Cationic lipids based on "ancide" buffer

Abstract

The present invention provides, in part, a second generation of "Gurd" buffer-based cationic lipid of formula (I) and its sub-formula: (I), or a pharmaceutically acceptable salt thereof. The compounds provided herein are useful, for example, for the delivery and expression of mRNA and encoded proteins as components of liposomal delivery vehicles, and are thus useful in the treatment of various diseases, disorders, and conditions, such as those associated with the lack of one or more proteins.

Description

This application is a divisional application of Chinese patent application No. 202380046217.3 (application date: 2023 / 4 / 13, invention title: cationic lipids based on "Good" buffer). Cross-reference to related applications

[0001] This application claims the benefits of Argentine patent application filed on April 13, 2022, with serial number P 22 01 00953; Taiwan patent application filed on April 14, 2022, with serial number 111114318; International patent application filed on April 15, 2022, with serial number PCT / US2022 / 025067; and European patent application filed on April 4, 2023, with serial number EP23305491.5, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] Nucleic acid delivery has been extensively explored as a potential therapeutic option for certain disease states. In particular, messenger RNA (mRNA) therapy has become an increasingly important option for the prevention and treatment of various diseases (e.g., for vaccines).

[0003] Efficient delivery of nucleic acids encapsulated in liposomes remains an active research area. The cationic lipid components of liposomes play a crucial role in promoting the efficient encapsulation of nucleic acids during liposome loading. Furthermore, cationic lipids play a vital role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. Various cationic lipids suitable for in vivo use have been identified. However, there is still a need to identify cationic lipids that can be synthesized efficiently and cost-effectively without forming potentially toxic byproducts. Furthermore, there is still a need to identify cationic lipids that exhibit improved biodegradability.

[0004] "Good" buffer (or "Good" buffer) was first selected and described by Norman Good and his colleagues for use in biochemical and biological research (Good, NE et al. (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry 5(2), 467-477). Most biological reactions occur at near-neutral pH between 6 and 8. Good therefore considered that the ideal buffer for biochemical or biological applications would have a pKa value within this range to provide maximum buffering capacity. Other selection criteria included high solubility, non-toxicity, limited interference with biochemical reactions, very low absorbance between 240 nm and 700 nm, enzyme and hydrolytic stability, minimal temperature and concentration-induced variations, limited effects of ionic or salt compositions in the solution, limited interactions with mineral cations, and limited permeability to biological membranes. Attached Figure Description

[0005] Figure 1 The lipid nanoparticles containing the lipids described herein were shown to be highly efficient in delivering hEPO mRNA and exhibited high levels of hEPO protein expression 6 hours after IM injection. Summary of the Invention

[0006] The aforementioned characteristics make "Good" buffers a particularly good starting point for synthesizing cationic lipids for use in the in vivo environment. Many "Good" buffers remain key tools in modern biochemistry and biological laboratories and are therefore readily available at low cost.

[0007] The inventors of this invention have surprisingly discovered that lipid nanoparticles containing second-generation cationic lipids derived from "Good" buffer (which contain ester moieties in the lipid tail and short (C3-C6) alkyl tails, such as butyl, isopropyl, and pent-3-yl following the ester moieties) exhibit improved properties compared to lipid nanoparticles containing other cationic lipids derived from "Good" buffer (e.g., in WO 2022 / 221688 A1 and WO2022 / 066916A1, both of which are incorporated herein by reference). For example, it is envisioned that lipid nanoparticles containing second-generation cationic lipids derived from "Good" buffer could exhibit improved in vivo degradation. It is also envisioned that lipid nanoparticles containing second-generation cationic lipids derived from "Good" buffer could also exhibit higher generalized polarization (GP) values ​​in Laurdan assays. Lower GP values ​​are associated with hydration and liquid membranes, while higher GP values ​​generally indicate fewer water molecules and more ordered lipid packaging. It is believed that additional ester and / or carbon branches in the lipid tails of second-generation cationic lipids derived from "Good" buffer can result in more tightly packed membranes compared to lipid nanoparticles containing other cationic lipids derived from "Good" buffer (such as those in WO 2022 / 221688A1 and WO 2022 / 066916 A1). It is believed that lipid nanoparticles with tighter bilayer packaging perform better in vivo by increasing the stability of lipid nanoparticles under physiological pH conditions. "Good" HEPES, HEPPS, and HEPBS buffers form the core of some of the cationic lipids of the present invention and are used to synthesize unique ionizable lipids containing different degradable moieties and carbon tails. The core structure with hydroxyl and sulfonic acid groups on both sides allows the ionizable lipids to contain both ester and disulfide degradable moieties. Preferably, the compounds are also characterized by asymmetric lipid tails on either arm of the final molecule and in the lipids of the present invention, these tails containing ester moieties to achieve greater degradability.

[0008] This invention particularly provides cationic lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids. The cationic lipids of this invention can be synthesized from readily available starting reagents such as “Good” buffers (see Table 1). The cationic lipids of this invention also contain cleavable groups (e.g., esters and disulfides) contemplated to improve biodegradability and thus contribute to their advantageous safety characteristics. It is envisioned that lipid nanoparticles containing these cationic lipid compounds can be delivered in vivo highly efficiently while maintaining advantageous safety characteristics. It is also envisioned that lipid nanoparticles containing these cationic lipid compounds can exhibit improved in vivo degradation. It is further envisioned that lipid nanoparticles containing these cationic lipid compounds can exhibit higher generalized polarization (GP) values.

[0009] On the one hand, this paper provides cationic lipids having a structure according to formula (I): Or its pharmaceutically acceptable salt, wherein: A 1 Selected from and -SS-, where the left side of each depicted structure is bound to -(CH2)a-; Z 1 Selected from and -SS-, where the right side of each depicted structure is bound to -(CH2)a-; Each 'a' is independently selected from 3 or 4; b is 1, 2, 3, 4, or 5; Each c, d, e, and f is independently selected from 3, 4, 5, or 6; and Each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C6) alkyl groups.

[0010] On the one hand, this article provides cationic lipids as pharmaceutically acceptable salts of formula (I).

[0011] In one aspect, this document provides compositions comprising the cationic lipids of the present invention or pharmaceutically acceptable salts thereof, and further comprising: (i) One or more non-cationic lipids (e.g., phospholipids, such as DOPE), (ii) one or more cholesterol-based lipids (e.g., cholesterol), and (iii) One or more lipids modified with PEG.

[0012] In one aspect, the composition is lipid nanoparticles, optionally liposomes.

[0013] In one respect, the composition comprising the cationic lipids of the present invention can be used in therapy.

[0014] In one aspect, the composition of the present invention is administered by intramuscular injection. Detailed Implementation definition

[0015] 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 on its practice are hereby incorporated by reference.

[0016] Amino acids: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, amino acids have the universal structure H₂N-C(H)(R)-COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids; in some embodiments, amino acids are d-amino acids; in some embodiments, amino acids are l-amino acids. "Standard amino acid" refers to any one of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids in a peptide (including carboxyl-terminal and / or amino-terminal amino acids) can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups, which may alter the cyclic half-life of the peptide without adversely affecting its activity. Amino acids can participate in disulfide bonds. Amino acids may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl, acetate, acetyl, phosphate, formyl moiety, isoprene-like group, sulfate group, polyethylene glycol moiety, lipid moiety, carbohydrate moiety, biotin moiety, etc.). The term "amino acid" is used interchangeably with "amino acid residue" and may refer to a free amino acid and / or an amino acid residue of a peptide. It will be clear from the context in which the term is used whether it refers to a free amino acid or a peptide residue.

[0017] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0018] Approximately or about: As used herein, the term “approximately” or “about” when applied to one or more intended values ​​refers to a value similar to the stated reference value. In some embodiments, unless otherwise stated or the context clearly indicates otherwise, the term “approximately” or “about” refers to a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value (except where this number would exceed 100% of the possible value).

[0019] Biologically active: As used herein, the term "biologically active" refers to the characteristic of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when applied to that organism is considered biologically active.

[0020] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, mRNA delivery includes situations where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also known as “local distribution” or “local delivery”); and situations where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient’s circulatory system (e.g., serum), and distributed systemically and absorbed by other tissues (also known as “systemic distribution” or “systemic delivery”).

[0021] Expression: As used herein, “expression” of a nucleic acid sequence refers to the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or post-translational modifications of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production” and their grammatical equivalents are used interchangeably.

[0022] Functionality: As used herein, a “functional” biomolecule is a biomolecule in such a form that it exhibits its characteristic properties and / or activities.

[0023] Half-life: As used herein, the term “half-life” is the time required for the amount of a substance, such as the concentration or activity of a nucleic acid or protein, to decrease to half of the value measured at the beginning of a period of time.

[0024] Supporting lipids: As used herein, the term "supporting lipid" refers to any neutral or zwitterionic lipid material containing cholesterol. Without wishing to be limited to any particular theory, supporting lipids can increase the stability, rigidity, and / or fluidity within lipid bilayers / nanoparticles.

[0025] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A “control subject” is a subject suffering from the same form of disease as the treated subject and of approximately the same age.

[0026] 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 in a multicellular organism.

[0027] In vivo: As used herein, the term "in vivo" refers to events occurring within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0028] Liposomes: As used herein, the term "liposome" refers to any layered, multilayered, or solid nanoparticle vesicle. Typically, liposomes as used herein can be formed by mixing one or more lipids or by mixing one or more lipids with one or more polymers. In some embodiments, liposomes suitable for use in the present invention contain one or more cationic lipids and optionally further include: (i) one or more non-cationic lipids, (ii) one or more cholesterol-based lipids, and / or (iii) One or more lipids modified with PEG.

[0029] Messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)” or “mRNA” refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA includes 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, generated and optionally purified using a recombinant expression system, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs (such as analogs of chemically modified bases or sugars), backbone modifications, etc. Unless otherwise indicated, mRNA sequences are presented in a 5' to 3' orientation. In some embodiments, the mRNA is or comprises a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine); or a nucleoside analogue (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylur ... Cytidine, 2-aminoadenosine, 7-deadenosine, 7-deadenosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., thiophosphates and 5'-N-phosphoramide linkages).

[0030] Nucleic Acids: 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 or potentially incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, “nucleic acid” refers to a single nucleic acid residue (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain containing a single nucleic acid residue. In some embodiments, “nucleic acid” includes 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 one or more of the following: 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), multimeric encoded nucleic acid (MCNA), polymeric encoded 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 can be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-concentrated 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 implementation schemes, RNA may take the following forms: messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (7SLRNA or SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cahalanosome-specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), Y RNA, telomerase RNA fraction (TERC), splicing leader RNA (SLRNA), antisense RNA (aRNA or asRNA), cis-native antisense transcript (cis-NAT), CRISPR RNA (crRNA), long non-coding 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 implementation schemes, the nucleic acid is mRNA encoding a protein (such as an enzyme).

[0031] Patient: As used herein, the term "patient" or "subject" means any organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Humans include both prenatal and postnatal forms.

[0032] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means a substance that, within reasonable medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0033] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SMBerge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by reacting amino groups with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or with organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid) or by using other methods used in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable alkalis include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. When appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Other pharmaceutically acceptable salts include those formed by quaternizing amines with a suitable electrophile (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0034] Whole-body distribution or delivery: As used herein, the terms “whole-body distribution” or “whole-body delivery” or their grammatical equivalents refer to a mechanism or method of delivery or distribution that affects the whole body or the entire organism. Generally, whole-body distribution or delivery is accomplished through the body’s circulatory system (e.g., blood flow). Compare this to the definition of “local distribution or delivery”.

[0035] Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a person presented to a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used interchangeably herein with “individual” or “patient.” A subject may suffer from or be susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0036] Essentially: As used herein, the term “essentially” refers to a qualitative situation that exhibits the overall or near-overall range or extent of a intended characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if ever) complete and / or proceed to the point of achieving or avoiding absolute results. Therefore, the term “essentially” is used herein to capture the inherent lack of completeness in many biological and chemical phenomena.

[0037] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some implementations, target tissue includes those tissues that exhibit disease-related symptoms, signs, or characteristics.

[0038] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of one or more symptoms of a disease, disorder, and / or condition when administered to a subject who has or is susceptible to such a disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0039] Treatment: As used herein, the terms “treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease for the purpose of reducing the risk of developing disease-related symptoms. Chemical definition

[0040] 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.

[0041] Aliphatic: As used in this article, the term aliphatic refers to (C1-C1) 50Hydrocarbons include both saturated and unsaturated hydrocarbons. Aliphatic hydrocarbons can be straight-chain, branched, and / or cyclic. For example, (C1-C2) hydrocarbons... 20 Aliphatic compounds can include (C1-C1) 20 )alkyl (e.g., straight-chain or branched (C1-C2) 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 compounds can include (C3-C) 20 ) Cyclic aliphatic (e.g., (C3-C) 20 )cycloalkyl, (C4-C 20 )cycloalkenyl or (C8-C 20 (Cycloalkyne). In some embodiments, the aliphatic group may comprise one or more cyclic aliphatic groups and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur), and may optionally be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). The aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, the aliphatic group may be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents), wherein each example of R" is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 R” is an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). In the embodiments, 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 embodiments, R” is independently an unsubstituted (C1-C3)alkyl. In embodiments, aliphatic is unsubstituted. In embodiments, aliphatic does not include any heteroatoms. Alkyl: As used herein, the term “alkyl” means acyclic straight-chain and branched hydrocarbon groups, such as “(C1-C3)alkyl”. 30"alkyl" refers to an alkyl group having 1 to 30 carbon atoms. Alkyl groups can be straight-chain or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, etc. The term "lower alkyl" means a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be apparent to those skilled in the art for the benefit of this disclosure. Alkyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, alkyl groups can be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents), wherein each example of R" is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 R” is an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). In the embodiments, 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 embodiments, "R" is independently an unsubstituted (C1-C3)alkyl. In embodiments, the alkyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkyl group is substituted with a -OH group and may also be referred to herein as "hydroxyalkyl", wherein the prefix indicates the -OH group and "alkyl" is as described herein.

[0042] 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 groups (") are present in some embodiments. In some embodiments, the alkyl group has 1 to 40 carbon atoms ("(C1-C1") 40 Alkyl groups (") are present in some embodiments. In some embodiments, the alkyl group has 1 to 30 carbon atoms ("(C1-C1") 30 Alkyl groups (") are present in some embodiments. In some embodiments, the alkyl group has 1 to 20 carbon atoms ("(C1-C2)"). 20 Alkyl groups (") are present in some embodiments. In some embodiments, the alkyl group has 1 to 10 carbon atoms ("(C1-C1")). 10In some embodiments, the alkyl group has 1 to 9 carbon atoms (“(C1-C9)alkyl”). In some embodiments, the alkyl group has 1 to 8 carbon atoms (“(C1-C8)alkyl”). In some embodiments, the alkyl group has 1 to 7 carbon atoms (“(C1-C7)alkyl”). In some embodiments, the alkyl group has 1 to 6 carbon atoms (“(C1-C6)alkyl”). In some embodiments, the alkyl group has 1 to 5 carbon atoms (“(C1-C5)alkyl”). In some embodiments, the alkyl group has 1 to 4 carbon atoms (“(C1-C4)alkyl”). In some embodiments, the alkyl group has 1 to 3 carbon atoms (“(C1-C3)alkyl”). In some embodiments, the alkyl group has 1 to 2 carbon atoms (“(C1-C2)alkyl”). In some embodiments, the alkyl group has 1 carbon atom (“C1 alkyl”). In some embodiments... In this embodiment, the alkyl group has 2 to 6 carbon atoms (“(C2-C6)alkyl”). In some embodiments, the alkyl group has 3 to 6 carbon atoms (“(C3-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). Further 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 a substituted (C1-C2) alkyl group. 50 )alkyl.

[0043] The suffix "-ene" is added to a group to indicate that the group is a divalent moiety. For example, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0044] Alkylene: As used herein, the term "alkylene" refers to a saturated divalent straight-chain or branched hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, etc. Similarly, the term "alkenylene" as used herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon double bonds (which may appear at any stable point along the chain), and the term "alkynylene" herein refers to an unsaturated divalent straight-chain or branched hydrocarbon group having one or more unsaturated carbon-carbon triple bonds (which may appear at any stable point along the chain). In some embodiments, the alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and may optionally be substituted with one or more substituents (such as alkyl, halogen, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide). For example, the alkylene, alkenylene, or ynylene group may be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR", -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents), wherein each example of R" is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 R” is an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). In the embodiments, 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 embodiments, R” is independently an unsubstituted (C1-C3)alkyl. In some embodiments, alkylene, alkenyl, or ynylene is unsubstituted. In some embodiments, alkylene, alkenyl, or ynylene does not include any heteroatoms. Alkenyl: As used herein, “alkenyl” means any straight or branched hydrocarbon chain having one or more unsaturated carbon-carbon double bonds (which can appear at any stable point along the chain), for example, (C2-C3)alkyl. 30"Alkenyl" refers to an alkenyl group having 2-30 carbon atoms. Examples of alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, etc. In embodiments, the alkenyl group comprises 1, 2, or 3 carbon-carbon double bonds. In embodiments, the alkenyl group comprises a single carbon-carbon double bond. In embodiments, multiple double bonds are concatenated (e.g., 2 or 3). The alkenyl group can be unsubstituted or unsubstituted. The alkenyl group may be substituted by one or more substituents as described herein. For example, the alkenyl group may be substituted by one or more of the following (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents): halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R", wherein each example of R" is independently (C1-C2). 20 Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 R” is an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). In the embodiments, 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 embodiments, "R" is independently an unsubstituted (C1-C3)alkyl. In embodiments, the alkenyl group is unsubstituted. In embodiments, the alkenyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkenyl group is substituted with a -OH group and may also be referred to herein as "hydroxyalkenyl", wherein the prefix indicates the -OH group and "alkenyl" is as described herein.

[0045] 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 In some embodiments, the alkenyl group has 2 to 40 carbon atoms (“(C2-C)”). 40 In some embodiments, the alkenyl group has 2 to 30 carbon atoms (“(C2-C)”). 30 In some embodiments, the alkenyl group has 2 to 20 carbon atoms (“(C2-C)”). 20 In some embodiments, the alkenyl group has 2 to 10 carbon atoms (“(C2-C)”). 10(C2-C9)alkenyl. In some embodiments, the alkenyl has 2 to 9 carbon atoms ("(C2-C8)alkenyl"). In some embodiments, the alkenyl has 2 to 7 carbon atoms ("(C2-C7)alkenyl"). In some embodiments, the alkenyl has 2 to 6 carbon atoms ("(C2-C6)alkenyl"). In some embodiments, the alkenyl has 2 to 5 carbon atoms ("(C2-C5)alkenyl"). In some embodiments, the alkenyl has 2 to 4 carbon atoms ("(C2-C4)alkenyl"). In some embodiments, the alkenyl has 2 to 3 carbon atoms ("(C2-C3)alkenyl"). In some embodiments, the alkenyl has 2 carbon atoms ("(C2)alkenyl"). One or more carbon atoms. The 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 (C2-C4)alkenyl groups described above, as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Further examples of alkenyl groups include heptenyl (C7), octenyl (C8), octetrinyl (C8), etc. Unless otherwise stated, each example of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is unsubstituted (C2-C4). 50 Alkenyl group. In some embodiments, the alkenyl group is substituted (C2-C). 50 )alkenyl.

[0046] Alkynyl: As used herein, "alkynyl" means any straight-chain or branched hydrocarbon chain having one or more carbon-carbon triple bonds (occurring at any stable point along the chain), for example, "(C2-C 30 "Alynyl" refers to an alkynyl group having 2-30 carbons. Examples of alkynyl groups include prop-2-alkynyl, but-2-alkynyl, but-3-alkynyl, pent-2-alkynyl, 3-methylpent-4-alkynyl, hex-2-alkynyl, hex-5-alkynyl, etc. In embodiments, the alkynyl group comprises a carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, the alkynyl group can be substituted with one or more of halogen, -COR", -CO2H, -CO2R", -CN, -OH, -OR", -OCOR', -OCO2R", -NH2, -NHR", -N(R")2, -SR", or -SO2R" (e.g., 1, 2, 3, 4, 5, or 6 independently chosen substituents), wherein each example of R" is independently (C1-C2). 20Aliphatic (e.g., (C1-C) 20 )alkyl, (C1-C 15 )alkyl, (C1-C 10 R” is an unsubstituted alkyl group (e.g., an unsubstituted (C1-C3)alkyl group). In the embodiments, 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 embodiments, R” is independently an unsubstituted (C1-C3)alkyl. In embodiments, the alkynyl group is unsubstituted. In embodiments, the alkynyl group is substituted (e.g., substituted by 1, 2, 3, 4, 5 or 6 substituents as described herein).

[0047] 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 ("alkynyl group") is also called "ene-alkynyl group" when it has one or more triple bonds and one or more double bonds. In some embodiments, the alkynyl group has 2 to 40 carbon atoms ("(C2-C2")). 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-C7) ynyl group"). 2) Alynyl group. 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) alynyl 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) alynyl groups as well as pentynyl (C5), hexynyl (C6), etc. Other examples of alynyl groups include heptyynyl (C7), octyynyl (C8), etc. Unless otherwise stated, each example of an alynyl group is independently unsubstituted (“unsubstituted alynyl”) or substituted with one or more substituents (“substituted alynyl”). In some embodiments, the alynyl group is unsubstituted (C2-C4). 50 ) alkynyl group. In some embodiments, the alkynyl group is substituted (C2-C... 50 ) yyn group.

[0048] Aryl: The term "aryl" used alone or as part of a larger portion (such as "araneyl") refers to a monocyclic, bicyclic, or tricyclic carbocyclic system having a total of six to fourteen ring members, wherein the ring system has a single point of attachment to the rest of the molecule, at least one ring in the system is aromatic, and each ring in the system contains four to seven ring members. In embodiments, the aryl group has six ring carbon atoms ("(C6)aryl", e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("(C6)aryl", e.g., C6)aryl). 10 aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“(C 14 "Aryl"; for example, anthracene. "Aryl" also includes ring systems in which an aromatic ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attached groups or dots are on the aryl ring, and in such cases, the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Exemplary aryl groups include phenyl, naphthyl, and anthracene.

[0049] As used herein, “aryl” also refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared π electrons in a cyclic arrangement) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“(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 an aromatic ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attached group or dot is on the aryl ring, and in such cases, the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Unless otherwise stated, each example of an aryl is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In some embodiments, the aryl group is unsubstituted (C6-C6). 14 aryl group. In some embodiments, the aryl group is substituted (C6-C6). 14 Aryl.

