heptadecano-9-yl)di(octanoate)5 (5.1 g, 5.3 mmol) and 10% Pd / C (0.7 g) in 20 mL of ethyl acetate was purged three times each with nitrogen and hydrogen, and then the reaction was stirred for 16 h at room temperature under a hydrogen gas chamber. After filtration and concentration, 8-di(heptadecano-9-yl)O was obtained as a colorless oil. '1 O1 -(3-hydroxypropane-1,2-diyl)bis(octanoate) (4.7 g, quantitative). Step 4: Synthesis of triethylammonium 2,3-bis((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)propylphosphonate (8) At 0°C, 8-di(heptadecano-9-yl)O '1 O 1 3-(3-hydroxypropane-1,2-diyl)bis(octanoate)6 (3.0 g, 3.4 mmol) was added dropwise to a solution of diphenyl phosphite 7 (3.9 mL, 20 mmol) in 10 mL of pyridine, and the resulting mixture was stirred at this temperature for 1 h. A mixture of triethylamine and water (10 mL, 1:1) was added, and the mixture was stirred at room temperature for another hour. After concentration, the crude product was partitioned between dichloromethane and saturated sodium bicarbonate solution. The combined organic layers were washed with brine and dried over Na2SO4, and then concentrated. The residue was purified by column chromatography (SiO2:95:5:0.5CHCl3-MeOH-NEt3) to give triethylammonium 2,3-bis((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)propylphosphonate (2.5 g, 70%) as a colorless oil. 1 ¹H NMR (400 MHz, CDCl₃): δ 7.61 (s, 0.5H), 6.04 (s, 0.5H), 5.24–5.13 (m, 1H), 4.83 (quintet, 2H), 4.35 (dd, 1H), 4.15 (dd, 1H), 3.98 (dd, 2H), 3.12–2.94 (m, 6H), 2.38–2.15 (m, 8H), 1.70–1.39 (m, 16H), 1.37–1.09 (m, 65H), 0.86 (t, 12H). 31 P NMR (162 MHz, CDCl3): δ 5.44. ESI-MS: m / z 1046.5 (M+H). Step 5: Synthesis of 2-(((2,3-bis((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)propoxy)-phosphoryl)oxy)-N,N,N-triethylethyl-1-ammonium iodide (10) A solution of triethylammonium 2,3-bis((8-(heptadecyl-9-yloxy)-8-oxooctanoyl)oxy)propylphosphonate 8 (1.0 g, 0.96 mmol) and N,N,N-triethyl-2-hydroxyethyl-1-ammonium iodide 9 (0.31 g, 1.1 mmol) in 10 mL of pyridine and 2 mL of dichloromethane was cooled to 0°C, and neopentanoyl chloride (0.15 mL, 1.12 mmol) was added dropwise. The resulting solution was stirred at this temperature for 1 h. After concentration, the residue was partitioned between dichloromethane and a saturated sodium bicarbonate solution. After washing with brine and drying with sodium sulfate, the solvent was removed, and the crude product was used for the next step without further purification. ESI-MS: m / z 1072.8 (M+H). Step 6: Synthesis of 2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(triethylammono)ethyl)phosphate (compound 34) A solution of 2-(((2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propoxy)-phosphoryl)oxy)-N,N,N-triethylethyl-1-ammonium iodide 10 (1.0 g crude) in 4.7 mL of 95% pyridine-water was cooled to 0°C, 800 mg of iodine was added, and the mixture was stirred at room temperature for 3 h. After concentration, the residue was partitioned between dichloromethane and a saturated sodium thiosulfate solution, and the organic layer was washed with saturated sodium bicarbonate and brine. After drying and concentration with sodium sulfate, the crude product was purified by column chromatography (SiO2: CHCl3 / MeOH / H2O 65:25:4) to give 2,3-bis((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(triethylammonium)ethyl)phosphate (107 mg, 10%) as a colorless oil. 1 ¹H NMR (400 MHz, CDCl₃): 5.24–5.15 (m, 1H), 4.83 (quintet, 2H), 4.39 (dd, 1H), 4.33–4.24 (m, 2H), 4.10 (dd, 1H), 3.96 (t, 2H), 3.66–3.57 (m, 2H), 3.55–3.43 (m, 6H), 2.41–2.06 (m, 10H), 1.69–1.43 (m, 16H), 1.42–1.11 (m, 63H), 0.86 (t, 12H). 31P NMR (162 MHz, CDCl3): δ 0.02. APCI-MS analysis: C61H118NO12P [M+H] calculated value = 1088.8, observed value = 1088.8. HPLC-ELSD: t R = 10.420 min (Method 1). Synthetic scheme of compound 31 Synthesis procedure for compound 31: Step 1: O' 1 O 1 Synthesis of -(3-(((2-(diethylamino)ethoxy)phosphoyl)oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (10) A solution of triethylammonium 2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propylphosphonate 8 (1.1 g, 1.1 mmol) and 2-(diethylamino)ethanol-1-ol 9 (0.15 g, 1.3 mmol) in 10 mL of pyridine was cooled to 0°C, and neopentanoyl chloride (0.77 mL, 6.3 mmol) was added dropwise. The resulting solution was stirred at this temperature for 1 h. After concentration, the residue was partitioned between dichloromethane and a saturated sodium bicarbonate solution. After washing with brine and drying with sodium sulfate, the solvent was removed, and the crude product was used for the next step without further purification. ESI-MS: m / z 1044.8 (M+H). Step 2: O' 1 O 1 Synthesis of -(3-(((2-(diethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (compound 31) O' 1 O 1A solution of 1.1 g crude 1,2-propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)10 in 10 mL of 95% pyridine-water was cooled to 0°C, iodine (800 mg, 3.2 mmol) was added, and the mixture was stirred at room temperature for 3 h. After concentration, the residue was partitioned between dichloromethane and saturated sodium thiosulfate solution, and the organic layer was washed with saturated sodium bicarbonate and brine. After drying and concentration with sodium sulfate, the crude product was purified by column chromatography (SiO2:CHC13 / MeOH / H2O 65:25:4) to give O' as a light brown gel. 1 O 1 -(3-(((2-(diethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (150 mg, 13%). 1 ¹H NMR (400 MHz, CDCl₃): 5.24–5.14 (m, 1H), 4.83 (quintet, 2H), 4.34 (dd, 1H), 4.24–4.16 (m, 2H), 4.15–4.07 (m, 1H), 4.03–3.94 (m, 2H), 3.20–3.06 (m, 6H), 2.32–2.16 (m, 8H), 1.66–1.39 (m, 16H), 1.36–1.10 (m, 63H), 0.84 (t, 12H). 31 P NMR (162 MHz, CDCl3): δ 1.74. APCI-MS analysis: C59H114NO12P [M+H] calculated value = 1060.8, observed value = 1060.8. HPLC-ELSD: t R = 10.372 min (Method 1). Synthesis scheme of compound 32 Synthetic procedure for compound 32: Step 1: Synthesis of pentadecane-7-ol (3) At 0°C, a solution of heptanal 1 (12.6 mL, 90 mmol) in 40 mL of diethyl ether was slowly added to a solution of octylmagnesium bromide 2 (2 M in diethyl ether, 52.5 mL, 0.105 mol) in 100 mL of diethyl ether, and the mixture was heated to room temperature and stirred overnight. The reaction was quenched with saturated ammonium chloride and extracted with diethyl ether; the organic layer was dried over sodium sulfate. After concentration, the crude product was purified by column chromatography (SiO2: 0%–20% EtOAc in hexane) to give the desired product (10.0 g, 50%) as a white waxy substance. Step 2: Synthesis of 8-oxo-8-(pentadecan-7-yloxy)octanoic acid (5) A solution of pentadecane-7-ol 3 (7.0 g, 30.8 mmol) in 35 mL of dichloromethane was slowly added to a mixture of octanoic acid 4 (12.8 g, 74 mmol), EDCI (14.1 g, 74 mmol), and DMAP (3.8 g, 30.8 mmol) in 40 mL of dichloromethane and 30 mL of DMF, and the resulting mixture was stirred overnight at room temperature. The clear solution was concentrated, and the residue was partitioned between a saturated ammonium chloride solution and hexane, and then ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give 8-oxo-8-(pentadecan-7-yloxy)octanoic acid (7.0 g, 59%) as a colorless oil. Step 3: O' 1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate) (7) A solution of 8-oxo-8-(pentadecano-7-yloxy)octanoic acid 5 (7.0 g, 18 mmol), 3-(benzyloxy)propane-1,2-diol 6 (1.5 g, 8.2 mmol), EDCI (9.5 g, 49 mmol), and DMAP (2.2 g, 18 mmol) in 50 mL of dichloromethane was stirred at room temperature for 48 h. After concentration, the residue was partitioned between a saturated ammonium chloride solution and ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give O' as a colorless oil. 1 O 1-(3-(benzyloxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate) (6.5 g, 86%). Step 4: O' 1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate) (8) O' 1 O 1 A mixture of 3-(benzyloxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate)7 (6.5 g, 7.1 mmol) and Pd / C (10 wt%, 700 mg) in 30 mL of ethyl acetate was hydrogenated in a gas chamber for 16 h. After filtration and concentration, O' was obtained as a colorless oil. 1 O 1 -(3-hydroxypropane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate) (5.9 g, quantitative). 1 ¹H NMR (400 MHz, CDCl₃) δ 5.06 (quintet, 1H), 4.85 (quintet, 2H), 4.30 (dd, 1H), 4.21 (dd, 1H), 3.71 (t, 2H), 2.40–2.20 (m, 8H), 2.19–2.09 (m, 1H), 1.70–1.54 (m, 8H), 1.53–1.41 (m, 8H), 1.38–1.15 (m, 48H), 0.86 (t, 12H). APCI-MS analysis: C49H92O9 [M+H] calculated value = 825.7, observed values ​​= 825.6, 807.6 (M+H-H2O). HPLC-ELSD: t R = 6.476 min (Method 2). Step 5: O' 1 O 1 Synthesis of -(3-((2-oxo-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate) (10) O' 1 O 1A solution of 1.5 g (1.8 mmol) of 3-hydroxypropane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate)8 and triethylamine (0.38 mL, 2.7 mmol) in 10 mL THF was cooled to 0°C, and then 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane (390 mg, 2.7 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. Step 6: O' 1 O 1 Synthesis of -(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-di(pentadecan-7-yl)di(octanoate) (compound 32) In the sealed tube, O' 1 O 1 A solution of 10-(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate)10 (crude, 1.8 mmol) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of dimethylamine (2 M in THF, 5.5 mL, 11 mmol) was added. The flask was sealed and heated at 85°C for 16 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 88 : 13 : 1) to give the desired product (800 mg, 45%) as a pale yellow waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.30–5.16 (m, 1H), 4.84 (quintet, 2H), 4.37 (dd, 1H), 4.27–4.19 (m, 2H), 4.13 (dd, 1H), 4.01 (t, 2H), 3.22–3.13 (m, 2H), 2.85 (s, 6H), 2.35–2.20 (m, 8H), 1.68–1.40 (m, 16H), 1.38–1.13 (m, 48H), 0.86 (t, 12H). 