"GOOD" Buffer-Based Cationic Lipids

Abstract

The present invention relates in part to second generation "good" buffer-based cationic lipids of formula (I) and subformulas thereof: [Formula 1] TIFF2025512049000432.tif43170 or a pharma- ceutically acceptable salt thereof. The compounds provided herein may be useful in the delivery and expression of mRNA and the encoded proteins, for example as components of liposomal delivery vehicles, and thus may be useful in treating a variety of diseases, disorders and conditions, such as those associated with a deficiency of one or more proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Argentine patent application having serial number P220100953, filed on April 13, 2022, Taiwanese patent application no. 111114318, filed on April 14, 2022, International patent application no. PCT / US2022 / 025067, filed on April 15, 2022, and European patent application no. 23305491.5, filed on April 4, 2023, each of which is incorporated by reference in its entirety. [Background technology]

[0002] The delivery of nucleic acids has been widely investigated 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 (e.g., the use of vaccines) of various diseases.

[0003] Efficient delivery of liposome-encapsulated nucleic acids remains an active area of ​​research. The cationic lipid component of liposomes plays an important role in promoting effective encapsulation of nucleic acids during liposome loading. In addition, cationic lipids may play an important role in the efficient release of nucleic acid cargo from liposomes into the cytoplasm of target cells. A variety of cationic lipids suitable for in vivo use have been discovered. However, there remains a need to identify cationic lipids that can be efficiently and inexpensively synthesized without the formation of potentially toxic by-products. There also remains a need to identify cationic lipids that exhibit improved biodegradability.

[0004] "Good's" buffers (or Good's buffers) are buffers for biochemical and biological research first selected and described by Norman Good and his colleagues (Good, NE, et al. (1966) Hydrogen Ion Buffers for Biological Research. Biochemistry 5(2), 467-477). Most biological reactions occur at a near-neutral pH between 6 and 8. Therefore, Good reasoned that an ideal buffer for a biochemical or biological application would have a pKa value in this region to provide maximum buffering capacity. Additional selection criteria included high solubility, lack of toxicity, limited interference with biochemical reactions, very low absorbance between 240 nm and 700 nm, enzymatic and hydrolytic stability, minimal change due to temperature and concentration, limited effects due to the ionic or salt composition of the solution, limited interaction with inorganic cations, and limited permeability of biological membranes. [Brief description of the drawings]

[0005] [Figure 1] We show that lipid nanoparticles comprising the lipids described herein are highly effective in delivering hEPO mRNA, demonstrating high levels of hEPO protein expression 6 hours after an IM injected dose. Summary of the Invention [Means for solving the problem]

[0006] The above characteristics make "Good's" buffers very good starting points for the synthesis of cationic lipids for use in in vivo situations. Many of the "Good's" buffers remain important tools in modern biochemistry and biology laboratories and are therefore readily available at low cost.

[0007] The inventors of the present invention have surprisingly found that the lipid tail contains an ester moiety followed by a short (C 3 ~C 6We have found that lipid nanoparticles containing second generation cationic lipids derived from "Good's" buffers containing alkyl tails exhibit improved properties compared to lipid nanoparticles containing other cationic lipids derived from "Good's" buffers, for example, in WO 2022 / 22168 A1 and WO 2022 / 066916 A1 (both of which are incorporated herein by reference). For example, it is believed that lipid nanoparticles containing second generation cationic lipids derived from "Good's" buffers may exhibit improved in vivo degradation. It is also believed that lipid nanoparticles containing second generation cationic lipids derived from "Good's" buffers may exhibit higher generalized polarization (GP) values ​​in the laurdan assay. Lower generalized polarization (GP) values ​​are associated with hydration and fluid membranes, while higher generalized polarization (GP) values ​​typically imply less water molecules and more ordered lipid packing. It is believed that the additional ester and / or carbon branching in the lipid tails of the second generation cationic lipids derived from "Good" buffers may result in tighter packed membranes compared to lipid nanoparticles containing other cationic lipids derived from "Good" buffers such as WO 2022 / 22168 A1 and WO 2022 / 066916 A1. It is believed that lipid nanoparticles with tighter bilayer packing may perform better in vivo by increasing the stability of the lipid nanoparticles under physiological pH conditions. "Good" HEPES, HEPPS and HEPBS buffers formed the core of some of the cationic lipids of the present invention and were used to synthesize unique ionizable lipids containing different degradable moieties and carbon tails. The core structure with hydroxyl and sulfonic acid groups on either side allows the ionizable lipids to contain both ester degradable and disulfide degradable moieties. Preferably, the compounds also feature asymmetric lipid tails on either arm of the final molecule, and in the lipids of the present invention, these tails contain ester moieties aimed at achieving higher degradability.

[0008] The present invention provides, inter alia, cationic lipid compounds for in vivo delivery of therapeutic agents, such as nucleic acids. The cationic lipids of the present invention can be synthesized from readily available starting reagents, such as "Good's" buffer (see Table 1). The cationic lipids of the present invention also contain cleavable groups (e.g., esters and disulfides) that are contemplated to contribute to their favorable safety profile by improving biodegradability. It is believed that lipid nanoparticles containing these cationic lipid compounds are capable of highly effective in vivo delivery while maintaining a favorable safety profile. It is also believed that lipid nanoparticles containing these cationic lipid compounds may exhibit improved in vivo degradation. In addition, it is believed that lipid nanoparticles containing these cationic lipid compounds may exhibit higher generalized polarization (GP) values.

[0009] In one embodiment, a cationic lipid having a structure according to formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: A 1 teeth, [ka] and -SS-, and the left side of each structure shown is selected from -(CH 2 ) bound to a-; Z 1 teeth, [ka] and -SS-, and the right side of each structure shown is selected from -(CH 2 ) bound to 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; Each R 1A , R 1B , R 1C and R1D is optionally substituted (C 3 ~C 6 ) alkyl.

[0010] In one embodiment, provided herein is a cationic lipid that is a pharma- ceutically acceptable salt of formula (I).

[0011] In one aspect, provided herein is a composition comprising a cationic lipid of the present invention, or a pharma- ceutically acceptable salt 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 PEG-modified lipids.

[0012] In one embodiment, the composition is a lipid nanoparticle, optionally a liposome.

[0013] In one embodiment, compositions comprising the cationic lipids of the present invention may be used therapeutically.

[0014] In one embodiment, the compositions of the invention are administered by intramuscular injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition In order that the present invention may be more readily understood, certain terms are first defined below. Further definitions of the following terms and other terms are set forth throughout the specification. Publications and other reference materials mentioned herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference.

[0016] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H 2The amino acid has NC(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a d-amino acid; in some embodiments, the amino acid is an l-amino acid. "Standard amino acid" refers to any of the 20 standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than the standard amino acids, whether it is prepared synthetically 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, including the carboxy- and / or amino-terminal amino acids in peptides, may be modified by methylation, amidation, acetylation, protecting groups, and / or substitutions with other chemical groups that may alter the circulating half-life of the peptides without adversely affecting their activity. The amino acids may participate in disulfide bonds. An amino acid may include one or post-translational modifications, such as association with one or more chemical entities (e.g., a methyl group, an acetate group, an acetyl group, a phosphate group, a formyl moiety, an isoprenoid group, a sulfate group, a polyethylene glycol moiety, a lipid moiety, a carbohydrate moiety, a 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. Whether the term refers to a free amino acid or a residue of a peptide will be clear from the context in which it is used.

[0017] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0018] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a value that falls within a range of 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 or less) of the stated reference value (except where such number exceeds 100% of a possible value), unless otherwise stated or clear from the context.

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

[0020] Delivery: As used herein, the term "delivery" encompasses both local and systemic delivery. For example, delivery of mRNA encompasses the situation where the mRNA is delivered to a target tissue, the encoded protein is expressed, and retained within the target tissue (also referred to as "local distribution" or "local delivery"), and the situation where the mRNA is delivered to a target tissue, the encoded protein is expressed, secreted into the patient's circulatory system (e.g., serum), distributed systemically, and absorbed by other tissues (also referred to 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 an intact protein (e.g., an enzyme), and / or the post-translational modification of a polypeptide or fully assembled protein (e.g., an enzyme). As used herein, the terms "expression" and "production," and their grammatical equivalents, are used interchangeably.

[0022] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0023] Half-life: As used herein, the term "half-life" is the time required for a quantity, such as the concentration or activity of a nucleic acid or protein, to fall to half of its value measured at the beginning of a period of time.

[0024] Helper lipid: As used herein, the term "helper lipid" refers to any neutral or zwitterionic lipid material, including cholesterol. Without wishing to be held to a particular theory, helper lipids may add stability, rigidity, and / or fluidity within the lipid bilayer / nanoparticle.

[0025] Improve, increase, or reduce: As used herein, the terms "improve," "increase" or "reduce," or grammatical equivalents, refer to a relative value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of a treatment described herein, or a measurement in a control subject (or control subjects) in the absence of a treatment described herein. A "control subject" is a subject who is about the same age as the subject being treated and who is afflicted with the same form of the disease as the subject being treated.

[0026] In vitro: As used herein, the term "in vitro" refers to events that take place not inside a multicellular organism, but in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc.

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

[0028] Liposome: As used herein, the term "liposome" refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, liposomes as used herein may be formed by mixing one or more lipids, or by mixing one or more lipids with a polymer. In some embodiments, liposomes suitable for the present invention include a cationic lipid, and optionally further include: (i) non-cationic lipids, (ii) cholesterol-based lipids, and / or (iii) Contains a PEG-modified lipid.

[0029] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" or "mRNA" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. The term "modified mRNA" refers to an mRNA that contains at least one chemically modified nucleotide. An mRNA may contain one or more coding and non-coding regions. An mRNA may be purified from a natural source, produced using a recombinant expression system, optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, an mRNA may contain nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, the mRNA is designed to be compatible with natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-amino The bases may be or contain: adenosine, 7-deazaadenosine, 7-deazaguanosine, 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., phosphorothioate and 5'-N-phosphoramidite linkages).

[0030] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain that comprises individual nucleic acid residues. In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, "nucleic acid" encompasses ribonucleic acid (RNA), including but not limited to any one or more of interfering RNA (RNAi), small interfering RNA (siRNA), short hairpin RNA (shRNA), antisense RNA (aRNA), messenger RNA (mRNA), modified messenger RNA (mRNA), long non-coding RNA (lncRNA), microRNA (miRNA), multimeric coding nucleic acid (MCNA), polymer-coated nucleic acid (PCNA), guide RNA (gRNA) and CRISPR RNA (crRNA). In some embodiments, "nucleic acid" encompasses deoxyribonucleic acid (DNA), including but not limited to any one or more of single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and complementary DNA (cDNA). In some embodiments, "nucleic acid" encompasses both RNA and DNA. In embodiments, the DNA may be in the form of antisense DNA, plasmid DNA, a portion of plasmid DNA, pre-condensed DNA, a product of polymerase chain reaction (PCR), a vector (e.g., P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives of these groups.In embodiments, the RNA is a messenger RNA (mRNA), a ribosomal RNA (rRNA), a signal recognition particle RNA (7 SL RNA or SRP RNA), a transcribed RNA (tRNA), a transcribed messenger RNA (tmRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a SmY RNA, a small Cajal body specific RNA (scaRNA), a guide RNA (gRNA), a ribonuclease P (RNase P), a Y RNA, a telomerase RNA component (TERC), a spliced ​​leader RNA (SL RNA), an antisense RNA (aRNA or asRNA), a cis natural antisense transcript (cis-NAT), a CRISPR RNA (crRNA), a long non-coding RNA (lncRNA), a microRNA (miRNA), a piwi interacting RNA (piRNA), a small interfering RNA (siRNA), a transactional siRNA (tasiRNA), a repeat associated siRNA (rasiRNA), a 73K It may be in the form of RNA, retrotransposons, viral genomes, viroids, satellite RNA, or derivatives of these groups. In some embodiments, the nucleic acid is an mRNA that codes for a protein, such as an enzyme.

[0031] Patient: As used herein, the term "patient" or "subject" refers to any organism to which provided compositions can be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. Human includes pre- and post-natal forms.

[0032] Pharmaceutically acceptable: The term "pharmacologically acceptable," as used herein, refers to a material that is suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable risk / benefit ratio.

[0033] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. The 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 the salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or 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 pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N-terminated salts, such as 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 109, 109, 109, 109, 109, + (C 1~4 Alkyl) 4Examples of pharmaceutically acceptable salts include salts of the amines and the amine salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, where appropriate, formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates, and arylsulfonates. Further pharmaceutically acceptable salts include salts formed from the quaternization of amines using suitable electrophiles, such as alkyl halides, to form quaternized alkylated amino salts.

[0034] Systemic distribution or delivery: As used herein, the term "systemic distribution" or "systemic delivery," or grammatical equivalents thereof, refers to a mechanism or method of delivery or distribution that affects the entire body or organ. Typically, systemic distribution or delivery is achieved via the body's circulatory system, e.g., the bloodstream. Compare with 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 prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, which refers to a human presenting to a health care provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0036] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the entire or nearly full extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical events.

[0037] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated. In some embodiments, a target tissue includes a tissue that exhibits a pathology, symptom, or characteristic associated with a disease.

[0038] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent symptoms, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0039] Treating: As used herein, the terms "treat", "treatment", or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. Treatment may be administered to subjects who do not show signs of the disease and / or who show only early signs of the disease in order to reduce the risk of developing pathologies associated with the disease.

[0040] chemical definition Acyl: As used herein, the term “acyl” refers to R Z -(C=O)-, R Zis, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene.

[0041] Aliphatic: As used herein, the term aliphatic means a cyclic alkyl group having a carboxylic acid moiety (C 1 ~C 50 Aliphatic groups refer to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic groups can be linear, branched, or cyclic. For example, (C 1 ~C 20 ) Aliphatic is (C 1 ~C 20 ) alkyl (e.g., linear or branched (C 1 ~C 20 ) saturated alkyl), (C 2 -C20) alkenyl (e.g., linear or branched (C 4 ~C 20 ) dienyl, linear or branched (C 6 ~C 20 )trienyl, etc.), and (C 2 ~C 20 ) alkynyl (e.g., linear or branched (C 2 ~C 20 ) alkynyl). 1 ~C 20 ) Aliphatic is (C 3 ~C 20 ) cycloaliphatic (e.g., (C 3 ~C 20 ) cycloalkyl, (C 4 ~C 20 ) cycloalkenyl, or (C 8 ~C 20 ) cycloalkynyl). In certain embodiments, an aliphatic can include one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and can be optionally substituted with one or more substituents, such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. An aliphatic group can be unsubstituted or substituted with one or more substituents, as described herein. For example, an aliphatic can include halogen, -COR", -CO 2 H, -CO 2 R”, ​​-CN, -OH, -OR”, -OCOR”, -OCO 2 R”, ​​-NH2 , -NHR”, -N(R”) 2 , -SR" or -SO 2 Each instance of R″ may be independently selected from the group consisting of 1, 2, 3, 4, 5, or 6 independently selected substituents. 1 ~C 20 ) aliphatic (e.g., (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl or (C 1 ~C 3 In embodiments, R″ is independently an unsubstituted alkyl (e.g., an unsubstituted (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl, or (C 1 ~C 3 In embodiments, R″ is independently unsubstituted (C 1 ~C 3 ) alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not contain any heteroatoms.