[0050] arylene: As used herein, the term "arylene" refers to a divalent aryl group (that is, having two sites that attach to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted or substituted phenylene).

[0051] Carbocyclic group: As used herein, "carbocyclic group" or "carbocyclic" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms in a non-aromatic ring system ("(C3-C...)"). 10 (C3-C8) carbocyclic group). In some embodiments, the carbocyclic group has 3 to 8 ring carbon atoms ("(C3-C7) carbocyclic group"). In some embodiments, the carbocyclic group has 3 to 7 ring carbon atoms ("(C3-C7) carbocyclic group"). In some embodiments, the carbocyclic group has 3 to 6 ring carbon atoms ("(C3-C6) carbocyclic group"). In some embodiments, the carbocyclic group has 4 to 6 ring carbon atoms ("(C4-C6) carbocyclic group"). In some embodiments, the carbocyclic group has 5 to 6 ring carbon atoms ("(C5-C6) carbocyclic group"). In some embodiments, the carbocyclic group has 5 to 10 ring carbon atoms ("(C5-C6) carbocyclic group"). 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-mentioned (C3-C6) carbocyclic groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptanetrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Exemplary (C3-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), bicyclo[2.2.2]octyl (C8), etc. 10 The carbocyclic group includes, but is not limited to, the (C3-C8) carbocyclic groups mentioned above, as well as cyclononyl (C9), cyclononenyl (C9), and cyclodecyl (C9). 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C9) 10 ), spiro[4.5]decyl(C 10 As illustrated in the foregoing examples, in some embodiments, the carbocyclic group is monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., containing fused, bridged, or spirocyclic systems, such as bicyclic systems (“bicyclic carbocyclic”) or tricyclic systems (“tricyclic carbocyclic”)) and may be saturated or may contain one or more carbon-carbon double or triple bonds. “Carbocyclic” also includes ring systems in which the carbocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the attachment point is on the carbocyclic ring, and in such cases, the number of carbons continues to specify the number of carbons in the carbocyclic system. Unless otherwise stated, each example of a carbocyclic group is independently unsubstituted (“unsubstituted carbocyclic”) or substituted with one or more substituents (“substituted carbocyclic”). In some embodiments, the carbocyclic group is an unsubstituted C3-C 10 Carbocyclic group. In some embodiments, the carbocyclic group is substituted (C3-C4). 10 ) carbon cyclic group.

[0052] In some embodiments, "carbocyclic group" or "carbocyclic" is referred to as "cycloalkyl," that is, a monocyclic saturated carbocyclic group ("(C3-C10") having 3 to 10 ring carbon atoms. 10(C3-C8)cycloalkyl. In some embodiments, the cycloalkyl has 3 to 8 ring carbon atoms ("(C3-C6)cycloalkyl"). In some embodiments, the cycloalkyl has 3 to 6 ring carbon atoms ("(C3-C6)cycloalkyl"). In some embodiments, the cycloalkyl has 4 to 6 ring carbon atoms ("(C4-C6)cycloalkyl"). In some embodiments, the cycloalkyl has 5 to 6 ring carbon atoms ("(C5-C6)cycloalkyl"). In some embodiments, the cycloalkyl has 5 to 10 ring carbon atoms ("(C5-C6)cycloalkyl"). 10 (C5-C6) cycloalkyl groups are cyclopentyl (C5) and cyclohexyl (C5). (C3-C6) cycloalkyl groups are cycloalkyl groups described above, as well as cyclopropyl (C3) and cyclobutyl (C4). (C3-C8) cycloalkyl groups are cycloalkyl groups described above, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise stated, each example of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In some embodiments, the cycloalkyl group is unsubstituted (C3-C6). 10 )cycloalkyl. In some embodiments, the cycloalkyl group is substituted (C3-C4). 10 )cycloalkyl.

[0053] Halogen: As used in this article, the term “halogen” means fluorine, chlorine, bromine or iodine.

[0054] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from N, O, S, and P. Heteroalkyl groups include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphate diesters, aminophosphates, sulfonamides, and disulfides. Heteroalkyl groups may optionally include monocyclic, bicyclic, or tricyclic rings, wherein each ring ideally has three to six members. Examples of heteroalkyl groups include polyethers such as methoxymethyl and ethoxyethyl.

[0055] Heteroalkylene: As used herein, the term “heteroalkylene” refers to the divalent form of a heteroalkylene as described herein.

[0056] Heteroaryl: As used herein, the term “heteroaryl” is a fully unsaturated heteroatom-containing ring in which at least one ring atom is a heteroatom (such as, but not limited to, nitrogen and oxygen).

[0057] As used herein, “heteroaryl” also refers to a group having a 5-14 member monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared π electrons in a cyclic arrangement) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In heteroaryls containing one or more nitrogen atoms, the attachment point can be a carbon or nitrogen atom if the valence allows. Heteroaryl polycyclic systems may include one or more heteroatoms in one or two rings. “Heteroaryl” includes ring systems in which the heteroaryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups, wherein the attachment point is on the heteroaryl ring, and in such cases, the number of ring members continues to specify the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused with one or more aryl groups, wherein the attachment site is on the aryl or heteroaryl ring, and in such cases, the number of ring members specifies the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. A polycyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) may have its attachment site on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

[0058] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system (“5-10 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatomum is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system (“5-8 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatomum is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system (“5-6 membered heteroaryl”) having a cyclic carbon atom provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (wherein each heteroatomum is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the 5-6-membered heteroaryl group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6-membered heteroaryl group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise stated, each example of a heteroaryl group is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some embodiments, the heteroaryl group is an unsubstituted 5-14-membered heteroaryl group. In some embodiments, the heteroaryl group is a substituted 5-14-membered heteroaryl group.

[0059] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrroloyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, aza-aryl. basic, oxygen oxepinyl and thiohexyl Thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolazinyl, and purinel. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridyl, quinolinyl, isoquinolinyl, cenolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridine, dibenzofuranyl, carbazole, acridineyl, phenothiazinyl, phenotoxazinyl, and phenothiazinyl.

[0060] As used herein, "heterocyclic group" or "heterocycle" refers to a group having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus) in a 3- to 14-membered non-aromatic ring system ("3-14-membered heterocyclic group"). In heterocyclic groups containing one or more nitrogen atoms, the attachment point can be a carbon or nitrogen atom, if 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 systems may include one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems in which a heterocyclic ring as defined above is fused with one or more carbocyclic groups, wherein the attachment point is on the carbocyclic or heterocyclic ring; or ring systems in which a heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups, wherein the attachment point is on the heterocyclic ring, and in such cases, the number of ring members continues to specify the number of ring members in the heterocyclic ring system. Unless otherwise stated, each example of a heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In some embodiments, the heterocyclic group is an unsubstituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a substituted 3-14 membered heterocyclic group.

[0061] In some embodiments, the heterocyclic group is a 5-10 membered non-aromatic ring system (“5-10 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heterocyclic group is a 5-8 membered non-aromatic ring system (“5-8 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the heterocyclic group is a 5-6 membered non-aromatic ring system (“5-6 membered heterocyclic group”) having a cyclic carbon atom and one or more (e.g., 1, 2, 3, or 4) cyclic heteroatoms (where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus). In some embodiments, the 5-6 membered heterocyclic group has one or more (e.g., 1, 2, or 3) cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one or two cyclic heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heterocyclic group has one cyclic heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0062] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxathiolanyl, oxathiolanyl, and dithiorenyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazineyl, morpholinyl, dithiaalkyl, and dioxaneyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptanyl, oxetaneheptyl, and thioheptanyl. Exemplary bicyclic heterocyclic groups include, but are not limited to, indololinyl, isoindololinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthidyl, decahydro-1,8-naphthidyl, octahydropyrrolo[3,2-b]pyrrole, indololinyl, phthalimidyl, naphthalimidyl, chromealkyl, chromenyl, 1H-benzo[e][1,4]diaza The compounds include 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolithyl, 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, and 1,2,3,4-tetrahydro-1,6-naphthidyl.

[0063] Heterocyclic alkyl: As used herein, the term "heterocyclic alkyl" is a non-aromatic ring in which at least one atom is a heteroatom (such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus) and the remaining atoms are carbon. The heterocyclic alkyl may be substituted or unsubstituted.

[0064] As understood from the foregoing, in some embodiments, alkyl, alkenyl, alkynyl, acyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups as defined herein are optionally substituted. Optionally substituted means that the group may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclic, "substituted" or "unsubstituted" heterocyclic, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). Generally, the term "substituted" means a substituent on which at least one hydrogen atom is permitted (e.g., substitution that produces a stable compound (e.g., does not self-substitute)). Substituents (such as those in compounds that undergo transformation through rearrangement, cyclization, elimination, or other reactions) are substituted. Unless otherwise indicated, a “substituted” group has substituents at one or more substituted positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of an organic compound, and any substituents described herein that result in the formation of a stable compound. Any and all such combinations are contemplated for obtaining stable compounds. For the purposes of this invention, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any suitable substituents that satisfy the valence state of the heteroatom as described herein and result in the formation of a stable moiety.

[0065] 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 groups, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl and heteroaryl group is independently bounded by 0, 1, 2, 3, 4 or 5 R groups. dd Group substitution; Alternatively, the two hydrogen atoms on the carbon atom can be replaced by the following groups: =O, =S, =NN(R) bb )2、=NNR bb C(=O)R aa =NNR bb C(=O)OR aa =NNR bb S(=O)2R aa =NR bb or = NOR cc ;

[0066] 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 rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0067] 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 rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0068] 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-C 14) 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 rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution;

[0069] 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)Ree 、-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 radicals dd Substituents can connect to form =O or =S;

[0070] 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;

[0071] 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 ff The groups, together with the heteroatoms to which they are attached, form 3-14 membered heterocyclic rings or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. ggGroup substitution; and 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-C 50 )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.

[0072] As used herein, the term “halogen” or “halogen” refers to fluorine (fluorinated, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0073] As used herein, a "counterion" is a negatively charged group associated with a positively charged quaternary ammonium in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F). - Cl- ,Br - I - NO3 - ClO4 - OH - H2PO4 - HSO4 - Sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonate-5-sulfonate, ethane-1-sulfonate-2-sulfonate, etc.) and carboxyl groups (e.g., acetate, ethanoate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0074] If the valence allows, the nitrogen atom can be substituted or unsubstituted, and includes primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen substituents include, but are not limited to, hydrogen, -OH, and -OR. aa -N(R) cc )2、-CN、-C(=O)R aa -C(=O)N(R) cc )2、-CO2R aa -SO2R aa -C(=NR) bb )R aa -C(=NR) cc OR aa -C(=NR) cc )N(R cc )2、-SO2N(R cc )2、-SO2R cc -SO2OR cc -SOR aa -C(=S)N(R) cc )2、-C(=O)SR cc -C(=S)SR cc -P(=O)2R aa -P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2、(C1-C 50 )alkyl, (C2-C 50 )alkenyl, (C2-C 50 ) ynyl group, (C3-C 10 ) carbocyclic group, 3-14 membered heterocyclic group, (C6-C 14 ) aryl and 5-14 heteroaryl, or two R ccThe alkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups together with the attached nitrogen atoms form 3-14 membered heterocyclic or 5-14 membered heteroaryl rings, wherein each alkyl, alkenyl, ynyl, carbocyclic, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R atoms. dd Group substitution, and wherein R aa R bb R cc and R dd It is as defined above.

[0075] In some embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also known as an amino protecting group). Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0076] For example, amide groups (e.g., -C(=O)R) aa The nitrogen protecting group of () includes, but is not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridinecarboxamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetylacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0077] Such as urethane groups (e.g., -C(=O)OR) aaThe nitrogen-protecting groups of ) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfonyl)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxybenzoylmethyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), and 1-(1-adamantyl)-1-methylethyl Carbamates (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethylcarbamate (TCBOC), 1-methyl-1-(4-biphenyl)ethylcarbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethylcarbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethylcarbamate (Pyoc), 2-(N,N-dicyclohexylcarbamate)ethylcarbamate, tert-butylcarbamate (BOC), 1-adamantylcarbamate (Adoc), carbamic acid Ethylene ester (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamon ester carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolinyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthraylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonyl Acyl ethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithiaalkyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphoethyl carbamate (Peoc), 2-triphenylphosphoisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolyl methyl carbamate, 2-(trifluoromethyl)-6-chromone methyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-Dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl)methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyylvinyl carbamate, o-(N,N-dimethylformamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodobenzyl Ethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicocarbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0078] Such as sulfonamide groups (e.g., -S(=O)2R) aa The nitrogen protecting groups of () include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylsomn-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracitesulfonamide, 4-(4',8'-dimethoxynaphthyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and benzoylmethylsulfonamide.

[0079] Other nitrogen-protecting groups include, but are not limited to, phenthiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazoline-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldimethylsilylazine pentane adduct (STABASE), and 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexanes. Hexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridinone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrololin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluoreneamine (PhF) N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferroceneylmethylamino (Fcm), N-2-pyridinemethylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylamine, N-p-methoxybenzylamine, N-diphenylmethyleneamine, N-[(2-pyridyl)trimethylmethyl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylamine, N-salicylamine, N-5-chlorosalicylamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl) Amines, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentaacylchromium or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitrosamines, N-nitrosamines, amine N-oxides, diphenylphosphamide (Dpp), dimethylthiophosphamide (Mpt), diphenylthiophosphamide (Ppt), dialkylphosphamide esters, dibenzylphosphamide esters, diphenylphosphamide esters, benzylsulfonamides, o-nitrobenzenesulfonamides (Nps), 2,4-dinitrobenzenesulfonamides, pentachlorobenzenesulfonamides, 2-nitro-4-methoxybenzenesulfonamides, triphenylmethylsulfonamides, and 3-nitropyridinesulfonamides (Npys).

[0080] In some embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also known as a hydroxy protecting group). Oxyprotecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0081] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacol methyl (GUM), tert-butoxymethyl, 4-pentenoxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), and tetrahydropyranyl (THP). 3-Bromotetrahydropyranyl, Tetrahydrothiopyranyl, 1-Methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-Methoxytetrahydrothiopyranyl, 4-Methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxane-2-yl, Tetrahydrofuranyl, Tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-bridged methylenebenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl 1-Methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenoethyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-pyridinemethyl, 4-pyridinemethyl, 3-methyl-2-pyridinemethyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl Di(p-methoxyphenyl)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-benzodicyclothioethane-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyl tert-hexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxy Acetates, phenoxyacetic acid esters, p-chlorophenoxyacetic acid esters, 3-phenylpropionate, 4-oxovalerate (acetylpropionate), 4,4-(ethylidene dithio)valerate (acetylpropionyl dithioacetal), neovalerate, adamantinate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzoate (mesitoate)), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2- (Triphenylphospho)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, p-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate 2,6-Dichloro-4-methylphenoxyacetic acid ester, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetic acid ester, 2,4-bis(1,1-dimethylpropyl)phenoxyacetic acid ester, dichlorophenylacetic acid ester, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, p-(methoxyyl)benzoate, α-naphthylcarbamate, nitrates, alkyl N,N,N',N'-tetramethylphosphinodiacetate, alkyl N-phenylcarbamate, borates, dimethylphosphinosulfonyl, alkyl 2,4-dinitrophenyl sulfenate, sulfates, methanesulfonate (or mesylate), benzyl sulfonate, and toluenesulfonate (TS).

[0082] In some embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Uts, 3rd edition, John Wiley & Sons, 1999, which are incorporated herein by reference.

[0083] Exemplary sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-pyridinemethyl, 2-quinolinylmethyl, 2-pyridinemethyl N-oxide, 9-anthraylmethyl, 9-fluorenylmethyl, xanthyl, ferroceneylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzocycloheptanyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, tert-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolyl, acetamylmethyl, trimethylacetamylmethyl, benzamidemethyl, allyloxycarbonylaminomethyl , phenylacetamidomethyl, phthalimidemethyl, 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, sulfenyl thiocarbonate, 3-nitro-2-pyridine sulfenyl sulfide, oxathioisoamyl ketone. The compounds of the present invention

[0084] Liposome-based mediators are considered attractive carriers for therapeutics and continue to be developed. While liposome-based mediators containing certain lipid components have shown promising results in terms of encapsulation, stability, and site localization, there remains a great need to improve liposome-based delivery systems. For example, significant drawbacks of liposome delivery systems involve the construction of liposomes with sufficient cell culture or in vivo stability to achieve the desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release their encapsulation materials into such target cells.

[0085] In particular, there remains a need for cationic lipids that can effectively deliver mRNA intramuscularly. There is also a need for improved lipid compounds that exhibit improved pharmacokinetic properties and can deliver macromolecules such as nucleic acids to a wide variety of cell types and tissues with enhanced efficiency. Importantly, there is still a particular need for novel lipid compounds characterized by improved safety profiles and the ability to efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues, and organs.

[0086] This article describes a novel class of cationic lipid compounds for improved in vivo delivery of therapeutic agents such as nucleic acids. In particular, the cationic lipids described herein can optionally be used in conjunction with other lipids to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic purposes such as disease treatment and prevention (vaccine) purposes.

[0087] In embodiments, the compounds of the present invention as described herein may provide one or more desired features or properties. That is, in some embodiments, the compounds of the present invention as described herein may be characterized by having one or more properties that provide such compounds with an advantage over other similar classes of lipids. For example, the compounds disclosed herein allow for the control and customization of the properties of liposome compositions (e.g., lipid nanoparticles), wherein the compounds disclosed herein are components of said liposome compositions (e.g., lipid nanoparticles). In particular, the compounds disclosed herein may be characterized by improved transfection efficiency and their ability to induce specific biological outcomes. Such outcomes may include, for example, increased cellular uptake, endosome / lysosome disruption, and / or promotion of intracellular release of encapsulated materials (e.g., polynucleotides). The compounds disclosed herein may also be characterized by achieving high levels of peptide or protein expression when delivered via intravenous, intrathecal, or intramuscular administration or via pulmonary delivery (optionally via nebulization) of mRNA encoding said peptide or protein. Additionally, the compounds disclosed herein have favorable pharmacokinetic properties, biodistribution, and efficiency.

[0088] This application demonstrates that the cationic lipids of the present invention are not only easy to synthesize from readily available starting materials, but also have unexpectedly high encapsulation efficiency.

[0089] Additionally, the cationic lipids of the present invention have cleavable groups such as ester groups. These cleavable groups (e.g., esters, disulfides) are envisioned to improve biodegradability and thus contribute to the advantageous safety characteristics of the lipids.

[0090] This document provides compounds as cationic lipids. For example, the cationic lipids of the present invention include compounds having a structure according to formula (I): Or its pharmaceutically acceptable salt, wherein: A 1 Selected from and -SS-, where the left side of each depicted structure is bound to -(CH2)a-; Z 1 Selected from and -SS-, where the right side of each depicted structure is bound to -(CH2)a-; Each 'a' is independently selected from 3 or 4; b is 1, 2, 3, 4, or 5; Each c, d, e, and f is independently selected from 3, 4, 5, or 6; and Each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C6) alkyl groups.

[0091] In the implementation scheme, the cationic lipid has a structure according to formula (Ia): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6.

[0092] In the implementation scheme, the cationic lipid has a structure according to formula (Ib): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6.

[0093] In the implementation scheme, the cationic lipid has a structure according to formula (Ic): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6.

[0094] In the implementation scheme, the cationic lipid has a structure according to formula (Id): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6.

[0095] In the implementation scheme, the cationic lipid has a structure according to formula (Ie): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6.

[0096] In the implementation scheme, the cationic lipid has a structure according to formula (If): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6.

[0097] In the implementation scheme, the cationic lipid has a structure according to formula (Ig): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6.

[0098] In the implementation scheme, the cationic lipid has a structure according to formula (Ih): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-; or (c) b is 2, A 1 yes The left side of the structure described is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6.

[0099] In the implementation scheme, the cationic lipid has a structure according to formula (Ii): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0100] In the implementation scheme, the cationic lipid has a structure according to formula (Ij): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0101] In the implementation scheme, the cationic lipid has a structure according to formula (Ik): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0102] In the implementation scheme, the cationic lipid has a structure according to formula (Im): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0103] In the implementation scheme, the cationic lipid has a structure according to formula (In): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0104] In the implementation scheme, the cationic lipid has a structure according to formula (Io): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0105] In the implementation scheme, the cationic lipid has a structure according to formula (Ip): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z 1 It is -SS-.

[0106] In the implementation scheme, the cationic lipid has a structure according to formula (Iq): Or a pharmaceutically acceptable salt thereof, optionally of which: (a) b is 2; or (b) b is 2, A 1 yes The structure described therein is bound to -(CH2)a- on the left and Z1 It is -SS-.

[0107] In the implementation plan, A 1 and Z 1 They are the same. In the implementation plan, A 1 and Z 1 They are different.

[0108] In the implementation plan, A 1 yes The left side of the depicted structure is bonded to -(CH2)a-. In the embodiment, A 1 yes The left side of the depicted structure is bonded to -(CH2)a-. In the embodiment, A 1 It is -SS-.

[0109] In the implementation plan, Z 1 yes The right side of the depicted structure is bonded to -(CH2)a-. In the implementation, Z 1 yes The right side of the depicted structure is bonded to -(CH2)a-. In the implementation, Z 1 It is -SS-.

[0110] In the implementation, b is 2, 3, or 4. In the implementation, b is 2 or 3. In the implementation, b is 1. In the implementation, b is 2. In the implementation, b is 3. In the implementation, b is 4. In the implementation, b is 5.

[0111] In the implementation scheme, the cationic lipid has a structure according to formula (Ir): Or a pharmaceutically acceptable salt thereof, wherein each of c, d, e and f is independently selected from 3, 4 or 6.

[0112] In one implementation, each a is 3. In another implementation, each a is 4. In one implementation, the value of a on the left side of the described expression is 3 and the value of a on the right side of the described expression is 4. In another implementation, the value of a on the left side of the described expression is 4 and the value of a on the right side of the described expression is 3.

[0113] In the implementation scheme, c is 3, 4, or 6. In the implementation scheme, c is 3. In the implementation scheme, c is 4. In the implementation scheme, c is 5. In the implementation scheme, c is 6.

[0114] In the implementation scheme, d is 3, 4, or 6. In the implementation scheme, d is 3. In the implementation scheme, d is 4. In the implementation scheme, d is 5. In the implementation scheme, d is 6.