31 P NMR (162 MHz, CDCl3): δ 0.38. APCI-MS analysis: C53H102NO12P [M+H] calculated value = 976.7, observed value = 976.7. HPLC-ELSD: t R = 8.133 min (Method 1). Synthesis of 2,3-bis((8-oxo-8-(pentadecan-7-yloxy)octanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 35) O' 1 O 1 A solution of 1,0-(3-hydroxypropane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate)8 (1.0 g, 1.2 mmol) and triethylamine (0.25 mL, 1.8 mmol) in 10 mL THF was cooled to 0°C, and then 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane (260 mg, 1.8 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. In the sealed tube, O' 1 O 1 A solution of -(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)8-bis(pentadecan-7-yl)bis(octanoate)10 (crude) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 3.5 mL, 7 mmol) was added. The flask was sealed and heated at 85°C for 48 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 88:13:1) to give the desired product (590 mg, 50%) as a colorless waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.25–5.15 (m, 1H), 4.83 (quintet, 2H), 4.43–4.32 (m, 3H), 4.11 (dd, 1H), 4.01–3.87 (m, 4H), 3.40 (s, 9H), 2.36–2.16 (m, 8H), 1.70–1.42 (m, 16H), 1.37–1.14 (m, 48H), 0.86 (t, 12H). 31 P NMR (162 MHz, CDCl3): δ -0.24. APCI-MS analysis: C54H104NO12P [M+H] calculated value = 990.7, observed value = 990.6. HPLC-ELSD: t R = 7.460 min (Method 1). Synthesis scheme of compound 33 Synthesis procedure for compound 33: Step 1: Synthesis of tridecane-5-ol (3) A solution of butyl magnesium chloride 2 (2 M in diethyl ether, 100 mL, 0.2 mol) was slowly added to a solution of nonanal 1 (23 g, 0.16 mol) in 200 mL THF at 0°C, and the mixture was heated to room temperature and stirred overnight. The reaction was quenched with saturated ammonium chloride and extracted with ethyl acetate; the organic layer was dried over sodium sulfate. After concentration, the crude product was purified by column chromatography (SiO2: 0%–20% EtOAc in hexane) to give the desired product (5.3 g, 16%) as a white waxy substance. Step 2: Synthesis of 8-oxo-8-(tetran-5-yloxy)octanoic acid (5) A solution of tridecane-5-ol 3 (5.3 g, 26 mmol) in 20 mL of dichloromethane was slowly added to a mixture of octanoic acid 4 (9.2 g, 53 mmol), EDCI (10 g, 53 mmol), and DMAP (3.2 g, 26 mmol) in 20 mL of dichloromethane and the resulting mixture was stirred overnight at room temperature. The clear solution was concentrated, and the residue was partitioned between a saturated ammonium chloride solution and hexane, and then ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 50% ethyl acetate in hexane) to give 8-oxo-8-(tetran-5-yloxy)octanoic acid (4.2 g, 45%) as a colorless oil. Step 3: O' 1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate) (7) A solution of 8-oxo-8-(tetran-5-yloxy)octanoic acid 5 (4.22 g, 11.8 mmol), 3-(benzyloxy)propane-1,2-diol 6 (0.93 g, 5.1 mmol), EDCI (5.9 g, 31 mmol), and DMAP (1.2 g, 10 mmol) in 60 mL of dichloromethane was stirred at room temperature for 17 h. After concentration, the residue was partitioned between a saturated ammonium chloride solution and ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give O' as a colorless oil. 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate) (3.9 g, 89%). Step 4: O' 1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate) (8) O' 1 O 1 A mixture of 3-(benzyloxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate)7 (3.9 g, 4.5 mmol) and Pd / C (10 wt%, 120 mg) in 20 mL of ethyl acetate was hydrogenated in a Par system (40 psi) for 16 h. After filtration and concentration, O' was obtained as a colorless oil. 1 O 1 -(3-hydroxypropane-1,2-diyl)8-bis(tetrane-5-yl)bis(octanoate) (3.4 g, 97%). 1 ¹H NMR (400 MHz, CDCl₃) δ 5.06 (quintet, 1H), 4.85 (quintet, 2H), 4.31 (dd, 1H), 4.22 (dd, 1H), 3.71 (t, 2H), 2.40–2.22 (m, 8H), 2.17–2.09 (m, 1H), 1.69–1.42 (m, 16H), 1.39–1.15 (m, 40H), 0.93–0.79 (m, 12H). APCI-MS analysis: C45H84O9 [M+H] calculated value = 769.6, observed values ​​= 769.1, 751.1 (M+H-H2O). HPLC-ELSD: tR = 6.668 min (Method 2). Step 5: O' 1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate) (10) O' 1 O 1 A solution of 3-hydroxypropane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate)8 (800 mg, 1.04 mmol) and triethylamine (0.36 mL, 2.6 mmol) in 8 mL THF was cooled to 0°C, and then 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane9 (370 mg, 2.6 mmol) was added dropwise. The reaction mixture was stirred overnight at room temperature. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. Step 6: O' 1 O 1 Synthesis of -(3-(((2-(dimethylamino)ethoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-di(tetran-5-yl)di(octanoate) (compound 33) In the sealed tube, O' 1 O 1 A solution of -(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate)10 (crude) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of dimethylamine (2 M in THF, 6 mL, 12 mmol) was added. The flask was sealed and heated at 85°C for 17 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 80:15:2) to give the desired product (535 mg, 56%) as a pale yellow waxy substance. 1¹H NMR (400 MHz, CDCl₃) δ 5.27–5.18 (m, 1H), 4.84 (quintet, 2H), 4.37 (dd, 1H), 4.27–4.18 (m, 2H), 4.14 (dd, 1H), 4.02 (t, 2H), 3.25–3.16 (m, 2H), 2.85 (s, 6H), 2.35–2.17 (m, 8H), 1.67–1.42 (m, 16H), 1.38–1.12 (m, 40H), 0.92–0.79 (m, 12H). 31 P NMR (162 MHz, CDCl3): δ 0.30. ESI-MS analysis: C49H94NO12P [M+H] calculated value = 920.6, observed value = 920.6. HPLC-ELSD: t R = 7.403 min (Method 1). Synthesis of 2,3-bis((8-oxo-8-(tetran-5-yloxy)octanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 36) O' 1 O 1 A solution of 3-hydroxypropane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate)8 (700 mg, 0.91 mmol) and triethylamine (0.32 mL, 2.28 mmol) in 8 mL THF was cooled to 0°C, and then 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane 9 (324 mg, 2.28 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 6 h. After filtration, the filtrate was concentrated under vacuum, and the residue was used for the next step without further purification. In the sealed tube, O' 1 O 1A solution of -(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)8-bis(tetran-5-yl)bis(octanoate)10 (crude) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 6 mL, 12 mmol) was added. The flask was sealed and heated at 85°C for 17 h. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 80:15:5) to give the desired product (535 mg, 56%) as a pale yellow waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.22–5.12 (m, 1H), 4.83 (quintet, 2H), 4.43–4.24 (m, 3H), 4.10 (dd, 1H), 4.02–3.70 (m, 4H), 3.37 (s, 9H), 2.37–2.16 (m, 8H), 1.68–1.39 (m, 16H), 1.39–1.11 (m, 40H), 0.94–0.76 (m, 12H). 31 P NMR (162 MHz, CDCl3): δ -0.42. ESI-MS analysis: C50H96NO12P [M+H] calculated value = 934.6, observed value = 934.6. HPLC-ELSD: t R = 7.292 min (Method 1). Synthetic scheme of compound 38 Synthesis procedure for compound 38: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 1 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 2 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: (R)-O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 4 (3.8 g, 20.9 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (19.5 g, 47.3 mmol), EDCI (27.2 g, 142 mmol), and DMAP (5.8 g, 47.5 mmol) in 300 mL of dichloromethane was stirred at room temperature for 15 h. TLC showed the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce a colorless oil (R)-O' 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (18.2 g, 90%). Step 3: (R)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) (R)-O '1 O 1A mixture of 3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)5 (6.3 g, 6.5 mmol) and 20% Pd(OH)2 / carbon (1 g) in 100 mL of ethyl acetate was hydrogenated using a Pal hydrogenator at 35–40 psi for 12 h. After filtration through silica gel and concentration, the product (5.6 g, 97%) was used for the next step without further purification. Step 4: (S)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(octanoate) (8) (R)-8-di(heptadecane-9-yl)O '1 O 1 A solution of 3-hydroxypropane-1,2-diyl)bis(octanoate)6 (705 mg, 0.8 mmol) and triethylamine (0.16 mL, 1.2 mmol) in 5 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 7 (214 mg, 1.2 mmol) in 1 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 5: Synthesis of (S)-2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-((3-hydroxypropyl)dimethylammonium)ethyl)phosphate (compound 38) To crude (S)-di(heptadecane-9-yl)O '1 O 13-((2-oxaphosphazenecyclopropane-2-yl)oxy)propane-1,2-diyl)bis(octanoate)8 (0.8 mmol) was added to a solution of 3-((tert-butyldimethylsilyl)oxy)-N,N-dimethylprop-1-amine 9 (2.5 g, 11.5 mmol) in 2 mL THF and 10 mL acetonitrile, and the resulting mixture was heated overnight in a sealed tube at 85°C–90°C. MS showed the formation of the desired product. After cooling to room temperature and concentration, the residue was dissolved in 10 