[0042] Alkyl: As used herein, the term “alkyl” refers to acyclic straight chain and branched hydrocarbon groups, such as “(C 1 ~C 30")alkyl" refers to an alkyl group having 1 to 30 carbons. The alkyl group may be straight chained or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentyl hexyl, isohexyl, and the like. The term "lower alkyl" refers to an alkyl group having 1 to 6 carbon atoms, straight chain or branched alkyl. Other alkyl groups will be readily apparent to the person of ordinary skill in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents, as described herein. For example, alkyl groups can be substituted with halogen, -COR", -CO 2 H, -CO 2 R”, ​​-CN, -OH, -OR”, -OCOR”, -OCO 2 R”, ​​-NH 2 , -NHR”, -N(R”) 2 , -SR" or -SO 2 Each instance of R″ may be independently selected from the group consisting of 1, 2, 3, 4, 5, or 6 independently selected substituents. 1 ~C 20 ) aliphatic (e.g., (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl or (C 1 ~C 3 In embodiments, R″ is independently an unsubstituted alkyl (e.g., an unsubstituted (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl, or (C 1 ~C 3 In embodiments, R″ is independently unsubstituted (C 1 ~C 3) alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkyl group is substituted with an -OH group, and may also be referred to herein as a "hydroxyalkyl" group, where the prefix represents the -OH group and "alkyl" is as described herein.

[0043] As used herein, "alkyl" also refers to a linear or branched saturated hydrocarbon group having 1 to 50 carbon atoms ("C 1 ~C 50 In some embodiments, an alkyl group has 1 to 40 carbon atoms ("C 1 ~C 40 In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C 1 ~C 30 In some embodiments, an alkyl group has 1 to 20 carbon atoms ("C 1 ~C 20 In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C 1 ~C 10 In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C 1 ~C 9 In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C 1 ~C 8 In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C 1 ~C 7 In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C 1 ~C 6 In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C 1 ~C 5 In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C 1 ~C 4In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C 1 ~C 3 In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C 1 ~C 2 In some embodiments, an alkyl group has one carbon atom ("C 1 In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C 2 ~C 6 In some embodiments, the alkyl group has 3 to 6 carbon atoms ("C 3 ~C 6 (C 1 ~C 6 Examples of alkyl groups include methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-Butyl (C 4 ), tert-Butyl (C 4 ), sec-Butyl (C 4 ), iso-butyl (C 4 ), n-pentyl (C 5 ), 3-pentanyl (C 5 ), Amyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butanyl (C 5 ), tertiary amyl (C 5 ), and n-hexyl (C 6 Further examples of alkyl groups include, but are not limited to, n-heptyl (C 7 ), n-octyl (C 8 Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents. In certain embodiments, an alkyl group is unsubstituted (C 1 ~C 50 ) alkyl. In certain embodiments, the alkyl group is a substituted (C 1 ~C 50) alkyl.

[0044] The addition of the suffix "-ene" to a base indicates that the group is a divalent moiety, for example, arylene is a divalent moiety of an aryl, and heteroarylene is a divalent moiety of a heteroaryl.

[0045] Alkylene: The term "alkylene" as used herein represents a saturated divalent straight or branched chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene, and the like. Similarly, the term "alkenylene" as used herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, and the term "alkynylene" as used herein represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur at any stable point along the chain. In certain embodiments, an alkylene, alkenylene, or alkynylene group may contain one or more cyclic aliphatic and / or one or more heteroatoms (such as oxygen, nitrogen, or sulfur) and may be optionally substituted with one or more substituents, such as alkyl, halo, alkoxy, hydroxy, amino, aryl, ether, ester, or amide. For example, an alkylene, alkenylene, or alkynylene may be selected from the group consisting of halogen, -COR", -CO 2 H, -CO 2 R”, ​​-CN, -OH, -OR”, -OCOR”, -OCO 2 R”, ​​-NH 2 , -NHR”, -N(R”) 2 , -SR" or -SO 2 Each instance of R″ may be independently selected from the group consisting of 1, 2, 3, 4, 5, or 6 independently selected substituents. 1 ~C 20 ) aliphatic (e.g., (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl or (C 1 ~C 3In embodiments, R″ is independently an unsubstituted alkyl (e.g., an unsubstituted (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl, or (C 1 ~C 3 In embodiments, R″ is independently unsubstituted (C 1 ~C 3 ) alkyl. In certain embodiments, the alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene does not contain any heteroatoms.

[0046] Alkenyl: As used herein, "alkenyl" refers to any straight or branched hydrocarbon chain with one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, e.g., "(C 2 ~C 30 ")Alkenyl" refers to an alkenyl group having 2 to 30 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, an alkenyl includes one, two, or three carbon-carbon double bonds. In embodiments, an alkenyl includes a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated. An alkenyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkenyl group can include halogen, -COR", -CO 2 H, -CO 2 R”, ​​-CN, -OH, -OR”, -OCOR”, -OCO 2 R”, ​​-NH 2 , -NHR”, -N(R”) 2 , -SR" or -SO 2 Each instance of R″ may be independently selected from the group consisting of 1, 2, 3, 4, 5, or 6 independently selected substituents. 1~C 20 ) aliphatic (e.g., (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl or (C 1 ~C 3 In embodiments, R″ is independently an unsubstituted alkyl (e.g., an unsubstituted (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl, or (C 1 ~C 3 In embodiments, R″ is independently unsubstituted (C 1 ~C 3 ) alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkenyl group is substituted with an -OH group and may also be referred to herein as a "hydroxyalkenyl" group, where the prefix represents the -OH group and "alkenyl" is as described herein.

[0047] 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) ("(C 2 ~C 50 In some embodiments, an alkenyl group has 2 to 40 carbon atoms ("C 2 ~C 40 In some embodiments, the alkenyl group has 2 to 30 carbon atoms ("C 2 ~C 30 In some embodiments, the alkenyl group has 2 to 20 carbon atoms ("C 2 ~C 20 In some embodiments, the alkenyl group has 2 to 10 carbon atoms ("C2 ~C 10 In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C 2 ~C 9 In some embodiments, the alkenyl group has 2 to 8 carbon atoms ("C 2 ~C 8 In some embodiments, the alkenyl group has 2 to 7 carbon atoms ("C 2 ~C 7 In some embodiments, the alkenyl group has 2 to 6 carbon atoms ("C 2 ~C 6 In some embodiments, the alkenyl group has 2 to 5 carbon atoms ("C 2 ~C 5 In some embodiments, the alkenyl group has 2 to 4 carbon atoms ("C 2 ~C 4 In some embodiments, the alkenyl group has 2 to 3 carbon atoms ("C 2 ~C 3 In some embodiments, the alkenyl group has two carbon atoms ("C 2 alkenyl). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). 2 ~C 4 Examples of alkenyl groups include, but are not limited to, ethenyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ) and others. (C 2 ~C 6 Examples of the alkenyl group include the above-mentioned (C 2 ~C 4 ) alkenyl groups, and pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6Further examples of alkenyl include heptenyl (C 7 ), octenyl (C 8 ), octatrienyl (C 8 Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted ("substituted alkenyl") with one or more substituents. In certain embodiments, an alkenyl group is unsubstituted (C 2 ~C 50 ) alkenyl. In certain embodiments, the alkenyl group is a substituted (C 2 ~C 50 ) alkenyl.

[0048] Alkynyl: As used herein, "alkynyl" refers to a hydrocarbon chain, either in a straight or branched configuration, with one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "(C 2 ~C 30 )Alkynyl" refers to an alkynyl group having 2 to 30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, an alkynyl group contains one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, alkynyl groups can include halogen, -COR", -CO 2 H, -CO 2 R”, ​​-CN, -OH, -OR”, -OCOR”, -OCO 2 R”, ​​-NH 2 , -NHR”, -N(R”) 2 , -SR" or -SO 2 Each instance of R″ may be independently selected from the group consisting of 1, 2, 3, 4, 5, or 6 independently selected substituents. 1 ~C 20 ) aliphatic (e.g., (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C1 ~C 10 ) alkyl or (C 1 ~C 3 In embodiments, R″ is independently an unsubstituted alkyl (e.g., an unsubstituted (C 1 ~C 20 ) alkyl, (C 1 ~C 15 ) alkyl, (C 1 ~C 10 ) alkyl, or (C 1 ~C 3 In embodiments, R″ is independently unsubstituted (C 1 ~C 3 ) alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents described herein).

[0049] 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) ("(C 2 ~C 50 (alkynyl) refers to the radical of an alkynyl group having one or more triple bonds and one or more double bonds, which are also referred to as "ene-ynes." In some embodiments, an alkynyl group has 2 to 40 carbon atoms ("C 2 ~C 40 In some embodiments, the alkynyl group has 2 to 30 carbon atoms ("C 2 ~C 30 In some embodiments, the alkynyl group has 2 to 20 carbon atoms ("C 2 ~C 20 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C 2 ~C 10 In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C 2 ~C 9In some embodiments, the alkynyl group has 2 to 8 carbon atoms ("C 2 ~C 8 In some embodiments, the alkynyl group has 2 to 7 carbon atoms ("C 2 ~C 7 In some embodiments, the alkynyl group has 2 to 6 carbon atoms ("C 2 ~C 6 In some embodiments, the alkynyl group has 2 to 5 carbon atoms ("C 2 ~C 5 In some embodiments, the alkynyl group has 2 to 4 carbon atoms ("C 2 ~C 4 In some embodiments, the alkynyl group has 2 to 3 carbon atoms ("C 2 ~C 3 In some embodiments, the alkynyl group has two carbon atoms ("C 2 alkynyl). The one or more carbon-carbon triple bonds can be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). 2 ~C 4 Examples of alkynyl groups include, but are not limited to, ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ) and others. (C 2 ~C 6 Examples of the alkynyl group include the above-mentioned (C 2 ~C 4 ) alkynyl groups, and pentynyl (C 5 ), Hexynyl (C 6 Further examples of alkynyl include heptynyl (C 7 ), Octynyl (C 8Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted ("substituted alkynyl") with one or more substituents. In certain embodiments, an alkynyl group is unsubstituted (C 2 ~C 50 In certain embodiments, the alkynyl group is a substituted (C 2 ~C 50 ) alkynyl.

[0050] Aryl: The term "aryl" used alone or as part of a larger moiety such as "aralkyl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of 6 to 14 ring members, said ring system having a single point of attachment to the remainder of the molecule, at least one ring of the system is aromatic, and each ring of the system contains 4 to 7 ring members. In embodiments, an aryl group has 6 ring carbon atoms ("(C 6 In some embodiments, an aryl group has 10 ring carbon atoms (e.g., "(C 10 )aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("(C 14 )aryl", e.g., anthracen. "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the radical or point of attachment is on the aryl ring, and in such cases the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Exemplary aryls include phenyl, naphthyl, and anthracene.

[0051] As used herein, "aryl" also refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10 or 14 pi electrons shared in a cyclic arrangement) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (e.g., having 6, 10 or 14 pi electrons shared in a cyclic arrangement). 6 ~C 14In some embodiments, an aryl group has 6 ring carbon atoms ("(C 6 In some embodiments, an aryl group has 10 ring carbon atoms ("(C 10 (C aryl)"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("(C 14 )aryl"; e.g., anthracyl). "Aryl" also includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, and the radical or point of attachment is on the aryl ring, and in such cases the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, an aryl group is unsubstituted (C 6 ~C 14 In certain embodiments, the aryl group is a substituted (C 6 ~C 14 ) aryl.

[0052] Arylene: As used herein, the term "arylene" refers to an aryl group that is divalent (i.e., has two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted or substituted phenylene).

[0053] Carbocyclyl: As used herein, "carbocyclyl" or "carbocyclic" refers to a ring system having 3 to 10 ring carbon atoms ("(C 3 ~C 10 )carbocyclyl" refers to the radical of a non-aromatic cyclic hydrocarbon group having 3 to 8 carbon atoms ("C 3 ~C 8 In some embodiments, the carbocyclyl group has 3 to 7 carbon atoms ("C 3 ~C 7In some embodiments, the carbocyclyl group has 3 to 6 carbon atoms ("C 3 ~C 6 In some embodiments, the carbocyclyl group has 4 to 6 carbon atoms ("C 4 ~C 6 In some embodiments, the carbocyclyl group has 5 to 6 carbon atoms ("C 5 ~C 6 In some embodiments, the carbocyclyl group has 5 to 10 carbon atoms ("C 5 ~C 10 Carbocyclyl). 3 ~C 6 ) Carbocyclyl groups include, but are not limited to, cyclopropyl (C 3) , cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4 ), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ) and others. 3 ~C 8 )Carbocyclyl groups include, but are not limited to, the above-mentioned (C 3 ~C 6 ) carbocyclyl group, and cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), bicyclo[2.2.1]heptanyl (C 7 ), bicyclo[2.2.2]octanyl (C 8 ) and others. 3 ~C 10 )Carbocyclyl groups include, but are not limited to, the above-mentioned (C3 ~C 8 ) carbocyclyl group, and cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 As the above examples indicate, in certain embodiments, a carbocyclyl group is either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., including bicyclic ("bicyclic carbocyclyl") or tricyclic (including fused, bridged, or spiro ring systems such as "tricyclic carbocyclyl"), may be saturated or may contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, where the radical or point of attachment is on the carbocyclyl ring, and in such cases the number of carbon atoms continues to specify the number of carbon atoms in the carbocyclyl ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, a carbocyclyl group is unsubstituted (C 3 ~C 10 In certain embodiments, the carbocyclyl group is a substituted (C 3 ~C 10 ) carbocyclyl.