[0115] In the implementation scheme, e is 3, 4, or 6. In the implementation scheme, e is 3. In the implementation scheme, e is 4. In the implementation scheme, e is 5. In the implementation scheme, e is 6.

[0116] In the implementation scheme, f is 3, 4, or 6. In the implementation scheme, f is 3. In the implementation scheme, f is 4. In the implementation scheme, f is 5. In the implementation scheme, f is 6.

[0117] In the implementation scheme, each of c, d, e, and f is independently selected from 3, 4, or 6.

[0118] In the implementation, c, d, e, and f are the same. In the implementation, c, d, e, and f are 3. In the implementation, c, d, e, and f are 4. In the implementation, c, d, e, and f are 5. In the implementation, c, d, e, and f are 6.

[0119] In the implementation, c and d are the same. In the implementation, c and d are 3. In the implementation, c and d are 4. In the implementation, c and d are 5. In the implementation, c and d are 6.

[0120] In the implementation, e and f are the same. In the implementation, e and f are 3. In the implementation, e and f are 4. In the implementation, e and f are 5. In the implementation, e and f are 6.

[0121] In one implementation, c and d are the same and e and f are the same, but c and d are different from e and f. In another implementation, c and d are 3 and e and f are 4. In another implementation, c and d are 3 and e and f are 5. In another implementation, c and d are 3 and e and f are 6. In another implementation, c and d are 4 and e and f are 3. In another implementation, c and d are 4 and e and f are 5. In another implementation, c and d are 4 and e and f are 6. In another implementation, c and d are 5 and e and f are 3. In another implementation, c and d are 5 and e and f are 4. In another implementation, c and d are 5 and e and f are 6. In another implementation, c and d are 6 and e and f are 3. In another implementation, c and d are 6 and e and f are 4. In another implementation, c and d are 6 and e and f are 5.

[0122] In the implementation plan, each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C4-C6) alkyl groups. In the embodiments, each R 1A R 1B R 1C and R1D Independently selected from optionally substituted (C5-C6) alkyl groups. In the embodiments, each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C5) alkyl groups. In the embodiments, each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C4) alkyl groups.

[0123] In the implementation plan, R 1A It is an optionally substituted C3 alkyl group. In the embodiments, R 1A It is an optionally substituted C4 alkyl group. In the embodiments, R 1A It is an optionally substituted C5 alkyl group. In the embodiments, R 1A It is an optional substituted C6 alkyl group.

[0124] In the implementation plan, R 1B It is an optionally substituted C3 alkyl group. In the embodiments, R 1B It is an optionally substituted C4 alkyl group. In the embodiments, R 1B It is an optionally substituted C5 alkyl group. In the embodiments, R 1B It is an optional substituted C6 alkyl group.

[0125] In the implementation plan, R 1C It is an optionally substituted C3 alkyl group. In the embodiments, R 1C It is an optionally substituted C4 alkyl group. In the embodiments, R 1C It is an optionally substituted C5 alkyl group. In the embodiments, R 1C It is an optional substituted C6 alkyl group.

[0126] In the implementation plan, R 1D It is an optionally substituted C3 alkyl group. In the embodiments, R 1D It is an optionally substituted C4 alkyl group. In the embodiments, R 1D It is an optionally substituted C5 alkyl group. In the embodiments, R 1D It is an optional substituted C6 alkyl group.

[0127] In the implementation plan, R 1A R 1B R 1C and R 1D They are the same. In the implementation plan, R 1A and R 1B They are the same. In the implementation plan, R 1Cand R 1D They are the same.

[0128] In the implementation plan, R 1A and R 1B They are the same and R 1C and R 1D They are the same, but R is different. 1A and R 1B With R 1C and R 1D different.

[0129] In the implementation plan, each R 1A R 1B R 1C and R 1D When it exists, it is selected independently: or

[0130] In the implementation plan, R 1A yes In the implementation plan, R 1A yes In the implementation plan, R 1A yes In the implementation plan, R 1A yes In the implementation plan, R 1A yes In the implementation plan, R 1A yes

[0131] In the implementation plan, R 1B yes In the implementation plan, R 1B yes In the implementation plan, R 1B yes In the implementation plan, R 1B yes In the implementation plan, R 1B yes In the implementation plan, R 1B yes

[0132] In the implementation plan, R 1C yes In the implementation plan, R 1C yes In the implementation plan, R 1C yes In the implementation plan, R 1C yes In the implementation plan, R 1C yes In the implementation plan, R 1C yes

[0133] In the implementation plan, R 1D yes In the implementation plan, R 1D yes In the implementation plan, R 1D yes In the implementation plan, R 1D yes In the implementation plan, R 1D yes In the implementation plan, R 1D yes

[0134] In the implementation scheme, c and d are 3 and R 1A and R 1B yes In the implementation scheme, c and d are 4 and R 1A and R 1B yes In the implementation scheme, c and d are 6 and R 1A and R 1B yes In the implementation scheme, c and d are 4 and R 1A and R 1B yes In the implementation scheme, c and d are 6 and R 1A and R 1B yes In the implementation scheme, c and d are 4 and R 1A and R 1B yes In the implementation scheme, c and d are 6 and R 1A and R 1B yes In the implementation scheme, c and d are 3 and R 1A and R 1B yes In the implementation scheme, c and d are 4 and R 1A and R 1B yes In the implementation scheme, c and d are 6 and R 1A and R 1B yes In the implementation scheme, c and d are 3 and R 1A and R 1B yes In the implementation scheme, c and d are 4 and R 1A and R 1Byes In the implementation scheme, c and d are 6 and R 1A and R 1B yes

[0135] In the implementation scheme, e and f are 3 and R 1C and R 1D yes In the implementation scheme, e and f are 4 and R 1C and R 1D yes In the implementation plan, e and f are 6 and R 1C and R 1D yes In the implementation scheme, e and f are 4 and R 1C and R 1D yes In the implementation plan, e and f are 6 and R 1C and R 1D yes In the implementation scheme, e and f are 4 and R 1C and R 1D yes In the implementation scheme, e and f are 6 and R 1C and R 1D yes In the implementation scheme, e and f are 3 and R 1C and R 1D yes In the implementation scheme, e and f are 4 and R 1C and R 1D yes In the implementation plan, e and f are 6 and R 1C and R 1D yes In the implementation scheme, e and f are 3 and R 1C and R 1D yes In the implementation scheme, e and f are 4 and R 1C and R 1D yes In the implementation plan, e and f are 6 and R 1C and R 1D yes

[0136] In the implementation plan, each a is 4, c and d are 6, and R... 1A and R 1B yes e and f are 4 and R 1C and R 1D yes

[0137] In the implementation scheme, the substituents are not optionally substituted.

[0138] In embodiments, the cationic lipids of the present invention have any one of the structures in Table A, Table B and / or Table C, or a pharmaceutically acceptable salt thereof.

[0139] In the embodiments, the cationic lipids of the present invention have any of the structures in the examples, or a pharmaceutically acceptable salt thereof.

[0140] In embodiments, this document provides compositions comprising the cationic lipids of the present invention, and further comprising: (i) One or more non-cationic lipids (e.g., phospholipids, such as DOPE), (ii) one or more cholesterol-based lipids (e.g., cholesterol), and (iii) One or more lipids modified with PEG.

[0141] In one embodiment, the composition is lipid nanoparticles, optionally liposomes. In another embodiment, the one or more cationic lipids constitute about 30 mol%-60 mol% of the lipid nanoparticles. In another embodiment, the one or more cationic lipids constitute about 31 mol%-59 mol% of the lipid nanoparticles. In another embodiment, the one or more cationic lipids constitute about 35 mol%-45 mol% of the lipid nanoparticles. In yet another embodiment, the one or more cationic lipids constitute about 40 mol% of the lipid nanoparticles.

[0142] In one embodiment, the one or more non-cationic lipids constitute about 10 mol% to 50 mol% of the lipid nanoparticles. In another embodiment, the one or more non-cationic lipids constitute about 11 mol% to 49 mol% of the lipid nanoparticles. In yet another embodiment, the one or more non-cationic lipids constitute about 20 mol% to 40 mol% of the lipid nanoparticles. In yet another embodiment, the one or more non-cationic lipids constitute about 25 mol% to 35 mol% of the lipid nanoparticles. In yet another embodiment, the one or more non-cationic lipids constitute about 30 mol% of the lipid nanoparticles.

[0143] In one embodiment, the one or more PEG-modified lipids constitute about 1 mol% to 10 mol% of the lipid nanoparticles. In another embodiment, the one or more PEG-modified lipids constitute about 1.1 mol% to 9 mol% of the lipid nanoparticles. In yet another embodiment, the one or more PEG-modified lipids constitute about 1 mol% to 5 mol% of the lipid nanoparticles. In yet another embodiment, the one or more PEG-modified lipids constitute about 1.5 mol% to 3 mol% of the lipid nanoparticles.

[0144] In one embodiment, cholesterol-based lipids constitute approximately 10 mol% to 50 mol% of the lipid nanoparticles. In another embodiment, cholesterol-based lipids constitute approximately 11 mol% to 49 mol% of the lipid nanoparticles. In yet another embodiment, cholesterol-based lipids constitute approximately 20 mol% to 40 mol% of the lipid nanoparticles. In yet another embodiment, cholesterol-based lipids constitute approximately 25 mol% to 35 mol% of the lipid nanoparticles. In yet another embodiment, cholesterol-based lipids constitute approximately 27 mol% to 28.5 mol% of the lipid nanoparticles.

[0145] In the embodiments, the one or more cationic lipids constitute about 31 mol%-59 mol% of the lipid nanoparticles, the one or more non-cationic lipids constitute about 11 mol%-49 mol% of the lipid nanoparticles, the one or more PEG-modified lipids constitute about 1.1 mol%-9 mol% of the lipid nanoparticles, and cholesterol-based lipids constitute about 11 mol%-49 mol% of the lipid nanoparticles.

[0146] In the embodiments, the one or more cationic lipids constitute about 35 mol%-45 mol% of the lipid nanoparticles, the one or more non-cationic lipids constitute about 25 mol%-35 mol% of the lipid nanoparticles, the one or more PEG-modified lipids constitute about 1 mol%-5 mol% of the lipid nanoparticles, and cholesterol-based lipids constitute about 25 mol%-35 mol% of the lipid nanoparticles.

[0147] In the embodiments, the one or more cationic lipids constitute about 40 mol% of the lipid nanoparticles, the one or more non-cationic lipids constitute about 30 mol% of the lipid nanoparticles, the one or more PEG-modified lipids constitute about 1.5 mol% to 3 mol% of the lipid nanoparticles, and cholesterol-based lipids constitute about 27 mol% to 28.5 mol% of the lipid nanoparticles.

[0148] In one embodiment, lipid nanoparticles encapsulate nucleic acids, optionally mRNA encoding peptides or proteins. In another embodiment, lipid nanoparticles encapsulate mRNA encoding peptides or proteins, optionally for use in a vaccine. In yet another embodiment, the peptide is an antigen.

[0149] As used herein, the phrase "encapsulation percentage" refers to the fraction of a therapeutic agent (e.g., mRNA) effectively encapsulated within a liposome-based medium (e.g., lipid nanoparticles) relative to an initial fraction 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 embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 90%. In embodiments, the encapsulation percentage of mRNA by the lipid nanoparticles is at least 95%. In the implementation, the encapsulation percentage is calculated by performing a Ribogreen assay (Invitrogen) with or without 0.1% Triton-X 100.

[0150] In an embodiment, the composition of the present invention is used in a therapeutic context.

[0151] In embodiments, the compositions of the present invention are used in methods of treating or preventing diseases that can be treated or prevented by peptides or proteins encoded by mRNA, optionally wherein the mRNA encodes an antigen and / or wherein the disease is (a) a protein deficiency, optionally wherein the protein deficiency affects the liver, lungs, brain or muscles, (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0152] In an implementation, 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 can be treated or prevented 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.

[0153] In some embodiments, the composition is administered intravenously, intrathecally, or intramuscularly, or via pulmonary delivery, optionally via nebulization. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intravenously. Exemplary compounds

[0154] In embodiments, the cationic lipids of the present invention comprise compounds selected from those depicted in Table A, or pharmaceutically acceptable salts thereof.

[0155] Exemplary compounds include those described in Table A, or pharmaceutically acceptable salts thereof. Table A

[0156] Any of the compounds 1-60 identified in Table A above may be provided in the form of pharmaceutically acceptable salts and such salts are intended to be covered by this invention.

[0157] Exemplary compounds include those described in Table B, or pharmaceutically acceptable salts thereof.

[0158] Any of the compounds identified in Table B above, 1-4, 6-24, 26-29, 31-54, 56-57, 59-130 and 155, may be provided in the form of pharmaceutically acceptable salts and such salts are intended to be covered by this invention.

[0159] Exemplary compounds include those described in Table C, or pharmaceutically acceptable salts thereof. Table C

[0160] Any of the compounds 131-154 identified in Table C above may be provided in the form of pharmaceutically acceptable salts and such salts are intended to be covered by this invention.

[0161] The compounds of the present invention as described herein can be prepared according to methods known in the art (including exemplary synthesis of the embodiments provided herein). Nucleic acid

[0162] The compounds of the present invention, as described herein, can be used to prepare compositions that can be used for the delivery of nucleic acids. Nucleic acid synthesis

[0163] Nucleic acids according to the invention can be synthesized according to any known method. For example, mRNA according to the invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using: a straight or circular DNA template containing a promoter, a library of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, mutant T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application.

[0164] In some embodiments, to prepare the mRNA of the present invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription (e.g., T3, T7, mutant T7, or SP6 promoter), followed by the desired nucleotide sequence of the desired mRNA and a termination signal.

[0165] One or more desired mRNA sequences 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 from a desired amino acid sequence (e.g., an enzyme sequence). An optimization algorithm can then be used to select appropriate codons. Typically, the G / C content can be optimized to achieve the highest possible G / C content, while the frequency of tRNA usage is considered to the maximum extent possible based on codon usage. The optimized RNA sequence can be constructed and displayed, for example, with the aid of a suitable display device, and compared with the initial (wild-type) sequence. Secondary structures can also be analyzed to calculate the stability and destabilization properties of the RNA or individual regions. Modified mRNA

[0166] In some embodiments, the mRNA according to the invention can be synthesized as unmodified or modified mRNA. Modified mRNA contains nucleotide modifications within the RNA. The modified mRNA according to the invention can therefore contain nucleotide modifications as, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, 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 nucleotide analogs or derivatives such as purines and pyrimidines, for example 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N-6-methyl-adenine, N-6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudourine Pyrimidine (5-uracil), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyluracil, 5-methoxy Aminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, methyl uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid (v), 1-methyl-pseudouracil, piracetamidine, β-D-mannosyl-piracetamidine, wybutoxosine, and aminophosphates, thiophosphates, peptide nucleotides, methylphosphonates, 7-dezoguanosine, 5-methylcytosine, and inosine. The preparation of such analogues is known to those skilled in the art, for example, by reference to 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 containing cationic lipids and nucleic acids

[0167] In some embodiments, the compounds of the present invention as described herein, and pharmaceutical and liposome compositions comprising such lipids, can be used in formulations to facilitate the delivery of encapsulated material (e.g., one or more polynucleotides such as mRNA) to one or more target cells and subsequently transfect said one or more target cells. For example, in some embodiments, the cationic lipids (and compositions comprising such lipids, such as liposome compositions) described herein are characterized by causing one or more of the following: receptor-mediated endocytosis, clathrin-mediated and pit-mediated endocytosis, phagocytosis and macropinocytosis, fusogenicity, endosome or lysosomal disruption, and / or release properties, which provide such compounds with an advantage over other similar classes of lipids.

[0168] According to the present invention, nucleic acids, such as mRNA encoding proteins (e.g., full-length, fragments, or portions of proteins), can be delivered via a delivery medium comprising compounds of the present invention as described herein.

[0169] As used herein, the terms “delivery medium,” “transfer medium,” “nanoparticle,” or their grammatical equivalents are used interchangeably.

[0170] For example, the present invention provides compositions comprising the 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 cholesterol-based lipids and / or (iv) One or more PEG-modified lipids.

[0171] 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 cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposome formulation comprising one or more polynucleotides encapsulated therein), such that the one or more target cells are transfected with the encapsulated material (e.g., one or more polynucleotides). As used herein, the term “transfect” or “transfection” refers to the intracellular introduction of one or more encapsulated materials (e.g., nucleic acids and / or polynucleotides) into cells (e.g., into target cells). The introduced polynucleotides can be stably or transiently maintained in the target cells. The term “transfection efficiency” refers to the relative amount of such encapsulated material (e.g., polynucleotides) absorbed by the transfected target cells, introduced into the transfected target cells, and / or expressed by the transfected target cells. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide products produced by the target cells after transfection. In some embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby improving the possibility of delivering an appropriate dose of encapsulated material (e.g., one or more polynucleotides) to a pathological site and subsequently expressing it, while minimizing potential systemic adverse effects or toxicities associated with the compounds or their encapsulated contents.

[0172] Transfection of one or more target cells with a polynucleotide, for example, encapsulated in one or more lipid nanoparticles including pharmaceutical or liposome compositions disclosed herein, can stimulate the production of products (e.g., peptides or proteins) encoded by such polynucleotides and enhance the ability of such target cells to express polynucleotides and produce, for example, the target peptide 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.

[0173] Additionally, 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, spleen, or muscle. In embodiments, delivery mediators described herein (e.g., liposome delivery mediators) can be prepared for preferential distribution to the lungs. In embodiments, delivery mediators described herein (e.g., liposome delivery mediators) can be prepared for preferential distribution to muscle tissue. 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 compounds or drug and liposome compositions described herein can be delivered to target cells or tissues and / or can be transfected into target cells or tissues. In some embodiments, the encapsulated polynucleotides (e.g., mRNA) are capable of being expressed by and produced by target cells (and in some cases secreted by target cells) into functional polypeptide products, thereby conferring beneficial properties, for example, to target cells or tissues. Such encapsulated polynucleotides (e.g., mRNA) can encode, for example, hormones, enzymes, receptors, polypeptides, peptides, or other target proteins. Liposome delivery mediators

[0174] In some embodiments, the composition is a suitable delivery medium. In one embodiment, the composition is a liposome delivery medium, such as lipid nanoparticles.

[0175] The terms “liposome delivery medium” and “liposome composition” are used interchangeably.

[0176] Enriching a liposome composition for one or more of the cationic lipids disclosed herein can be used as a means of improving safety profiles or otherwise conferring one or more desired properties to such enriched liposome compositions (e.g., 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 cationic lipids disclosed herein, particularly liposome compositions, are also contemplated.

[0177] Therefore, in some embodiments, the compounds of the present invention as described herein can be used as components of liposome compositions to facilitate or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic agents) to one or more target cells (e.g., by penetrating or fusing with the lipid membrane of such target cells).

[0178] As used herein, liposome delivery mediators (e.g., lipid nanoparticles) are typically characterized as microvesicles having an internal water space isolated from an external medium by one or more bilayer membranes. The bilayer membranes of liposomes are typically formed of amphiphilic molecules, such as synthetic or naturally derived lipids, which contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membranes of liposomes can also be formed of amphiphilic polymers and surfactants (e.g., polymerosomes, lipid vesicles, etc.). In the context of this invention, liposome delivery mediators are generally used to transport desired mRNA to target cells or tissues.

[0179] In some embodiments, materials (such as one or more bioactive polynucleotides (e.g., mRNA)) are loaded into such compositions (e.g., liposome compositions) or such compositions (e.g., liposome compositions) are otherwise encapsulated in these materials.

[0180] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises mRNA encoding a peptide or protein encapsulated within liposomes. In some embodiments, the liposomes comprise: (i) one or more cationic lipids, (ii) one or more non-cationic lipids, (iii) One or more cholesterol-based lipids and (iv) One or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the present invention as described herein.

[0181] 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).

[0182] In some embodiments, the composition (e.g., a pharmaceutical composition) comprises nucleic acids encapsulated in liposomes, wherein the liposomes contain compounds described herein.

[0183] In some embodiments, the nucleic acid is mRNA encoding a peptide or protein. In some embodiments, the mRNA encodes a peptide or protein for delivery to or treatment of the lungs or lung cells of a subject. In some embodiments, the mRNA encodes a peptide or protein for delivery to or treatment of the liver or hepatocytes of a subject. In some embodiments, the mRNA encodes a peptide or protein for delivery to or treatment of muscle cells. In some embodiments, the mRNA encodes a peptide or protein for delivery to or treatment of immune cells. Other exemplary mRNAs are still described herein.

[0184] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net positive charge.

[0185] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net negative charge.

[0186] In the implementation scheme, the liposome delivery medium (e.g., lipid nanoparticles) may have a net neutral charge.

[0187] In some embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein.

[0188] For example, the amount of the compounds of the present invention in a composition as described herein can be described as a percentage (“wt%”) of the combined dry weight of all lipids in the composition (e.g., the combined dry weight of all lipids present in a liposome composition).

[0189] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in amounts of about 0.5 wt% to about 30 wt% (e.g., about 0.5 wt% to about 20 wt%) of the combined dry weight of all lipids present in the composition (e.g., liposome composition).

[0190] In embodiments, the compounds of the present invention as described herein are present in amounts of about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, about 1 wt% to about 15 wt%, about 1 wt% to about 10 wt%, or about 5 wt% to about 25 wt% of the combined dry weight of all lipids present in the composition (e.g., a liposome composition). In embodiments, the compounds of the present invention as described herein are present in amounts of about 0.5 wt% to about 5 wt%, about 1 wt% to about 10 wt%, about 5 wt% to about 20 wt%, or about 10 wt% to about 20 wt% of the combined dry weight of all lipids present in the composition (e.g., a liposome delivery medium).

[0191] In embodiments, the compounds of the present invention as described herein are present in amounts of at least about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in the composition (e.g., a liposome composition).

[0192] In embodiments, the amount of the compounds of the present invention as described herein is not more than about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, or about 99 wt% of the combined dry weight of total lipids in the composition (e.g., a liposome composition).

[0193] In some embodiments, the composition (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises about 0.1 wt% to about 20 wt% (e.g., about 0.1 wt% to about 15 wt%) of the compounds described herein. In some embodiments, the delivery medium (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, or about 10 wt% of the compounds described herein. In some embodiments, the delivery medium (e.g., a liposome delivery medium, such as lipid nanoparticles) comprises up to about 0.5 wt%, about 1 wt%, about 3 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt% of the compounds described herein. In some embodiments, the percentages result in improved beneficial effects (e.g., improved delivery to target tissues such as the liver, lungs, or muscle).