mL THF and transferred to a polytetrafluoroethylene round-bottom flask, 3 mL of pyridine and 1.5 mL of a pyridine hydrofluoride complex were added, and the mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 and extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 1) to give the desired product (55 mg, 5%) as a pale yellow waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.26–5.21 (m, 1H), 4.83 (quintet, 2H), 4.43–4.23 (m, 3H), 4.15–4.03 (m, 1H), 3.99–3.82 (m, 3H), 3.79–3.60 (m, 6H), 3.35–3.17 (s, 6H), 2.36–2.19 (m, 8H), 2.09–1.94 (m, 2H), 1.69–1.42 (m, 18H), 1.41–1.15 (m, 60H), 0.86 (t, 12H). APCI-MS analysis: C60H116NO13P [M+H] calculated value = 1090.8, observed value = 1090.8. HPLC-ELSD: t R = 8.426 min (Method 1). Synthesis scheme of compound 28 Synthesis procedure for compound 28: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 1 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 2 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: (R)-O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 4 (3.8 g, 20.9 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (19.5 g, 47.3 mmol), EDCI (27.2 g, 142 mmol), and DMAP (5.8 g, 47.5 mmol) in 300 mL of dichloromethane was stirred at room temperature for 15 h. TLC showed the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce a colorless oil (R)-O' 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (18.2 g, 90%). Step 3: (R)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) (R)-O '1 O 1A mixture of 3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)5 (6.3 g, 6.5 mmol) and 20% Pd(OH)2 / carbon (1 g) in 100 mL of ethyl acetate was hydrogenated using a Pal hydrogenator at 35–40 psi for 12 h. After filtration through silica gel and concentration, the product (5.6 g, 97%) was used for the next step without further purification. Step 4: (S)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(octanoate) (8) (R)-8-di(heptadecane-9-yl)O '1 O 1 A solution of 3-hydroxypropane-1,2-diyl)bis(octanoate)6 (2.24 g, 2.5 mmol) and triethylamine (0.40 mL, 2.86 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 7 (408 mg, 2.86 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 5: 8-Di(heptadecane-9-yl)O' 1 O 1 Synthesis of -((2S)-3-((hydroxy(2-((3-hydroxypropyl)(methyl)amino)ethoxy)phosphoyl)oxy)propane-1,2-diyl)di(octanoate) (compound 28) To crude (S)-di(heptadecane-9-yl)O '1 O 13-((2-oxaphosphazenecyclopropane-2-yl)oxy)propane-1,2-diyl)di(octanoate)8 (2.5 mmol) was added to a solution of 3 mL THF and 15 mL acetonitrile, followed by the addition of 4.1 g (20 mmol) of 3-((tert-butyldimethylsilyl)oxy)-N-methylprop-1-amine 9. The resulting mixture was heated overnight in a sealed tube at 85°C–90°C. MS showed the formation of the desired product. After cooling to room temperature and concentration, the residue was dissolved in 25 mL THF and transferred to a polytetrafluoroethylene round-bottom flask. 10 mL of pyridine and 5 mL of a pyridine hydrofluoride complex were added, and the mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 and extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32:13:1) to give the desired product (1.03 g, 37%) as a grayish-white waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.26–5.16 (m, 1H), 4.83 (quintet, 2H), 4.43–4.31 (m, 1H), 4.29–4.18 (m, 2H), 4.18–4.06 (m, 1H), 4.03–3.92 (m, 2H), 3.80–3.67 (m, 2H), 3.37–3.14 (m, 4H), 2.81 (s, 3H), 2.38–2.17 (m, 8H), 2.02–1.88 (m, 2H), 1.69–1.41 (m, 16H), 1.37–1.13 (m, 58H), 0.85 (t, 12H). APCI-MS analysis: C59H114NO13P [M+H] calculated value = 1076.8, observed value = 1076.8. 8-Di(heptadecane-9-yl)O' 1 O 1 Synthesis of -((2S)-3-((hydroxy(2-((2-hydroxyethyl)(methyl)amino)ethoxy)phosphoyl)oxy)propane-1,2-diyl)di(octanoate) (compound 26) To crude (S)-di(heptadecane-9-yl)O '1 O 13.4 mmol of 2-((tert-butyldimethylsilyl)oxy)propane-1,2-diyl)bis(octanoate)8 was added to a solution of 3-(3-(2-oxo-1,3,2-dioxaphosphazenecyclopropane-2-yl)oxy)propane-1,2-diyl)bis(octanoate)8 in 5 mL THF and 20 mL acetonitrile, and the resulting mixture was heated overnight in a sealed tube at 85°C–90°C. MS showed the formation of the desired product. After cooling to room temperature and concentrating, the residue was dissolved in 50 mL THF and transferred to a polytetrafluoroethylene round-bottom flask, 8 mL of pyridine and 5 mL of a pyridine hydrofluoride complex were added, and the mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 and extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32:13:1) to give the desired product (1.05 g, 29%) as a pale yellow waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.27–5.15 (m, 1H), 4.83 (quintet, 2H), 4.45–4.06 (m, 4H), 4.04–3.82 (m, 4H), 3.43–3.07 (m, 2H), 2.88 (s, 3H), 2.37–2.16 (m, 8H), 1.69–1.40 (m, 16H), 1.38–1.12 (m, 60H), 0.85 (t, 12H). API-MS analysis: C58H112NO13P [M+H] calculated value = 1062.8, observed value = 1062.9. Synthetic scheme of compound 40 Synthesis procedure for compound 40: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 1 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 2 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: (R)-O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 4 (3.8 g, 20.9 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (19.5 g, 47.3 mmol), EDCI (27.2 g, 142 mmol), and DMAP (5.8 g, 47.5 mmol) in 300 mL of dichloromethane was stirred at room temperature for 15 h. TLC showed the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce a colorless oil (R)-O' 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (18.2 g, 90%). Step 3: (R)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) (R)-O '1 O 1A mixture of 3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)5 (6.3 g, 6.5 mmol) and 20% Pd(OH)2 / carbon (1 g) in 100 mL of ethyl acetate was hydrogenated using a Pal hydrogenator at 35–40 psi for 12 h. After filtration through silica gel and concentration, the product (5.6 g, 97%) was used for the next step without further purification. Step 4: (S)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(octanoate) (8) (R)-8-di(heptadecane-9-yl)O '1 O 1 A solution of 3-hydroxypropane-1,2-diyl)bis(octanoate)6 (2.5 g, 2.84 mmol) and triethylamine (788 mg, 5.68 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 7 (809 mg, 5.68 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 5: Synthesis of (S)-2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-((2-hydroxyethyl)dimethylammonium)ethyl)phosphate (compound 40) To crude (S)-di(heptadecane-9-yl)O '1 O 12-((tert-butyldimethylsilyl)oxy)propane-1,2-diyl)bis(octanoate)8 (2.84 mmol) was reacted with 2-((tert-butyldimethylsilyl)oxy)-N,N-dimethylethyl-1-amine9 (4 g, 19.7 mmol) in a solution of 5 mL THF and 10 mL acetonitrile, and the resulting mixture was heated in a sealed tube at 85°C–90°C for 3 days. MS showed the formation of the desired product. After cooling to room temperature and concentration, the residue was dissolved in 50 mL THF and transferred to a polytetrafluoroethylene round-bottom flask, 7 mL of pyridine and 5 mL of a pyridine hydrofluoride complex were added, and the mixture was stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 and extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O32 : 13 : 1) to give the desired product (100 mg, 6%) as a pale yellow waxy substance. 1 ¹H NMR (400 MHz, CDCl₃) δ 6.68–6.33 (m, 1H), 4.82 (quintet, 2H), 4.58–3.46 (m, 12H), 3.31 (s, 6H), 2.43–2.09 (m, 8H), 1.67–1.39 (m, 16H), 1.37–1.10 (m, 56H), 0.85 (t, 12H). ESI-MS analysis: C59H114NO13P [M+H] calculated value = 1076.8, observed values ​​= 1076.8, 1098.8 (M+Na). HPLC-ELSD: t R = 6.539 min (Method 1). Synthesis scheme of compound 41 Synthesis procedure for compound 41: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 1 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 2 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: (S)-O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A mixture of (R)-3-(benzyloxy)propane-1,2-diol 4 (3.46 g, 19 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (17.6 g, 42.75 mmol), EDCI (19.2 g, 100 mmol), and DMAP (5.2 g, 42.75 mmol) in 150 mL of dichloromethane was stirred at room temperature for 2 days. TLC showed that the reaction was complete. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce (S)-O' as a colorless oil. 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (17.4 g, 94%). Step 3: (S)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) (S)-O '1 O 1A mixture of 17.4 g (17.8 mmol) and 20% Pd(OH)₂ / carbon (4 g) in 200 mL of ethyl acetate was hydrogenated using a Pal hydrogenator at 35–40 psi for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (15.7 g, 99%). Step 4: (R)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(octanoate) (8) (S)-8-di(heptadecane-9-yl)O '1 O 1 A solution of 15.7 g (17.8 mmol) of 3-hydroxypropane-1,2-diyl)bis(octanoate)6 and triethylamine (3.5 mL, 25.45 mmol) in 50 mL of THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 7 (3.6 g, 25.45 mmol) in 10 mL of THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 5: Synthesis of (R)-2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 41) In a sealed tube, (R)-8-di(heptadecane-9-yl)O' 1 O 1A solution of -(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)di(octanoate)8 (crude) in 60 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (1 M in THF, 56 mL, 56 mmol) was added. The flask was sealed and heated at 65°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O32 : 13 : 0.4) to give the desired product (5.5 g, 30%) as a white solid. 