[0054] In some embodiments, "carbocyclyl" or "carbocyclic" refers to "cycloalkyl", i.e., a monocyclic saturated carbocyclyl group having 3 to 10 ring carbon atoms ("(C 3 ~C 10 In some embodiments, a cycloalkyl group has 3 to 8 carbon atoms ("C 3 ~C 8In some embodiments, the cycloalkyl group has 3 to 6 carbon atoms ("C 3 ~C 6 In some embodiments, the cycloalkyl group has 4 to 6 carbon atoms ("C 4 ~C 6 In some embodiments, the cycloalkyl group has 5 to 6 carbon atoms ("C 5 ~C 6 In some embodiments, the cycloalkyl group has 5 to 10 carbon atoms ("C 5 ~C 10 (Cycloalkyl). 5 ~C 6 Examples of cycloalkyl groups include cyclopentyl (C 5 ) and cyclohexyl (C 5 ) are listed. (C 3 ~C 6 Examples of the cycloalkyl group include the above-mentioned (C 5 ~C 6 ) cycloalkyl groups, as well as cyclopropyl (C 3 ) and cyclobutyl (C 4 ) are listed. (C 3 ~C 8 Examples of the cycloalkyl group include the above-mentioned (C 3 ~C 6 ) cycloalkyl groups, as well as cycloheptyl (C 7 ) and cyclooctyl (C 8 Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted ("substituted cycloalkyl") with one or more substituents. In certain embodiments, a cycloalkyl group is unsubstituted (C 3 ~C 10 )cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted (C 3 ~C 10 ) cycloalkyl.

[0055] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0056] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1-14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyl groups can optionally include monocyclic, bicyclic, or tricyclic rings, each ring desirably having 3-6 ring members. Examples of heteroalkyls include polyethers such as methoxymethyl and ethoxyethyl.

[0057] Heteroalkylene: The term "heteroalkylene," as used herein, refers to a divalent form of the heteroalkyl groups described herein.

[0058] Heteroaryl: The term "heteroaryl," as used herein, refers to 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.

[0059] As used herein, "heteroaryl" also refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4 ring heteroatoms) ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more carbocyclyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case 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, and the point of attachment is on the aryl or heteroaryl ring, and in such cases the number of ring members designates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., either in the ring with a heteroatom (e.g., 2-indolyl) or in the ring without a heteroatom (e.g., 5-indolyl).

[0060] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms provided in the aromatic ring system and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each heteroatom being independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl"). In some embodiments, a 5-6 membered heteroaryl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5-6 membered heteroaryl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5-6 membered heteroaryl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl.In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

[0061] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, 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 tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0062] As used herein, "heterocyclyl" or "heterocyclic" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each heteroatom independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "3- to 14-membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment may be at a carbon atom or a nitrogen atom, where valence permits. Heterocyclyl groups can be either monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., bicyclic ("bicyclic heterocyclyl") or tricyclic (fused, bridged, or spiro ring systems such as "tricyclic heterocyclyl"), can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also refers to ring systems in which a heterocyclyl ring, as defined above, is fused to one or more carbocyclyl groups, and the point of attachment is on the carbocyclyl ring or on the heterocyclyl ring, or Heterocyclyl rings as defined above include ring systems fused with one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring, and in such instances the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of a heterocyclyl group is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

[0063] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each of which is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each of which is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, each of which is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5- to 6-membered heterocyclyl"). In some embodiments, the 5- to 6-membered heterocyclyl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heterocyclyl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5- to 6-membered heterocyclyl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0064] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyldinyl, decahydro-1,8-naphthyldinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7- These include, but are not limited to, tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

[0065] Heterocycloalkyl: The term "heterocycloalkyl," as used herein, refers to a non-aromatic ring in which at least one atom is a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocycloalkyl groups can be substituted or unsubstituted.

[0066] As will be understood from the above, the alkyl, alkenyl, alkynyl, acyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups defined herein are, in certain embodiments, optionally substituted. "Optionally substituted" refers to a group that may be substituted or unsubstituted (e.g., a "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl group. In general, the term "substituted" refers to at least one hydrogen present on a group that is capable of being substituted with an acceptable substituent, e.g., a compound that is stable upon substitution, such as by spontaneous transformation, e.g., rearrangement, cyclization, elimination, or other reaction). It means that the group is substituted with a substituent that results in a compound that is free of any of the substituents described herein. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that result in the formation of a stable compound. The present invention contemplates any and all such combinations to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0067] Exemplary carbon atom substituents include halogen, -CN, -NO 2 , -N 3 , -SO 2 , -SO 3 H, -OH, -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 、-CO 2 H、-CHO、-C(OR cc ) 2 、-CO 2 R aa 、-OC(=O)R aa 、-OCO 2 R aa 、-C(=O)N(R bb ) 2 、-OC(=O)N(R bb ) 2 、-NR bb C(=O)R aa 、-NR bb CO 2 R 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 SO 2 R aa 、-NR bb SO 2 R aa 、-SO 2 N(R bb ) 2 、-SO 2 R aa 、-SO 2 ORaa 、-OSO 2 R 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) 2 R aa 、-OP(=O) 2 R aa 、-P(=O)(R aa ) 2 、-OP(=O)(R aa ) 2 、-OP(=O)(OR cc ) 2 、-P(=O) 2 N(R bb ) 2 、-OP(=O) 2 N(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 ), (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 14 ) carbocyclyl, 3-14 membered heterocyclyl, (C 6 ~C 14 ) aryl, and 5- to 14-membered heteroaryl, each of which may be selected from the group consisting of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl, and each of which may be selected from the group consisting of 0, 1, 2, 3, 4, or 5 R dd is independently substituted with; or the two geminal hydrogens on the carbon atom are groups =O, =S, =NN(R bb ) 2 , =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O) 2 R aa , =NR bb , or =NOR cc Replaced by;

[0068] R aa Each example of 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3-14 membered heterocyclyl, (C 6 ~C 14 aryl, and 5- to 14-membered heteroaryl, or two R aagroups combine to form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may be selected from 0, 1, 2, 3, 4, or 5 R dd independently substituted with groups;

[0069] R bb Each instance of is independently hydrogen, -OH, -OR aa , -N(R cc ) 2 , -CN, -C(=O)R aa , -C(=O)N(R cc ) 2 , -CO 2 R aa , -SO 2 R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc ) 2 , -SO 2 N(R cc ) 2 , -SO 2 R cc , -SO 2 OR cc , -SOR aa , -C(=S)N(R cc ) 2 , -C(=O)SR cc , -C(=S)SR cc , -P(=O) 2 R aa , -P(=O)(R aa ) 2 , -P(=O) 2 N(R cc ) 2 , -P(=O)(NR cc ) 2 , (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3-14 membered heterocyclyl, (C 6~C 14 aryl, and 5- to 14-membered heteroaryl, or two R bb The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may contain 0, 1, 2, 3, 4, or 5 R dd is independently substituted with;

[0070] R cc Each instance of is independently hydrogen, (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3-14 membered heterocyclyl, (C 6 ~C1 4 aryl, and 5- to 14-membered heteroaryl, or two R cc groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl or a 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups;

[0071] R dd Each example is independently a halogen, -CN, -NO 2 , -N 3 , -SO 2 H, -SO 3 H, -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)Ree 、-CO 2 H、-CO 2 R ee 、-OC(=O)R ee 、-OCO 2 R ee 、-C(=O)N(R ff ) 2 、-OC(=O)N(R ff ) 2 、-NR ff C(=O)R ee 、-NR ff CO 2 R 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 SO 2 R ee 、-SO 2 N(R ff ) 2 、-SO 2 R ee 、-SO 2 OR ee 、-OSO 2 R ee 、-S(=O)R ee 、-Si(R ee ) 3 、-OSi(R ee ) 3 、-C(=S)N(R ff ) 2 、-C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O) 2R ee , -P(=O)(R ee ) 2 , -OP(=O)(R ee ) 2 ,-OP(=O)(OR ee ) 2 , (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2~ C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3- to 10-membered heterocyclyl, (C 6 ~C 10 ) aryl, and 5-10 membered heteroaryl, each of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl being selected from 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd The substituents are linked to form =O or =S;

[0072] R ee Each example of 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, (C 6 ~C 10 ) aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, each of alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl being selected from 0, 1, 2, 3, 4, or 5 R gg independently substituted with groups; R ff Each instance of is independently hydrogen, (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3- to 10-membered heterocyclyl, (C 6 ~C 10 aryl and 5-10 membered heteroaryl, or two R ff The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may have 0, 1, 2, 3, 4, or 5 R ff Independently substituted with R gg Examples of -CN, -NO 2 , -N 3 , -SO 2 H, -SO 3 H, -OH, -O(C 1 ~C 50 ) alkyl, -ON((C 1 ~C 50 )Alkyl) 2 , -N((C 1 ~C 50 )Alkyl) 2 , -N((C 1 ~C 50 )Alkyl) 3 +X - , -NH((C 1 ~C 50 )Alkyl) 2 +X - , -NH 2 ((C 1 ~C 50 )Alkyl)+X - , -NH 3 +X - , -N(O(C 1 ~C 50 ) alkyl) ((C 1 ~C 50 ) alkyl), -N(OH)((C 1 ~C 50 ) alkyl), -NH(OH), -SH, -S(C 1 ~C 50 ) alkyl, -SS((C 1 ~C 50 ) alkyl), -C(=O)((C 1 ~C50 ) alkyl), -CO 2 H, -CO 2 ((C 1 ~C 50 ) alkyl), -OC(=O)((C 1 ~C 50 ) alkyl), -OCO 2 ((C 1 ~C 50 ) alkyl), -C(=O)NH 2 , -C(=O)N((C 1 ~C 50 )Alkyl) 2 , -OC(=O)NH((C 1 ~C 50 ) alkyl), -NHC(=O)((C 1 ~C 50 ) alkyl), -N((C 1 ~C 50 ) alkyl)C(=O)((C 1 ~C 50 ) alkyl), -NHCO 2 ((C 1 ~C 50 ) alkyl), -NHC(=O)N((C 1 ~C 50 )Alkyl) 2 , -NHC(=O)NH((C 1 ~C 50 ) alkyl), -NHC(=O)NH 2 , -C(=NH)O((C 1 ~C 50 ) alkyl), -OC(=NH)((C 1~ C 50 ) alkyl), -OC(=NH)O(C 1 ~C 50 ) alkyl, -C(=NH)N((C 1 ~C 50 )Alkyl) 2 , -C(=NH)NH((C 1 ~C 50 ) alkyl), -C(=NH)NH 2 , -OC(=NH)N((C 1 ~C 50 )Alkyl) 2 , -OC(NH)NH((C 1 ~C 50) alkyl), -OC(NH)NH 2 , -NHC(NH)N((C 1 ~C 50 )Alkyl) 2 , -NHC(=NH)NH 2 , -NHSO 2 ((C 1 ~C 50 ) alkyl), -SO 2 N((C 1 ~C 50 )Alkyl) 2 , -SO 2 NH((C 1 ~C 50 ) alkyl), -SO 2 NH 2 , -SO 2 ((C 1 ~C 50 ) alkyl), -SO 2 O((C 1 ~C 50 ) alkyl), -OSO 2 ((C 1 ~C 6 ) alkyl), -SO((C 1 ~C 6 ) alkyl), -Si((C 1 ~C 50 )Alkyl) 3 , -OSi((C 1 ~C 6 )Alkyl) 3 , -C(=S)N((C 1 ~C 50 )Alkyl) 2 , C(=S)NH((C 1 ~C 50 ) alkyl), C(=S)NH 2 , -C(=O)S((C 1 ~C 6 ) alkyl), -C(=S)S((C 1 ~C 6 ) alkyl), -SC(=S)S((C 1 ~C 6 ) alkyl), -P(=O) 2 ((C 1 ~C 50 ) alkyl), -P(=O)((C 1~ C 50 )Alkyl)2 , -OP(=O)((C 1 ~C 50 )Alkyl) 2 , -OP(=O)(O(C 1 ~C 50 )Alkyl) 2 , (C 1 ~C 50 ) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, (C 6 ~C 10 ) aryl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl; or two geminal R gg The substituents may be linked to form =O or =S; X - is the counter ion.

[0073] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0074] As used herein, a "counterion" is a negatively charged group that is attached to a positively charged quaternary amine to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F - , Cl - , Br - , I - ), NO 3 - , ClO 4 - , O.H. - , H 2 PO 4 - , HSO 4 -, sulfonate ions (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, 10-camphorsulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1-sulfonic acid-5-sulfonic acid, ethane-1-sulfonic acid-2-sulfonic acid, etc.) and carboxylate ions (e.g., acetic acid, ethanoic acid, propanoic acid, benzoic acid, glyceric acid, lactic acid, tartaric acid, glycolic acid, etc.).

[0075] Nitrogen atoms may be substituted or unsubstituted where valence permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include hydrogen, -OH, -OR aa , -N(R cc ) 2 , -CN, -C(=O)R aa , -C(=O)N(R cc ) 2 , -CO 2 R aa , -SO 2 R aa , -C(=NR bb )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc ) 2 , -SO 2 N(R cc ) 2 , -SO 2 R cc , -SO 2 OR cc , -SOR aa , -C(=S)N(R cc ) 2、 -C(=O)SR cc , -C(=S)SR cc , -P(=O) 2 R aa , -P(=O)(R aa ) 2 , -P(=O) 2 N(R cc ) 2 , -P(=O)(NR cc ) 2 , (C 1 ~C 50) alkyl, (C 2 ~C 50 ) alkenyl, (C 2 ~C 50 ) alkynyl, (C 3 ~C 10 ) carbocyclyl, 3-14 membered heterocyclyl, (C 6 ~C 14 ) aryl, and 5-14 membered heteroaryl, or two R cc The groups, together with the N atom to which they are attached, form a 3- to 14-membered heterocyclyl or 5- to 14-membered heteroaryl ring, and each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl may contain 0, 1, 2, 3, 4, or 5 R dd are independently substituted with R aa , R bb , R cc and R dd is as defined above.

[0076] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also called 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 PG M Huts, 3rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0077] For example, an amide group (e.g., -C(=O)R aaNitrogen protecting groups such as acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0078] Carbamate groups (e.g., -C(=O)OR aa), nitrogen protecting groups such as methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthy]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Tro c), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate t-Bumeoc, 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyl carbamate (Alc), 1-methyl- ... Benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzoisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chloro bromomethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2 ,2-Dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-phenylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, 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-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,Examples include, but are not limited to, 6-trimethylbenzyl carbamate.

[0079] Sulfonamide groups (e.g., -S(=O) 2 R aa ) and nitrogen protecting groups such as 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-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0080] Other nitrogen protecting groups include phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-naphthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, and N-1,1,4,4-tetramethyldisilylazacyclo. Pentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrolin-3 -yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethyl Thiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidates, diphenyl phosphoramidates, benzenesulfenamides, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

[0081] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also called a hydroxyl protecting group).Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PG M Huts, 3rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0082] Exemplary oxygen protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), and the like. ), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), 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-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuanyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethoxyethyl, ethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyl t-hexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate Acetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl aryl 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-naphthotyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl Examples of suitable alkyl esters include, but are not limited to, alkyl-2-butenoates, o-(methoxyacyl)benzoates, α-naphthoates, nitrates, alkyl N,N,N',N'-tetramethylphosphorodiamidates, alkyl N-phenylcarbamates, borates, dimethylphosphinothioyls, alkyl 2,4-dinitrophenylsulfenates, sulfates, methanesulfonates (mesylates), benzylsulfonates, and tosylates (Ts).