[0194] The amount of the compounds of the present invention as described herein in a composition can be described as a percentage (“mol%”) of the total molar amount of lipids in the composition (e.g., the total molar amount of all lipids present in a liposome delivery medium).

[0195] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention as described herein are present in amounts of about 0.5 mol% to about 50 mol% (e.g., about 0.5 mol% to about 20 mol%) of the combined molar amount of all lipids present in the composition (e.g., liposome delivery mediator).

[0196] In embodiments, the compounds of the present invention as described herein are present in amounts of about 0.5 mol% to about 5 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 15 mol% to about 30 mol%, about 20 mol% to about 35 mol%, about 25 mol% to about 40 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 55 mol%, or about 45 mol% to about 60 mol%, representing the combined molar amounts of all lipids present in the composition (e.g., a liposome delivery medium). In embodiments, the compounds of the present invention as described herein are present in amounts of about 1 mol% to about 60 mol%, 1 mol% to about 50 mol%, 1 mol% to about 40 mol%, 1 mol% to about 30 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 5 mol% to about 55 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 35 mol%, or about 5 mol% to about 25 mol%.

[0197] In some embodiments, the compounds of the present invention as described herein may comprise about 0.1 mol% to about 50 mol% of the total lipids in the composition (e.g., a liposome delivery medium), or about 1 mol% to about 50 mol%, or about 5 mol% to about 50 mol%, or about 10 mol% to about 50 mol%, or about 15 mol% to about 50 mol%, or about 20 mol% to about 50 mol%, or about 25 mol% to about 50 mol%, or about 30 mol% to about 50 mol%.

[0198] In some embodiments, the compounds of the present invention as described herein may constitute a greater than about 0.1 mol%, or greater than about 0.5 mol%, or greater than about 1 mol%, or greater than about 5 mol%, or greater than about 10 mol%, or greater than about 20 mol%, or greater than about 30 mol%, or greater than about 40 mol% of the total lipids in the lipid nanoparticles.

[0199] In some embodiments, the compounds described herein may constitute less than about 60 mol%, or less than about 55 mol%, or less than about 50 mol%, or less than about 45 mol%, or less than about 40 mol%, or less than about 35 mol%, or less than about 30 mol%, or less than about 25 mol%, or less than about 10 mol%, or less than about 5 mol%, or less than about 1 mol% of the total lipids in the composition (e.g., a liposome delivery medium).

[0200] In embodiments, the compounds of the present invention as described herein are present in amounts of at least about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol% of the combined molar amount of total lipids in the composition (e.g., a liposome composition).

[0201] In embodiments, the amount of the compounds of the present invention as described herein is such that the combined molar amount of total lipids in the composition (e.g., a liposome composition) is not more than about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about 96 mol%, about 97 mol%, about 98 mol%, or about 99 mol%.

[0202] In the implementation, the percentage results in improved beneficial effects (e.g., improved delivery to target tissues such as liver, lungs, or muscle, optionally muscle).

[0203] In a typical embodiment, 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 cholesterol-based lipids, and (iv) One or more PEG-modified lipids, wherein at least one cationic lipid is a compound of the present invention as described herein.

[0204] For example, compositions suitable for practicing the present invention have four lipid components, including compounds of the present invention as described herein as cationic lipid components, and further include: (i) Non-cationic lipids, (ii) Cholesterol-based lipids and (iii) PEG-modified lipids.

[0205] Non-cationic lipids can be DOPE or DEPE. Cholesterol-based lipids can be cholesterol. PEG-modified lipids can be DMG-PEG2K.

[0206] In another embodiment, the pharmaceutical (e.g., liposome) composition comprises one or more of PEG-modified lipids, non-cationic lipids, and cholesterol lipids. In yet another embodiment, such pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids, one or more non-cationic lipids, and one or more cholesterol lipids. In yet another embodiment, such pharmaceutical (e.g., liposome) composition comprises one or more PEG-modified lipids and one or more cholesterol lipids.

[0207] In embodiments, compositions encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) (e.g., lipid nanoparticles) comprise one or more compounds of the present invention as described herein, and one or more lipids selected from cationic lipids, non-cationic lipids, and PEGylated lipids.

[0208] In embodiments, the composition encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) (e.g., lipid nanoparticles) comprises one or more compounds of the present invention as described herein; one or more lipids selected from cationic lipids, non-cationic lipids, and PEGylated lipids; and further comprises cholesterol-based lipids. Typically, such a composition has four lipid components, including compounds of the present invention as described herein as cationic lipid components, and further comprises: (i) Non-cationic lipids (e.g., DOPE). (ii) Cholesterol-based lipids (e.g., cholesterol) and (iii) PEG-modified lipids (e.g., DMG-PEG2K).

[0209] In the embodiments, the lipid nanoparticles encapsulating nucleic acids (e.g., mRNA encoding peptides or proteins) comprise one or more compounds of the present invention as described herein, and one or more lipids selected from the following: (i) cationic lipids, (ii) Non-cationic lipids, (iii) PEGylated lipids and (iv) Cholesterol-based lipids.

[0210] According to various embodiments, the selection of cationic lipids, non-cationic lipids, and / or PEG-modified lipids constituting the lipid nanoparticles, and the relative molar ratios of such lipids relative to each other, is based on the characteristics of one or more selected lipids, the properties of the intended target cells, and the characteristics of the mRNA to be delivered. Other considerations include, for example, the saturation of the alkyl chain, and the size, charge, pH, pKa, fusion properties, and toxicity of one or more selected lipids. Therefore, the molar ratios can be adjusted accordingly.

[0211] In one embodiment, the lipid nanoparticles of the present invention have a diameter of about 120 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 60 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 70 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 80 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 90 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 100 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 110 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 115 nm to 125 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 60 nm to 130 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 70 nm to 130 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 80 nm to 130 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of about 90 nm to 130 nm. In one embodiment, the lipid nanoparticles of the present invention have a diameter of approximately 100 nm to 130 nm. In another embodiment, the lipid nanoparticles of the present invention have a diameter of approximately 110 nm to 130 nm. In yet another embodiment, the diameter of the lipid nanoparticles is determined using dynamic light scattering (DLS). Dynamic light scattering (DLS) measurements can be performed using a Malvern Instruments Zetasizer (Worcestershire, UK) with a 173° backscatter detector angle and a 4-mW, 633-nm He-Ne laser. Samples can be analyzed by diluting them in 10% trehalose and measuring the diameter in an optical-grade polystyrene cuvette. cationic lipids

[0212] In addition to any of the compounds of the present invention as described herein, the composition may also contain one or more additional cationic lipids.

[0213] In some embodiments, liposomes may comprise one or more additional cationic lipids. As used herein, the phrase "cationic lipid" refers to any of a variety of lipid species that have 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.

[0214] Suitable additional cationic lipids for use in the composition include cationic lipids as described in the literature. assist lipids

[0215] Compositions (e.g., liposome compositions) may also contain one or more accessory lipids. Such accessory lipids include noncationic lipids. As used herein, the phrase "noncationic lipid" refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipids that carry a net negative charge at a selected pH, such as physiological pH. Noncationic lipids include, but are not limited to, distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylethanolamine (…). Dioleoylphosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearate-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or mixtures thereof. A suitable non-cationic lipid or co-lipid for practicing this invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE) can be used as a non-cationic lipid or co-lipid.

[0216] 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.

[0217] In some embodiments, non-cationic lipids may be present in the following molar ratios (mol%): about 5% to about 90% of the total lipids present in the composition, 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, total non-cationic lipids may be present in the following molar ratios (mol%): about 5% to about 90% of the total lipids present in the composition, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40%. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipids in the liposomes may not exceed about 5 mol%, not exceed about 10 mol%, not exceed about 20 mol%, not exceed about 30 mol%, or not exceed about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may not exceed about 5 mol%, not exceed about 10 mol%, not exceed about 20 mol%, not exceed about 30 mol%, or not exceed about 40 mol%.

[0218] In some embodiments, non-cationic lipids may be present in the following weight percentages (wt%): 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 weight percentages (wt%): about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non-cationic lipids in the liposomes may not exceed about 5 wt%, not exceed about 10 wt%, not exceed about 20 wt%, not exceed about 30 wt%, or not exceed about 40 wt%. In some embodiments, the percentage of total non-cationic lipids in the liposomes may not exceed about 5 wt%, not exceed about 10 wt%, not exceed about 20 wt%, not exceed about 30 wt%, or not exceed about 40 wt%. Cholesterol-based lipids

[0219] In some embodiments, the composition comprising the cationic lipids of the present invention (e.g., liposome compositions) further comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for practicing the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylformamidocholesterol), 1,4-bis(3-N-oleoylaminopropyl)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:

[0220] In some embodiments, cholesterol-based lipids may be present in the following molar ratios (mol%): from about 1% to about 30% or from about 5% to about 20% of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may not exceed about 5 mol%, not exceed about 10 mol%, not exceed about 20 mol%, not exceed about 30 mol%, or not exceed about 40 mol%.

[0221] In some embodiments, cholesterol-based lipids may be present in the following weight percentages (wt%): from about 1% to about 30% or from about 5% to about 20% of the total lipids present in the liposomes. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipids in the lipid nanoparticles may not exceed about 5 wt%, not exceed about 10 wt%, not exceed about 20 wt%, not exceed about 30 wt%, or not exceed about 40 wt%. PEGylated lipids

[0222] 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-racemic-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0223] For example, the present invention also envisions the use of polyethylene glycol (PEG) modified phospholipids and derivatized lipids (such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000](C8 PEG-2000 ceramide)) in combination with one or more compounds of the present invention as described herein and other lipids constituting liposomes in some embodiments. 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.

[0224] Other envisioned PEG-modified lipids (also referred to herein as PEGylated lipids, the term being used interchangeably with PEG-modified lipids) include, but are not limited to, covalently attached to lipids having one or more (C6-C) lines. 20The lipids are alkyl chains of up to 5 kDa polyethylene glycol chains. In some embodiments, the PEG-modified lipids or PEGylated lipids are PEGylated cholesterol or PEG-2K. The addition of such components can prevent complex aggregation and can also provide a means of increasing the cycle life of the lipid-nucleic acid composition and increasing its delivery to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they can be selected for rapid exchange from the formulation in vivo (see U.S. Patent No. 5,885,613).

[0225] 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

[0226] The compounds of the present invention as described herein can be used in the preparation of compositions (e.g., to construct liposome compositions) that promote or enhance the delivery and release of encapsulated materials (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeating or fusing with the lipid membrane of such target cells).

[0227] For example, when a liposome composition (e.g., lipid nanoparticles) contains or is otherwise enriched with one or more compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate the delivery of encapsulated materials (e.g., one or more therapeutic polynucleotides encapsulated in lipid nanoparticles) to one or more target cells.

[0228] 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 varies with the high transfection efficiency associated with the compositions disclosed herein, making it possible to administer a reduced amount of this composition to a subject to achieve a desired therapeutic response or outcome.

[0229] In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomes characterized by efficient intramuscular delivery of mRNA. In some embodiments, the compounds of the present invention as described herein can be used to prepare liposomes characterized by achieving high levels of expression of said peptide or protein when delivered via intramuscular delivery of mRNA encoding said peptide or protein.

[0230] Therefore, pharmaceutical formulations comprising the compounds described herein and the nucleic acids provided herein can be used for various disease treatment and / or disease prevention purposes. To facilitate in vivo delivery of nucleic acids, the compounds and nucleic acids described herein can be formulated in combination with one or more additional drug carriers, targeting ligands, or stabilizing agents. In some embodiments, the compounds described herein can be formulated via a premixed lipid solution. In other embodiments, compositions comprising the compounds described herein can be formulated into lipid membranes of nanoparticles using a post-insertion technique. Techniques for drug formulation and administration can be found in the following literature: “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.

[0231] Suitable routes of administration include, for example, oral, rectal, vaginal, mucosal, pulmonary (including tracheal or inhalation), or enteral administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intravenous, intravenous, intraperitoneal, or intranasal administration. In certain embodiments, intramuscular administration is administration to muscles selected from skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, 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).

[0232] A common route of administration for the liposome compositions of the present invention is intravenous delivery, particularly when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamate (OTC) deficiency). Alternatively, the liposome compositions may be administered via pulmonary delivery, depending on the disease or disorder to be treated (e.g., for the treatment of cystic fibrosis). For vaccination, the liposome compositions of the present invention are typically administered intramuscularly. Diseases or disorders affecting the eye can be treated by intravitreal administration of the liposome compositions of the present invention.

[0233] Alternatively or additionally, the pharmaceutical formulations of the present invention can be administered locally rather than systemically, for example, by direct injection of the pharmaceutical formulation into a target tissue (e.g., in a sustained-release formulation). Local delivery can be affected in a variety of ways, depending on the tissue to be targeted. Exemplary tissues in which mRNA is delivered and / or expressed include, but are not limited to, the liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In an embodiment, the tissue to be targeted is in the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); the composition of the present invention can be injected into, for example, sites of injury, disease manifestation, or pain; the composition can be provided in tablet form for oral, tracheal, or esophageal application; it can be supplied in liquid, tablet, or capsule form for administration to the stomach or intestine; it can be supplied in suppository form for rectal or vaginal application; or it can even be delivered to the eye using cream, drops, or even injection.

[0234] The compositions described herein may contain mRNA encoding peptides, including those described herein (e.g., polypeptides, such as proteins).

[0235] In the implementation plan, the mRNA encodes a polypeptide.

[0236] In this implementation, the mRNA encodes a peptide. In this implementation, the peptide is an antigen.

[0237] In the implementation plan, mRNA encodes a protein.

[0238] The present invention provides a method for delivering a composition having a full-length mRNA molecule encoding a target peptide or protein, the method being used in a treatment subject (e.g., a human subject) or in cells of a human subject, or in cells of a human subject that have been treated and delivered to the human subject. delivery method

[0239] 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 methods involve intranasal, intratracheal, or pulmonary administration by aerosolization, nebulization, or instillation of a composition comprising mRNA encoding a therapeutic peptide or protein in a suitable transfection or lipocarrier medium as described above. In some embodiments, the peptide or protein is encapsulated in liposomes. In some embodiments, the liposomes comprise lipids that are compounds of this invention. As used herein, administration of the compounds of this invention includes administration of a composition comprising the compounds of this invention.

[0240] While localized lung cells and tissues can serve as potential targets for the production and secretion of mRNA-encoded proteins, the applicants have found that administration of the compounds of the present invention to the lungs via aerosolization, nebulization, or infusion results in the distribution of non-secreting proteins even outside lung cells. Without wishing to be bound by any particular theory, it is envisioned that the nanoparticle compositions of the present invention cross the lung airway-blood barrier, thereby resulting in the translation of intact nanoparticles into non-lung cells and tissues (e.g., heart, liver, spleen, muscle), where this leads to the production of encoded peptides or proteins in these non-lung tissues. Therefore, the use of the compounds of the present invention and the methods of the present invention not only produces therapeutic proteins in lung cells and lung tissues but can also be used for delivery to non-lung target cells and / or tissues. They can be used to manage and treat a wide range of diseases. In some embodiments, the compounds of the present invention used in the methods of the present invention result in the distribution of mRNA-encapsulated nanoparticles and the production of encoded peptides or proteins in liver, spleen, heart, muscle, and / or other non-lung cells. For example, the compounds of the present invention, when administered to the lungs via aerosolization, nebulization, or instillation, result in the detection of the composition itself and its peptide or protein products (e.g., antigens or functional proteins) 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.

[0241] In some embodiments, the compounds of the invention may be employed in the methods of the invention to specifically target peripheral cells or tissues. Following pulmonary delivery, it is envisioned that the compounds of the invention cross the pulmonary airway-blood barrier and distribute to cells outside the local lung cells. Therefore, the compounds disclosed herein can be administered to a subject via the pulmonary administration route using various methods known to those skilled in the art (e.g., by inhalation) and distributed to local target cells and tissues of the lungs as well as peripheral non-pulmonary 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-pulmonary cells can act as biological reservoirs or repositories capable of producing and / or secreting translational products encoded by one or more polynucleotides. Therefore, the invention is not limited to treating lung diseases or conditions, but can be used as a non-invasive means of promoting the delivery of polynucleotides or the production of peptides or proteins encoded therefrom in peripheral organs, tissues, and cells (e.g., hepatocytes), which in other cases is only 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.

[0242] 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 peripheral target tissues. The amount of peptide or protein product necessary to achieve a therapeutic effect varies depending on the condition to be treated, the encoded peptide or protein, and the patient's condition. For example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 days or longer after administration of the compound to a subject, a peptide or protein product at concentrations (e.g., therapeutic concentrations) of at least 0.025 μg / ml to 1.5 μg / ml (e.g., at least 0.050 μg / ml) can be detected in peripheral target tissues. (ml, at least 0.075μg / ml, at least 0.1μg / ml, at least 0.2μg / ml, at least 0.3μg / ml, at least 0.4μg / ml, at least 0.5μg / ml, at least 0.6μg / ml, at least 0.7μg / ml, at least 0.8μg / ml, at least 0.9μg / ml, at least 1.0μg / ml, at least 1.1μg / ml, at least 1.2μg / ml, at least 1.3μg / ml, at least 1.4μg / ml, or at least 1.5μg / ml).

[0243] Nucleic acids have been demonstrated to be delivered to the lungs via intratracheal administration of liquid suspensions of compounds, aerosol vapor generated by inhalation of liquid nebulizers, or the use of dry powder devices (such as those described in U.S. Patent 5,780,014, which is incorporated herein by reference).

[0244] In some embodiments, the compounds of the present invention can be formulated such that they can be aerosolized or otherwise delivered as particulate liquids or solids before or during administration to a subject. Such compounds can be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions) to produce particles easily breathed 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 (e.g., about 0.5 mg / kg mRNA per dose) to a subject. 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 containing the compound and a suitable propellant. In some embodiments, the compounds of the present invention can be formulated as particulate powders (e.g., inhalable dry granules) intended for inhalation. In some embodiments, the sizes of the compositions of the invention, formulated as breathable particles, are suitably designed such that they can be breathed by a subject or delivered using a suitable device (e.g., average D50 or D90 particle sizes less than about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm, or smaller). In yet other embodiments, the compounds of the invention are formulated to contain one or more pulmonary surfactants (e.g., lamellar bodies). In some embodiments, the compounds of the present invention are administered to a subject in a single dose at concentrations of at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, and so on. Less than 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg / kg body weight.In some embodiments, the compound of the invention is administered to a subject such that one or more doses are given in total amounts of: at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg mRNA. Synthesis of the compounds of the present invention

[0245] Cationic lipid MC3 is the current gold standard for in vivo delivery of, for example, siRNA (see WO2010 / 144740). However, the synthesis of this lipid involves a six-step process and requires handling Grignard reagents. In contrast, the present invention provides cationic lipids that can be prepared from readily available starting reagents such as “Good” buffer (see Table 1 below). These starting reagents can be coupled to cationic head groups and lipid tails using coupling reactions such as sulfonation, acetylation, and alkylation (see, for example, Table 2 below). Table 1: Examples of "Good" buffer solutions Table 2: Examples of lipid chains suitable for use in this invention

[0246] In implementations, the cationic lipids described herein can be prepared by conjugating a “Good” buffer with a lipid (e.g., a carboxylic acid of the lipid) under suitable conditions. Exemplary “Good” buffers are described in Table 1, and exemplary lipid chains are described in Table 2. Therefore, suitable cationic lipids include cationic lipids derived from any combination of precursors described in Tables 1 and 2.

[0247] In some embodiments, the sulfonic acid group of a compound (such as “Good” buffer) can be derivatized by forming a sulfonyl chloride using a reagent (such as oxaloyl chloride). The resulting sulfonyl chloride can undergo a variety of reactions, including but not limited to reduction with Zn / HCl to form the corresponding thiol and coupling to a nucleophile (such as an amine or alcohol) to form the corresponding sulfonamide and sulfonate ester (see, for example, Scheme A below):

[0248] Using the chemistry outlined in Scheme A, sulfonic acid starting reagents can be derivatized with a range of suitable cationic lipid head groups and lipid chains.

[0249] Furthermore, compounds such as the "Good" buffer can be readily synthesized, for example, by nucleophilic ring-opening of cyclic sulfides with piperazine (see, for example, Scheme B below).