1 ¹H NMR (400 MHz, CDCl₃) δ 5.25–5.11 (m, 1H), 4.83 (quintet, 2H), 4.43–4.25 (m, 3H), 4.16–4.04 (m, 1H), 4.00–3.76 (m, 4H), 3.36 (s, 9H), 2.36–2.18 (m, 8H), 1.69–1.39 (m, 16H), 1.38–1.08 (m, 56H), 0.85 (t, 12H). ESI-MS analysis: C58H112NO12P [M+H] calculated value = 1046.8, observed value = 1046.8. HPLC-ELSD: t R = 6.762 min (Method 1). Synthesis scheme of compound 42 Synthesis procedure for compound 42: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 1 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 2 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: (R)-O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 4 (4.7 g, 25.7 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (23.8 g, 58 mmol), EDCI (26 g, 135 mmol), and DMAP (7.0 g, 58 mmol) in 200 mL of dichloromethane was stirred at room temperature for 15 h. TLC showed the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce a colorless oil (R)-O' 1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (24.5 g, 98%). Step 3: (R)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) (R)-O '1 O 1A mixture of 12 g (12.3 mmol) of 3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)5 (20% Pd(OH)2 / carbon) in 80 mL of ethyl acetate was hydrogenated at 35–40 psi for 12 h using a Pal hydrogenator. After filtration through silica gel and concentration, the product was used for the next step without further purification (10.8 g, quantified). Step 4: (S)-8-bis(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-((2-Oxy-1,3,2-dioxaphosphane-2-yl)oxy)propane-1,2-diyl)di(octanoate) (8) (R)-8-di(heptadecane-9-yl)O '1 O 1 A solution of 3-hydroxypropane-1,2-diyl)bis(octanoate)6 (4.4 g, 5 mmol) and triethylamine (1.12 mL, 8 mmol) in 20 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 7 (1.14 mg, 8 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 5: Synthesis of (S)-2,3-bis((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 42) In a sealed tube, (S)-8-bis(heptadecane-9-yl)O' 1 O 1A solution of 3-(((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)bis(octanoate)8 (crude, 5 mmol) in 50 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 30 mL, 60 mmol) was added. The flask was sealed and heated at 85°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (3.0 g, 57%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 5.28-5.10 (m, 1H), 4.92-4.76 (m, 2H), 4.46-4.24 (m, 3H), 4.18-4.07 (m, 1H), 4.04-3.76 (m, 4H), 3.38 (s, 9H), 2.54-2.00(m, 8H), 1.70-1.42 (m, 12H), 1.39-1.11 (m, 60H), 0.86 (t, 12H). ESI-MS analysis: C58H112NO12P [M+H] calculated value = 1046.8, observed values ​​= 1046.7, 1069.8 (M+Na). HPLC-ELSD: t R = 6.780 min (Method 1). Synthesis scheme of compound 44 Synthesis procedure for compound 44: Step 1: Synthesis of 10-(heptadecano-9-yloxy)-10-oxodecanoic acid (3) DMAP (5.2 g, 43.4 mmol) and EDCI (18 g, 95.5 mmol) were added to a homogenized solution of succinic acid 2 (26.3 g, 130 mmol) in 150 mL DMF:DCM (1:2, v / v) and the mixture was stirred at room temperature for 10 min. Then, a homogenized solution of 9-heptadecanool 1 (11 g, 43.4 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 10-(heptadecano-9-yloxy)-10-oxodecanoic acid (14.5 g, 76%) as a colorless oil. Step 2: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (5) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 4 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 3: Synthesis of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecane-9-yl)octanoate (7) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 6 (1.1 g, 6 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 5 (1.65 g, 4 mmol), EDCI (1.15 g, 6 mmol), and DMAP (488 mg, 4 mmol) in 20 mL of dichloromethane was stirred at room temperature for 15 h. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–40% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecane-9-yl)octanoic acid ester (635 mg, 27%) as a colorless oil. Step 4: Synthesis of (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)10-(heptadecano-9-yl) sebacate (8) A mixture of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecyl-9-yl)octanoate 7 (750 mg, 1.3 mmol), 10-(heptadecyl-9-yloxy)-10-oxodecanoic acid 3 (860 mg, 1.95 mmol), EDCI (591 mg, 4 mmol), and DMAP (244 mg, 2 mmol) in 40 mL of dichloromethane was stirred at room temperature for 15 h. TLC showed the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%-15% ethyl acetate in hexane to produce (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)10-(heptadecano-9-yl) sebacate (1.2 g, 92%), which is a colorless oil. Step 5: Synthesis of (R)-1-(heptadecano-9-yl)10-(1-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl-2-yl) sebacate (9) A mixture of (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl-2-yl)10-(heptadecano-9-yl) sebacate 8 (1.1 g, 1.1 mmol) and 10% palladium / carbon (500 mg) in 20 mL of ethyl acetate was hydrogenated using a pneumatic bladder for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (1.0 g, quantification). Step 6: Synthesis of (S)-1-(heptadecano-9-yl)10-(1-((8-(heptadecano-9-yloxy)-8-oxo-octanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)prop-2-yl) sebacate (11) A solution of (R)-1-(heptadecano-9-yl)10-(1-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl-2-yl) sebacate 9 (910 mg, 1 mmol) and triethylamine (0.21 mL, 1.5 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 10 (214 mg, 1.5 mmol) in 1 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-2-((10-(heptadecano-9-yloxy)-10-oxodecanoyl)oxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 44) In a sealed tube, (S)-8-bis(heptadecane-9-yl)O' 1 O 1 A solution of -(3-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)di(octanoate)11 (crude product) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 5 mL, 10 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (320 mg, 30%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 5.25-5.11 (m, 1H), 4.90-4.77 (m, 2H), 4.43-4.24 (m, 3H), 4.18-4.04 (m, 1H), 4.00-3.86 (m, 2H), 3.84-3.72 (m, 2H), 3.34(s, 9H), 2.35-2.18 (m, 8H), 1.69-1.40 (m, 16H), 1.37-1.11 (m, 60H), 0.86 (t,12H). ESI-MS analysis: C60H116NO12P [M+H] calculated value = 1074.8, observed values ​​= 1074.8, 1096.8 (M+Na). HPLC-ELSD: t R = 6.906 min (Method 1). Synthetic scheme of compound 46 Synthetic procedure for compound 46: Step 1: Synthesis of 9-(heptadecane-9-yloxy)-9-oxonanoic acid (3) DMAP (0.26 g, 2.15 mmol) and EDCI (9.8 g, 51.5 mmol) were added to a homogenized solution of azelaic acid 2 (18 g, 94.4 mmol) in 100 mL DMF:DCM (1:1, v / v) and the mixture was stirred at room temperature for 10 min. Then, a homogenized solution of 9-heptadecanool 1 (11 g, 43.4 mmol) in 50 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 9-(heptadecano-9-yloxy)-9-oxononanoic acid (12.5 g, 70%) as a colorless oil. Step 2: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (5) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 4 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 3: Synthesis of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecane-9-yl)octanoate (7) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 6 (1.1 g, 6 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 5 (1.65 g, 4 mmol), EDCI (1.15 g, 6 mmol), and DMAP (488 mg, 4 mmol) in 20 mL of dichloromethane was stirred at room temperature for 15 h. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–40% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecane-9-yl)octanoic acid ester (635 mg, 27%) as a colorless oil. Step 4: Synthesis of (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)9-(heptadecano-9-yl)azelate (8) A mixture of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)8-(heptadecyl-9-yl)octanoate 7 (635 mg, 1.1 mmol), 10-(heptadecyl-9-yloxy)-10-oxodecanoic acid 3 (640 mg, 1.5 mmol), EDCI (576 mg, 3 mmol), and DMAP (183 mg, 1.5 mmol) in 40 mL of dichloromethane was stirred at room temperature for 2 days. TLC showed that the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%-15% ethyl acetate in hexane to produce (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)9-(heptadecano-9-yl)azelate (980 mg, 92%), which is a colorless oil. Step 5: Synthesis of (R)-1-(heptadecano-9-yl)9-(1-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl-2-yl)azelate (9) A mixture of (R)-1-(1-(benzyloxy)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)prop-2-yl)9-(heptadecano-9-yl)azelate 8 (900 mg, 0.91 mmol) and 10% palladium / carbon (500 mg) in 50 mL of ethyl acetate was hydrogenated using a Pal hydrogenator at 35–40 psi for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (893 mg, quantified). Step 6: Synthesis of (S)-1-(heptadecano-9-yl)9-(1-((8-(heptadecano-9-yloxy)-8-oxo-octanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)prop-2-yl)azelate (11) A solution of (R)-1-(heptadecano-9-yl)-9-(1-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl-2-yl)azelate 9 (893 mg, 0.91 mmol) and triethylamine (0.19 mL, 1.37 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 10 (194 mg, 1.37 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-3-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-2-((9-(heptadecano-9-yloxy)-9-oxononanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 46) In a sealed tube, a solution of (S)-1-(heptadecano-9-yl)9-(1-((8-(heptadecano-9-yloxy)-8-oxo-octanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)prop-2-yl)azelate 11 (crude) in 12 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 5.0 mL, 10 mmol) was added. The flask was sealed and heated at 85°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (530 mg, 50%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.25-5.10 (m, 1H), 4.90-4.75 (m, 2H), 4.45-4.22 (m, 3H), 4.18-4.04 (m, 1H), 4.00-3.84 (m, 2H), 3.83-3.70 (m, 2H), 3.34(s, 9H), 2.34-2.16 (m, 8H), 1.70-1.41 (m, 16H), 1.39-1.08 (m, 58H), 0.85 (t,12H). ESI-MS analysis: C59H114NO12P [M+H] calculated value = 1060.8, observed values ​​= 1060.8, 1082.8 (M+Na). HPLC-ELSD: t R = 6.845 min (Method 1). Synthesis scheme of compound 48 Synthesis procedure for compound 48: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 2 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: Synthesis of 6-(heptadecane-9-yloxy)-6-oxohexanoic acid (5) DMAP (5.2 g, 43.4 mmol) and EDCI (18 g, 95.4 mmol) were added to a homogenized solution of adipic acid 4 (19 g, 130 mmol) in 100 mL DMF:DCM (1:1, v / v) and the mixture was stirred at room temperature for 10 minutes. Then, a homogenized solution of 9-heptadecanool 1 (11 g, 43.4 mmol) in 50 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 2 days. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 6-(heptadecano-9-yloxy)-6-oxohexanoic acid (10.3 g, 62%) as a colorless oil. Step 3: Synthesis of (R)-3-(benzyloxy)-2-hydroxypropylheptadecane-9-yl adipate (7) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 6 (1.1 g, 6 mmol), 6-(heptadecane-9-yloxy)-6-oxohexanoic acid 5 (1.5 g, 4 mmol), EDCI (2.2 g, 12 mmol), and DMAP (488 mg, 4 mmol) in 30 mL of dichloromethane was stirred at room temperature for 15 h. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–40% ethyl acetate in hexane to produce (R)-3-(benzyloxy)-2-hydroxypropylheptadecane-9-yl adipate (620 mg, 26%) as a colorless oil. Step 4: Synthesis of (R)-1-(1-(benzyloxy)-3-((6-(heptadecano-9-yloxy)-6-oxohexanoyl)oxy)prop-2-yl)8-(heptadecano-9-yl)octanoate (8) A mixture of (R)-3-(benzyloxy)-2-hydroxypropylheptadecane-9-yl adipate 7 (620 mg, 1.13 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (700 mg, 1.7 mmol), EDCI (326 mg, 1.7 mmol), and DMAP (138 mg, 1.13 mmol) in 40 mL of dichloromethane was stirred overnight at room temperature. TLC showed that the reaction was complete. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce (R)-1-(1-(benzyloxy)-3-((6-(heptadecane-9-yloxy)-6-oxohexanoyl)oxy)propyl-2-yl)8-(heptadecane-9-yl)octanoic acid ester (840 mg, 79%) as a colorless oil. Step 5: Synthesis of (R)-1-(heptadecano-9-yl)8-(1-((6-(heptadecano-9-yloxy)-6-oxohexanoyl)oxy)-3-hydroxypropyl-2-yl)octanoate (9) A mixture of (R)-1-(1-(benzyloxy)-3-((6-(heptadecano-9-yloxy)-6-oxohexanoyl)oxy)propyl-2-yl)8-(heptadecano-9-yl)octanoate 8 (840 mg, 0.89 mmol) and 10% palladium / carbon (500 mg) in 40 mL of ethyl acetate was hydrogenated in a pneumatic chamber for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (750 mg, 98%). Step 6: Synthesis of (S)-1-(heptadecano-9-yl)8-(1-((6-(heptadecano-9-yloxy)-6-oxo-hexanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)prop-2-yl)octanoate (11) A solution of (R)-1-(heptadecano-9-yl)8-(1-((6-(heptadecano-9-yloxy)-6-oxohexanoyl)oxy)-3-hydroxypropyl-2-yl)octanoate 9 (750 mg, 0.88 mmol) and triethylamine (0.18 mL, 1.32 mmol) in 5 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 10 (188 mg, 1.32 mmol) in 1 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-3-((6-(heptadecano-9-yloxy)-6-oxohexanoyl)oxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 48) In a sealed tube, a solution of (S)-1-(heptadecano-9-yl)8-(1-((6-(heptadecano-9-yloxy)-6-oxo-hexanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)prop-2-yl)octanoate 11 (crude) in 10 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 5.0 mL, 10 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (625 mg, 69%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.25-5.12 (m, 1H), 4.89-4.74 (m, 2H), 4.44-4.24 (m, 3H), 4.19-4.05 (m, 1H), 4.00-3.88 (m, 2H), 3.84-3.74 (m, 2H), 3.35 (s, 9H), 2.36-2.16 (m, 8H), 1.69-1.39 (m, 16H), 1.36-1.12 (m, 52H), 0.85 (t,12H). ESI-MS analysis: C56H108NO12P [M+H] calculated value = 1018.8, observed value = 1018.8, 1040.7 (M+Na). HPLC-ELSD: t R = 7.626 min (Method 1). Synthesis scheme of compound 50 Synthesis procedure for compound 50: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 2 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: Synthesis of 9-(heptadecane-9-yloxy)-9-oxonanoic acid (5) DMAP (0.26 g, 2.15 mmol) and EDCI (9.8 g, 51.5 mmol) were added to a homogenized solution of azelaic acid 4 (18 g, 94.4 mmol) in 100 mL DMF:DCM (1:1, v / v) and the mixture was stirred at room temperature for 10 min. Then, a homogenized solution of 9-heptadecanool 1 (11 g, 43.4 mmol) in 50 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 9-(heptadecano-9-yloxy)-9-oxononanoic acid (12.5 g, 70%) as a colorless oil. Step 3: Synthesis of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)9-(heptadecane-9-yl)azelate (7) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 6 (1.0 g, 6 mmol), 9-(heptadecane-9-yloxy)-9-oxononanoic acid 5 (1.7 g, 4 mmol), EDCI (2.2 g, 12 mmol), and DMAP (0.48 g, 4 mmol) in 30 mL of dichloromethane was stirred at room temperature for 15 h. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–40% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-hydroxypropyl)9-(heptadecane-9-yl)azelate (680 mg, 29%) as a colorless oil. Step 4: Synthesis of (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)9-(heptadecano-9-yl)azelate (8) A mixture of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)9-(heptadecano-9-yl)azelate 7 (680 mg, 1.15 mmol), 8-(heptadecano-9-yloxy)-8-oxooctanoic acid 3 (712 mg, 1.73 mmol), EDCI (1.2 g, 6 mmol), and DMAP (366 mg, 3 mmol) in 40 mL of dichloromethane was stirred overnight at room temperature. TLC showed that the reaction was complete. After concentration, the crude product was ground with hexane and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)9-(heptadecano-9-yl)azelate (830 mg, 81%) as a colorless oil. Step 5: Synthesis of (R)-1-(heptadecano-9-yl)9-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl)azelate (9) A mixture of (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)9-(heptadecano-9-yl)azelate 8 (830 mg, 0.87 mmol) and 10% palladium / carbon (500 mg) in 30 mL of ethyl acetate was hydrogenated using a pneumatic tube for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (750 mg, quantified). Step 6: Synthesis of (S)-1-(heptadecano-9-yl)9-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)propyl)azelate (11) A solution of (R)-1-(heptadecano-9-yl)-9-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl)azelate 9 (750 mg, 0.87 mmol) and triethylamine (178 mg, 1.74 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 10 (248 mg, 1.74 mmol) in 1 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-2-((8-(heptadecane-9-yloxy)-8-oxooctanoyl)oxy)-3-((9-(heptadecane-9-yloxy)-9-oxononanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 50) In a sealed tube, a solution of (S)-1-(heptadecano-9-yl)9-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)propyl)azelate 11 (crude) in 15 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 5.0 mL, 10.0 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (60 mg, 6%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.26-5.09 (m, 1H), 4.90-4.74 (m, 2H), 4.44-4.25 (m, 3H), 4.24-4.03 (m, 1H), 4.01-3.85 (m, 2H), 3.85-3.66 (m, 2H), 3.32(s, 9H), 2.34-2.15 (m, 8H), 1.68-1.39 (m, 16H), 1.37-1.07 (m, 58H), 0.85 (t,12H). ESI-MS analysis: C59H114NO12P [M+H] calculated value = 1060.8, observed values ​​= 1060.9, 1082.8 (M+Na). HPLC-ELSD: t R = 6.516 min (Method 1). Synthesis scheme of compound 52 Synthesis procedure for compound 52: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 2 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: Synthesis of 10-(heptadecano-9-yloxy)-10-oxodecanoic acid (5) DMAP (5.2 g, 43.4 mmol) and EDCI (18 g, 95.5 mmol) were added to a homogenized solution of succinic acid 4 (26.3 g, 130 mmol) in 150 mL DMF:DCM (1:1, v / v) and the mixture was stirred at room temperature for 10 min. Then, a homogenized solution of 9-heptadecanool 1 (11 g, 43.4 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 10-(heptadecano-9-yloxy)-10-oxodecanoic acid (14.5 g, 76%) as a colorless oil. Step 3: Synthesis of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)10-(heptadecane-9-yl) sebacate (7) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 6 (1.0 g, 6 mmol), 10-(heptadecano-9-yloxy)-10-oxodecanoic acid 5 (1.76 g, 4 mmol), EDCI (2.2 g, 12 mmol), and DMAP (0.48 g, 4 mmol) in 30 mL of dichloromethane was stirred at room temperature for 15 h. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–40% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-hydroxypropyl)10-(heptadecano-9-yl) sebacate (1.0 g, 41%) as a colorless oil. Step 4: Synthesis of (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)10-(heptadecano-9-yl) sebacate (8) A mixture of (R)-1-(3-(benzyloxy)-2-hydroxypropyl)10-(heptadecane-9-yl) sebacate 7 (980 mg, 1.62 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid 3 (1.25 g, 3.0 mmol), EDCI (0.6 g, 3 mmol), and DMAP (183 mg, 1.5 mmol) in 40 mL of dichloromethane was stirred overnight at room temperature. TLC showed that the reaction was complete. After concentration, the crude product was ground with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%-15% ethyl acetate in hexane to produce (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)10-(heptadecano-9-yl) sebacate (1.55 g, 90%), which is a colorless oil. Step 5: Synthesis of (R)-1-(heptadecano-9-yl)10-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl) sebacate (9) A mixture of (R)-1-(3-(benzyloxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl)10-(heptadecano-9-yl) sebacate 8 (1.55 g, 1.54 mmol) and 10% palladium / carbon (500 mg) in 30 mL of ethyl acetate was hydrogenated using a pneumatic tube for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (1.4 g, quantification). Step 6: Synthesis of (S)-1-(heptadecano-9-yl)10-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)propyl) sebacate (11) A solution of (R)-1-(heptadecano-9-yl)10-(2-(((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-hydroxypropyl) sebacate 9 (1.36 g, 1.5 mmol) and triethylamine (0.42 mL, 3 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 10 (428 mg, 3 mmol) in 1 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-3-((10-(heptadecano-9-yloxy)-10-oxodecanoyl)oxy)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 52) In a sealed tube, a solution of (S)-1-(heptadecano-9-yl)10-(2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-((2-oxaphosphacyclopropane-2-yl)oxy)propyl)sepiacetate 11 (crude) in 15 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 7.0 mL, 14.0 mmol) was added. The flask was sealed and heated at 95°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (765 mg, 49%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.26-5.12 (m, 1H), 4.90-4.76 (m, 2H), 4.43-4.26 (m, 3H), 4.18-4.04 (m, 1H), 4.02-3.88 (m, 2H), 3.87-3.76 (m, 2H), 3.36 (s, 9H), 2.35-2.17 (m, 8H), 1.67-1.39 (m, 16H), 1.36-1.10 (m, 60H), 0.86 (t,12H). ESI-MS analysis: C60H116NO12P [M+H] calculated value = 1074.8, observed value = 1074.7. HPLC-ELSD: t R = 7.496 min (Method 1). Synthesis scheme of compound 54 Synthesis procedure for compound 54: Step 1: Synthesis of 2-octyldecanoic acid (2) A suspension of sodium hydride (60% in mineral oil, 2.56 g, 64 mmol) in 300 mL THF was cooled to 0°C, and a solution of 1-decanoic acid (10 g, 58 mmol) in 100 mL THF was slowly added, followed by a solution of lithium diisopropylamino (2 M in THF, 34.8 mL, 69.6 mmol). The reaction mixture was heated to room temperature for 30 min. After the addition of 1-octyl iodide (16.7 g, 69.6 mmol), the resulting mixture was heated to reflux overnight. The reaction was cooled to room temperature and adjusted to pH 2 with 4 N HCl, followed by extraction with ethyl acetate. The combined organic layers were concentrated and purified by column chromatography (SiO2: 0% to 40% ethyl acetate in hexane) to give 2-octyldecanoic acid (7.4 g, 45%) as a white solid. Step 2: Synthesis of 6-hydroxyhexyl-2-octyldecanoate (4) A mixture of 2-octyldecanoic acid 2 (3.0 g, 10.5 mmol), hexane-1,6-diol 3 (2.5 g, 21 mmol), EDCI (4.0 g, 21 mmol), and DMAP (1.28 g, 10.5 mmol) in 150 mL of dichloromethane was stirred at room temperature for 3 days. The clear solution was diluted with water and extracted with dichloromethane. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 40% ethyl acetate in hexane) to give 6-hydroxyhexyl-2-octyldecanoate (3.4 g, 68%) as a colorless oil. Step 3: Synthesis of 6-((2-octyldecanoyl)oxy)hexanoic acid (5) Jones' reagent was added to a solution of 6-hydroxyhexyl-2-octyldecanoate 4 (3.7 g, 9.6 mmol) in 50 mL of acetone until the orange color persisted, and the resulting mixture was stirred for 30 min. Excess Jones' reagent was consumed by adding a few drops of 2-propanol, and the blue solution was then diluted with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine, dried, and concentrated to give the desired product (3.77 g, quantified), which was used for the next step without further purification. Step 4: Synthesis of (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (7) A solution of 6-((2-octyldecanoyl)oxy)hexanoic acid 5 (3.6 g, 9 mmol), (S)-3-(benzyloxy)propane-1,2-diol 6 (730 mg, 4 mmol), EDCI (4.0 g, 21 mmol), and DMAP (1.0 g, 9 mmol) in 60 mL of dichloromethane was stirred at room temperature for 48 h. After concentration, the residue was partitioned between a saturated ammonium chloride solution and ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (3.12 g, 79%) as a colorless oil. Step 5: Synthesis of (R)-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (8) In a Parr reactor, a mixture of (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate)7 (1.0 g, 1.06 mmol) and 10% palladium / carbon (700 mg) in 60 mL of ethyl acetate was hydrogenated at 40 psi for 16 h. After filtration and concentration, (R)-9-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (937 mg, quantified) was obtained as a colorless oil. Step 6: Synthesis of (S)-((3-((2-oxo-1,3,2-dioxaphosphane propane-2-yl)oxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate) (10) A solution of (R)-9-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate)8 (900 mg, 1.06 mmol) and triethylamine (0.29 mL, 2.12 mmol) in 10 mL THF was cooled to 0°C, and then a solution of 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane9 (0.30 g, 2.12 mmol) in 2 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-2,3-bis((6-((2-octyldecanoyl)oxy)hexanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 54) In a sealed tube, a solution of (S)-((3-((2-oxaphosphacyclopropane-2-yl)oxy)propane-1,2-diyl)bis(oxy))bis(6-oxohexane-6,1-diyl)bis(2-octyldecanoate)10 (crude) in 6 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 6.0 mL, 12 mmol) was added. The flask was sealed and heated at 95°C for 3 days. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (611 mg, 54%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 5.30-5.05 (m, 1H), 4.45-4.28 (m, 3H), 4.15-4.08 (m, 1H), 4.03 (t, 4H), 3.99-3.90 (m, 2H), 3.86-3.77 (m, 2H), 3.36 (s,9H), 2.42-2.18 (m, 6H), 1.70-1.49 (m, 12H), 1.47-1.32 (m, 8H), 1.31-1.12 (m,48H), 0.86 (t, 12H). 31 P NMR (162 MHz, CDCl3) δ -0.24. ESI-MS analysis: C50H108NO12P [M+H] calculated value = 1018.8, observed value = 1018.6. HPLC-ELSD: t R = 6.851 min (Method 1). Synthesis scheme of compound 56 Synthesis procedure for compound 56: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 4 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: Synthesis of (R)-3-(benzyloxy)-2-hydroxypropyl palmitate (6) A mixture of (S)-3-(benzyloxy)propane-1,2-diol 5 (1.0 g, 6 mmol) and 2,4,6-trimethylpyridine (2.6 mL, 20 mmol) in 50 mL of dichloromethane was cooled to 0°C, and palmitoyl chloride 4 (1.2 mL, 4 mmol) was added dropwise. The reaction mixture was then stirred at this temperature for 2 h. The mixture was diluted with dichloromethane and washed with 1 N HCl, water, and brine. The combined organic layers were dried over sodium sulfate. After filtration and concentration, the crude product was purified by column chromatography, eluting with 0%–20% ethyl acetate in hexane to produce (R)-3-(benzyloxy)-2-hydroxypropyl palmitate (1.38 g, 82%) as a colorless oil. Step 3: Synthesis of (R)-1-(1-(benzyloxy)-3-(palmitoyloxy)prop-2-yl)8-(heptadecyl-9-yl)octanoate (7) A mixture of (R)-3-(benzyloxy)-2-hydroxypropyl palmitate 6 (840 mg, 2.0 mmol), 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid 3 (1.25 g, 3.0 mmol), EDCI (0.6 g, 3 mmol), and DMAP (183 mg, 1.5 mmol) in 25 mL of dichloromethane was stirred overnight at room temperature. TLC showed that the reaction was complete. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce (R)-1-(1-(benzyloxy)-3-(palmitoyloxy)propyl-2-yl)8-(heptadecyl-9-yl)octanoic acid ester (1.5 g, 93%) as a colorless oil. Step 4: Synthesis of (R)-1-(heptadecano-9-yl)8-(1-hydroxy-3-(palmitoyloxy)prop-2-yl)octanoate (8) A mixture of (R)-1-(1-(benzyloxy)-3-(palmitoyloxy)propyl-2-yl)8-(heptadecane-9-yl)octanoate 7 (1.5 g, 1.8 mmol) and 10% palladium / carbon (600 mg) in 25 mL of ethyl acetate was hydrogenated using a pneumatic tube for 12 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (1.3 g, quantification). Step 5: Synthesis of (S)-1-(heptadecano-9-yl)8-(1-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)-3-(palmitoyloxy)prop-2-yl)octanoate (10) A solution of (R)-1-(heptadecane-9-yl)-8-(1-hydroxy-3-(palmitoyloxy)prop-2-yl)octanoate 8 (1.3 g, 1.8 mmol) and triethylamine (0.42 mL, 3 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 9 (427 mg, 3 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 6: Synthesis of (S)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 56) In a sealed tube, a solution of (S)-1-(heptadecane-9-yl)8-(1-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)-3-(palmitoyloxy)prop-2-yl)octanoate 10 (crude) in 15 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 10 mL, 20 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O32 : 13 : 0.4) to give the desired product (1.13 g, 69%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.24-5.12 (m, 1H), 4.88-4.77 (m, 1H), 4.45-4.22 (m, 3H), 4.16-4.04 (m, 1H), 3.98-3.76 (m, 4H), 3.37 (s, 9H), 2.37-2.18(m, 6H), 1.69-1.42 (m, 8H), 1.36-1.14 (m, 54H), 0.86 (t, 9H). 