[0083] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also called 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 PG M Huts, 3rd edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0084] Exemplary sulfur protecting groups include alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxide, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosuberyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbonylaminomethyl, phenyl, acetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carboethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylprop-2-yl, acetyl, benzoyl, trimethylsilyl, methyltri ... These include, but are not limited to, trifluoroacetyl, N-[[(p-biphenylyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, sulfenylthiocarbonate, 3-nitro-2-pyridine sulfenyl sulfide, and oxathiolone.

[0085] Compounds of the Invention Liposome-based vehicles are considered as attractive carriers for therapeutic agents and are the subject of ongoing development efforts.Although liposome-based vehicles containing certain lipid components have shown promising results in terms of encapsulation, stability and site localization, there is still a great need for improved liposome-based delivery systems.For example, a significant drawback of liposome delivery systems relates to the construction of liposomes with sufficient cell culture or in vivo stability to reach desired target cells and / or intracellular compartments, and the ability of such liposome delivery systems to efficiently release encapsulated materials to such target cells.

[0086] In particular, there remains a need for cationic lipids that are effective for intramuscular delivery of mRNA.There remains 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 remains a need to identify novel lipid compounds that are characterized as having improved safety profiles and being able to efficiently deliver encapsulated nucleic acids and polynucleotides to target cells, tissues and organs.

[0087] Described herein is a novel class of cationic lipid compounds for improving the in vivo delivery of therapeutic agents such as nucleic acids.In particular, the cationic lipids described herein, optionally together with other lipids, can be used to formulate lipid-based nanoparticles (e.g., liposomes) for encapsulating therapeutic agents such as nucleic acids (e.g., DNA, siRNA, mRNA, microRNA) for therapeutic use, such as disease treatment and prevention (vaccine) purposes.

[0088] In embodiments, the compounds of the invention described herein can provide one or more desirable features or characteristics. That is, in certain embodiments, the compounds of the invention described herein can be characterized as having one or more properties that provide such compounds with advantages over other similarly classified lipids. For example, the compounds disclosed herein can allow for control and tuning of the properties of the liposomal composition (e.g., lipid nanoparticles) of which it is a component. In particular, the compounds disclosed herein can be characterized by enhanced transfection efficiency and their ability to induce specific biological outcomes. Such outcomes may include, for example, enhanced cellular uptake, endosomal / lysosomal disruption capabilities and / or enhanced intracellular release of encapsulated materials (e.g., polynucleotides). The compounds disclosed herein can also be characterized by achieving high levels of peptide or protein expression when delivering mRNA encoding said peptide or protein by intravenous, intramedullary or intramuscular administration, or optionally by pulmonary delivery via nebulization. Additionally, the compounds disclosed herein have advantageous pharmacokinetic properties, biodistribution and efficiency.

[0089] The present application demonstrates that the cationic lipids of the present invention are not only synthetically easy to handle from readily available starting materials, but also have unexpectedly high encapsulation efficiency.

[0090] In addition, the cationic lipids of the present invention have cleavable groups, such as ester groups. These cleavable groups (e.g., esters, disulfides) are intended to improve biodegradability and thus contribute to the favorable safety profile of the lipids.

[0091] Compounds that are cationic lipids are provided herein. For example, the cationic lipids of the present invention have the formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: A 1 teeth, [ka] and -SS-, and the left side of each structure shown is selected from -(CH 2 ) bound to a-; Z 1 teeth, [ka] and -SS-, and the right side of each structure shown is selected from -(CH 2 ) bound to 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; Each R 1A , R 1B , R 1C and R 1D is optionally substituted (C 3 ~C 6 ) alkyl.

[0092] In embodiments, the cationic lipid has formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; and each c and d is independently selected from 3, 4, or 6.

[0093] In embodiments, the cationic lipid has formula (Ib): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS- and each e and f is independently selected from 3, 4, or 6.

[0094] In embodiments, the cationic lipid has formula (Ic): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; and each c and d is independently selected from 3, 4, or 6.

[0095] In embodiments, the cationic lipid has formula (Id): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS- and each e and f is independently selected from 3, 4, or 6.

[0096] In embodiments, the cationic lipid has formula (Ie): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; and each c and d is independently selected from 3, 4, or 6.

[0097] In embodiments, the cationic lipid has the formula (If): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS- and each e and f is independently selected from 3, 4, or 6.

[0098] In embodiments, the cationic lipid has the formula (Ig): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; and each c and d is independently selected from 3, 4, or 6.

[0099] In embodiments, the cationic lipid has the formula (Ih): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (c) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS- and each e and f is independently selected from 3, 4, or 6.

[0100] In embodiments, the cationic lipid has formula (Ii): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0101] In embodiments, the cationic lipid has formula (Ij): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0102] In embodiments, the cationic lipid has the formula (Ik): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH2 ) a- and Z 1 is -SS-.

[0103] In embodiments, the cationic lipid has the formula (Im): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0104] In embodiments, the cationic lipid has the formula (In): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0105] In embodiments, the cationic lipid has the formula (Io): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0106] In embodiments, the cationic lipid has the formula (Ip); [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0107] In embodiments, the cationic lipid has the formula (Iq): [ka] or a pharma- ceutically acceptable salt thereof, optionally having the structure: (a) b is 2; or (b) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-.

[0108] In an embodiment, A 1 and Z 1 are the same. In an embodiment, A 1 and Z 1 is different.

[0109] In an embodiment, A 1teeth [ka] The left side of the illustrated structure is -(CH 2 ) a-. In an embodiment, A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a-. In an embodiment, A 1 is -SS-.

[0110] In an embodiment, Z 1 teeth [ka] and on the right side of the illustrated structure is -(CH 2 )a-. In an embodiment, Z 1 teeth [ka] and on the right side of the illustrated structure is -(CH 2 )a-. In an embodiment, Z 1 is -SS-.

[0111] In embodiments, b is 2, 3 or 4. In embodiments, b is 2 or 3. In embodiments, b is 1. In embodiments, b is 2. In embodiments, b is 3. In embodiments, b is 4. In embodiments, b is 5.

[0112] In embodiments, the cationic lipid has the formula (Ir): [ka] or a pharma- ceutically acceptable salt thereof; and optionally each c, d, e and f is independently selected from 3, 4, or 6.

[0113] In an embodiment, each a is 3. In an embodiment, each a is 4. In an embodiment, the value of a on the left side of the illustrated equation is 3 and the value of a on the right side of the illustrated equation is 4. In an embodiment, the value of a on the left side of the illustrated equation is 4 and the value of a on the right side of the illustrated equation is 3.

[0114] In embodiments, c is 3, 4 or 6. In embodiments, c is 3. In embodiments, c is 4. In embodiments, c is 5. In embodiments, c is 6.

[0115] In embodiments, d is 3, 4 or 6. In embodiments, d is 3. In embodiments, d is 4. In embodiments, d is 5. In embodiments, d is 6.

[0116] In embodiments, e is 3, 4 or 6. In embodiments, e is 3. In embodiments, e is 4. In embodiments, e is 5. In embodiments, e is 6.

[0117] In embodiments, f is 3, 4 or 6. In embodiments, f is 3. In embodiments, f is 4. In embodiments, f is 5. In embodiments, f is 6.

[0118] In an embodiment, each c, d, e, and f is independently selected from 3, 4, or 6.

[0119] In an embodiment, c, d, e and f are the same. In an embodiment, c, d, e and f are 3. In an embodiment, c, d, e and f are 4. In an embodiment, c, d, e and f are 5. In an embodiment, c, d, e and f are 6.

[0120] In an embodiment, c and d are the same. In an embodiment, c and d are 3. In an embodiment, c and d are 4. In an embodiment, c and d are 5. In an embodiment, c and d are 6.

[0121] In an embodiment, e and f are the same. In an embodiment, e and f are 3. In an embodiment, e and f are 4. In an embodiment, e and f are 5. In an embodiment, e and f are 6.

[0122] In an embodiment, c and d are the same and e and f are the same, but c and d are different from e and f. In an embodiment, c and d are 3 and e and f are 4. In an embodiment, c and d are 3 and e and f are 5. In an embodiment, c and d are 3 and e and f are 6. In an embodiment, c and d are 4 and e and f are 3. In an embodiment, c and d are 4 and e and f are 5. In an embodiment, c and d are 4 and e and f are 6. In an embodiment, c and d are 5 and e and f are 3. In an embodiment, c and d are 5 and e and f are 4. In an embodiment, c and d are 5 and e and f are 6. In an embodiment, c and d are 6 and e and f are 3. In an embodiment, c and d are 6 and e and f are 4. In an embodiment, c and d are 6 and e and f are 5.

[0123] In an embodiment, each R 1A , R 1B , R 1C and R 1D is optionally substituted (C 4 ~C 6 ) alkyl. In embodiments, each R 1A , R 1B , R 1C and R 1D is optionally substituted (C 5 ~C 6 ) alkyl. In embodiments, each R 1A , R 1B , R 1C and R 1D is optionally substituted (C 3 ~C 5 ) alkyl. In embodiments, each R 1A , R 1B , R 1C and R 1D is optionally substituted (C3 ~C 4 ) alkyl.

[0124] In embodiments, R 1A is an optionally substituted C 3 In embodiments, R 1A is an optionally substituted C 4 In embodiments, R 1A is an optionally substituted C 5 In embodiments, R 1A is an optionally substituted C 6 It is an alkyl.

[0125] In embodiments, R 1B is an optionally substituted C 3 In embodiments, R 1B is an optionally substituted C 4 In embodiments, R 1B is an optionally substituted C 5 In embodiments, R 1B is an optionally substituted C 6 It is an alkyl.

[0126] In embodiments, R 1C is an optionally substituted C 3 In embodiments, R 1C is an optionally substituted C 4 In embodiments, R 1C is an optionally substituted C 5 In embodiments, R 1C is an optionally substituted C 6 It is an alkyl.

[0127] In embodiments, R 1D is an optionally substituted C 3 In embodiments, R 1D is an optionally substituted C 4 In embodiments, R 1Dis an optionally substituted C 5 In embodiments, R 1D is an optionally substituted C 6 It is an alkyl.

[0128] In embodiments, R 1A , R 1B , R 1C and R 1D are the same. In an embodiment, R 1A and R 1B are the same. In an embodiment, R 1C and R 1D is the same.

[0129] In embodiments, R 1A and R 1B is the same as R 1C and R 1D is the same, but R 1A and R 1B is R 1C and R 1D is different.

[0130] In embodiments, when present, each R 1A , R 1B , R 1C and R 1D are independently selected from: [ka]

[0131] In embodiments, R 1A teeth, [ka] In an embodiment, R 1A teeth, [ka] In an embodiment, R 1A teeth, [ka] In an embodiment, R 1A teeth, [ka] In an embodiment, R 1A teeth, [ka] In an embodiment, R 1A teeth, [ka] It is.

[0132] In embodiments, R 1B teeth, [ka] In an embodiment, R 1B teeth, [ka] In an embodiment, R 1B teeth, [ka] In an embodiment, R 1B teeth, [ka] In an embodiment, R 1B teeth, [ka] In an embodiment, R 1B teeth, [ka] It is.

[0133] In embodiments, R 1C teeth, [ka] In an embodiment, R 1C teeth, [ka] In an embodiment, R 1C teeth, [ka] In an embodiment, R 1C teeth, [ka] In an embodiment, R 1C teeth, [ka] In an embodiment, R 1C teeth, [ka] It is.

[0134] In embodiments, R 1D teeth, [ka] In an embodiment, R 1D teeth, [ka] In an embodiment, R 1D teeth, [ka] In an embodiment, R 1D teeth, [ka] In an embodiment, R 1D teeth, [ka] In an embodiment, R 1D teeth, [ka] It is.

[0135] In an embodiment, c and d are 3 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 4 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 6 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 4 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 6 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 4 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 6 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 3 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 4 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 6 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 3 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 4 and R 1A and R 1B teeth [ka] In an embodiment, c and d are 6 and R 1A and R 1B teeth [ka] It is.

[0136] In an embodiment, e and f are 3 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 4 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 6 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 4 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 6 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 4 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 6 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 3 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 4 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 6 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 3 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 4 and R 1C and R 1D teeth [ka] In an embodiment, e and f are 6 and R 1C and R 1D teeth [ka] It is.

[0137] In an embodiment, each a is 4, c and d are 6, and R 1A and R 1B teeth [ka] where e and f are 4, and R 1C and R 1D teeth [ka] It is.

[0138] In embodiments, the substituents are optionally unsubstituted.

[0139] In embodiments, the cationic lipids of the present invention have any one of the structures of Table A, Table B and / or Table C, or a pharma- ceutically acceptable salt thereof.

[0140] In embodiments, the cationic lipid of the present invention has any one of the structures in the examples, or a pharma- ceutically acceptable salt thereof.

[0141] In embodiments, provided herein are compositions comprising a cationic lipid 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 PEG-modified lipids.

[0142] In an embodiment, the composition is a lipid nanoparticle, optionally a liposome. In an embodiment, the one or more cationic lipids comprise about 30 mol% to 60 mol% of the lipid nanoparticle. In an embodiment, the one or more cationic lipids comprise about 31 mol% to 59 mol% of the lipid nanoparticle. In an embodiment, the one or more cationic lipids comprise about 35 mol% to 45 mol% of the lipid nanoparticle. In an embodiment, the one or more cationic lipids comprise about 40 mol% of the lipid nanoparticle.

[0143] In embodiments, the one or more non-cationic lipids comprise about 10 mol% to 50 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids comprise about 11 mol% to 49 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids comprise about 20 mol% to 40 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids comprise about 25 mol% to 35 mol% of the lipid nanoparticle. In embodiments, the one or more non-cationic lipids comprise about 30 mol% of the lipid nanoparticle.

[0144] In an embodiment, the one or more PEG-modified lipids comprise about 1 mol% to 10 mol% of the lipid nanoparticle. In an embodiment, the one or more PEG-modified lipids comprise about 1.1 mol% to 9 mol% of the lipid nanoparticle. In an embodiment, the one or more PEG-modified lipids comprise about 1 mol% to 5 mol% of the lipid nanoparticle. In an embodiment, the one or more PEG-modified lipids comprise about 1.5 mol% to 3 mol% of the lipid nanoparticle.

[0145] In embodiments, the cholesterol-based lipid comprises about 10 mol% to 50 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipid comprises about 11 mol% to 49 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipid comprises about 20 mol% to 40 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipid comprises about 25 mol% to 35 mol% of the lipid nanoparticle. In embodiments, the cholesterol-based lipid comprises about 27 mol% to 28.5 mol% of the lipid nanoparticle.