[0250] The compounds of the present invention as described herein can be prepared according to methods known in the art (including exemplary synthesis of the embodiments provided herein). Example

[0251] While certain compounds, compositions, and methods of the present invention have been specifically described according to some embodiments, the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0252] Any compounds identified in the examples may be provided in the form of pharmaceutically acceptable salts and such salts are intended to be covered by this invention. List of abbreviations: APCI-MS: Atmospheric Pressure Chemical Ionization Mass Spectrometry EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: Ethyl acetate MS: Mass spectrometry Na2SO4: Sodium sulfate SiO2: Silicon dioxide TLC = Thin-layer chromatography Example 1: Synthesis of the compound of the present invention

[0253] For example, the compounds of the present invention can be prepared according to schemes 1 and 2. Option 1: Synthesis scheme of intermediates Scheme 2: Synthesis scheme for compound 48 Synthesis procedure for intermediate 3: Step 1: Synthesis of 2-(3-(triphenylmethylthio)propyl)isoindoline-1,3-dione (2)

[0254] As described in Scheme 1: Triphenylmethanethiol (200 g, 0.724 mol) was added in portions to a mixture of sodium hydride (30 g, 1.08 mol, 60% dispersion in mineral oil) in 600 mL of N,N-dimethylformamide at 0 °C. After stirring for 1 h, a solution of N-(3-bromopropyl)phthalimide 1 (194.1 g, 0.724 mol) in 400 mL of N,N-dimethylformamide was slowly added, and the resulting mixture was slowly warmed to room temperature and stirred overnight. The reaction mixture was poured into 6 L of ice-cold water, the solution was poured off, and the solid was dissolved in ethyl acetate and washed with brine. The organic layer was dried over Na₂SO₄ and concentrated to give 2-(3-(triphenylmethylthio)propyl)isoindoline-1,3-dione (252 g, 75%) as a white solid, which was used for the next step without further purification. Step 2: Synthesis of 3-(triphenylmethylthio)prop-1-amine (3)

[0255] As described in Scheme 1: A mixture of 2-(3-(triphenylmethylthio)propyl)isoindoline-1,3-dione 2 (252 g, 0.54 mol) and hydrazine hydrate (112 mL, 2.7 mol) in ethanol (3 L) was heated to slight reflux overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered through diatomaceous earth and then washed with ethanol. The combined filtrates were concentrated under reduced pressure, and the residue was dissolved in chloroform. After stirring for 15 min, the mixture was filtered and concentrated, and the crude product was purified by rapid column chromatography (SiO2: 0 to 15% methanol in dichloromethane) to give 3-(triphenylmethylthio)propyl-1-amine (100 g, 55%) as an oil. Synthesis procedure for epoxide 8: Step 1: Synthesis of non-8-enoic acid (5)

[0256] As described in Scheme 1: A solution of nonan-8-en-1-ol 4 (100 g, 0.7 mol) was added to a solution of periodic acid (353 g, 1.55 mol) in 2 L of acetonitrile at 0 °C, followed by dropwise addition of a solution of pyridinium chlorochromate (3.23 g, 15 mmol) in 500 mL of acetonitrile over 2 h. The resulting turbid mixture was stirred overnight at room temperature. TLC showed complete reaction. The reaction mixture was diluted with 1 L of EtOAc, and the solution was washed with water and brine. After drying with sodium sulfate, the organic layer was concentrated, and the crude product was purified by column chromatography (SiO2: 0 to 50% ethyl acetate in hexane) to give nonan-8-enoic acid (88 g, 80%) as a pale yellow oil. Step 2: Synthesis of 2-ethylbutyl nonanoic acid (7)

[0257] As described in Scheme 1: EDCI (73.6 g, 0.384 mol) and dimethylaminopyridine (7.8 g, 64 mmol) were added to a mixture of nonanoic acid 5 (50 g, 0.32 mol) and 2-ethylbutanol 6 (39.2 g, 0.384 mol) in 250 mL of dichloromethane, and the reaction mixture was stirred overnight. MS and TLC analyses showed complete reaction. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, water, and 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 20% ethyl acetate in hexane) to give 2-ethylbutane nonanoic acid (71 g, 92%) as a colorless oil. Step 3: Synthesis of 2-ethylbutyl 7-(ethylene oxide-2-yl)heptanoate (8)

[0258] As described in Scheme 1: A solution of 7-(71 g, 0.295 mol) 2-ethylbutyl nonanoic acid in 500 mL of dichloromethane was cooled to 0 °C, and 3-chloroperbenzoic acid (99.3 g, 0.443 mol) was added. The reaction mixture was stirred overnight at this temperature. The suspension was filtered, and a 1.2 M sodium bisulfite solution was added to the filtrate. After stirring for 1 h, the organic layer was separated and washed with sodium bicarbonate solution and brine. After drying with sodium sulfate, the solvent was evaporated to give 7-(ethylene oxide-2-yl)heptanic acid 2-ethylbutyl ester (70 g, 92%) as a colorless oil, which was used for the next step without purification. Synthesis procedure for TIM-3-E9Es6: Step 1: Synthesis of bis(2-ethylbutyl)9,9'-((3-(triphenylmethylthio)propyl)azanediyl)bis(8-hydroxynonanoate) (9)

[0259] As described in Scheme 1: A mixture of 3-(triphenylmethylthio)propyl-1-amine 3 (3.9 g, 11.7 mmol) and 2-ethylbutyl 7-(ethylene oxide-2-yl)heptanoate 8 (8.0 g, 35.1 mmol) in 30 mL of isopropanol was heated to slight reflux overnight under a nitrogen atmosphere. The reaction mixture was concentrated, and the crude product was purified by rapid column chromatography (SiO2: 0 to 10% methanol in dichloromethane) to give bis(2-ethylbutyl)9,9'-((3-(triphenylmethylthio)propyl)azaalkyldiyl)bis(8-hydroxynonanoate) (5.3 g, 53%) as a yellow oil. Step 2: Synthesis of bis(2-ethylbutyl)9,9'-((3-mercaptopropyl)azanidinediyl)bis(8-hydroxynonanoate) (TIM-3-E9Es6)

[0260] As described in Scheme 1: Trifluoroacetic acid (0.1 mL, 1.0 mmol) was added to a solution of bis(2-ethylbutyl)9,9'-((3-(triphenylmethylthio)propyl)azonidinediyl)bis(8-hydroxynonanoate)9 (169 mg, 0.20 mmol) and triethylsilane (0.1 mL, 0.6 mmol) in 10 mL of dichloromethane at 0 °C. The resulting reaction mixture was warmed to room temperature and stirred for 1 h. MS showed complete reaction. The volatiles were evaporated, and the residue was evaporated three times with toluene under vacuum. The crude product was used for the next step without further purification. Synthesis procedure for AIM-3-E9Es6: Step 1: Synthesis of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (11)

[0261] As described in Scheme 1: A solution of tert-butyl 4-aminobutyrate 10 (3.3 g, 15.9 mmol), 2-ethylbutyl 7-(ethylene oxide-2-yl)heptanoate 8 (9.0 g, 35.1 mmol), and diisopropylethylamine (5 mL, 28.7 mmol) in 5 mL of isopropanol was heated to reflux for 3 days. MS showed complete reaction. After concentration to dryness, the residue was purified by rapid column chromatography (SiO2: 0 to 100% ethyl acetate in hexane) to obtain bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (6.0 g, 56%) as a colorless oil. Step 2: Synthesis of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate)(12)

[0262] As described in Scheme 1: tert-butyldimethylchlorosilyl (5.3 g, 35 mmol), imidazole (0.6 g, 8.7 mmol), and dimethylaminopyridine (1.1 g, 8.7 mmol) were added to a solution of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azonidinediyl)bis(8-hydroxynonanoate)11 (6.0 g, 8.7 mmol) in 50 mL of dichloromethane, and the resulting mixture was heated to reflux for 48 h. MS showed complete reaction. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with water and brine. The combined organic layers were dried over sodium sulfate. After concentration, the residue was purified by rapid column chromatography (SiO2: 0 to 30% ethyl acetate in hexane) to obtain bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanidinediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) (4.9 g, 61%), which was a colorless oil. Step 7: Synthesis of 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxonyl)amino)butyric acid (AIM-3-E9Es6)

[0263] As described in Scheme 1: A solution of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azonidinediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate)12 (4.9 g, 5.36 mmol) in 15 mL of dichloromethane was cooled to 0 °C, and trifluoroacetic acid (20 mL, 0.13 mol) was added dropwise. The resulting mixture was stirred overnight at room temperature. MS showed complete reaction. Saturated sodium bicarbonate solution was added to adjust the solution to pH 7, and the mixture was extracted with dichloromethane. After drying with sodium sulfate, the solvent was removed under vacuum, and the residue was purified by rapid column chromatography (SiO2: 0 to 10% methanol in dichloromethane) to obtain 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxonyl)amino)butyric acid (4.1 g, 89%) as a colorless oil. Synthetic procedure for disulfide intermediate 16: Synthesis of 2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethyl-1-ol (16)

[0264] As described in Scheme 1: In a 2 L round-bottom flask, 18 g (0.3 mol) of cyclothioethane was added to a solution of 2-(piperazin-1-yl)ethyl-1-ol 13 (30.0 g, 0.23 mol) in 1500 mL of dichloromethane, and the mixture was stirred at room temperature for 72 h. Pyridyl disulfide 15 (60.8 g, 0.276 mol) was added, and the reaction mixture was stirred at room temperature for 24 h. MS and TLC analyses indicated that the reaction was complete. The reaction mixture was concentrated, and the residue was purified by rapid column chromatography (SiO2: 0 to 10% methanol in dichloromethane) to obtain 2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethyl-1-ol (37 g, 53%) as a pale yellow oil. Synthesis procedure of compound 48 Step 1: Synthesis of bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithio)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate)(17)

[0265] As described in Scheme 2: EDCI (0.79 g, 4.11 mmol) and dimethylaminopyridine (67 mg, 0.54 mmol) were added to a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxonyl)amino)butyric acid AIM-3-E9Es6 (2.2 g, 2.74 mmol) in 30 mL of dichloromethane, followed by the addition of a solution of 2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethanol-1-ol 16 (0.98 g, 3.29 mmol) in 5 mL of dichloromethane. The reaction mixture was stirred overnight. MS and TLC analyses showed complete reaction. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, water, and 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 100% ethyl acetate containing 1% triethylamine in hexane, followed by 10% triethylamine in ethyl acetate, followed by 25% triethylamine in ethyl acetate) to give bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) (1.8 g, 60%). Step 2: Synthesis of bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate) (18)

[0266] As described in Scheme 2: Bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azonidyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate)17 (1.8 g, 1.6 mmol) was added to a solution of bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azonidyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate)17 in 30 mL of tetrahydrofuran / dichloromethane (1:1) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h. MS and TLC analyses indicated complete reaction. The reaction was quenched by slow pouring into saturated sodium bicarbonate, and the resulting mixture was then extracted with dichloromethane. The combined organic layers were washed with brine and dried over sodium sulfate. After concentration, the crude product was purified by rapid column chromatography (SiO2: 0 to 100% ethyl acetate containing 1% triethylamine in hexane, followed by 10% triethylamine in ethyl acetate, followed by 25% triethylamine in ethyl acetate) to obtain bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate) (1.06 g, 73%), which was a pale yellow oil. Step 3: Synthesis of bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)-bis(8-hydroxynonanoate) (compound 48)

[0267] As described in Scheme 2: A solution of crude bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldithioalkyl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate)18 (90 mg, 0.10 mmol) in 5 mL of chloroform was added to the solution of crude bis(2-ethylbutyl)9,9'-((3-mercaptopropyl)azanediyl)bis(8-hydroxynonanoate)TIM-3-E9Es6 (0.20 mmol). The reaction mixture was purged three times with nitrogen and then stirred at room temperature for 2 h. MS and TLC analyses indicated complete reaction. The reaction mixture was concentrated to dryness, and the crude product was purified by rapid column chromatography (SiO2: 0 to 100% ethyl acetate containing 1% triethylamine in hexane containing 1% triethylamine, followed by 10% triethylamine in ethyl acetate) to give a pale yellow oil bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (82 mg, 58%).

[0268] Other lipids of the present invention were prepared according to the representative procedures stated in Schemes 1 and 2 and described above. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 1)

[0269] 1H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.65(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,52H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C66H126N4O14S2[M+H] Calculated value = 1263.8, observed value = 1263.9. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 18)

[0270] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.62(m,4H),2.85-2.23(m,38H),1.79-1.25(m,56H),0.91(d,24H). APCI-MS analysis: C69H132N4O14S2[M+H] Calculated value = 1305.9, observed value = 1305.9. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 49)

[0271] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.85-2.23(m,40H),1.79-1.25(m,52H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1334.0. Dibutyl 9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 10)

[0272] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.05(t,4H),3.64(m,4H),2.86-2.23(m,40H),1.75-1.23(m,64H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1333.7. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 39)

[0273] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.62(m,4H),2.85-2.23(m,38H),1.79-1.25(m,58H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1320.0. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 13)

[0274] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.85–2.22 (m, 38H), 1.85–1.24 (m, 44H), 1.22 (d, 12H), 0.91 (d, 12H). APCI-MS analysis: C64H122N4O14S2[M+H] Calculated value = 1235.8, observed value = 1235.9. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 19)

[0275] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.62(m,4H),2.85-2.23(m,38H),1.91-1.25(m,54H),0.91(d,24H). APCI-MS analysis: C68H130N4O14S2[M+H] Calculated value = 1291.9, observed value = 1292.0. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 4)

[0276] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.64(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.23(m,64H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1319.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 7)

[0277] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.84-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.25(m,54H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C67H128N4O14S2[M+H] Calculated value = 1277.9, observed value = 1278.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 15)

[0278] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85–2.22 (m, 38H), 1.78–1.24 (m, 46H), 1.22 (d, 12H), 0.91 (d, 12H). APCI-MS analysis: C₆₅H₁₂₄N₄O₁₄S₂[M+H] Calculated value = 1249.8, observed value = 1249.9. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 14)

[0279] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.84–2.35 (m, 30H), 2.28 (q, 8H), 1.92–1.74 (m, 5H), 1.68–1.56 (m, 9H), 1.54–1.26 (m, 32H), 1.22 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C65H124N4O14S2[M+H] Calculated value = 1249.8, observed value = 1249.7. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (Compound 20)

[0280] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.08(t,4H),3.98(d,4H),3.65(m,4H),2.84-2.32(m,32H),2.2 9(dt,8H),1.92-1.74(m,5H),1.72-1.56(m,9H),1.54-1.26(m,36H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C69H132N4O14S2[M+H] Calculated value = 1305.9, observed value = 1305.8. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (Compound 40)

[0281] 1 ¹H NMR (300MHz, CDCl₃) δ 4.21 (t, 2H), 3.97 (d, 8H), 3.78 (m, 6H), 2.94–2.39 (m, 28H), 2.29 (dt, 8H), 1.92–1.74 (m, 4H), 1.72–1.26 (m, 52H), 0.88 (t, 24H). APCI-MS analysis: C₇¹H¹³⁶N₄O¹⁴S₂[M+H] Calculated value = 1334.0, observed value = 1333.8. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-oxo-9-propoxynonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 5)

[0282] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.05(t,4H),3.99(d,4H),3.65(bs,4H),2.84-2.39(m,28H),2.29(t, 4H),2.28(t,4H),1.92-1.74(m,6H),1.68-1.55(m,14H),1.52-1.24(m,44H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1334.0. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)-amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 29)

[0283] 1H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.67(m,4H),2.85-2 .25(m,38H),1.92-1.78(m,4H),1.74-1.26(m,56H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C73H140N4O14S2[M+H] Calculated value = 1362.0, observed value = 1362.0. Bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 48)

[0284] 1 ¹H NMR (300MHz, CDCl₃) δ 4.20 (t, 2H), 3.98 (d, 8H), 3.67 (m, 4H), 2.88–2.35 (m, 30H), 2.29 (t, 8H), 1.96–1.78 (m, 4H), 1.70–1.28 (m, 60H), 0.88 (t, 24H). APCI-MS analysis: C₇₅H₁₄₄N₄O₁₄S₂[M+H] Calculated value = 1390.1, observed value = 1390.1. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 3)

[0285] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.63(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,62H),0.93(t,6H),0.88(t,12H). APCI-MS analysis: C68H130N4O14S2[M+H] Calculated value = 1291.9, observed value = 1291.9. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 21)

[0286] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.24(m,58H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1320.0. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 41)

[0287] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.63(m,4H),2.86-2.46(m,22H),2.45-2.23(m,16H),1.88-1.24(m,62H),0.88(t,24H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1348.0. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 16)

[0288] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.66 (m, 4H), 2.85–2.24 (m, 40H), 1.86–1.75 (m, 4H), 1.70–1.26 (m, 46H), 1.22 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C66H126N4O14S2[M+H] Calculated value = 1263.8, observed value = 1263.9. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 50)

[0289] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.61(m,4H),2.84-2.26(m,36H),1.83-1.28(m,64H),0.88(t,24H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1348.0. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 11)

[0290] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.84-2.25(m,36H),1.83-1.28(m,70H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1348.0. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 38)

[0291] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.85-2.25(m,38H),1.85-1.24(m,62H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C74H142N4O14S2[M+H] Calculated value = 1376.0, observed value = 1376.1. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 60)

[0292] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.62(m,4H),2.85-2.25(m,36H),1.85-1.24(m,72H),0.88(t,24H). APCI-MS analysis: C76H146N4O14S2[M+H] Calculated value = 1404.1, observed value = 1404.0. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 9)

[0293] 1H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.23(m,58H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C69H132N4O14S2[M+H] Calculated value = 1305.9, observed value = 1306.0. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 51)

[0294] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.61(m,4H),2.86-2.46(m,22H),2.45-2.23(m,16H),1.75-1.24(m,60H),0.88(t,24H). APCI-MS analysis: C73H140N4O14S2[M+H] Calculated value = 1362.0, observed value = 1362.0. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 12)

[0295] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.63(m,4H),2.86-2.23(m,38H),1.70-1.23(m,62H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1362.0, observed value = 1361.5. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 31)

[0296] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.24(m,60H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1333.9. Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 6)

[0297] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.23(m,68H),0.92(t,6H),0.88(d,12H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1346.9. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 2)

[0298] 1H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(t,4H),3.98(d,4H),3.64(m,4H),2.84-2.24(m,40H),1.92-1.26(m,56H),0.92(d,6H),0.88(t,12H). APCI-MS analysis: C67H128N4O14S2[M+H] Calculated value = 1277.8, observed value = 1277.9. Bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 8)

[0299] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.84-2.26(m,34H),1.85-1.28(m,60H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C68H130N4O14S2[M+H] Calculated value = 1291.9, observed value = 1291.9. Bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 17)

[0300] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.85–2.23 (m, 38H), 1.83–1.25 (m, 50H), 1.22 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C₆₇H₁₂₈N₄O₁₄S₂[M+H] Calculated value = 1277.9, observed value = 1277.9. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 27)

[0301] 1 H NMR (300MHz, CDCl3) δ 4.77 (pentane, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.65 (m, 4H), 2.85–2.25 (m, 38H), 1.90–1.24 (m, 46H), 0.91 (d, 12H), 0.86 (t, 12H). APCI-MS analysis: C65H124N4O14S2[M+H] Calculated value = 1249.8, observed value = 1249.8. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 36)

[0302] 1 H NMR (300MHz, CDCl3) δ4.77(penta,2H),4.19(t,2H),4.08(t,4H),3.62(m,4H),2.85-2.25(m,36H),1.86-1.24(m,54H),0.91(d,12H),0.86(t,12H). APCI-MS analysis: C66H126N4O14S2[M+H] Calculated value = 1263.8, observed value = 1263.9. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 24)

[0303] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.65 (m, 4H), 2.85–2.24 (m, 40H), 1.92–1.78 (m, 4H), 1.72–1.26 (m, 36H), 1.23 (d, 12H), 0.91 (t, 12H). APCI-MS analysis: C63H120N4O14S2[M+H] Calculated value = 1221.7, observed value = 1221.8. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 34)

[0304] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85–2.24 (m, 36H), 1.86–1.27 (m, 46H), 1.22 (d, 12H), 0.91 (t, 12H). APCI-MS analysis: C₆₄H₁₂₂N₄O₁₄S₂[M+H] Calculated value = 1235.8, observed value = 1235.9. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 25)

[0305] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 8H), 3.63 (m, 4H), 2.85–2.24 (m, 40H), 1.95–1.27 (m, 40H), 1.22 (d, 12H), 0.91 (t, 12H). APCI-MS analysis: C₆₄H₁₂₂N₄O₁₄S₂[M+H] Calculated value = 1235.8, observed value = 1235.9. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 26)

[0306] 1H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.63(m,4H),2.84-2.46(m,22H),2.43-2.23(m,16H),1.91-1.29(m,48H),0.91(d,24H). APCI-MS analysis: C67H128N4O14S2[M+H] Calculated value = 1277.9, observed value = 1277.9. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 35)

[0307] 1 HNMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.61(m,4H),2.85-2.21(m,38H),1.85-1.25(m,50H),0.91(d,24H). APCI-MS analysis: C68H130N4O14S2[M+H] Calculated value = 1291.9, observed value = 1291.8. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(8-hydroxynonanoate) (Compound 28)

[0308] 1 ¹H NMR (300MHz, CDCl₃) δ 4.20 (t, 2H), 4.08 (t, 4H), 3.98 (d, 4H), 3.68 (m, 4H), 2.88–2.45 (m, 22H), 2.42–2.24 (m, 16H), 1.95–1.26 (m, 52H), 0.95–0.86 (m, 24H). APCI-MS analysis: C₆₁₃₂N₄O₁₄S₂[M+H] Calculated value = 1305.9, observed value = 1305.8. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate))(compound 30)

[0309] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.63(m,4H),2.85-2.25(m,40H),1.95-1.24(m,52H),0.91(t,12H),0.88(t,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1320.0. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)-azanediyl)bis(8-hydroxynonanoate) (Compound 37)

[0310] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.84-2.28(m,38H),1.95-1.22(m,54H),0.95-0.86(m,24H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1319.8. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 22)

[0311] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(m,8H),3.64(m,4H),2.88-2.45(m,22H),2.42-2.24(m,16H),1.95-1.26(m,58H),0.95-0.88(m,18H). APCI-MS analysis: C69H132N4O14S2[M+H] Calculated value = 1305.9, observed value = 1305.8. Dibutyl9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 32)

[0312] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(m,8H),3.61(m,4H),2.82-2.46(m,22H),2.43-2.25(m,16H),1.90-1.22(m,60H),0.93-0.85(m,18H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1319.8. Dibutyl9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)-amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 33)

[0313] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.85-2.25(m,38H),1.80-1.25(m,62H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1334.0. Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxononyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 23)

[0314] 1H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.25(m,16H),1.91-1.25(m,60H),0.92(t,6H),0.91(d,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1319.9, observed value = 1319.9. Bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 46)

[0315] 1 H NMR (300MHz, CDCl3) δ4.75(penta,2H),4.19(t,2H),3.98(d,4H),3.64(m,4H),2.85-2.25(m,40H),1.90-1.24(m,52H),0.88(d,12H),0.86(t,12H). APCI-MS analysis: C67H128N4O14S2[M+H] Calculated value = 1277.9, observed value = 1277.9. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 59)

[0316] 1 H NMR (300MHz, CDCl3) δ4.75(penta,2H),4.19(t,2H),3.98(d,4H),3.61(m,4H),2.85-2.25(m,38H),1.86-1.24(m,52H),0.88(t,12H),0.86(t,12H). APCI-MS analysis: C68H130N4O14S2[M+H] Calculated value = 1291.9, observed value = 1291.9. Bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 44)

[0317] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (t, 4H), 3.64 (m, 4H), 2.85–2.24 (m, 36H), 1.90–1.78 (m, 4H), 1.68–1.26 (m, 44H), 1.22 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C65H124N4O14S2[M+H] Calculated value = 1249.8, observed value = 1249.9. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 55)

[0318] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.64 (m, 4H), 2.85–2.24 (m, 40H), 1.78–1.29 (m, 48H), 1.21 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C₆₇H₁₂₈N₄O₁₄S₂[M+H] Calculated value = 1277.9, observed value = 1277.0. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 54)

[0319] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85–2.24 (m, 38H), 1.86–1.27 (m, 48H), 1.22 (d, 12H), 0.88 (t, 12H). APCI-MS analysis: C₆₆H₁₂₆N₄O₁₄S₂[M+H] Calculated value = 1263.8, observed value = 1263.9. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 56)

[0320] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .45(m,22H),2.44-2.25(m,16H),1.83-1.28(m,54H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C70H134N4O14S2[M+H] Calculated value = 1318.9, observed value = 1319.0. Bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 45)

[0321] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.29(m,52H),0.91(d,12H),0.88(t,12H). APCI-MS analysis: C69H132N4O14S2[M+H] Calculated value = 1305.9, observed value = 1305.9. Bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(8-hydroxynonanoate) (Compound 47)

[0322] 1 H NMR (300MHz, CDCl3) δ4.21(t,2H),3.98(d,8H),3.74(m,4H),2.82-2.46(m,22H),2.43-2.25(m,16H),1.99-1.25(m,56H),0.93-0.85(m,24H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1133.8. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazine-1-l)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 57)

[0323] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.74(m,4H),2.83-2.25(m,38H),1.90-1.22(m,58H),0.88(t,24H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1347.9. Bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 58)

[0324] 1H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.63(m,4H),2.85-2.25(m,40H),1.90-1.24(m,62H),0.88(t,24H). APCI-MS analysis: C73H140N4O14S2[M+H] Calculated value = 1362.0, observed value = 1361.2. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 42)

[0325] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.04(t,4H),3.98(d,4H),3.72(m,4H),2.84-2.28(m,38H),1.95-1.22(m,62H),0.95-0.85(m,18H). APCI-MS analysis: C71H136N4O14S2[M+H] Calculated value = 1334.0, observed value = 1333.8. Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 52)

[0326] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.79(m,4H),2.84-2.28(m,38H),1.95-1.22(m,64H),0.95-0.85(m,18H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1347.9. Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 53)

[0327] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.05(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .45(m,22H),2.44-2.25(m,16H),1.77-1.26(m,66H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C73H140N4O14S2[M+H] Calculated value = 1362.0, observed value = 1362.0. Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 43)

[0328] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,64H),0.92(t,6H),0.88(t,12H). APCI-MS analysis: C72H138N4O14S2[M+H] Calculated value = 1348.0, observed value = 1348.0. Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate) (Compound 92)

[0329] 1 ¹H NMR (300MHz, methanol-d⁴) δ 4.21 (t, 2H), 4.01 (d, 8H), 3.62 (m, 4H), 2.88–2.50 (m, 22H), 2.45–2.28 (m, 16H), 1.89–1.73 (m, 4H), 1.64 (m, 8H), 1.56–1.45 (m, 12H), 1.37 (m, 24H), 0.91 (t, 24H). APCI-MS analysis: C67H128N4O14S2[M+H] Calculated value = 1277.9, observed value = 1277.8. Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 78)

[0330] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.18 (t, 2H), 4.05 (t, 4H), 3.64 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.28 (m, 46H), 1.22 (d, 12H), 0.90 (t, 6H). APCI-MS analysis: C₆₂H₁₁₈N₄O₁₄S₂[M+H] Calculated value = 1207.8, observed value = 1207.8. Dibutyl7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Es6-DS-3-E7-E4)

[0331] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.64(m,4H),2.84-2.45(m,22H),2.42- 2.24(m,16H),1.85-1.73(m,4H),1.72-1.46(m,20H),1.45-1.29(m,22H),0.93(t,6H),0.88(t,12H).