31 P NMR (162 MHz, CDCl3) δ -0.24. ESI-MS analysis: C49H96NO10P [M+H] calculated value = 890.6, observed value = 890.6, 912.5 (M+Na). HPLC-ELSD: t R = 6.244 min (Method 1). Synthesis scheme of compound 57 Synthesis procedure for compound 57: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) DMAP (7.13 g, 59 mmol) and EDCI (25 g, 130 mmol) were added to a homogeneous solution of octanoic acid 2 (30 g, 172 mmol) in 200 mL DMF:DCM (1:1, v / v) and stirred at room temperature for 10 min. Then, a homogeneous solution of 9-heptadecanool 1 (15 g, 59 mmol) in 100 mL DMF:DCM (1:1, v / v) was added dropwise over an 8-hour period via a feeding funnel. The mixture was stirred at room temperature for 10 h. After removing volatiles under vacuum, the residual DMF solution was partitioned between water and EtOAc. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by column chromatography, eluting with 0%-20% EtOAc in hexane to give 8-(heptadecano-9-yloxy)-8-oxooctanoic acid (19.5 g, 81%) as a colorless oil. Step 2: Synthesis of (S)-3-(benzyloxy)-2-hydroxypropyl palmitate (6) A mixture of (R)-3-(benzyloxy)propane-1,2-diol 5 (1.0 g, 6 mmol) and 2,4,6-trimethylpyridine (2.6 mL, 20 mmol) in 50 mL of dichloromethane was cooled to 0°C, and palmitoyl chloride 4 (1.2 mL, 4 mmol) was added dropwise. The reaction mixture was then stirred at this temperature for 2 h. The mixture was diluted with dichloromethane and washed with 1 N HCl, water, and brine. The combined organic layers were dried over sodium sulfate. After filtration and concentration, the crude product was purified by column chromatography, eluting with 0%–30% ethyl acetate in hexane to produce (S)-3-(benzyloxy)-2-hydroxypropyl palmitate (1.35 g, 81%) as a colorless oil. Step 3: Synthesis of (S)-1-(1-(benzyloxy)-3-(palmitoyloxy)prop-2-yl)8-(heptadecano-9-yl)octanoate (7) A mixture of (S)-3-(benzyloxy)-2-hydroxypropyl palmitate 6 (840 mg, 2.0 mmol), 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid 3 (1.25 g, 3.0 mmol), EDCI (0.6 g, 3 mmol), and DMAP (183 mg, 1.5 mmol) in 25 mL of dichloromethane was stirred overnight at room temperature. TLC showed that the reaction was complete. After concentration, the crude product was ground together with hexane, and the solvent was removed. The crude product was purified by column chromatography, eluting with 0%–15% ethyl acetate in hexane to produce (S)-1-(1-(benzyloxy)-3-(palmitoyloxy)propyl-2-yl)8-(heptadecyl-9-yl)octanoic acid ester (1.5 g, 93%) as a colorless oil. Step 4: Synthesis of (S)-1-(heptadecano-9-yl)8-(1-hydroxy-3-(palmitoyloxy)prop-2-yl)octanoate (8) A mixture of (S)-1-(1-(benzyloxy)-3-(palmitoyloxy)prop-2-yl)8-(heptadecane-9-yl)octanoate 7 (1.5 g, 1.8 mmol) and 10% palladium / carbon (600 mg) in 25 mL of ethyl acetate was hydrogenated at 40 psi for 26 h. After filtration through silica gel and concentration, the product was used for the next step without further purification (1.3 g, quantification). Step 5: Synthesis of (R)-1-(heptadecano-9-yl)8-(1-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)-3-(palmitoyloxy)prop-2-yl)octanoate (10) A solution of (S)-1-(heptadecane-9-yl)-8-(1-hydroxy-3-(palmitoyloxy)prop-2-yl)octanoate 8 (1.3 g, 1.8 mmol) and triethylamine (0.50 mL, 3.6 mmol) in 15 mL THF was cooled to 0°C, and then a solution of 2-chloro-1,3,2-dioxaphosphacyclopropane 2-oxide 9 (513 mg, 3.6 mmol) in 5 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 6: Synthesis of (R)-2-((8-(heptadecano-9-yloxy)-8-oxooctanoyl)oxy)-3-(palmitoyloxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 57) In a sealed tube, a solution of (R)-1-(heptadecyl-9-yl)8-(1-((2-oxo-1,3,2-dioxaphosphacyclopropane-2-yl)oxy)-3-(palmitoyloxy)prop-2-yl)octanoate 10 (crude) in 15 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 10 mL, 20 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O32 : 13 : 0.4) to give the desired product (0.95 g, 59%) as a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 5.23-5.13 (m, 1H), 4.89-4.77 (m, 1H), 4.47-4.26 (m, 3H), 4.16-4.04 (m, 1H), 4.00-3.79 (m, 4H), 3.37 (s, 9H), 2.37-2.16(m, 6H), 1.68-1.42 (m, 8H), 1.37-1.08 (m, 54H), 0.86 (t, 9H). 31 P NMR (162 MHz, CDCl3) δ -0.27. ESI-MS analysis: C49H96NO10P [M+H] calculated value = 890.6, observed values ​​= 890.5, 912.5 (M+Na). HPLC-ELSD: t R = 6.234 min (Method 1). Synthesis scheme of compound 59 Synthesis procedure for compound 59: Step 1: Synthesis of 2-octyldecanoic acid (2) A suspension of sodium hydride (60% in mineral oil, 2.56 g, 64 mmol) in 300 mL THF was cooled to 0°C, and a solution of 1-decanoic acid (10 g, 58 mmol) in 100 mL THF was slowly added, followed by a solution of lithium diisopropylamino (2 M in THF, 34.8 mL, 69.6 mmol). The reaction mixture was heated to room temperature for 30 min. After the addition of 1-octyl iodide (16.7 g, 69.6 mmol), the resulting mixture was heated to reflux overnight. The reaction was cooled to room temperature and adjusted to pH 2 with 4 N HCl, followed by extraction with ethyl acetate. The combined organic layers were concentrated and purified by column chromatography (SiO2: 0% to 40% ethyl acetate in hexane) to give 2-octyldecanoic acid (7.4 g, 45%) as a white solid. Step 2: Synthesis of 7-hydroxyheptyl-2-octyldecanoate (4) A mixture of 2-octyldecanoic acid 2 (4.4 g, 15.5 mmol), heptane-1,7-diol 3 (4.0 g, 31 mmol), EDCI (6.0 g, 31 mmol), and DMAP (1.8 g, 15.5 mmol) in 150 mL of dichloromethane was stirred at room temperature for 3 days. The clear solution was diluted with water and extracted with dichloromethane. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 40% ethyl acetate in hexane) to give 7-hydroxyheptyl-2-octyldecanoate (4.4 g, 72%) as a colorless oil. Step 3: Synthesis of 7-((2-octyldecanoyl)oxy)heptanoic acid (5) Jones' reagent was added to a solution of 4-hydroxyheptyl-2-octyldecanoate 4 (4.4 g, 11 mmol) in 50 mL of acetone until the orange color persisted, and the resulting mixture was stirred for 30 min. Excess Jones' reagent was consumed by adding a few drops of 2-propanol, and the blue solution was then diluted with water (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine, dried, and concentrated to give the desired product (4.53 g, quantified), which was used for the next step without further purification. Step 4: Synthesis of (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (7) A solution of 7-((2-octyldecanoyl)oxy)heptanoic acid 5 (4.5 g, 11 mmol), (S)-3-(benzyloxy)propane-1,2-diol 6 (900 mg, 4.9 mmol), EDCI (4.8 g, 24.5 mmol), and DMAP (1.34 g, 11 mmol) in 60 mL of dichloromethane was stirred at room temperature for 48 h. After concentration, the residue was partitioned between a saturated ammonium chloride solution and ethyl acetate. The combined organic layers were concentrated under vacuum, and the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (3.45 g, 74%) as a colorless oil. Step 5: Synthesis of (R)-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (8) In a Parr reactor, a mixture of (R)-((3-(benzyloxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate)7 (1.23 g, 1.27 mmol) and 10% palladium / carbon (600 mg) in 30 mL of ethyl acetate was hydrogenated by a gasket for 16 h. After filtration and concentration, (R)-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (1.15 g, quantified) was obtained as a colorless oil. Step 6: Synthesis of (S)-((3-((2-oxo-1,3,2-dioxaphosphazenecyclopropane-2-yl)oxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate) (10) A solution of (R)-((3-hydroxypropane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate)8 (1.12 g, 1.27 mmol) and triethylamine (0.45 mL, 3.2 mmol) in 10 mL THF was cooled to 0°C, and then a solution of 2-chloro-2-oxo-1,3,2-dioxaphosphacyclopropane9 (456 mg, 3.2 mmol) in 2 mL THF was added dropwise. The mixture was slowly heated to room temperature and stirred for 15 h. TLC showed that the reaction was complete. The triethylamine hydrochloride formed in the reaction was carefully filtered off under nitrogen, the filtrate was concentrated on a rotary evaporator at 10°C, and the residual oil was dried under high vacuum for 2 h. The crude product was used for the next step without further purification. Step 7: Synthesis of (S)-2,3-bis((7-((2-octyldecanoyl)oxy)heptanoyl)oxy)propyl(2-(trimethylammonium)ethyl)phosphate (compound 59) In a sealed tube, a solution of (S)-((3-((2-oxaphosphazenecyclopropane-2-yl)oxy)propane-1,2-diyl)bis(oxy))bis(7-oxoheptane-7,1-diyl)bis(2-octyldecanoate)10 (crude) in 16 mL of acetonitrile was cooled in an ice bath, and a solution of trimethylamine (2 M in THF, 8.0 mL, 16 mmol) was added. The flask was sealed and heated overnight at 95°C. After cooling to room temperature, the volatiles were evaporated under vacuum, and the crude product was purified by column chromatography (SiO2:CHCl3 / MeOH / H2O 32 : 13 : 0.4) to give the desired product (550 mg, 42%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 5.26-5.14 (m, 1H), 4.45-4.26 (m, 3H), 4.14-4.08 (m, 1H), 4.03 (t, 4H), 3.99-3.89 (m, 2H), 3.85-3.76 (m, 2H), 3.35 (s,9H), 2.46-2.15 (m, 6H), 1.69-1.48 (m, 12H), 1.46-1.14 (m, 60H), 0.86 (t,12H). 