[0146] In embodiments, the one or more cationic lipids comprise between about 31 mol% and 59 mol% of the lipid nanoparticle, the one or more non-cationic lipids comprise between about 11 mol% and 49 mol% of the lipid nanoparticle, the one or more PEG-modified lipids comprise between about 1.1 mol% and 9 mol% of the lipid nanoparticle, and the cholesterol-based lipids comprise between about 11 mol% and 49 mol% of the lipid nanoparticle.

[0147] In embodiments, the one or more cationic lipids comprise about 35 mol% to 45 mol% of the lipid nanoparticle, the one or more non-cationic lipids comprise about 25 mol% to 35 mol% of the lipid nanoparticle, the one or more PEG-modified lipids comprise about 1 mol% to 5 mol% of the lipid nanoparticle, and the cholesterol-based lipids comprise about 25 mol% to 35 mol% of the lipid nanoparticle.

[0148] In embodiments, the one or more cationic lipids constitute about 40 mol% of the lipid nanoparticle, the one or more non-cationic lipids constitute about 30 mol% of the lipid nanoparticle, the one or more PEG-modified lipids constitute about 1.5 mol% to 3 mol% of the lipid nanoparticle, and the cholesterol-based lipids constitute about 27 mol% to 28.5 mol% of the lipid nanoparticle.

[0149] In embodiments, the lipid nanoparticles encapsulate a nucleic acid, optionally an mRNA encoding a peptide or protein. In embodiments, the lipid nanoparticles encapsulate an mRNA encoding a peptide or protein, optionally for use in a vaccine. In embodiments, the peptide is an antigen.

[0150] As used herein, the phrase "encapsulation rate" refers to the percentage of therapeutic agent (e.g., mRNA) that is effectively encapsulated within a liposome-based vehicle (e.g., lipid nanoparticle) relative to the initial percentage of therapeutic agent present in the lipid phase. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 50% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 55% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 60% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 65% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 70% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 75% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 80% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 85% mRNA. In embodiments, the lipid nanoparticles have an encapsulation rate of at least 90% mRNA. In an embodiment, the lipid nanoparticles have an encapsulation rate of at least 95% mRNA. In an embodiment, the encapsulation rate is calculated by performing a Ribogreen assay (Invitrogen) with or without the presence of 0.1% Triton-X 100.

[0151] In an embodiment, the compositions of the invention are for use in therapy.

[0152] In an embodiment, the compositions of the invention are for use in a method of treating or preventing a disease suitable for treatment or prevention by a peptide or protein encoded by an mRNA, optionally where the mRNA encodes an antigen and / or the disease is (a) a protein deficiency (optionally where the protein deficiency affects the liver, lungs, brain or muscle), (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0153] In embodiments, a method for treating or preventing a disease is provided, the method comprising administering a composition of the invention to a subject in need thereof, and the disease is amenable to treatment or prevention with a peptide or protein encoded by the mRNA, optionally wherein the mRNA encodes an antigen, and / or the disease is (a) a protein deficiency (optionally wherein the protein deficiency affects the liver, lungs, brain or muscles), (b) an autoimmune disease, (c) an infectious disease, or (d) cancer.

[0154] In embodiments, the compositions are administered intravenously, intrathecally, intramuscularly, or by pulmonary delivery, optionally via nebulization. In embodiments, the compositions are administered intramuscularly. In embodiments, the compositions are administered by intravenous administration.

[0155] Exemplary Compounds In embodiments, the cationic lipid of the present invention comprises a compound selected from those shown in Table A, or a pharma- ceutically acceptable salt thereof.

[0156] Exemplary compounds include those set forth in Table A, or a pharma- ceutically acceptable salt thereof.

[0157] [Table 1]

[0158] [Table 2]

[0159] [Table 3]

[0160] [Table 4]

[0161] [Table 5]

[0162] [Table 6]

[0163] [Table 7]

[0164] [Table 8]

[0165] [Table 9]

[0166] [Table 10]

[0167] Any of compounds 1-60 identified in Table A above may be provided in the form of a pharma- ceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0168] Exemplary compounds include those set forth in Table B, or a pharma- ceutically acceptable salt thereof.

[0169] [Table 11]

[0170] [Table 12]

[0171] [Table 13]

[0172] [Table 14]

[0173] Any of compounds 1-4, 6-24, 26-29, 31-54, 56-57, 59-130 and 155 identified in Table B above may be provided in the form of a pharma- ceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0174] Exemplary compounds include those set forth in Table C, or a pharma- ceutically acceptable salt thereof.

[0175] [Table 15]

[0176] [Table 16]

[0177] Any of compounds 131-154 identified in Table C above may be provided in the form of a pharma- ceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0178] The compounds of the invention described herein can be prepared according to methods known in the art, including the exemplary syntheses of the Examples provided herein.

[0179] nucleic acid The compounds of the invention described herein can be used to prepare compositions useful for the delivery of nucleic acids.

[0180] Nucleic Acid Synthesis The nucleic acid according to the present invention can be synthesized according to any known method. For example, the mRNA according to the present disclosure can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, mutant T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and / or RNAse inhibitor. The exact conditions vary according to the specific application.

[0181] In some embodiments, for the preparation of mRNA according to the present invention, a DNA template is transcribed in vitro. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7, mutant T7 or SP6 promoter, followed by the desired nucleotide sequence for the desired mRNA and a termination signal.

[0182] The desired mRNA sequence according to the invention can be determined and incorporated into a DNA template using standard methods. For example, starting from the desired amino acid sequence (e.g., enzyme sequence), virtual back-translation is carried out based on the degenerate genetic code. An optimization algorithm can then be used to select suitable codons. Typically, the G / C content is optimized on the one hand to achieve the highest possible G / C content, and on the other hand to take into account the maximum possible frequency of tRNAs according to codon usage. The optimized RNA sequence can be established and displayed, for example, using a suitable display device, and compared with the original (wild-type) sequence. The secondary structure can also be analyzed to calculate the stabilizing and destabilizing properties, or regions of the RNA, respectively.

[0183] modified mRNA In some embodiments, the mRNA according to the present invention can be synthesized as unmodified or modified mRNA. Modified mRNA comprises nucleotide modifications in RNA. Thus, modified mRNA according to the present invention can comprise nucleotide modifications, such as backbone modifications, sugar modifications or base modifications. In some embodiments, the mRNA is composed of 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)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 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, inos ... Leu-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine (wybutoxosine), and can be synthesized as phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.The preparation of such analogs is known to those skilled in the art, for example from U.S. Pat. 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 by reference in their entireties.

[0184] Pharmaceutical formulations of cationic lipids and nucleic acids In certain embodiments, the compounds of the invention described herein, and pharmaceutical and liposomal compositions comprising such lipids, can be used in formulations to facilitate delivery of encapsulated materials (e.g., one or more polynucleotides, such as mRNA) to one or more target cells, and subsequent transfection of one or more target cells. For example, in certain embodiments, the cationic lipids described herein (and liposomal compositions comprising such lipids, etc.) are characterized by providing one or more of receptor-mediated endocytosis, clathrin-mediated and caveolae-mediated endocytosis, phagocytosis and macropinocytosis, fusogenicity, endosomal or lysosomal disruption, and release properties that provide such compounds with advantages over other similarly classified lipids.

[0185] In accordance with the present invention, a nucleic acid described herein, e.g., an mRNA encoding a protein described herein (e.g., a full-length, fragment, or portion of a protein), can be delivered via a delivery vehicle comprising a compound of the invention described herein.

[0186] As used herein, the terms "delivery vehicle," "transfer vehicle," "nanoparticle," or grammatical equivalents are used interchangeably.

[0187] For example, the present invention provides compositions (e.g., pharmaceutical compositions) comprising a compound described herein and one or more polynucleotides. The compositions (e.g., pharmaceutical compositions) can further include: (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.

[0188] In certain embodiments, the compositions exhibit enhanced (e.g., increased) ability to transfect one or more target cells. Accordingly, methods of transfecting one or more target cells are also provided herein. Such methods generally include contacting one or more target cells with a cationic lipid and / or pharmaceutical composition disclosed herein (e.g., a liposomal formulation comprising a compound described herein encapsulating one or more polynucleotides), thereby transfecting one or more target cells with the encapsulated material (e.g., one or more polynucleotides) therein. 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 a cell (e.g., a target cell). The introduced polynucleotides may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of such encapsulated material (e.g., polynucleotides) taken up by, introduced into, and / or expressed by a target cell subject to transfection. In practice, transfection efficiency can be estimated by the amount of reporter polynucleotide product produced by target cells after transfection. In certain embodiments, the compounds and pharmaceutical compositions described herein exhibit high transfection efficiency, thereby improving the likelihood that an adequate dose of the encapsulated material (e.g., one or more polynucleotides) is delivered to the pathology site and subsequently expressed, while minimizing potential systemic adverse effects or toxicity associated with the compound or its encapsulated contents.

[0189] For example, after transfection of one or more target cells with a polynucleotide encapsulated in one or more lipid nanoparticles, including the pharmaceutical or liposomal compositions disclosed herein, the production of the product (e.g., polypeptide or protein) encoded by such polynucleotide may be stimulated, and the ability of such target cells to express the polynucleotide and, for example, produce a polypeptide or protein of interest is enhanced. For example, transfection of target cells with one or more compounds or pharmaceutical compositions encapsulating an mRNA will enhance (i.e., increase) the production of the protein or enzyme encoded by such mRNA.

[0190] Additionally, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to distribute preferentially to other target tissues, cells or organs, such as the heart, lung, kidney, spleen or muscle. In embodiments, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to distribute preferentially to the lung. In embodiments, the delivery vehicles described herein (e.g., liposomal delivery vehicles) can be prepared to distribute preferentially to muscle tissue. In embodiments, the lipid nanoparticles of the present invention can be prepared to achieve enhanced delivery to target cells and tissues. For example, a polynucleotide (e.g., mRNA) encapsulated in one or more of the compounds or pharmaceutical compositions and liposomal compositions described herein can be delivered and / or transfected into a targeted cell or tissue. In some embodiments, the encapsulated polynucleotide (e.g., mRNA) can be expressed by the target cell and a functional polypeptide product can be produced (and optionally excreted), thereby conferring beneficial properties to the target cell or tissue, for example. Such an encapsulated polynucleotide (eg, mRNA) can encode, for example, a hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest.

[0191] Liposomal Delivery Vehicles In some embodiments, the composition is a suitable delivery vehicle. In some embodiments, the suitable delivery vehicle is a liposomal delivery vehicle, such as a lipid nanoparticle.

[0192] The terms "liposome delivery vehicle" and "liposome composition" are used interchangeably.

[0193] Concentrating liposomal compositions with one or more of the cationic lipids disclosed herein can be used as a means to improve safety profile or to impart one or more desired properties to such concentrated liposomal compositions (e.g., improving delivery of encapsulated polynucleotides to one or more target cells and / or reducing the in vivo toxicity of the liposomal composition).Therefore, pharmaceutical compositions, particularly liposomal compositions, that include one or more of the cationic lipids disclosed herein are also contemplated.

[0194] Thus, in certain embodiments, the compounds of the invention described herein may 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 permeabilizing or fusing the lipid membranes of such target cells).

[0195] As used herein, liposome delivery vehicles, such as lipid nanoparticles, are generally characterized as microscopic vesicles with an internal aqueous space separated from the external medium by one or more bilayer membranes.The bilayer membrane of liposomes is generally formed by amphiphilic molecules such as synthetic or natural lipids that contain hydrophilic and hydrophobic domains spaced apart (Lasic, Trends Biotechnol., 16:307-321, 1998).The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).In the context of the present invention, liposome delivery vehicles are generally useful for transporting desired mRNA to target cells or tissues.

[0196] In certain embodiments, such compositions (eg, liposomal compositions) are loaded or encapsulated with a material, such as, for example, one or more biologically active polynucleotides (eg, mRNA).

[0197] In embodiments, the composition (e.g., pharmaceutical composition) comprises an mRNA encoding a peptide or protein encapsulated within a liposome. In embodiments, the liposome comprises: (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 the one or more cationic lipids are compounds of the invention as described herein.

[0198] In embodiments, the composition comprises an mRNA that encodes a peptide or protein (e.g., any peptide or protein described herein). In embodiments, the composition comprises an mRNA that encodes a peptide (e.g., any peptide described herein). In embodiments, the composition comprises an mRNA that encodes a protein (e.g., any protein described herein).

[0199] In embodiments, a composition (eg, a pharmaceutical composition) comprises a nucleic acid encapsulated within a liposome, and the liposome comprises a compound described herein.

[0200] In embodiments, the nucleic acid is an mRNA that encodes a peptide or protein. In embodiments, the mRNA encodes a peptide or protein for delivery to or use in treatment of the lung or lung cells of a subject. In embodiments, the mRNA encodes a peptide or protein for delivery to or use in treatment of the liver or liver cells of a subject. In embodiments, the mRNA encodes a peptide or protein for delivery to or use in treatment of muscle cells. In embodiments, the mRNA encodes a peptide or protein for delivery to or use in treatment of immune cells. Still other exemplary mRNAs are described herein.

[0201] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) may have a net positive charge.

[0202] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) may have a net negative charge.

[0203] In embodiments, the liposome delivery vehicle (eg, lipid nanoparticle) may have a net neutral charge.

[0204] In embodiments, lipid nanoparticles encapsulating a nucleic acid (eg, an mRNA encoding a peptide or protein) comprise one or more compounds of the invention as described herein.

[0205] For example, the amount of a compound of the invention described herein in a composition can be described as a percentage ("wt %") of the total dry weight of all lipids of the composition (e.g., the total dry weight of all lipids present in a liposome composition).

[0206] In embodiments of the pharmaceutical compositions described herein, a compound of the invention described herein is present in an amount that is about 0.5% to about 30% by weight (e.g., about 0.5% to about 20% by weight) of the total dry weight of all lipids present in the composition (e.g., a liposomal composition).

[0207] In embodiments, the compounds of the invention described herein are present in an amount that is about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 5% to about 25% by weight of the total dry weight of all lipids present in a composition (e.g., a liposomal composition). In embodiments, the compounds of the invention described herein are present in an amount that is about 0.5% to about 5%, about 1% to about 10%, about 5% to about 20%, or about 10% to about 20% by weight of the total dry weight of all lipids present in a composition, such as a liposomal delivery vehicle.

[0208] In embodiments, the amount of a compound of the invention described herein is present in an amount that is at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% by weight of the total dry weight of the total lipid in the composition (e.g., liposomal composition).

[0209] In embodiments, the amount of a compound of the invention described herein is present in an amount that is about 5%, about 10%, about 15%, about 20%, about 25% or less by weight of the total dry weight of the total lipid in the composition (e.g., liposomal composition), and is present in an amount that is about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% by weight or less.