[0332] APCI-MS analysis: C63H120N4O14S2[M+H] calculated value = 1221.8, observed value = 1221.7. bis(2-ethylbutyl)7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Es6-DS-4-E7-Ei3)

[0333] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.83–2.21 (m, 38H), 1.85–1.24 (m, 40H), 1.22 (d, 12H), 0.86 (t, 12H).

[0334] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.8, observed value = 1207.7. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-3-E7-Ei5)

[0335] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.70(m,4H),2.84-2.46(m,22H),2.43-2.26(m,16H),1.96-1.25(m,50H),0.95-0.88(m,18H).

[0336] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.8. Dibutyl9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-4-E7-Ei5)

[0337] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(m,8H),3.75(m,4H),2.84-2.46(m,22H),2.43-2.25(m,16H),1.92-1.25(m,52H),0.95-0.88(m,18H).

[0338] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.9, observed value = 1263.7. bis(2-ethylbutyl)7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Es6-DS-3-E7-Ei3)

[0339] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.65 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.28 (m, 38H), 1.22 (d, 12H), 0.89 (t, 12H).

[0340] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.6. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-3-E7-Ei3)

[0341] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.04 (d, 4H), 3.62 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.28 (m, 44H), 1.22 (d, 12H), 0.89 (t, 6H).

[0342] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.7. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Es6-DS-4-E9-E4)

[0343] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.64(m,4H),2.84-2 .45(m,22H),2.42-2.24(m,16H),1.90-1.28(m,56H),0.93(t,6H),0.88(t,12H).

[0344] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.8. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Es6-DS-3-E9-Ei5)

[0345] 1 H NMR(300MHz, CDCl3)δ4.20(t,2H),4.08(t,4H),3.98(d,4H),3.68(m,4H),2.8 8-2.45(m,22H),2.42-2.24(m,16H),1.95-1.26(m,52H),0.95-0.86(m,24H).

[0346] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1305.8. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-3-E9-Ei5)

[0347] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(m,8H),3.64(m,4H),2.88-2.45(m,22H),2.42-2.24(m,16H),1.95-1.26(m,58H),0.95-0.88(m,18H).

[0348] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1305.8. bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Es6-DS-3-E9-Es6)

[0349] 1 H NMR (300MHz, CDCl3) δ4.21(t,2H),3.98(d,8H),3.74(m,4H),2.82-2.46(m,22H),2.43-2.25(m,16H),1.99-1.25(m,56H),0.93-0.85(m,24H).

[0350] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1133.8. Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E6-Es5-DS-3-E7-Ei3)

[0351] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.74 (pentane, 2H), 4.19 (t, 2H), 3.65 (m, 4H), 3.32–3.00 (bs, 4H), 2.83–2.24 (m, 38H), 1.91–1.74 (m, 2H), 1.70–1.36 (m, 30H), 1.22 (d, 12H), 0.86 (t, 12H).

[0352] APCI-MS analysis: C57H108N4O14S2[M+H] calculated value = 1137.6, observed value = 1137.6. Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei3)

[0353] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 4H), 4.19 (t, 2H), 3.68 (m, 4H), 3.32–3.00 (bs, 4H), 2.87–2.35 (m, 30H), 2.26 (t, 8H), 1.93–1.74 (m, 4H), 1.70–1.56 (m, 8H), 1.54–1.33 (m, 16H), 1.22 (d, 24H).

[0354] APCI-MS analysis: C55H104N4O14S2[M+H] calculated value = 1109.5, observed value = 1109.6. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-Es6-DS-3-E7-Ei3)

[0355] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.84–2.35 (m, 30H), 2.28 (q, 8H), 1.92–1.74 (m, 5H), 1.68–1.56 (m, 9H), 1.54–1.26 (m, 32H), 1.22 (d, 12H), 0.88 (t, 12H).

[0356] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.7. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-3-E9-Es6)

[0357] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.04(t,4H),3.98(d,4H),3.72(m,4H),2.84-2.28(m,38H),1.95-1.22(m,62H),0.95-0.85(m,18H).

[0358] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1333.8. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Es6-DS-4-E9-Ei5)

[0359] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.84-2.28(m,38H),1.95-1.22(m,54H),0.95-0.86(m,24H).

[0360] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1319.8. Dibutyl9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-4-E9-Ei5)

[0361] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(m,8H),3.61(m,4H),2.82-2.46(m,22H),2.43-2.25(m,16H),1.90-1.22(m,60H),0.93-0.85(m,18H).

[0362] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1319.8. Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei5)

[0363] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.64 (m, 4H), 2.82–2.35 (m, 24H), 2.28 (t, 8H), 1.92–1.74 (m, 6H), 1.72–1.56 (m, 12H), 1.50 (q, 8H), 1.44–1.32 (m, 14H), 1.22 (d, 12H), 0.91 (d, 12H).

[0364] APCI-MS analysis: C59H112N4O14S2[M+H] calculated value = 1165.6, observed value = 1165.7. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-Es6-DS-3-E7-Ei5)

[0365] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.08(t,4H),3.98(d,4H),3.65(m,4H),2.84-2.32(m,32H),2.2 9(dt,8H),1.92-1.74(m,5H),1.72-1.56(m,9H),1.54-1.26(m,36H),0.91(d,12H),0.88(t,12H).

[0366] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1305.8. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazine-1-l)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Es6-DS-4-E9-Es6)

[0367] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.74(m,4H),2.83-2.25(m,38H),1.90-1.22(m,58H),0.88(t,24H).

[0368] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1347.9. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-E4-DS-4-E9-Es6)

[0369] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.79(m,4H),2.84-2.28(m,38H),1.95-1.22(m,64H),0.95-0.85(m,18H).

[0370] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1347.9. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Ei5-DS-3-E7-Ei3)

[0371] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.26 (m, 34H), 1.22 (d, 12H), 0.92 (d, 12H).

[0372] APCI-MS analysis: C59H112N4O14S2[M+H] calculated value = 1165.7, observed value = 1165.8. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9-E4-DS-3-E7-Ei3)

[0373] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.18 (t, 2H), 4.05 (t, 4H), 3.64 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.28 (m, 46H), 1.22 (d, 12H), 0.90 (t, 6H).

[0374] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.8, observed value = 1207.8. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Ei5-DS-4-E7-Ei3)

[0375] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.83–2.23 (m, 38H), 1.84–1.30 (m, 36H), 0.92 (d, 12H), 0.86 (d, 12H).

[0376] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.8. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate)(GL(GL-HEPES-E4-E9-E4-DS-4-E7-Ei3)

[0377] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.60 (m, 4H), 2.86–2.21 (m, 38H), 1.86–1.25 (m, 48H), 1.21 (d, 12H), 0.92 (t, 6H).

[0378] APCI-MS analysis: C63H120N4O14S2[M+H] calculated value = 1221.8, observed value = 1221.8. bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7-Ei3-DS-3-E7-Es6)

[0379] 1 ¹H NMR (300MHz, CDCl₃) δ 4.98 (septet, 2H), 4.21 (t, 2H), 3.97 (d, 4H), 3.74 (m, 6H), 2.92–2.38 (m, 28H), 2.29 (dt, 8H), 1.98–1.78 (m, 4H), 1.72–1.29 (m, 34H), 1.21 (d, 12H), 0.88 (t, 12H).

[0380] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.7. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate)(GL-HEPES-E3-E9-Es6-DS-3-E7-Es6)

[0381] 1 H NMR(300MHz, CDCl3)δ4.21(t,2H),3.97(d,8H),3.78(m,6H),2.94-2.39(m ,28H),2.29(dt,8H),1.92-1.74(m,4H),1.72-1.26(m,52H),0.88(t,24H).

[0382] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1333.8. Dibutyl9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxohepyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)-azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Ei3-DS-3-E9-E4)

[0383] 1 ¹H NMR (300MHz, CDCl₃) δ 4.98 (septet, 2H), 4.20 (t, 2H), 4.05 (t, 4H), 3.63 (bs, 4H), 2.82–2.24 (m, 36H), 1.92–1.74 (m, 6H), 1.68–1.55 (m, 12H), 1.50–1.27 (m, 28H), 1.22 (d, 12H), 0.92 (t, 6H).

[0384] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.8. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-oxo-9-propoxynonyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E3-E9-Es6-DS-3-E9-E4)

[0385] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.05(t,4H),3.99(d,4H),3.65(bs,4H),2.84-2.39(m,28H),2.29(t, 4H),2.28(t,4H),1.92-1.74(m,6H),1.68-1.55(m,14H),1.52-1.24(m,44H),0.92(t,6H),0.88(t,12H).

[0386] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1334.0. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentoxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9-E4-DS-3-E7-Ei5)

[0387] 1H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.62(m,4H),2.83-2.23(m,38H),1.91-1.22(m,52H),0.95-0.86(m,18H).

[0388] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.9, observed value = 1263.9. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7-Ei5-DS-4-E9-E4)

[0389] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.61(m,4H),2.85-2.23(m,38H),1.89-1.25(m,52H),0.95-0.86(m,18H).

[0390] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.9, observed value = 1263.9. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei5-DS-3-E9E4)

[0391] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.62(m,4H),2.85-2.23(m,38H),1.90-1.25(m,50H),0.95-0.86(m,18H).

[0392] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.9. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E6Es5-DS-3-E9Ei5)

[0393] 1 H NMR (300MHz, CDCl3) δ4.77(penta,2H),4.19(t,2H),4.08(t,4H),3.65(m,4H),2.85-2.25(m,38H),1.90-1.24(m,46H),0.91(d,12H),0.86(t,12H).

[0394] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.8. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei3-DS-3-E9Ei5)

[0395] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.65 (m, 4H), 2.85–2.24 (m, 40H), 1.92–1.78 (m, 4H), 1.72–1.26 (m, 36H), 1.23 (d, 12H), 0.91 (t, 12H).

[0396] APCI-MS analysis: C63H120N4O14S2[M+H] calculated value = 1221.7, observed value = 1221.8. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)-amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E3-E9Es6-DS-3-E9Ei5)

[0397] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.67(m,4H),2.85-2 .25(m,38H),1.92-1.78(m,4H),1.74-1.26(m,56H),0.91(d,12H),0.88(t,12H).

[0398] APCI-MS analysis: C73H140N4O14S2[M+H] calculated value = 1362.0, observed value = 1362.0. Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentoxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei5-DS-4-E7Ei5)

[0399] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.63(m,4H),2.84-2.46(m,22H),2.43-2.26(m,16H),1.84-1.33(m,42H),0.91(d,24H).

[0400] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Dibutyl9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-4-E7Ei5)

[0401] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.05(t,4H),3.62(m,4H),2.86-2 .46(m,22H),2.45-2.25(m,16H),1.79-1.25(m,54H),0.92(t,6H),0.90(d,12H).

[0402] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1277.9. bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei5-DS-3-E7Es6)

[0403] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.64(m,4H),2.84-2 .45(m,22H),2.44-2.25(m,16H),1.85-1.28(m,44H),0.91(d,12H),0.88(t,12H).

[0404] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.9. bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E6Es5-DS-3-E9Es6)

[0405] 1 H NMR (300MHz, CDCl3) δ4.75(penta,2H),4.19(t,2H),3.98(d,4H),3.64(m,4H),2.85-2.25(m,40H),1.90-1.24(m,52H),0.88(d,12H),0.86(t,12H).

[0406] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1277.9. bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei3-DS-3-E9Es6)

[0407] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (t, 4H), 3.64 (m, 4H), 2.85–2.24 (m, 36H), 1.90–1.78 (m, 4H), 1.68–1.26 (m, 44H), 1.22 (d, 12H), 0.88 (t, 12H).

[0408] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.9. 7-O-Heptyl)Amino)Propyl)Dithioalkyl)Ethyl)Piperazin-1-yl)Ethoxy)-5-O-pentyl)-Zanedidyl)Bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E7Es6)

[0409] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.85-2 .45(m,22H),2.44-2.24(m,16H),1.92-1.25(m,58H),0.92(t,6H),0.88(t,12H).

[0410] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.9. bis(2-ethylbutyl)7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei5-DS-4-E7Es6)

[0411] 1H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .45(m,22H),2.44-2.25(m,16H),1.86-1.28(m,46H),0.91(d,12H),0.88(t,12H).

[0412] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. Dibutyl9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-4-E7Es6)

[0413] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.85-2 .45(m,22H),2.44-2.24(m,16H),1.78-1.26(m,58H),0.92(t,6H),0.88(m,12H).

[0414] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1306.0. Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei5-DS-3-E9Ei5)

[0415] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.63(m,4H),2.84-2.46(m,22H),2.43-2.23(m,16H),1.91-1.29(m,48H),0.91(d,24H).

[0416] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1277.9. Dibutyl9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentoxy)-9-oxononyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-3-E9Ei5)

[0417] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.25(m,16H),1.91-1.25(m,60H),0.92(t,6H),0.91(d,12H).

[0418] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1319.9. bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-3-E9Es6)

[0419] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),3.98(d,8H),3.67(m,4H),2.88-2.35(m ,30H),2.29(t,8H),1.96-1.78(m,4H),1.70-1.28(m,60H),0.88(t,24H).

[0420] APCI-MS analysis: C75H144N4O14S2[M+H] calculated value = 1390.1, observed value = 1390.1. Diisopropyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei3-DS-4-E7Ei3)

[0421] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 4H), 4.20 (t, 2H), 3.65 (m, 4H), 2.85–2.34 (m, 28H), 2.27 (t, 8H), 1.92–1.32 (m, 36H), 1.22 (d, 24H).

[0422] APCI-MS analysis: C56H106N4O14S2[M+H] calculated value = 1123.6, observed value = 1123.7. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-4-E7Ei3)

[0423] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.66 (m, 4H), 2.85–2.24 (m, 40H), 1.86–1.75 (m, 4H), 1.70–1.26 (m, 46H), 1.22 (d, 12H), 0.88 (t, 12H).

[0424] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. Dibutyl7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei5-DS-3-E7E4)

[0425] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.64(m,4H),2.86-2 .46(m,22H),2.45-2.25(m,16H),1.91-1.28(m,42H),0.92(t,6H),0.91(d,12H).

[0426] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.9. Dibutyl9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-3-E7E4)

[0427] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.66(m,4H),2.85-2.46(m,22 H),2.43-2.23(m,16H),1.90-1.70(m,4H),1.69-1.25(m,54H),0.92(t,12H).

[0428] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Dibutyl7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei5-DS-4-E7E4)

[0429] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.62(m,4H),2.82-2 .46(m,22H),2.43-2.25(m,16H),1.86-1.22(m,44H),0.92(t,6H),0.91(d,12H).

[0430] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.8. Dibutyl9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-4-E7E4)

[0431] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.61(m,4H),2.83-2.25(m,38H),1.85-1.25(m,56H),0.92(t,12H).

[0432] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.9. Dibutyl7,7'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Es6-DS-4-E7E4)

[0433] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.84-2.25(m,38H),1.90-1.22(m,48H),0.92(t,6H),0.88(t,12H).

[0434] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Dibutyl9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9E4-DS-4-E7E4)

[0435] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.61(m,4H),2.83-2.25(m,38H),1.85-1.25(m,54H),0.92(t,12H).

[0436] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Diisopentyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei3-DS-4-E7Ei5)

[0437] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 4H), 4.19 (t, 2H), 4.08 (t, 4H), 3.63 (m, 4H), 2.84–2.24 (m, 36H), 1.84–1.32 (m, 40H), 1.22 (d, 12H), 0.91 (d, 12H).

[0438] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.8. Dibutyl7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei3-DS-3-E7E4)

[0439] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 4H), 4.20 (t, 2H), 4.06 (t, 4H), 3.64 (m, 4H), 2.87–2.24 (m, 36H), 1.90–1.32 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0440] APCI-MS analysis: C57H108N4O14S2[M+H] calculated value = 1136.7, observed value = 1137.8. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-3-E7E4)

[0441] 1 H NMR (300MHz, CDCl3) δ4.20(t,2H),4.06(t,4H),3.98(d,4H),3.64(m,4H),2.84-2.24(m,40H),1.92-1.26(m,56H),0.92(d,6H),0.88(t,12H).

[0442] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.8, observed value = 1277.9. bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei5-DS-3-E9Es6)

[0443] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.29(m,52H),0.91(d,12H),0.88(t,12H).

[0444] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1305.9. Dibutyl9,9'-((5-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-3-E9Es6)

[0445] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,64H),0.92(t,6H),0.88(t,12H).

[0446] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1348.0. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei5-DS-4-E9Ei5)

[0447] 1 HNMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.61(m,4H),2.85-2.21(m,38H),1.85-1.25(m,50H),0.91(d,24H).

[0448] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.8. Dibutyl7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E6Es5-DS-4-E7E4)

[0449] 1H NMR (300MHz, CDCl3) δ4.75 (penta, 2H), 4.20 (t, 2H), 4.06 (t, 4H), 3.63 (m, 4H), 2.85-2.28 (m, 30H), 1.84-1.33 (m, 54H), 0.92 (d, 6H), 0.86 (t, 12H).

[0450] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.8. Dibutyl7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei3-DS-4-E7E4)

[0451] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.84–2.24 (m, 34H), 1.85–1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 12H).

[0452] APCI-MS analysis: C58H110N4O14S2[M+H] calculated value = 1151.6, observed value = 1151.7. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-4-E7E4)

[0453] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.84-2.26(m,34H),1.85-1.28(m,60H),0.92(t,6H),0.88(t,12H).

[0454] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.9. Dibutyl9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxononyl)-amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)-bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-4-E9Ei5)

[0455] NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.85-2.25(m,38H),1.80-1.25(m,62H),0.92(t,6H),0.91(d,12H).

[0456] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1334.0. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei5-DS-4-E9Es6)

[0457] 1 HNMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.84-2 .45(m,22H),2.44-2.25(m,16H),1.83-1.28(m,54H),0.91(d,12H),0.88(t,12H).

[0458] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1318.9, observed value = 1319.0. Dibutyl9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-4-E9Es6)

[0459] 1 HNMR(300MHz, CDCl3)δ4.19(t,2H),4.05(t,4H),3.98(d,4H),3.61(m,4H),2.84- 2.45(m,22H),2.44-2.25(m,16H),1.77-1.26(m,66H),0.92(t,6H),0.88(t,12H).

[0460] APCI-MS analysis: C73H140N4O14S2[M+H] calculated value = 1362.0, observed value = 1362.0. bis(2-ethylbutyl)7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7Ei3-DS-4-E7Es6)

[0461] 1 ¹H NMR (300MHz, CDCl₃) δ 5.01 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.63 (m, 4H), 2.83–2.24 (m, 34H), 1.82–1.29 (m, 44H), 1.22 (d, 12H), 0.88 (t, 12H).

[0462] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.8. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-4-E7Es6)

[0463] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.61(m,4H),2.84-2.26(m,36H),1.83-1.28(m,64H),0.88(t,24H).

[0464] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1348.0. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei3-DS-4-E9E4)

[0465] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.61 (m, 4H), 2.83–2.24 (m, 36H), 1.82–1.27 (m, 52H), 1.22 (d, 12H), 0.93 (t, 6H).

[0466] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.8. Dibutyl9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-4-E9E4)

[0467] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.62(m,4H),2.84-2.25(m,36H),1.83-1.28(m,70H),0.92(t,6H),0.88(t,12H).