31 P NMR (162 MHz, CDCl3) δ -0.16. ESI-MS analysis: C58H112NO12P [M+H] calculated value = 1046.8, observed values ​​= 1046.8, 1068.6 (M+Na). HPLC-ELSD: t R = 6.508 min (Method 1). Synthesis scheme of compound 61 Synthesis procedure for compound 61: Step 1: Synthesis of 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (3) A mixture of octanedioic acid 6 (22.0 g, 0.126 mol), 1-octylnonanol 14 (16.2 g, 63.1 mmol), EDCI (24.0 g, 0.126 mol), and DMAP (771 mg, 6.3 mmol) in 400 mL of dichloromethane was stirred overnight at room temperature. TLC showed the presence of ethanol. The reaction mixture was diluted with water, extracted with dichloromethane, and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the residue was purified by rapid column chromatography (SiO2: 0% to 40% EtOAc in hexane) to give 8-(heptadecan-9-yloxy)-8-oxooctanoic acid (13.2 g, 50%) as a colorless oil. Step 2: O '1 O 1 Synthesis of -(3-(benzyloxy)propane-1,2-diyl)8-bis(heptadecane-9-yl)bis(octanoate) (5) A solution of 8-(heptadecano-9-yloxy)-8-oxooctanoic acid 3 (13.2 g, 32 mmol), 3-(benzyloxy)propane-1,2-diol 4 (2.65 g, 14.5 mmol), EDCI (6.17 g, 32 mmol), and DMAP (0.39 g, 3.2 mmol) in 200 mL of dichloromethane was stirred overnight at room temperature. TLC showed the desired product accompanied by a monoester intermediate. The reaction mixture was diluted with water, extracted with dichloromethane, and the combined organic layers were washed with brine. After drying and concentration with sodium sulfate, the crude product was purified by rapid column chromatography (SiO2: 0% to 20% ethyl acetate in hexane) to give an O2- as a colorless oil. '1 O 1 -(3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (9.55 g, 67%). Step 3: 8-Di(heptadecane-9-yl)O '1 O 1 Synthesis of -(3-hydroxypropane-1,2-diyl)bis(octanoate) (6) O '1 O 1 A mixture of 16 g (16.4 mmol) of 3-(benzyloxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)5 and 10% Pd / C (1.6 g) in 100 mL of ethyl acetate was purged three times with nitrogen and three times with hydrogen, respectively. The reaction was then stirred at room temperature under a hydrogen gas chamber for 16 h. After filtration and concentration, 8-di(heptadecane-9-yl)O was obtained as a colorless oil. '1 O 1 -(3-hydroxypropane-1,2-diyl)bis(octanoate) (13.67 g, 94%). Step 4: O' 1 O 1 Synthesis of -(3-(((3-bromopropoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (8) At 0°C, towards 8-di(heptadecano-9-yl)O' 1 O 1POCl3 (123 mg, 0.8 mmol) was slowly added to a solution of 3-(3-hydroxypropane-1,2-diyl)bis(octanoate)6 (500 mg, 0.51 mmol) and triethylamine (0.11 mL, 0.8 mmol) in 8 mL of diethyl ether, and the resulting mixture was stirred at room temperature for 2 h. TLC showed the disappearance of ethanol. The volatiles were evaporated under vacuum, and the residue was redissolved in 5 mL of toluene. After cooling to 0°C, a solution of 3-bromoprop-1-ol7 (88 mg, 0.63 mmol) and triethylamine (0.11 mL, 0.8 mmol) in 2 mL of toluene was added, and the mixture was stirred at room temperature overnight. 2 mL of water was added, and the mixture was stirred for 2 h. After concentration, the crude product was purified by column chromatography (SiO2: 0%-10% methanol in dichloromethane) to give the desired product containing triethylamine, which was then partitioned between water and dichloromethane to give a pure product in a waxy state (170 mg, 25%). Step 5: O' 1 O 1 Synthesis of -(3-(((3-(dimethylamino)propoxy)(hydroxy)phosphoryl)oxy)-propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate) (compound 61) O' 1 O 1 -(3-(((3-bromopropoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl)8-di(heptadecane-9-yl)di(octanoate)8 (170 mg, 0.13 mmol) was heated in a sealed tube at 85°C–90°C for 3 h in 5 m...

Claims

1. A compound having a structure according to formula (I): (I) Or its pharmaceutically acceptable salt, wherein: Y is selected from C, which can be substituted at will. 2- C6 alkylene or optionally substituted C 4- C6-olefinic; R a Does not exist, or R a C is arbitrarily replaced 1- C 10 Alkyl, wherein, when R a When it exists, with R a The bonded nitrogen carries a positive charge; Each R1 is independently selected from hydrogen or optionally substituted C. 1- C 10 alkyl; Each R2 is selected independently from: (i) Optional substitution of C4-C 24 Alkyl, optionally substituted C4-C 24 alkenyl, and optionally substituted C4-C 24 alkynyl group; (ii) , where each R A Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z A Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 alkenyl; (iii) , where each R B Independently selected from optionally substituted C1-C 31 Alkyl, optionally substituted C2-C 31 alkenyl groups, and optionally substituted C2-C groups 31 alkynyl group; and Each Z B Independently selected from optionally substituted C1-C 10 Alkylenes and optionally substituted C2-C 10 Alkenyl group.

2. The compound of claim 1, wherein the compound has a structure according to formula (II), (IIa), (IIb), (IIc), (IId) or (III): (II) (IIa) (IIb) (IIc) (IId) (III), Or its pharmaceutically acceptable salt.

3. The compound of claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein Y is selected from (i) optionally substituted C 2-6 Alkylene, or (ii) optionally substituted C 4-6 Alkenyl groups, for example, where Y is -CH2CH2-.

4. The compound of claim 1, wherein the compound has a structure according to formula (IV), (V), (Va), (Vb), (Vc), or (Vd): (IV) (V) (Va) (Vb) (Vc) (Vd), Or its pharmaceutically acceptable salt.

5. The compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof. in (i) The stereochemistry of the carbon atom located between R2C(=O)O-CH2- and -CH2-OP(=O)(OH)-O- is shown in the following structure: ,or (ii) The stereochemistry of the carbon atom located between R2C(=O)O-CH2- and -CH2-OP(=O)(OH)-O- is shown in the following structure: 。 6. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein each R2 is independently selected from... (i) Each n is independently selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23. (ii) , (iii) , (iv) , (v) ， (we) , (vii) , (viii) , (ix) , (x) ， (xi) , (xii) , (xiii) , (xiv) , (xv) ,or (xvi) 。 7. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein each R A Or R B Independently selected where present: (i) Each n is independently selected from 0, 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, and 30. (ii) , (iii) , (iv) , (v) ， (we) , (vii) , (viii) , (ix) , (x) ， (xi) , (xii) (xiii) , (xiv) , (xv) ,or (xvi) 。 8. The compound as claimed in any of the preceding claims, wherein: (i) Each R A In the case that it exists Then each Z A It is a C7 alkylene group; (ii) Each R A In the case that it exists Then each Z A It is a C6 alkylene group; (iii) Each R A In the case that it exists Then each Z A It is a C2 alkylene group; (iv) Each R A In the case that it exists Then each Z A Independently selected from C5-C7 alkylene groups, for example, each Z A It is a C6 alkylene group; (v) Each R A In the case that it exists Then each Z A It is a C6 alkylene group; (vi) Each R A In the case that it exists Then each Z A It is a C6 subene group; (vii) Each R A In the case that it exists Then each Z A It is a C6 alkylene group; (viii) Each R A In the case that it exists Then each Z A It is a C6 alkylene group; (ix) Each R B In the case that it exists Then each Z B It is a C5 alkylene; or (x) Each R B In the case that it exists Then each Z B It is a C6 alkylene group.

9. A compound or a pharmaceutically acceptable salt thereof, the compound being selected from the compounds listed in Table A.

10. A composition comprising one or more lipids or pharmaceutically acceptable salts thereof as described in any one of claims 1-9, and further comprising: (i) One or more cationic lipids, (ii) one or more sterol-based lipids, and (iii) One or more PEG-modified lipids, Optionally, one or more of the sterol-based lipids are cholesterol-based lipids, such as cholesterol.

11. The composition of claim 10, wherein the composition is lipid nanoparticles, optionally wherein: (i) The one or more cationic lipids constitute approximately 20 mol% to approximately 60 mol% of the lipid nanoparticles. (ii) The lipids, one or more as described in any one of claims 1-9, constitute about 10 mol% to about 50 mol% of the lipid nanoparticles. (iii) The one or more PEG-modified lipids constitute approximately 1 mol% to approximately 4 mol% of the lipid nanoparticles; and / or (iv) The one or more sterol-based lipids comprise about 10 mol% to about 50 mol% of the lipid nanoparticles.

12. The composition of claim 11, wherein the lipid nanoparticles encapsulate mRNA encoding a peptide or protein, optionally for use in a vaccine.

13. The composition of claim 12, for use in a therapeutic context.

14. The composition of claim 12, for use in a method of treating or preventing a disease that can be treated or prevented by a peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen, and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain, or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer, optionally wherein the composition is administered intravenously, intrathecally, or intramuscularly, or delivered via the lungs, optionally via nebulization.

15. A method for treating or preventing a disease, wherein the method comprises administering the composition of claim 12 to a subject in need, and wherein the disease is treatable or preventable by a peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen, and / or the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain, or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer, optionally wherein the composition is administered intravenously, intrathecally, or intramuscularly, or via pulmonary delivery, optionally via nebulization.

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