[0210] In embodiments, the composition (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.1% to about 20% by weight (e.g., about 0.1% to about 15% by weight) of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises about 0.5%, about 1%, about 3%, about 5%, or about 10% by weight of a compound described herein. In embodiments, the delivery vehicle (e.g., a liposomal delivery vehicle such as a lipid nanoparticle) comprises up to about 0.5%, about 1%, about 3%, about 5%, about 10%, about 15%, or about 20% by weight of a compound described herein. In embodiments, the percentage results in an improved beneficial effect (e.g., improved delivery to target tissues such as liver, lung, or muscle).

[0211] The amount of a compound of the invention described herein in a composition can also be described as a percentage ("mol %) of the total molar amount of the total lipid of the composition (e.g., the total molar amount of all lipids present in a liposome delivery vehicle).

[0212] In embodiments of the pharmaceutical compositions described herein, the compounds of the invention described herein are present in an amount that is from about 0.5 mol % to about 50 mol % (e.g., from about 0.5 mol % to about 20 mol %) of the total molar amount of all lipids present in the composition, such as a liposome delivery vehicle.

[0213] In embodiments, the compounds of the invention described herein are present in an amount that is 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% of the total molar amount of all lipids present in a composition, such as a liposome delivery vehicle. In embodiments, the compounds of the invention described herein are present in an amount that is from 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% of the total molar amount of all lipids present in a composition, such as a liposome delivery vehicle.

[0214] In certain embodiments, the compounds of the invention described herein can comprise from about 0.1 mol% to about 50 mol%, or from 0.5 mol% to about 50 mol%, or from about 1 mol% to about 50 mol%, or from about 5 mol% to about 50 mol%, or from about 10 mol% to about 50 mol%, or from about 15 mol% to about 50 mol%, or from about 20 mol% to about 50 mol%, or from about 25 mol% to about 50 mol%, or from about 30 mol% to about 50 mol% of the total amount of lipid in a composition (e.g., a liposomal delivery vehicle).

[0215] In certain embodiments, the compounds of the invention described herein may constitute more than 0.1 mol%, or more than about 0.5 mol%, or more than about 1 mol%, or more than about 5 mol%, or more than about 10 mol%, or more than about 20 mol%, or more than about 30 mol%, or more than about 40 mol% of the total amount of lipid in the lipid nanoparticle.

[0216] In certain embodiments, the described compounds 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 amount of lipid in a composition (e.g., a liposome delivery vehicle).

[0217] In embodiments, the amount of a compound of the invention described herein is present in an amount that is 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 total molar amount of total lipid in a composition (e.g., a liposomal composition).

[0218] In embodiments, the amount of a compound of the invention described herein is present in an amount that is less than or equal to 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 total molar amount of total lipid in a composition (e.g., a liposomal composition).

[0219] In an embodiment, the percentage results in an improved beneficial effect (eg, improved liver, lung or muscle, optionally to a target tissue such as muscle).

[0220] In an exemplary embodiment, a composition of the invention (e.g., a liposomal composition) comprises: (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 the one or more cationic lipids are compounds of the invention as described herein.

[0221] For example, a composition suitable for practicing the present invention has four lipid components that include the compounds of the present invention described herein as cationic lipid components, and further includes: (i) non-cationic lipids, (ii) cholesterol-based lipids and (iii) PEG-modified lipids.

[0222] The non-cationic lipid can be DOPE or DEPE. The cholesterol-based lipid can be cholesterol. The PEG-modified lipid can be DMG-PEG2K.

[0223] In further embodiments, the pharmaceutical (e.g., liposomal) composition comprises one or more of a PEG-modified lipid, a non-cationic lipid, and a cholesterol lipid. In other embodiments, such a pharmaceutical (e.g., liposomal) composition comprises: one or more PEG-modified lipids; one or more non-cationic lipids; and one or more cholesterol lipids. In further embodiments, such a pharmaceutical (e.g., liposomal) composition comprises: one or more PEG-modified lipids and one or more cholesterol lipids.

[0224] In embodiments, a composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprises one or more compounds of the invention described herein and one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids.

[0225] In embodiments, a composition (e.g., lipid nanoparticle) encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprises one or more compounds of the invention described herein; one or more lipids selected from the group consisting of cationic lipids, non-cationic lipids, and PEGylated lipids, and further comprises a cholesterol-based lipid. Typically, such a composition has four lipid components that include a compound of the invention described herein as the cationic lipid component, 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).

[0226] In embodiments, lipid nanoparticles encapsulating a nucleic acid (e.g., an mRNA encoding a peptide or protein) comprise one or more compounds of the invention described herein and one or more lipids selected from the group consisting of: (i) a cationic lipid, (ii) non-cationic lipids, (iii) PEGylated lipids, and (iv) Cholesterol-based lipids.

[0227] According to various embodiments, the cationic lipid, non-cationic lipid and / or PEG-modified lipid that constitute lipid nanoparticles and the relative molar ratio of these lipids to each other are based on the characteristics of selected lipid, the nature of the intended target cell, and the characteristics of the nucleic acid to be delivered.Additional considerations include, for example, the saturation of alkyl chain, and the size, charge, pH, pKa, membrane fusogenicity and toxicity of selected lipid.Therefore, the molar ratio can be adjusted accordingly.

[0228] In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 120 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 60 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 70 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 80 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 90 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 100 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 110 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 115 to 125 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 60 to 130 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 70 to 130 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 80 to 130 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 90 to 130 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 100-130 nm. In an embodiment, the lipid nanoparticles of the present invention have a diameter of about 110-130 nm. In an 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 backscatter detector angle of 173° and a 4 mW, 633 nm He-Ne laser. Samples can be analyzed by diluting with 10% trehalose and measuring the diameter in an optical grade polystyrene cuvette.

[0229] Cationic lipids In addition to any of the compounds of the invention described herein, the compositions may include one or more additional cationic lipids.

[0230] In some embodiments, liposome can contain one or more additional cationic lipids.As used herein, the phrase "cationic lipid" refers to any of several lipid species that have a net positive charge at a selected pH, such as physiological pH.Several cationic lipids have been described in the literature, and many of them are commercially available.

[0231] Additional cationic lipids suitable for use in the compositions include those cationic lipids described in the literature.

[0232] Helper lipids The composition (e.g., liposome composition) may also include one or more helper lipids. Such helper lipids include non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of several lipid species that have a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl -phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or mixtures thereof. A suitable non-cationic or helper lipid for the implementation of the present invention is dioleoylphosphatidylethanolamine (DOPE). Alternatively, 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE) can be used as the non-cationic or helper lipid.

[0233] In some embodiments, the non-cationic lipids are neutral lipids, ie, lipids that have no net charge in the conditions in which the composition is formulated and / or administered.

[0234] In some embodiments, the non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes can 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 can 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 can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%. In some embodiments, the percentage of total non-cationic lipids in the liposomes can be less than about 5 mol%, less than about 10 mol%, less than about 20 mol%, less than about 30 mol%, or less than about 40 mol%.

[0235] In some embodiments, the non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10% to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in the composition. In some embodiments, the percentage of non-cationic lipids in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be greater than about 5% by weight, greater than about 10% by weight, greater than about 20% by weight, greater than about 30% by weight, or greater than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight. In some embodiments, the percentage of total non-cationic lipids in the liposomes may be less than about 5% by weight, less than about 10% by weight, less than about 20% by weight, less than about 30% by weight, or less than about 40% by weight.

[0236] Cholesterol-Based Lipids In some embodiments, the composition (e.g., liposome composition) comprising the cationic lipid of the present invention further comprises one or more cholesterol-based lipids. For example, a suitable cholesterol-based lipid for carrying out the present invention is cholesterol. Other suitable cholesterol-based lipids include, for example, DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem.Biophys.Res.Comm.179,280(1991); Wolf et al. BioTechniques 23,139(1997); U.S. Pat. No. 5,744,335), β-sitosterol, or imidazole cholesterol ester (ICE) having the following structure: [ka]

[0237] In some embodiments, the cholesterol-based lipid may be present in a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle 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 lipid in the lipid nanoparticle may be about 5 mol% or less, about 10 mol% or less, about 20 mol% or less, about 30 mol% or less, or about 40 mol% or less.

[0238] In some embodiments, the cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipid present in the liposome. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be about 5 wt% or less, about 10 wt% or less, about 20 wt% or less, about 30 wt% or less, or about 40 wt% or less.

[0239] PEGylated lipids In some embodiments, the composition (e.g., liposomal composition) comprises one or more additional PEGylated lipids. A suitable PEG-modified or PEGylated lipid for carrying out the present invention is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[0240] The use of derivatized lipids, such as, for example, polyethylene glycol (PEG) modified phospholipids and derivatized ceramides (PEG-CER) containing N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), in combination with one or more compounds of the invention, and in some embodiments, in combination with other lipids that together comprise liposomes, is also contemplated by the invention. In some embodiments, particularly useful exchangeable lipids have shorter acyl chains (e.g., C 14 Or C 18 ) is a PEG-ceramide.

[0241] Additional PEG-modified lipids (also referred to herein as PEGylated lipids, a term interchangeable with PEG-modified lipids) contemplated include: 6 ~C 20) long alkyl chains covalently attached to lipids. In some embodiments, the PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means to increase circulation lifetime and delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268(1):235-237), or they may be selected to be rapidly cleared from the formulation in vivo (see U.S. Pat. No. 5,885,613).

[0242] The PEG-modified phospholipids and derivatized lipids of the present invention may be present in a molar ratio (mol %) of about 0% to about 10%, about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, about 3% to about 5%, about 1% to about 5%, or about 1.5% to about 3% of the total lipid present in the composition (e.g., liposome composition).

[0243] Pharmaceutical Formulations and Therapeutic Uses The compounds of the invention described herein may be used in the preparation of compositions (e.g., to construct liposomal compositions) that facilitate or enhance the delivery and release of an encapsulated material (e.g., one or more therapeutic polynucleotides) to one or more target cells (e.g., by permeabilizing or fusing with the lipid membrane of such target cells).

[0244] For example, when a liposome composition (e.g., lipid nanoparticle) comprises or is enriched with one or more of the compounds disclosed herein, a phase transition in the lipid bilayer of one or more target cells can facilitate delivery of the encapsulated material (e.g., one or more therapeutic polynucleotides encapsulated in the lipid nanoparticle) to one or more target cells.

[0245] Similarly, in certain embodiments, the compounds of the invention described herein may be used to prepare liposomal vehicles that are characterized by reduced toxicity in vivo, in certain embodiments, the reduced toxicity is a function of the high transfection efficiency associated with the compositions disclosed herein, and thus, small amounts of such compositions may be administered to a subject to achieve a desired therapeutic response or outcome.

[0246] In certain embodiments, the compounds of the present invention described herein can be used to prepare liposomal vehicles characterized by effective intramuscular delivery of mRNA.In certain embodiments, the compounds of the present invention described herein can be used to prepare liposomal vehicles characterized by achieving high levels of peptide or protein expression when mRNA encoding said peptide or protein is delivered by intramuscular delivery.

[0247] Thus, pharmaceutical formulations containing the compounds described and the nucleic acids provided by the present invention can be used for various therapeutic disease and / or disease prevention purposes. To facilitate in vivo delivery of the nucleic acid, the compounds and nucleic acids described herein can be formulated in combination with one or more additional pharmaceutical carriers, targeting ligands, or stabilizing reagents. In some embodiments, the compounds described herein can be formulated via a premixed lipid solution. In other embodiments, compositions containing the compounds described herein can be formulated using post-insertion techniques into the lipid membrane of nanoparticles. Techniques for drug formulation and administration can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.

[0248] Suitable routes of administration include, for example, oral, rectal, vaginal, mucosal, intratracheal or pulmonary, including inhalation, or intestinal administration; intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal. In certain embodiments, intramuscular administration is to a muscle selected from the group consisting of skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, administration results in delivery of the nucleic acid to a muscle cell. In some embodiments, administration results in delivery of the nucleic acid to a hepatocyte (i.e., liver cell).

[0249] A common route for administering the liposomal composition of the present invention may be intravenous delivery, especially when treating metabolic disorders, especially those affecting the liver (e.g., ornithine transcarbamylase (OTC) deficiency). Alternatively, depending on the disease or disorder being treated, the liposomal composition may be administered via pulmonary delivery (e.g., for the treatment of cystic fibrosis). For vaccination, the liposomal composition of the present invention is typically administered intramuscularly. Diseases or disorders affecting the eye may be treated by administering the liposomal composition of the present invention intravitreally.

[0250] Alternatively or in addition, the pharmaceutical formulation of the present invention can be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical formulation directly into the tissue to be targeted (e.g., in a sustained release formulation). Local delivery can be affected in various ways, depending on the tissue to be targeted. Exemplary tissues to which mRNA can be delivered and / or expressed include, but are not limited to, liver, kidney, heart, spleen, serum, brain, skeletal muscle, lymph nodes, skin, and / or cerebrospinal fluid. In embodiments, the tissue targeted is the liver. For example, an aerosol containing the composition of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); for example, the composition of the present invention can be injected at the site of injury, disease manifestation, or pain; the composition can be provided in a troche for oral, tracheal, or esophageal application; can be provided in liquid, tablet, or capsule form for administration to the stomach or intestinal tract, or in suppository form for rectal or vaginal application; or can be delivered to the eye by using a cream, drop, or injection.

[0251] The compositions described herein can include mRNA that encodes a peptide (eg, a polypeptide such as a protein), including those described herein.

[0252] In embodiments, the mRNA encodes a polypeptide.

[0253] In embodiments, the mRNA encodes a peptide. In embodiments, the peptide is an antigen.

[0254] In an embodiment, the mRNA encodes a protein.

[0255] The present invention provides a method for delivering a composition having a full length mRNA molecule encoding a peptide or protein of interest for use in treating a subject, e.g., a human subject, or a cell of a human subject, or a cell that is treated and delivered to a human subject.

[0256] Delivery method The delivery route used in the method of the present invention allows non-invasive self-administration of the compound of the present invention. In some embodiments, the method includes intranasal, intratracheal or pulmonary administration by aerosolization, nebulization or injection of a composition comprising mRNA encoding a therapeutic peptide or protein in a suitable transfection or lipid carrier vehicle as described above. In some embodiments, the peptide or protein is encapsulated in a liposome. In some embodiments, the liposome comprises a lipid that is a compound of the present invention. As used herein below, administration of a compound of the present invention includes administration of a composition comprising a compound of the present invention.