[0468] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1348.0. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-3-E7Ei3)

[0469] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.62 (m, 4H), 2.85–2.22 (m, 38H), 1.85–1.24 (m, 44H), 1.22 (d, 12H), 0.91 (d, 12H).

[0470] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Dibutyl9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9E4-DS-3-E7Ei3)

[0471] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.18 (t, 2H), 4.05 (t, 4H), 3.64 (m, 4H), 2.86–2.21 (m, 38H), 1.90–1.28 (m, 46H), 1.22 (d, 12H), 0.90 (t, 6H).

[0472] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.8, observed value = 1207.8. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-4-E7Ei3)

[0473] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85–2.22 (m, 38H), 1.78–1.24 (m, 46H), 1.22 (d, 12H), 0.91 (d, 12H).

[0474] APCI-MS analysis: C65H124N4O14S2[M+H] calculated value = 1249.8, observed value = 1249.9. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E6Es5-DS-4-E9Ei5)

[0475] 1 H NMR (300MHz, CDCl3) δ4.77(penta,2H),4.19(t,2H),4.08(t,4H),3.62(m,4H),2.85-2.25(m,36H),1.86-1.24(m,54H),0.91(d,12H),0.86(t,12H).

[0476] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei3-DS-4-E9Ei5)

[0477] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 4H), 3.61 (m, 4H), 2.85–2.24 (m, 36H), 1.86–1.27 (m, 46H), 1.22 (d, 12H), 0.91 (t, 12H).

[0478] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. bis(2-ethylbutyl)9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E3-E9Es6-DS-4-E9Ei5)

[0479] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.85-2.25(m,38H),1.85-1.24(m,62H),0.91(d,12H),0.88(t,12H).

[0480] APCI-MS analysis: C74H142N4O14S2[M+H] calculated value = 1376.0, observed value = 1376.1. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pent-3-yloxy)hexyl)-amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E6Es5-DS-4-E9Es6)

[0481] 1 H NMR (300MHz, CDCl3) δ4.75(penta,2H),4.19(t,2H),3.98(d,4H),3.61(m,4H),2.85-2.25(m,38H),1.86-1.24(m,52H),0.88(t,12H),0.86(t,12H).

[0482] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.9. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E4-E9Es6-DS-4-E7Ei3

[0483] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.85–2.23 (m, 38H), 1.83–1.25 (m, 50H), 1.22 (d, 12H), 0.88 (t, 12H).

[0484] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1277.9. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-3-E7E4)

[0485] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.65(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,52H),0.92(t,6H),0.91(d,12H).

[0486] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-3-E7E4)

[0487] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.63(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.91-1.23(m,62H),0.93(t,6H),0.88(t,12H).

[0488] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.9. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E9Es6-DS-4-E9Es6)

[0489] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.62(m,4H),2.85-2.25(m,36H),1.85-1.24(m,72H),0.88(t,24H).

[0490] APCI-MS analysis: C76H146N4O14S2[M+H] calculated value = 1404.1, observed value = 1404.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-4-E7E4)

[0491] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.61(m,4H),2.84-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.25(m,54H),0.92(t,6H),0.91(d,12H).

[0492] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1278.0. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-4-E7E4)

[0493] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.23(m,58H),0.92(t,6H),0.88(t,12H).

[0494] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1306.0. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-3-E7Ei5)

[0495] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.62(m,4H),2.85-2.23(m,38H),1.91-1.25(m,54H),0.91(d,24H).

[0496] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1292.0. Diisopropyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Ei3-DS-3-E7Ei3)

[0497] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 4H), 4.19 (t, 2H), 3.62 (m, 4H), 2.85–2.24 (m, 34H), 1.95–1.32 (m, 38H), 1.22 (d, 24H).

[0498] APCI-MS analysis: C56H106N4O14S2[M+H] calculated value = 1123.6, observed value = 1123.7. bis(2-ethylbutyl)7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Es6-DS-3-E7Ei3)

[0499] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.65 (m, 4H), 2.85–2.23 (m, 40H), 1.88–1.29 (m, 42H), 1.22 (d, 12H), 0.88 (t, 12H).

[0500] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.9. Dibutyl7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Ei3-DS-3-E7E4)

[0501] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.85–2.24 (m, 40H), 1.95–1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0502] APCI-MS analysis: C58H110N4O14S2[M+H] calculated value = 1151.6, observed value = 1151.8. Dibutyl7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Es6-DS-3-E7E4)

[0503] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.65(m,4H),2.85-2.28(m,38H),1.95-1.24(m,52H),0.92(t,6H),0.88(t,12H).

[0504] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.8. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E4-E9Es6-DS-3-E7Ei5)

[0505] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.24(m,58H),0.91(d,12H),0.88(t,12H).

[0506] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1320.0. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-4-E7Ei5)

[0507] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,8H),3.62(m,4H),2.85-2.23(m,38H),1.79-1.25(m,56H),0.91(d,24H).

[0508] APCI-MS analysis: C69H132N4O14S2[M+H] calculated value = 1305.9, observed value = 1305.9. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E4-E9Es6-DS-4-E7Ei5)

[0509] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.80-1.24(m,60H),0.91(d,12H),0.88(t,12H).

[0510] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1333.9. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E7Ei3-DS-3-E9Ei5)

[0511] 1¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 8H), 3.63 (m, 4H), 2.85–2.24 (m, 40H), 1.95–1.27 (m, 40H), 1.22 (d, 12H), 0.91 (t, 12H).

[0512] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.9. Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E4-E7Es6-DS-3-E9Ei5)

[0513] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.63(m,4H),2.85-2.25(m,40H),1.95-1.24(m,52H),0.91(t,12H),0.88(t,12H).

[0514] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1320.0. Dibutyl7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-4-E7Ei3)

[0515] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.63 (m, 4H), 2.85–2.22 (m, 38H), 1.85–1.28 (m, 38H), 1.22 (d, 12H), 0.92 (t, 6H).

[0516] APCI-MS analysis: C58H110N4O14S2[M+H] calculated value = 1151.6, observed value = 1151.1. Dibutyl7,7'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7E4-DS-4-E7Ei3)

[0517] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.06 (t, 4H), 3.62 (m, 4H), 2.85–2.22 (m, 38H), 1.85–1.24 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0518] APCI-MS analysis: C59H112N4O14S2[M+H] calculated value = 1165.6, observed value = 1165.2. Dibutyl7,7'-((4-((2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyryl)-oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-4-E7E4)

[0519] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.62(m,4H),2.85-2.22(m,38H),1.85-1.24(m,50H),0.92(t,12H).

[0520] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.0. Dibutyl7,7'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-4-E7Es6)

[0521] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.63(m,4H),2.85-2.22(m,38H),1.85-1.24(m,36H),0.92(t,6H),0.88(t,12H).

[0522] APCI-MS analysis: C64H122N4O14S2[M+H] calculated value = 1235.8, observed value = 1235.0. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E7Ei3-DS-4-E9Es6)

[0523] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.64 (m, 4H), 2.85–2.24 (m, 40H), 1.78–1.29 (m, 48H), 1.21 (d, 12H), 0.88 (t, 12H).

[0524] APCI-MS analysis: C67H128N4O14S2[M+H] calculated value = 1277.9, observed value = 1277.0. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E7Es6-DS-4-E9Es6)

[0525] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.63(m,4H),2.85-2.25(m,40H),1.90-1.24(m,62H),0.88(t,24H).

[0526] APCI-MS analysis: C73H140N4O14S2[M+H] calculated value = 1362.0, observed value = 1361.2. Dibutyl9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-3-E9E4)

[0527] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.64(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.23(m,64H),0.92(t,6H),0.91(d,12H).

[0528] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1319.0. Dibutyl9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-3-E9E4)

[0529] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.61(m,4H),2.86-2 .46(m,22H),2.45-2.23(m,16H),1.90-1.23(m,68H),0.92(t,6H),0.88(d,12H).

[0530] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1346.9. Dibutyl7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)propyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-3-E7Ei5)

[0531] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.64(m,4H),2.87-2 .46(m,22H),2.45-2.26(m,16H),1.91-1.31(m,46H),0.92(t,6H),0.91(d,12H).

[0532] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.4. Dibutyl7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentoxy)-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-4-E7Ei5)

[0533] 1 H NMR(300MHz, CDCl3)δ4.19(t,2H),4.08(t,4H),4.06(t,4H),3.63(m,4H),2.86-2 .46(m,22H),2.45-2.25(m,16H),1.91-1.28(m,48H),0.92(t,6H),0.91(d,12H).

[0534] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.4. Dibutyl9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentoxy)-9-oxonyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-4-E9E4)

[0535] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),4.05(t,4H),3.64(m,4H),2.86-2.23(m,40H),1.75-1.23(m,64H),0.92(t,6H),0.91(d,12H).

[0536] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1333.7. Dibutyl9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxonyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-4-E9E4)

[0537] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,4H),3.98(d,4H),3.63(m,4H),2.86-2.23(m,38H),1.70-1.23(m,62H),0.92(t,6H),0.88(t,12H).

[0538] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1362.0, observed value = 1361.5. Dibutyl7,7'-((3-((2-(4-(2-((5-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7E4-DS-3-E7E4)

[0539] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.64(m,4H),2.86-2.46(m,22H),2.45-2.26(m,16H),1.91-1.30(m,50H),0.92(t,12H).

[0540] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.4. Dibutyl7,7'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7E4-DS-4-E7E4)

[0541] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.06(t,8H),3.63(m,4H),2.86-2.46(m,22H),2.45-2.26(m,16H),1.81-1.30(m,52H),0.92(t,12H).

[0542] APCI-MS analysis: C61H116N4O14S2[M+H] calculated value = 1193.7, observed value = 1193.5. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate))(GL-HEPES-E4-E9Es6-DS-3-E7Ei3)

[0543] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.62 (m, 4H), 2.86–2.22 (m, 38H), 1.85–1.24 (m, 40H), 1.22 (d, 12H), 0.88 (t, 12H).

[0544] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. bis(2-ethylbutyl)9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)butyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E3-E7Ei3-DS-4-E9Es6)

[0545] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 4H), 3.61 (m, 4H), 2.85–2.24 (m, 38H), 1.86–1.27 (m, 48H), 1.22 (d, 12H), 0.88 (t, 12H).

[0546] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Ei3-DS-3-E7Ei5)

[0547] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.08 (t, 8H), 3.62 (m, 4H), 2.85–2.24 (m, 40H), 1.95–1.27 (m, 34H), 1.21 (d, 12H), 0.91 (d, 12H).

[0548] APCI-MS analysis: C60H114N4O14S2[M+H] calculated value = 1179.7, observed value = 1179.8. Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate))(GL-HEPES-E4-E7Es6-DS-3-E7Ei5)

[0549] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.63(m,4H),2.85-2.25(m,40H),1.95-1.24(m,48H),0.91(d,12H),0.88(t,12H).

[0550] APCI-MS analysis: C66H126N4O14S2[M+H] calculated value = 1263.8, observed value = 1263.9. Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-3-E7Es6)

[0551] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.62(m,4H),2.85-2.23(m,38H),1.79-1.25(m,58H),0.91(d,12H),0.88(t,12H).

[0552] APCI-MS analysis: C70H134N4O14S2[M+H] calculated value = 1319.9, observed value = 1320.0. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-3-E7Es6)

[0553] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.63(m,4H),2.86-2.46(m,22H),2.45-2.23(m,16H),1.88-1.24(m,62H),0.88(t,24H).

[0554] APCI-MS analysis: C72H138N4O14S2[M+H] calculated value = 1348.0, observed value = 1348.0. bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Ei3-DS-3-E7Es6)

[0555] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 3.98 (d, 8H), 3.64 (m, 4H), 2.85–2.24 (m, 40H), 1.95–1.29 (m, 38H), 1.21 (d, 12H), 0.88 (t, 12H).

[0556] APCI-MS analysis: C62H118N4O14S2[M+H] calculated value = 1207.7, observed value = 1207.8. bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)valeryl)oxy)ethyl)piperazin-1-yl)ethyl)dithioalkyl)propyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7Es6-DS-3-E7Es6)

[0557] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.63(m,4H),2.85-2.25(m,40H),1.95-1.24(m,52H),0.88(t,24H).

[0558] APCI-MS analysis: C68H130N4O14S2[M+H] calculated value = 1291.9, observed value = 1291.9. Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Ei5-DS-4-E7Es6)

[0559] 1H NMR (300MHz, CDCl3) δ4.19(t,2H),4.08(t,4H),3.98(d,4H),3.61(m,4H),2.85-2.23(m,40H),1.79-1.25(m,52H),0.91(d,12H),0.88(t,12H).

[0560] APCI-MS analysis: C71H136N4O14S2[M+H] calculated value = 1334.0, observed value = 1334.0. bis(2-ethylbutyl)9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate)(GL-HEPES-E4-E9Es6-DS-4-E7Es6)

[0561] 1 H NMR (300MHz, CDCl3) δ4.19(t,2H),3.98(d,8H),3.61(m,4H),2.86-2.46(m,22H),2.45-2.23(m,16H),1.75-1.24(m,60H),0.88(t,24H).

[0562] APCI-MS analysis: C73H140N4O14S2[M+H] calculated value = 1362.0, observed value = 1362.0. Dibutyl7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E3-E7E4-DS-3-E7Ei3)

[0563] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.63 (m, 4H), 2.86–2.46 (m, 22H), 2.45–2.23 (m, 16H), 1.88–1.30 (m, 40H), 1.22 (d, 12H), 0.92 (t, 6H).

[0564] APCI-MS analysis: C57H108N4O14S2[M+H] calculated value = 1137.6, observed value = 1137.7. Dibutyl7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-dithioalkyl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate)(GL-HEPES-E4-E7E4-DS-3-E7Ei3)

[0565] 1 ¹H NMR (300MHz, CDCl₃) δ 4.99 (septet, 2H), 4.19 (t, 2H), 4.05 (t, 4H), 3.63 (m, 4H), 2.86–2.46 (m, 22H), 2.45–2.23 (m, 16H), 1.88–1.30 (m, 42H), 1.22 (d, 12H), 0.92 (t, 6H).

[0566] APCI-MS analysis: C58H110N4O14S2[M+H] calculated value = 1151.6, observed value = 1151.7.

[0567] The HEPBS-based cationic lipids described in this paper can also be prepared according to scheme 3: Option 3 Intermediate [3]:

[0568] A solution of NaOH (1.44 g, 36.16 mmol) in 40 mL of water was added to a solution of triphenylmethanethiol (5.0 g, 18.08 mmol) in EtOH (40 mL) and water (40 mL). The reaction mixture was stirred for 10 min and a solution of 1,4-dibromobutane (3.65 g, 18.08 mmol) in 40 mL of EtOH was added to the reaction mixture. The reaction mixture was stirred at room temperature for 4 h. The progress of the reaction was monitored by TLC (5% EtOAc / hexane). The reaction mixture was diluted with DCM and an aqueous solution of sodium bicarbonate, and the organic layer was washed with brine. The organic layer was dried over sodium sulfate and concentrated under vacuum to give a crude compound. MeOH (15 mL) was added to the crude product and stirred at 0 °C–10 °C for 15 min. The solid compound was filtered and dried under vacuum to give a white solid [3] (5.1 g, 69%).

[0569] result:

[0570] ¹H NMR (400MHz, CDCl₃): δ 7.42–7.39 (m, 6H), 7.30–7.26 (m, 6H), 7.23–7.19 (m, 3H), 3.24 (t, 2H), 2.17 (t, 2H), 1.82–1.77 (m, 2H), 1.55–1.50 (m, 2H). LCMS: Purity 84.99% (low ionization). Intermediate [5]:

[0571] K₂CO₃ (6.72 g, 48.62 mmol) was added to solutions of [3] (5.0 g, 12.16 mmol) and [4] (3.16 g, 24.32 mmol) in ACN (75 mL). The reaction mixture was heated at 40 °C for 48 h. The reaction progress was monitored by TLC (2.5% MeOH in DCM). The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under vacuum to give a crude product. The crude product was purified by rapid chromatography (0 to 2.5% MeOH in DCM) to give [5] (2.6 g, 46%) as a white solid.

[0572] result:

[0573] 1H NMR (400MHz, DMSO-d6): δ7.41(d,6H),7.28(d,6H),7.20(t,3H),3.59(t,2H),2.73(brs,1H),2.53-2.39(m,10H),2.20-2.14(m,4H),1.41(brs,4H). LCMS: 98% purity

[0574] ESI-MS analysis: C29H37N2OS[M+H] calculated value = 461.26, observed value = 461.29 intermediate [7]:

[0575] Add to a solution of [5] (0.613 g, 1.33 mmol) in DCM (7 mL) [6] (1.0 g, 1.26 mmol), EDC (0.364 g, 1.90 mmol), DMAP (31 mg, 0.253 mmol), and DIPEA (0.442 mL, 2.54 mmol) in DCM (8 mL) and stir at room temperature for 14 hours. After the reaction was completed as monitored by MS, the reaction mixture was diluted with DCM and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20% EtOAc in hexane) to obtain a pure compound [7] (0.77 g, 49%) as a colorless oil. This was confirmed by MS analysis.

[0576] result:

[0577] ESI-MS analysis: C 71 H 119 Calculated value of N3O8SSi2[M+H] = 1230.98, observed value = 1230.8 Intermediate [8]:

[0578] TFA (3 mL) was slowly added to a solution of [7] (0.77 g, 0.625 mmol) in DCM (3 mL) at room temperature and stirred for 0.5 h at room temperature. Triethylsilane (0.124 mL, 0.782 mmol) was then slowly added and stirred for 1 h. After the reaction was completed as monitored by MS, the reaction mixture was concentrated to obtain the crude product [8] (quantitative). This was confirmed by MS analysis.

[0579] result:

[0580] ESI-MS analysis: C 52 H 105 Calculated value of N3O8SSi2[M+H] = 988.66, observed value = 988.66 intermediate

[10] :

[0581] [9] (0.234 g, 1.06 mmol) was added to a solution of [8] (quantitatively) in MeOH (4 mL) at room temperature and stirred for 2 hours. After the reaction was completed as monitored by MS, the reaction mixture was concentrated and the crude compound was purified (eluting buffer: 100% ethyl acetate, then 0-20% methanol in ethyl acetate) to obtain the pure product

[10] (0.691 g, quantitative yield). This was confirmed by MS analysis.

[0582] result:

[0583] ESI-MS analysis: C 57 H 108 Calculated value of N4O8S2Si2[M+H] = 1097.80, observed value = 1097.8 Intermediate

[12] :

[0584] Triethylamine (0.266 ml, 1.91 mmol) was added to the solutions of

[10] (0.350 g, 0.319 mmol) and

[11] (0.322 g, 0.574 mmol) in chloroform and allowed to react at room temperature for 2.5 h. After the reaction was complete, the reaction mixture was concentrated and used without purification for the next step (0.800 g crude material).

[0585] ESI-MS analysis: C82H 162 N4O 14 Calculated value of S2Si2[M+H] = 1548.50; Observed value = 1548.8 GL-HEPBS-E3(C6-Es-C1-3;5)-DS-4-(C6-Es-C1-3;5)

[13] :

[0586] Add

[12] (crude material, 0.800 g) and 4 mL of dry tetrahydrofuran to a 20 mL polypropylene scintillation vial. Cool the vial to 0 °C–5 °C and add HF / pyridine (2.0 mL, 76.33 mmol) dropwise. After addition, allow the reaction vial to warm to room temperature and stir for 18 hours. Then, cool the reaction mixture back to 0 °C and neutralize with solid sodium bicarbonate, dilute with ethyl acetate, and wash with NaHCO3 solution, water, and brine. Dry the organic layer with anhydrous Na2SO4 and concentrate. Purify the crude product to obtain compound

[13] (0.196 g, 46% after two steps). This was obtained by 1 H NMR and MS analyses were used for confirmation.

[0587] result:

[0588] 1 H NMR(400MHz, CDCl3)4.19(t,2H),3.97(d,8H),3.64(br,4H),2.76–2.22(m,36H),1.86– 1.74(m,2H),1.73–1.56(m,15H),1.55–1.44(m,9H),1.43–1.26(m,28H),0.87(t,24H).

[0589] ESI-MS analysis: C 70 H 134 N4O 14 Calculated value of S2[M+H] = 1319.98; Observed value = 1319.8

[0590] The HEPBS-based cationic lipids described in this paper can also be prepared according to scheme 4: Option 4

[0591] Use the same procedure as in Scheme 3 to synthesize intermediate 5. Intermediate [7]:

[0592] Add to the solution of [5] (0.613 g, 1.33 mmol) in DCM (7 mL) in DCM [6] (1.0 g, 1.26 mmol), EDC (0.364 g, 1.90 mmol), DMAP (31 mg, 0.253 mmol), and DIPEA (0.442 mL, 2.54 mmol) were added to 8 mL of the mixture and stirred at room temperature for 14 hours. After the reaction was completed as monitored by MS, the reaction mixture was diluted with DCM and washed with NaHCO3 solution, water, and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and the crude compound was purified (eluent: 20% EtOAc in hexane) to obtain a pure compound [7] (0.77 g, 49%) as a colorless oil. This was confirmed by MS analysis.

[0593] result:

[0594] ESI-MS analysis: C 71 H 119 Calculated value of N3O8SSi2[M+H] = 1230.98, observed value = 1230.8 Intermediate [8]:

[0595] TFA (3 mL) was slowly added to a solution of [7] (0.77 g, 0.625 mmol) in DCM (3 mL) at room temperature and stirred for 0.5 h at room temperature. Triethylsilane (0.124 mL, 0.782 mmol) was then slowly added and stirred for 1 h. After the reaction was completed as monitored by MS, the reaction mixture was concentrated to obtain the crude product [8] (quantitative). This was confirmed by MS analysis.

[0596] result:

[0597] ESI-MS analysis: C 52 H 105 Calculated value of N3O8SSi2[M+H] = 988.66, observed value = 988.66 intermediate

[10] :

[0598] [9] (0.234 g, 1.06 mmol) was added to a solution of [8] (quantitatively) in MeOH (4 mL) at room temperature and stirred for 2 hours. After the reaction was completed as monitored by MS, the reaction mixture was concentrated and the crude compound was purified (eluting buffer: 100% ethyl acetate, then 0-20% methanol in ethyl acetate) to obtain the pure product

[10] (0.691 g, quantitative yield). This was confirmed by MS analysis.