[0257] While local lung cells and tissues are potential targets that can serve as biological depots or reservoirs for the production and secretion of proteins encoded by mRNA, applicants have discovered that administering the compounds of the invention to the lungs via aerosolization, nebulization, or injection results in the distribution of non-secreted proteins even outside of lung cells. Without wishing to be bound by a particular theory, it is contemplated that the nanoparticle compositions of the invention cross the lung airway blood barrier, resulting in the migration of intact nanoparticles to non-lung cells and tissues, such as the heart, liver, spleen, muscle, resulting in the production of the encoded peptide or protein in these non-lung tissues. Thus, the utility of the compounds of the invention and the methods of the invention extends beyond the production of therapeutic proteins in lung cells and lung tissues, and can be used for delivery to non-lung target cells and / or tissues. They are useful for the management and treatment of a number of diseases. In certain embodiments, the compounds of the invention used in the methods of the invention result in the distribution of mRNA-encapsulated nanoparticles and the production of the encoded peptide or protein in the liver, spleen, heart, muscle, and / or other non-lung cells. For example, administration of a compound of the invention by aerosolization, nebulization or injection into the lungs will result in the composition itself and its peptide or protein products (e.g., antigens or functional proteins) being detectable in both local cells and tissues of the lung, as well as in peripheral target cells, tissues and organs as a result of transfer of the mRNA and delivery vehicle to non-pulmonary cells.

[0258] In certain embodiments, the compounds of the present invention may be used in the methods of the present invention to specifically target peripheral cells or tissues. It is contemplated that after pulmonary delivery, the compounds of the present invention will cross the pulmonary airway blood barrier and distribute to cells other than local lung cells. Thus, the compounds disclosed herein are administered to a subject by a pulmonary administration route (e.g., by inhalation) using various approaches known to those skilled in the art, and distributed to both local target cells and tissues in the lung, as well as peripheral non-lung cells and tissues (e.g., liver, spleen, kidney, heart, skeletal muscle cells, lymph nodes, brain, cerebrospinal fluid, and plasma). As a result, both local cells in the lung and peripheral non-lung cells can function as biological reservoirs or depots that can produce and / or secrete translation products encoded by one or more polynucleotides. Thus, the present invention is not limited to the treatment of pulmonary diseases or conditions, but can be used as a non-invasive means to facilitate the delivery of polynucleotides or the production of peptides or proteins encoded thereby in end organs, tissues, and cells (e.g., liver cells) that would otherwise be achieved only by systemic administration. Exemplary peripheral non-pulmonary cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, bone cells, stem cells, mesenchymal cells, neuronal cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiac myocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells.

[0259] After administration of the composition to a subject, the peptide or protein product (e.g., a functional protein or enzyme) encoded by the mRNA is detectable in peripheral target tissues for at least about 1-7 days or more after administration of the compound to the subject. The amount of peptide or protein product necessary to achieve a therapeutic effect will vary depending on the condition being treated, the encoded peptide or protein, and the condition of the patient. For example, the peptide or protein product may be at least 0.025-1.5 μg / ml (e.g., at least 0.050 μg / ml, at least 0.075 μg / ml, at least 0.1 μg / ml, at least 0.2 μg / ml, at least 0.3 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.7 μg / ml, at least 0.8 μg / ml, at least 0.9 μg / ml, at least 1.0 μg / ml, at least 1.5 ... In some embodiments, the compound may be detectable in a distal target tissue at a concentration (e.g., a therapeutic concentration) of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or more days after administration of the compound to a subject.

[0260] It has been demonstrated that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the compound and inhalation of the aerosol mist produced by a liquid nebulizer, or by use of a dry powder device such as that described in U.S. Pat. No. 5,780,014, incorporated herein by reference.

[0261] In certain embodiments, the compounds of the present invention may be formulated so that they can be delivered as aerosolized or particulate liquids or solids before or at the time of administration to a subject. Such compounds may be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions) to generate particles that are easily respirable or inhalable by a subject. In some embodiments, such devices (e.g., metered dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers or insufflators) facilitate administration of a predetermined mass, volume or dose of the composition (e.g., about 0.5 mg / kg of mRNA per dose) to a subject. For example, in certain embodiments, the compounds of the present invention are administered to a subject using a metered dose inhaler that includes a suspension or solution that includes the compound and a suitable propellant. In certain embodiments, the compounds of the present invention may be formulated as particulate powders (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the invention formulated as respirable particles are respirable by a subject or delivered using a suitable device (e.g., average D50 or D90 particle size of about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or less). In yet other embodiments, compounds of the invention are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).In some embodiments, the compounds of the invention are administered at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least The subject is administered a concentration of 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 administered in a single dose. In some embodiments, a compound of the invention is administered to a subject such that a total amount 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 of mRNA is administered in one or more doses.

[0262] Synthesis of Compounds of the Invention Cationic lipid MC3 is the current gold standard for in vivo delivery of, for example, siRNA (see WO 2010 / 144740). However, the synthesis of this lipid involves a six-step process and requires the handling of Grignard reagents. In contrast, the present invention provides cationic lipids that can be prepared from readily available starting reagents such as "Good's" buffer (see Table 1 below). These starting reagents can be attached to cationic head groups and lipid tails using coupling reactions such as sulfonylation, acetylation and alkylation (see, for example, Table 2 below).

[0263] [Table 17]

[0264] [Table 18]

[0265] [Table 19]

[0266] [Table 20]

[0267] [Table 21]

[0268] In embodiments, the cationic lipids described herein can be prepared by conjugating a lipid, such as a carboxylic acid of a lipid, with a "Good's" buffer under suitable conditions. Exemplary "Good's" buffers are listed in Table 1, and exemplary lipid chains are listed in Table 2. Thus, suitable cationic lipids include those resulting from any combination of the precursors listed in Tables 1 and 2.

[0269] In some embodiments, the sulfonic acid groups of a compound, such as "Good's" buffer, can be derivatized by forming a sulfonyl chloride using a reagent such as oxalyl chloride. The resulting sulfonyl chloride can undergo a number of reactions, including but not limited to reduction with Zn / HCl to form the corresponding thiols, and coupling to nucleophiles such as amines and alcohols to form the corresponding sulfonamides and sulfonates (see, for example, Scheme A below): [ka]

[0270] Using the chemistry outlined in Scheme A, the sulfonic acid starting reagents can be derivatized with a range of suitable cationic lipid head groups and lipid chains.

[0271] Additionally, compounds such as "Good's" buffers can be readily synthesized, for example, via nucleophilic ring opening of episulfides with piperazine (see, for example, Scheme B below). [ka]

[0272] The compounds of the invention described herein can be prepared according to methods known in the art, including the exemplary syntheses of the Examples provided herein. EXAMPLES

[0273] While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples are merely illustrative of the compounds of the present invention and are not intended to be limiting thereof.

[0274] Any of the compounds specified in the examples may be provided in the form of a pharma- ceutically acceptable salt, and such salts are intended to be encompassed by the present invention.

[0275] List of abbreviations: APCI-MS: Atmospheric pressure chemical ionization mass spectrometry EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EtOAc: ethyl acetate MS: Mass spectrometry Na 2 SO 4 : Sodium sulfate SiO 2 :Silicon dioxide TLC: Thin Layer Chromatography iv.

[0276] Example 1 Synthesis of compounds of the present invention For example, compounds of the present invention can be prepared according to Schemes 1 and 2.

[0277] Scheme 1: Synthetic scheme for intermediates [ka]

[0278] Scheme 2: Synthesis scheme of compound 48 [ka]

[0279] Synthesis procedure for intermediate 3: Step 1: Synthesis of 2-(3-(tritylthio)propyl)isoindoline-1,3-dione (2) [ka] As shown in Scheme 1: To a mixture of sodium hydride (30 g, 1.08 mol, 60% dispersion in mineral oil) in 600 mL of N,N-dimethylformamide, triphenylmethanethiol (200 g, 0.724 mol) was added portionwise 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 allowed to warm slowly to room temperature and stirred overnight. The reaction mixture was poured into 6 L of ice-cold water, the solution was decanted, the solid was dissolved in ethyl acetate, and washed with brine. The organic layer was extracted with Na 2 SO 4 Drying and concentration afforded 2-(3-(tritylthio)propyl)isoindoline-1,3-dione as a white solid (252 g, 75%) which was used in the next step without further purification.

[0280] Step 2: Synthesis of 3-(tritylthio)propan-1-amine (3) [ka] As shown in Scheme 1: A mixture of 2-(3-(tritylthio)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 gentle reflux overnight under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was filtered through Celite, which was then washed with ethanol. The combined filtrate was concentrated under reduced pressure, and the residue was dissolved in chloroform. After stirring for 15 min, the mixture was filtered, concentrated, and the crude material was purified by flash column chromatography (SiO 2 Purification by chromatography (HPLC: 0-15% methanol in dichloromethane) afforded 3-(tritylthio)propan-1-amine as an oil (100 g, 55%).

[0281] Procedure for the synthesis of epoxide 8: Step 1: Synthesis of nona-8-enoic acid (5) [ka] As shown in Scheme 1: To a solution of periodic acid (353 g, 1.55 mol) in 2 L of acetonitrile was added a solution of non-8-en-1-ol 4 (100 g, 0.7 mol) at 0° C., followed by the dropwise addition of a solution of pyridinium chlorochromate (3.23 g, 15 mmol) in 500 mL of acetonitrile in 2 h. The resulting cloudy mixture was stirred at room temperature overnight. TLC indicated a complete reaction. The reaction mixture was diluted with 1 L of EtOAc, and the solution was washed with water and brine. After drying over sodium sulfate, the organic layer was concentrated and the crude material was purified by column chromatography (SiO 2 Purification by chromatography (C: 0 to 50% ethyl acetate in hexanes) afforded non-8-enoic acid as a pale yellow oil (88 g, 80%).

[0282] Step 2: Synthesis of 2-ethylbutylnon-8-enoate (7) [ka] As shown in Scheme 1: To a mixture of non-8-enoic acid 5 (50 g, 0.32 mol) and 2-ethylbutanol 6 (39.2 g, 0.384 mol) in 250 mL of dichloromethane, EDCI (73.6 g, 0.384 mol) and dimethylaminopyridine (7.8 g, 64 mmol) were added, and the reaction mixture was then stirred overnight. MS and TLC analysis indicated a complete reaction. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, water, and brine. After drying over sodium sulfate, the solvent was evaporated under vacuum and the crude material was purified by flash column chromatography (SiO 2 Purification by chromatography (C: 0-20% ethyl acetate in hexanes) afforded 2-ethylbutylnon-8-enoate as a colorless oil (71 g, 92%).

[0283] Step 3: Synthesis of 2-ethylbutyl 7-(oxiran-2-yl)heptanoate (8) [ka] As shown in Scheme 1: A solution of 2-ethylbutyl non-8-enoate 7 (71 g, 0.295 mol) 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 at this temperature overnight. The suspension was filtered and 1.2 M sodium bisulfite solution was added to the filtrate. After stirring for 1 h, the organic layer was separated and then washed with sodium bicarbonate solution and brine. After drying over sodium sulfate, the solvent was evaporated to give 2-ethylbutyl 7-(oxiran-2-yl)heptanoate as a colorless oil (70 g, 92%), which was used in the next step without purification.

[0284] Synthesis procedure for TIM-3-E9Es6: Step 1: Synthesis of bis(2-ethylbutyl) 9,9'-((3-(tritylthio)propyl)azanediyl) bis(8-hydroxynonanoate) (9) [ka] As shown in Scheme 1: A mixture of 3-(tritylthio)propan-1-amine 3 (3.9 g, 11.7 mmol) and 2-ethylbutyl 7-(oxiran-2-yl)heptanoate 8 (8.0 g, 35.1 mmol) in 30 mL of isopropanol was heated to gentle reflux overnight under a nitrogen atmosphere. The reaction mixture was concentrated and the crude material was purified by flash column chromatography (SiO 2 Purification by chromatography (HPLC: 0-10% methanol in dichloromethane) gave bis(2-ethylbutyl) 9,9'-((3-(tritylthio)propyl)azanediyl) bis(8-hydroxynonanoate) as a yellow oil (5.3 g, 53%).

[0285] Step 2: Synthesis of bis(2-ethylbutyl) 9,9'-((3-mercaptopropyl)azanediyl) bis(8-hydroxynonanoate) (TIM-3-E9Es6) [ka] As shown in Scheme 1: To a solution of bis(2-ethylbutyl)9,9'-((3-(tritylthio)propyl)azanediyl)bis(8-hydroxynonanoate)9 (169 mg, 0.20 mmol) and triethylsilane (0.1 mL, 0.6 mmol) in 10 mL of dichloromethane, trifluoroacetic acid (0.1 mL, 1.0 mmol) was added slowly at 0°C. The resulting reaction mixture was allowed to warm to room temperature and stirred for 1 h. MS indicated complete reaction. The volatiles were evaporated and the residue was co-evaporated under vacuum with toluene three times. The crude material was used in the next step without further purification.

[0286] Synthesis procedure of AIM-3-E9Es6: Step 1: Synthesis of bis(2-ethylbutyl) 9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (11) [ka] As shown in Scheme 1: A solution of tert-butyl 4-aminobutanoate 10 (3.3 g, 15.9 mmol), 2-ethylbutyl 7-(oxiran-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 indicated a complete reaction. After concentration to dryness, the residue was purified by flash column chromatography (SiO 2 Purification by elution with ethyl acetate (0 to 100% ethyl acetate in hexanes) gave bis(2-ethylbutyl) 9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl) bis(8-hydroxynonanoate) as a colorless oil (6.0 g, 56%).

[0287] Step 2: Synthesis of bis(2-ethylbutyl) 9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) (12) [ka] As shown in Scheme 1: To a solution of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) 11 (6.0 g, 8.7 mmol) in 50 mL of dichloromethane, tert-butyldimethyl chloride (5.3 g, 35 mmol), imidazole (0.6 g, 8.7 mmol) and dimethylaminopyridine (1.1 g, 8.7 mmol) were added and the resulting mixture was heated to reflux for 48 h. MS indicated a 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 flash column chromatography (SiO 2 Purification by elution with ethyl acetate (0-30% ethyl acetate in hexanes) gave bis(2-ethylbutyl) 9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl) bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) as a colorless oil (4.9 g, 61%).

[0288] Step 7: Synthesis of 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxononyl)amino)butanoic acid (AIM-3-E9Es6) [ka] As shown in Scheme 1: A solution of bis(2-ethylbutyl)9,9'-((4-(tert-butoxy)-4-oxobutyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) 12 (4.9 g, 5.36 mmol) in 15 mL of dichloromethane was cooled to 0 °C, trifluoroacetic acid (20 mL, 0.13 mol) was added dropwise, and the resulting mixture was stirred at room temperature overnight. MS indicated a complete reaction. The solution was adjusted to pH 7 by adding saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane. After drying over sodium sulfate, the solvent was removed under vacuum, and the residue was purified by flash column chromatography (SiO 2: 0-10% methanol in dichloromethane) to give 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxononyl)amino)butanoic acid as a colorless oil (4.1 g, 89%).