[0599] result:

[0600] ESI-MS analysis: C 57 H 108 Calculated value of N4O8S2Si2[M+H] = 1097.80, observed value = 1097.8 Intermediate

[12] :

[0601] Triethylamine (0.243 ml, 1.75 mmol) was added to the solutions of

[10] (0.320 g, 0.291 mmol) and

[11] (0.287 g, 0.525 mmol) in chloroform and allowed to react at room temperature for 2.5 h. After the reaction was complete, the reaction mixture was concentrated and used without purification for the next step (0.800 g crude material).

[0602] ESI-MS analysis: C 81 H 160 N4O 14 Calculated value of S2Si2[M+H] = 1534.48; Observed value = 1534.8 GL-HEPBS-E3(C6-Es-C1-3;5)-DS-3-(C6-Es-C1-3;5)

[13] :

[0603] Add

[12] (crude material, 0.800 g) and 4 mL of dry tetrahydrofuran to a 20 mL polypropylene scintillation vial. Cool the vial to 0 °C–5 °C and add HF / pyridine (2.0 mL, 77.03 mmol) dropwise. After the addition, allow the reaction vial to warm to room temperature and stir for 18 hours. Then, cool the reaction mixture back to 0 °C and neutralize with solid sodium bicarbonate, dilute with ethyl acetate, and wash with NaHCO3 solution, water, and brine. Dry the organic layer with anhydrous Na2SO4 and concentrate. Purify the crude product to obtain compound

[13] (0.211 g, 55% by two steps). This was obtained by 1 H NMR and MS analyses were used for confirmation.

[0604] result:

[0605] 1 H NMR(400MHz, CDCl3)4.19(t,2H),3.97(d,8H),3.64(br,4H),2.85–2.23(m,36H),1.89– 1.74(m,4H),1.73–1.55(m,12H),1.55–1.44(m,8H),1.43–1.28(m,30H),0.87(t,24H).

[0606] ESI-MS analysis: C 69 H 132 N4O 14 S2[M+H] calculated value = 1305.95; observed value = 1305.8 Example 2: Lipid Nanoparticle Formulation

[0607] The cationic lipids described herein can be used to prepare lipid nanoparticles according to methods known in the art. Suitable methods include those described in International Publication No. WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[0608] Process A using WO 2018 / 089801 (see, for example, Example 1 of WO 2018 / 089801 and...) Figure 1The lipid nanoparticles in the embodiments of the present invention are formulated. Process A (“A”) relates to a conventional method of encapsulating mRNA by mixing mRNA with a lipid mixture, without requiring the lipids to be pre-formed into lipid nanoparticles first. In an exemplary process, an ethanolic solution of a mixture of lipids (cationic lipids, phosphatidylethanolamine, cholesterol, and polyethylene glycol-lipids) is combined with an aqueous buffer solution of the target mRNA under controlled conditions at an acidic pH at a fixed lipid-to-mRNA ratio to obtain a homogeneous suspension of LNPs. After ultrafiltration and percolation into a suitable diluent system, the resulting nanoparticle suspension is diluted to the final concentration, filtered, and frozen at -80°C until use.

[0609] The lipid nanoparticle formulations listed in Table 3 were prepared using process A. All lipid nanoparticle formulations contained hEPO mRNA and different lipids (cationic lipids: DMG-PEG2000: cholesterol: DOPE / DSPC) in the mol% ratios indicated in Table 3. Table 3. Characteristics of exemplary lipid nanoparticles The N / P ratio is defined as the ratio of the amount of nitrogen in cationic lipids to the amount of phosphate in nucleic acids.

[0610] The cationic lipids of the present invention were evaluated together with lipid nanoparticle formulation 1. MC3 was evaluated together with lipid nanoparticle formulation 2, which is a typical MC3 formulation. Example 3: Delivery of hEPO mRNA via intramuscular administration Mouse studies

[0611] In summary, lipid screening was conducted using 6-8 week old female BALB / cJ mice. Mice were administered 0.1 μg of LNP in 30 μL via a single intramuscular (IM) injection into the gastrocnemius leg muscle. Blood samples were collected at 6 and 24 hours post-injection, and serum hEPO levels were measured using an ELISA assay according to the manufacturer's protocol. WO 2022 / 099003 A1 also describes in vivo assays for intramuscular administration (e.g., page 46, paragraph

[00206] ).

[0612] Further details of the intramuscular experiments conducted in this application are provided below. Research Design Table An. = animal; TA = test item; Conc. = concentration; ROA = route of administration; IM = intramuscular. Test materials and treatment plans During loading into the syringe, the test material remains free of RNase (if applicable).

[0613] Test item compound category: Oligonucleotides

[0614] ABSL-1 Treatment regimen: On day 1, animals from groups 1–13 were administered the drug intramuscularly under mild isoflurane anesthesia according to the study design table above. Animals in groups 1–13 were injected with EPO mRNA LNP only in the right leg. Animals in group 1 received the MC3 control. The cationic lipid MC3 is the current gold standard for in vivo delivery of drugs such as siRNA (see WO 2010 / 144740). Study animals animal : Species / Sex Mice / Female strain BALB / cJ(Jax#000651) serial number N＝112 age 6-8 weeks

[0615] Adaptation: Allow the animal to adapt to the testing facility for at least 24 hours.

[0616] Householding: All animals are housed together in polycarbonate cages with contact mats in the animal housing.

[0617] Food and water: The animals are provided with unlimited food (Envigo's irradiated 2918 diet) and filtered tap water. Survival observation and measurement

[0618] Animal health check: Animals should undergo a cage-side health check and observation at least once a day.

[0619] Clinical observation: All animals were clinically observed on day 1 before dose administration and before euthanasia. If animals exhibited abnormal clinical signs during the study, clinical observation was performed more frequently.

[0620] Weight: Record weight before applying the test material. Round the weight to the nearest 0.1g.

[0621] Provisional sample collection: Interim whole blood (approximately 50 μL) was collected via tail clipping or saphenous vein at 6 and 24 hours (±5%) following dose administration. Blood samples were collected into serum separation tubes and allowed to clot at room temperature for at least 10 minutes. The samples were then centrifuged at a minimum of 1000 g for 10 minutes at ambient temperature and serum was extracted. All serum samples were stored at nominal -70°C until hEPO analysis was performed at the testing facility. The results of the EPO analysis were included in the data submission. Survival Sample Collection Form No. = Number Termination of Procedure

[0622] Euthanasia: On day 2, 24 hours after administration, all animals were euthanized by CO2 asphyxiation, followed by thoracotomy and final blood collection.

[0623] Final blood collection: Whole blood was collected via cardiac puncture into a serum separation tube, allowed to clot at room temperature for at least 10 minutes, centrifuged at a minimum of 1000g for 10 minutes at ambient temperature, and serum was extracted. Serum samples were stored at nominal -70°C until hEPO was analyzed using testing facilities. Final Sample Collection Table No. = Number; MOV = Maximum obtainable volume. In vitro assay:

[0624] ELISA assay: Determine the level of human erythropoietin (hEPO) in serum samples using an ELISA kit (R&D system, catalog number DEP-00) according to the manufacturer's instructions and include the results in the data submission. Use the "shaker" protocol. Dilute serum samples between 1:40 and 1:100. Report and data retention

[0625] Data submission: Submit a summary table of data for this study, including the time of dosing and euthanasia, body weight, clinical observation, in vitro analysis and mortality (if applicable), animal allocation, individual and group means (if applicable). Table 4 Results of hEPO mRNA delivery studies - Intramuscular administration of hEPO mRNA lipid formulations containing protected cationic lipids. Example 4: Laurdan determination for measuring generalized polarization (GP) values

[0626] The Laurdan probe was used to compare lipid packaging in lipid nanoparticles containing second-generation cationic lipids derived from the "Good" buffer of the present invention with lipid nanoparticles containing other cationic lipids derived from the "Good" buffer.

[0627] The formulation was diluted to buffer solutions with pH values ​​of 4.5, 5.5, 6.5, or 7.5, and Laurdan molecules were added to a final Laurdan concentration of 1 μM. The solutions were incubated at room temperature in the dark for three hours. GP values ​​were calculated based on fluorescence values ​​to understand the lipid membrane packaging of the formulation. Samples were analyzed using a SpectraMax M5 multimode microplate reader. Fluorescence excitation wavelength of 340 nm and emission wavelengths of 440 nm and 490 nm were used. GP values ​​were calculated using the following equation: GP = (AUC 440 -AUC 490 ) / (AUC 440 +AUC 490 ).

[0628] Further details of the Laurdan determination for determining generalized polarization (GP) values ​​are provided in 1) Koitabashi, K.; Nagumo, H.; Nakao, M.; Machida, T.; Yoshida, K.; Sakai-Kato, K. Acidic PH-Induced Changes in Lipid Nanoparticle Membrane Packing. Biochimica Et Biophysica Acta Bba-Biomembr 2021, 1863(8), 183627 and 2) Parasassi, T.; Stasio, GD; Ravagnan, G.; Rusch, RM; Gratton, E. Quantitation of Lipid Phases in Phospholipid Vesicles by the Generalized Polarization of Laurdan Fluorescence. Biophys J 1991, 60(1), 179-189, which are incorporated herein by reference.

[0629] The Laurdan probe is uniformly inserted into the hydrophilic / hydrophobic surface of the lipid bilayer and used to measure polarity changes in the bilayer environment, which may be related to lipid membrane packaging and orderliness. Generalized polarization (GP) values ​​were calculated based on the change in fluorescence intensity from 440 nm to 490 nm when the Laurdan probe interacts with water molecules in the lipid membrane. Lower GP values ​​are associated with hydration and liquid membranes, while higher GP values ​​generally indicate fewer water molecules and more ordered lipid packaging. GP values ​​of lipid nanoparticles (LNPs) were measured in buffers at pH 7.5, 6.5, 5.5, and 4.5 to simulate the endosomal pH changes that occur when cells absorb the particles. It was envisioned that for all formulations tested, lower pH levels (4.5 and 5.5) could result in lower GP values ​​compared to pH 6.5 and 7.5. This suggests that lipid nanoparticles (LNPs) become more mobile and less ordered as the pH environment decreases. Lipid nanoparticles containing second-generation cationic lipids derived from "Good" buffer are envisioned to have a generally higher GP value compared to lipid nanoparticles containing other cationic lipids derived from "Good" buffer. Additional ester and / or carbon branching in the lipid tails of second-generation cationic lipids derived from "Good" buffer are envisioned to result in a more tightly packed membrane compared to other cationic lipids derived from "Good" buffer. A positive trend is envisioned between the GP value and the amount of hEPO produced in mice at pH 6.5 for lipid nanoparticles containing second-generation cationic lipids derived from "Good" buffer. One hypothesis for the envisioned correlation between GP value and protein production is that particles with a tighter bilayer packaging perform better in vivo by increasing the stability of lipid nanoparticles (LNPs) under physiological pH conditions.

[0630] In summary, lipid nanoparticles comprising the second-generation cationic lipids derived from the "Good" buffer of the present invention are envisioned to have a generally higher generalized polarization (GP) value compared to other cationic lipids derived from the "Good" buffer. A positive linear correlation is envisioned between the Laurdan GP value and the amount of EPO produced in mice at 6 hours. An increase in GP value is envisioned to be associated with an increase in EPO protein in a pH 6.5 solution. Example 5: In vitro degradation study In vitro lipid degradation of mouse / human lung S9 Measurement format - 4 or 5 time points, in triplicate. I. Measurement Procedure: 1) Plan the experiments, compounds and reagents. 2) Dissolve each lipid in DMSO or IPA to prepare a 5 mM stock solution, and then dilute it to a 200 μM working solution with IPA. 3) Thaw mouse and human lung S9. 4) Prepare the combined incubation mixture as shown in the following reaction formula on ice. 5) Distribute 495 μL of the incubation mixture prepared in step #4 equally into each well of a 2 mL 96-well plate. 6) Add 5 μL of the compound to each well to begin the reaction. Take the t0 sample (as in step #8). 7) Cover the plate with two layers of breathable sealant and incubate it in a 37°C CO2 incubator on a fixed-track shaker at 150 rpm. 8) At each time point, pipette to mix the incubation mixture 5 times, then transfer 70 μL of the incubation mixture to a new plate. Store immediately in a -20°C freezer. 9) Add 210 μL (3x volume) of the cold termination solution to each well of the collected sample plate. Mix on a track-mounted shaker at 600 rpm for 15 min. 10) Centrifuge the quenched plate at 3800 rpm for 10 min at 4℃ and transfer the supernatant to a new plate. 11) Load the supernatant onto the filter plate and centrifuge again at 3800 rpm for 5 min at 4 °C. Collect the final sample in a new plate for LC / MS. II. Time Process and Termination Solution: 4-5 time points (hours): for example, 0, 4, 8, 24, 48h Termination solution: 1:1:1 ACN / MeOH / IPA (v / v / v), with propranolol and MC3 as internal standards. Store at 4°C. III. Reaction components and formula: Mouse / human lung S9 Example 6: RiboGreen Measurement

[0631] The encapsulation efficiency of mRNA in lipid nanoparticles can be determined using the Invitrogen RiboGreen assay kit. Unencapsulated mRNA is detected directly. Total mRNA is measured after lysis of lipid nanoparticles in the presence of 0.45% w / v Triton X-100. Encapsulation efficiency is calculated as (total mRNA - unencapsulated mRNA) / total mRNA x 100%.

[0632] RiboGreen assay is a fluorescence-based method for use in Quant-iT in lipid nanoparticles containing mRNA. TM RNA reagents are used to determine mRNA concentration (total and free) and encapsulation. Materials / Reagents Triton-X, 98%, for molecular biology, free of DNase, RNase and protease, Acros Organics, catalog number AC327371000 UltraPure DNA / RNase-free distilled water, Life Technologies, catalog number 10977-023 · RNase Detergent Solution, Life Technologies, Catalog No. AM9784 ·Quant-iT TM RNA reagents, Life Technologies, catalog number R11491 or Quant-iT TM RNA Assay Kit, Life Technologies, Catalog No. R11490 • RNase-free 20X TE buffer, Life Technologies, catalog number T11493 · RNase Detergent Solution, Life Technologies, Catalog No. AM9784 equipment Gemini EM microplate reader (molecular device) • RNase-free microcentrifuge tubes (2.0 mL) • RNase-free long-necked narrow-mouth tubes (15mL and 50mL) · Vortex mixer • With transparent background 96-well special optical microplate (catalog number 3615) Preparation of mRNA standards Standard products 0 0.02ug / mL 0.05ug / mL 0.2ug / mL 0.4ug / mL 0.6ug / mL blank mRNA-1 mRNA-2 mRNA-3 mRNA-4 mRNA-5 10XTE buffer 950μL 930μL 900μL 750μL 550μL 350μL 4% Triton 50μL 50μL 50μL 50μL 50μL 50μL 2X mRNA dilution 0μL 20μL 50μL 200μL 400μL 600μL 200x RG 1000μL 1000μL 1000μL 1000μL 1000μL 1000μL Sample preparation 200x RiboGreen dye formulation program • Add 1.0 mL of 200x Ribogreen reagent solution to each standard (blank, mRNA-1, mRNA-2, mRNA-3, mRNA-4, mRNA-5) and sample (free mRNA and total mRNA) and mix gently by inverting. This is a 2X dilution. • Using reverse pipetting, add 200 μL of each standard and sample in triplicate to 96 wells of a Costa Black plate with a transparent background. Ensure there are no air bubbles in the plate before fluorescence reading. • Use the following instrument parameters to read the fluorescence signal: • Reading type: Fluorescence, bottom reading • Excitation: 485nm; Cutoff: 515nm; Emission: 530nm • Board type: 96-hole Costa Black with transparent background Data Analysis

[0633] The average fluorescence from each calibration standard was plotted against concentration to generate a linear calibration curve using MS Excel software. The coefficient of determination (R²) of the calibration curve was then calculated. 2 It must be R 2 >0.99. The generated linear equations can be interpreted as follows: y = mx + c in, Y = Average fluorescence value m: slope x: Concentration (μg / mL) c: y-intercept • Using linear equations, the concentrations of free mRNA and total mRNA in the test samples are calculated by replacing the y-values ​​in the equations with the average fluorescence values ​​of each corresponding sample. • After determining the concentration, the actual concentration in the sample can be calculated by multiplying the concentration in the test sample by the dilution factor (DF), as follows: Free mRNA concentration = Free mRNA concentration in the test sample x 800 (DF) Total mRNA concentration = Total mRNA concentration in the test sample x 4000 (DF) • The concentration of encapsulated mRNA can be determined by subtracting the concentration of free mRNA from the total mRNA concentration. Encapsulation can then be calculated by taking the ratio of encapsulated mRNA to total mRNA and multiplying the result by 100. Example 7: Delivery of human erythropoietin (hEPO) mRNA via intramuscular (IM) administration

[0634] Lipid nanoparticles (LNPs) encapsulating hEPO mRNA were prepared using process A as described above for IM application. The applied LNP composition contained 1.5% PEG, 40% cationic lipids, 28.5% cholesterol, and 30% DOPE, with an N / P ratio of 4. After LNP formulation, the nanoparticles were initially buffer-exchanged with 20% EtOH, followed by a final buffer-exchanged with 10% trehalose. The LNPs were characterized for size, PDI, encapsulation, and mRNA concentration. For hEPO animal dosing studies, the LNPs were diluted to 3.33 μg / mL in 10% trehalose. 0.1 μg in a 30 μL volume was administered intramuscularly to the right gastrocnemius muscle of mice. Blood samples were collected at 6 and 24 hours post-injection to measure the amount of hEPO protein produced in serum. The amount of EPO protein was detected using an ELISA assay from a commercially available kit. Figure 1 The lipid nanoparticles containing the lipids described herein were shown to be highly efficient in delivering hEPO mRNA and exhibited high levels of hEPO protein expression 6 hours after IM injection.

[0635] The polydispersity index (PdI) of lipid nanoparticles can be determined by diluting the formulation in 10% trehalose at a concentration of approximately 0.1 mg / ml mRNA and then measuring the size on a Malvern zetasizer.

[0636] Lipid nanoparticle sizes can be obtained using Malvern zetasizer Nano-ZS.

[0637] As can be seen from the foregoing description, those skilled in the art can readily determine the essential features of the present invention, and various changes and modifications can be made to the present invention to adapt it to various uses and conditions without departing from its spirit and scope.

[0638] All references, patents, or applications from the United States or other countries or regions cited in this application are hereby incorporated in their entirety as if they were written herein. In the event of any inconsistency, the material disclosed herein shall prevail. Implementation plan with numbering 1. A compound having a structure according to formula (I): Or its pharmaceutically acceptable salt, wherein: A 1 Selected from and -SS-, where the left side of each depicted structure is bound to -(CH2)a-; Z 1 Selected from and -SS-, where the right side of each depicted structure is bound to -(CH2)a-; Each 'a' is independently selected from 3 or 4; b is 1, 2, 3, 4, or 5; Each c, d, e, and f is independently selected from 3, 4, 5, or 6; and Each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C6) alkyl groups. 2. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ia): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6. 3. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ib): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6. 4. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ic): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6. 5. The compound according to embodiment 1, wherein the compound has a structure according to formula (Id): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6. 6. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ie): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6. 7. The compound according to embodiment 1, wherein the compound has a structure according to formula (If): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6. 8. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ig): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each c and d is independently selected from 3, 4 or 6. 9. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ih): Or a pharmaceutically acceptable salt thereof, optionally of which: (d)b is 2; (e)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-; or (f)b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a-, Z 1 It is -SS- and each e and f is independently selected from 3, 4 or 6. 10. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ii): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 11. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ij): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 12. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ik): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 13. The compound according to embodiment 1, wherein the compound has a structure according to formula (Im): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 14. The compound according to embodiment 1, wherein the compound has a structure according to formula (In): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z1 It is -SS-. 15. The compound according to embodiment 1, wherein the compound has a structure according to formula (Io): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 16. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ip): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 17. The compound according to embodiment 1, wherein the compound has a structure according to formula (Iq): Or a pharmaceutically acceptable salt thereof, optionally of which: (c)b is 2; or (d) b is 2, A 1 yes The left side of the described structure is bonded to -(CH2)a- and Z 1 It is -SS-. 18. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-17, wherein A 1 and Z 1 They are the same. 193. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-17, wherein A 1 and Z 1 They are different. 20. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-19, wherein A 1 yes The left side of the described structure is bonded to -(CH2)a-. 21. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-19, wherein A 1 yes The left side of the described structure is bonded to -(CH2)a-. 22. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-19, wherein A 1 It is -SS-. 23. The compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein Z 1 yes The right side of the described structure is bonded to -(CH2)a-. 24. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein Z 1 yes The right side of the described structure is bonded to -(CH2)a-. 25. A compound or a pharmaceutically acceptable salt thereof according to any one of embodiments 1-22, wherein Z 1 It is -SS-. 26. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-25 of the numbered scheme, wherein b is 2. 27. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-25 of the numbered scheme, wherein b is 3. 28. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-25 of the numbered scheme, wherein b is 4. 29. The compound according to embodiment 1, wherein the compound has a structure according to formula (Ir): Or a pharmaceutically acceptable salt thereof, wherein each of c, d, e and f is independently selected from 3, 4 or 6. 30. A compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-29, wherein each a is 3. 31. The compound or a pharmaceutically acceptable salt thereof according to any one of the embodiments 1-29, wherein each a is 4. ...

Claims

1. A compound having a structure according to formula (I): Or its pharmaceutically acceptable salt, wherein: A 1 Selected from and -SS-, where the left side of each depicted structure is bound to -(CH2)a-; Z 1 Selected from and -SS-, where the right side of each depicted structure is bound to -(CH2)a-; Each 'a' is independently selected from 3 or 4; b is 1, 2, 3, 4, or 5; Each c, d, e, and f is independently selected from 3, 4, 5, or 6; and Each R 1A R 1B R 1C and R 1D Independently selected from optionally substituted (C3-C6) alkyl groups.

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

3. The compound according to claim 1, wherein the compound has a structure according to formula (Ir): Or a pharmaceutically acceptable salt thereof, wherein each of c, d, e and f is independently selected from 3, 4 or 6.

4. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein each a is 3.

5. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein each a is 4.

6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the value of a on the left side of the described formula is 3 and the value of a on the right side of the described formula is 4.

7. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the value of a on the left side of the described formula is 4 and the value of a on the right side of the described formula is 3.

8. The compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein each R 1A R 1B R 1C and R 1D Selected independently from: or 9. A composition comprising a cationic lipid according to any one of claims 1-8, and further comprising: (i) one or more non-cationic lipids, (ii) one or more cholesterol-based lipids, and (iii) One or more PEG-modified lipids.

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

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