[0289] Procedure for the synthesis of disulfide intermediate 16: Synthesis of 2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethan-1-ol (16) [ka] As shown in Scheme 1: In a 2 L round bottom flask, ethylene sulfide (18 g, 0.3 mol) was added to a solution of 2-(piperazin-1-yl)ethan-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 analysis indicated the completion of the reaction. The reaction mixture was concentrated, and the residue was purified by flash column chromatography (SiO 2 Purification by chromatography (C: 0-10% methanol in dichloromethane) afforded 2-(4-(2-(pyridin-2-yldisulfanyl)ethyl)piperazin-1-yl)ethan-1-ol as a pale yellow oil (37 g, 53%).

[0290] Synthesis procedure for compound 48 Step 1: Synthesis of bis(2-ethylbutyl) 9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) (17) [ka] As shown in Scheme 2: To a solution of 4-(bis(2-((tert-butyldimethylsilyl)oxy)-9-(2-ethylbutoxy)-9-oxononyl)amino)butanoic acid AIM-3-E9Es6 (2.2 g, 2.74 mmol) in 30 mL of dichloromethane, EDCI (0.79 g, 4.11 mmol) and dimethylaminopyridine (67 mg, 0.54 mmol) were added, followed by 2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethan-1-ol 16 (0.98 g, 3.29 mmol) in 5 mL of dichloromethane. The reaction mixture was stirred overnight. MS and TLC analysis indicated a complete reaction. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, water and brine. After drying over sodium sulfate, the solvent was evaporated under vacuum and the crude material was purified by flash column chromatography (SiO 2 Purification by: 0-100% ethyl acetate with 1% triethylamine in hexanes with 1% triethylamine, then 10% triethylamine in ethyl acetate, then 25% triethylamine in ethyl acetate) to give bis(2-ethylbutyl) 9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) as a colorless oil (1.8 g, 60%).

[0291] Step 2: Synthesis of bis(2-ethylbutyl) 9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate) (18) [ka] As shown in Scheme 2: To a solution of bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-((tert-butyldimethylsilyl)oxy)nonanoate) 17 (1.8 g, 1.6 mmol) in 30 mL of tetrahydrofuran / dichloromethane (1:1) was added hydrogen fluoride pyridine (70% HF, 1 mL, 34.5 mmol) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. MS and TLC analysis indicated a complete reaction. The reaction was quenched by slowly 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 material was purified by flash column chromatography (SiO 2 Purification by chromatography (80° C.: 0-100% ethyl acetate with 1% triethylamine in hexanes with 1% triethylamine, then 10% triethylamine in ethyl acetate, then 25% triethylamine in ethyl acetate) gave bis(2-ethylbutyl) 9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate) as a pale yellow oil (1.06 g, 73%).

[0292] Step 3: Synthesis of bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)-bis(8-hydroxynonanoate) (compound 48) [ka] As shown in Scheme 2: To a solution of bis(2-ethylbutyl)9,9'-((4-oxo-4-(2-(4-(2-(pyridin-2-yldisulfanayl)ethyl)piperazin-1-yl)ethoxy)butyl)azanediyl)bis(8-hydroxynonanoate) 18 (90 mg, 0.10 mmol) in 5 mL chloroform was added a 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 with nitrogen three times and then stirred at room temperature for 2 h. MS and TLC analysis indicated a complete reaction. The reaction mixture was concentrated to dryness and the crude material was purified by flash column chromatography (SiO 2 Purification with: 0 to 100% ethyl acetate with 1% triethylamine in hexanes with 1% triethylamine, then 10% triethylamine in ethyl acetate) to give bis(2-ethylbutyl)9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) as a pale yellow oil (82 mg, 58%).

[0293] Other lipids of the present invention were prepared according to the representative procedures depicted in Schemes 1 and 2 and explained above.

[0294] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 1) [ka] 1H NMR(300MHz,CDCl3)δ 4.19(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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0295] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 18) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.9.

[0296] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 49) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0297] Dibutyl 9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 10) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.7.

[0298] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 39) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0299] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 13) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0300] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 19) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1292.0.

[0301] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.0.

[0302] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 7) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1278.0.

[0303] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 15) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0304] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (compound 14) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.7.

[0305] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (compound 20) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(t,2H),4.08(t,4H),3.98(d,4H),3.65(m,4H),2.84-2.32(m,32H),2.29(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0306] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (compound 40) [ka] 1H NMR (300 MHz, CDCl 3 )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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.8.

[0307] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-oxo-9-propoxynonyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0308] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)-amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 29) [ka] 1H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0309] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 48) [ka] 1 H NMR (300 MHz, CDCl 3 )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: calculated for C75H144N4O14S2 [M+H] = 1390.1, observed = 1390.1.

[0310] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0311] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 21) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0312] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 41) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0313] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 16) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,2H),4.19(t,2H),3.98(d,4H),3.66(m,4H),2.85-2.24(m,4 0H),1.86-1.75(m,4H),1.70-1.26(m,46H),1.22(d,12H),0.88(t,12H). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0314] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 50) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0315] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 11) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0316] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 38) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C74H142N4O14S2 [M+H] = 1376.0, observed = 1376.1.

[0317] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 60) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C76H146N4O14S2 [M+H] = 1404.1, observed = 1404.0.

[0318] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 9) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1306.0.

[0319] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 51) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0320] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 12) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1362.0, observed = 1361.5.

[0321] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 31) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.9.

[0322] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1346.9.

[0323] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 2) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(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: calculated for C67H128N4O14S2 [M+H] = 1277.8, observed = 1277.9.

[0324] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 8) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0325] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 17) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0326] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 27) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.77(pent,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.8.

[0327] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 36) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.77(pent,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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0328] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 24) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),4.08(t,4H),3.65(m,4H),2.85-2.24(m,4 0H),1.92-1.78(m,4H),1.72-1.26(m,36H),1.23(d,12H),0.91(t,12H). APCI-MS analysis: calculated for C63H120N4O14S2 [M+H] = 1221.7, observed = 1221.8.

[0329] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 34) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0330] Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 25) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0331] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (Compound 26) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0332] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 35) [ka] 1 HNMR (300 MHz, CDCl 3) δ 4.19 (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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.8.

[0333] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(8-hydroxynonanoate) (Compound 28) [ka] 1 H NMR (300 MHz, CDCl 3 )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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0334] Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate)) (compound 30) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0335] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)-azanediyl)bis(8-hydroxynonanoate) (compound 37) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.8.

[0336] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 22) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.20(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0337] Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (Compound 32) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.8.

[0338] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)-amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)-bis(8-hydroxynonanoate) (compound 33) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0339] Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 23) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.9.

[0340] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 46) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.75(pent,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0341] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 59) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.75(pent,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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0342] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 44) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),3.98(t,4H),3.64(m,4H),2.85-2.24(m,3 6H),1.90-1.78(m,4H),1.68-1.26(m,44H),1.22(d,12H),0.88(t,12H). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0343] 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)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 55) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.0.

[0344] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 54) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0345] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 56) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1318.9, observed = 1319.0.

[0346] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (compound 45) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.9.

[0347] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(8-hydroxynonanoate) (compound 47) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.21(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1133.8.

[0348] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazine-1-l)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 57) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1347.9.

[0349] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (compound 58) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1361.2.

[0350] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (compound 42) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.8.

[0351] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (Compound 52) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1347.9.

[0352] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 53) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0353] Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (compound 43) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0354] Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (Compound 92) [ka] 1 H NMR (300MHz, Methanol-d 4 )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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.8.

[0355] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (Compound 78) v. [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C62H118N4O14S2 [M+H] = 1207.8, observed = 1207.8.

[0356] Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-3-E7-E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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,16 H),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). APCI-MS analysis: calculated for C63H120N4O14S2 [M+H] = 1221.8, observed = 1221.7.

[0357] Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-4-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.8, observed = 1207.7.

[0358] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-3-E7-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.8.

[0359] Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-4-E7-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.9, observed = 1263.7.

[0360] Bis(2-ethylbutyl) 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Es6-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.6.

[0361] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.7.

[0362] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Es6-DS-4-E9-E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.8.

[0363] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Es6-DS-3-E9-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0364] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-3-E9-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0365] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Es6-DS-3-E9-Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.21(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1133.8.

[0366] Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6-Es5-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,2H),4.74(pent,2H),4.19(t,2H),3.65(m,4H),3.32-3.00(bs,4H),2.8 3-2.24(m,38H),1.91-1.74(m,2H),1.70-1.36(m,30H),1.22(d,12H),0.86(t,12H). APCI-MS analysis: calculated for C57H108N4O14S2 [M+H] = 1137.6, observed = 1137.6.

[0367] Diisopropyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C55H104N4O14S2 [M+H] = 1109.5, observed = 1109.6.

[0368] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.7.

[0369] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-3-E9-Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.8.

[0370] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Es6-DS-4-E9-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.8.

[0371] Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-4-E9-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.8.

[0372] Diisopentyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C59H112N4O14S2 [M+H] = 1165.6, observed = 1165.7.

[0373] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(t,2H),4.08(t,4H),3.98(d,4H),3.65(m,4H),2.84-2.32(m,32H),2.29(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.8.

[0374] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazine-1-l)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Es6-DS-4-E9-Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1347.9.

[0375] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-butoxy-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-E4-DS-4-E9-Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1347.9.

[0376] Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei5-DS-3-E7-Ei3) [ka] 1H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C59H112N4O14S2 [M+H] = 1165.7, observed = 1165.8.

[0377] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9-E4-DS-3-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C62H118N4O14S2 [M+H] = 1207.8, observed = 1207.8.

[0378] Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei5-DS-4-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.8.

[0379] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (GL(GL-HEPES-E4-E9-E4-DS-4-E7-Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C63H120N4O14S2 [M+H] = 1221.8, observed = 1221.8.

[0380] Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E7-Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.98(hept,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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.7.

[0381] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)-bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-Es6-DS-3-E7-Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.8.

[0382] Dibutyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)-azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Ei3-DS-3-E9-E4) [ka] 1 H NMR (300 MHz, CDCl3 )δ 4.98(hept,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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.8.

[0383] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-oxo-9-propoxynonyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9-Es6-DS-3-E9-E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0384] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentyloxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9-E4-DS-3-E7-Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.9, observed = 1263.9.

[0385] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7-Ei5-DS-4-E9-E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.9, observed = 1263.9.

[0386] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0387] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.77(pent,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.8.

[0388] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),4.08(t,4H),3.65(m,4H),2.85-2.24(m,4 0H),1.92-1.78(m,4H),1.72-1.26(m,36H),1.23(d,12H),0.91(t,12H). APCI-MS analysis: calculated for C63H120N4O14S2 [M+H] = 1221.7, observed = 1221.8.

[0389] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)-amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0390] Diisopentyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopentyloxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl3 )δ 4.19(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). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0391] Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0392] Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0393] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-3-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.75(pent,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0394] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-3-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),3.98(t,4H),3.64(m,4H),2.85-2.24(m,3 6H),1.90-1.78(m,4H),1.68-1.26(m,44H),1.22(d,12H),0.88(t,12H). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0395] 7-Oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)-azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0396] Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0397] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1306.0.

[0398] Diisopentyl 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0399] Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.9.

[0400] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-3-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3)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: calculated for C75H144N4O14S2 [M+H] = 1390.1, observed = 1390.1.

[0401] Diisopropyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C56H106N4O14S2 [M+H] = 1123.6, observed = 1123.7.

[0402] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),3.98(d,4H),3.66(m,4H),2.85-2.24(m,4 0H),1.86-1.75(m,4H),1.70-1.26(m,46H),1.22(d,12H),0.88(t,12H). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0403] Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.9.

[0404] Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(t,2H),4.06(t,8H),3.66(m,4H),2.85-2.46(m,22H),2.43-2.23(m,16H),1.90-1.70(m,4H),1.69-1.25(m,54H),0.92(t,12H). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0405] Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei5-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.8.

[0406] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0407] Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Es6-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0408] Dibutyl 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9E4-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0409] Diisopentyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.8.

[0410] Dibutyl 7,7'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C57H108N4O14S2 [M+H] = 1136.7, observed = 1137.8.

[0411] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.20(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: calculated for C67H128N4O14S2 [M+H] = 1277.8, observed = 1277.9.

[0412] Bis(2-ethylbutyl) 9,9'-((3-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-3-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.9.

[0413] Dibutyl 9,9'-((5-(2-(4-(2-((3-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0414] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-4-E9Ei5) [ka] 1 HNMR (300 MHz, CDCl 3) δ 4.19 (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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.8.

[0415] Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E6Es5-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.75(pent,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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.8.

[0416] Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C58H110N4O14S2 [M+H] = 1151.6, observed = 1151.7.

[0417] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0418] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)-amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)-bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E9Ei5) [ka] NMR (300 MHz, CDCl 3) δ 4.19 (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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0419] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei5-DS-4-E9Es6) [ka] 1 HNMR (300MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1318.9, observed = 1319.0.

[0420] Dibutyl 9,9'-((5-(2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-4-E9Es6) [ka] 1 HNMR (300MHz, CDCl3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0421] Bis(2-ethylbutyl) 7,7'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7Ei3-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 5.01(hept,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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.8.

[0422] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl3 )δ 4.19(t,2H),3.98(d,8H),3.61(m,4H),s 2.84-2.26(m,36H),1.83-1.28(m,64H),0.88(t,24H). APCI-MS analysis: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0423] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-4-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.8.

[0424] Dibutyl 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0425] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0426] Dibutyl 9,9'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-pentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9E4-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C62H118N4O14S2 [M+H] = 1207.8, observed = 1207.8.

[0427] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C65H124N4O14S2 [M+H] = 1249.8, observed = 1249.9.

[0428] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-4-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.77(pent,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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0429] Diisopentyl 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-4-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0430] Bis(2-ethylbutyl) 9,9'-((4-(2-(4-(2-((4-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C74H142N4O14S2 [M+H] = 1376.0, observed = 1376.1.

[0431] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-6-oxo-6-(pentan-3-yloxy)hexyl)-amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E6Es5-DS-4-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.75(pent,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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0432] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.9.

[0433] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0434] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0435] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E9Es6-DS-4-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C76H146N4O14S2 [M+H] = 1404.1, observed = 1404.0.

[0436] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1278.0.

[0437] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1306.0.

[0438] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl3 )δ 4.19(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: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1292.0.

[0439] Diisopropyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C56H106N4O14S2 [M+H] = 1123.6, observed = 1123.7.

[0440] Bis(2-ethylbutyl)7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.9.

[0441] Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C58H110N4O14S2 [M+H] = 1151.6, observed = 1151.8.

[0442] Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.8.

[0443] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0444] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C69H132N4O14S2 [M+H] = 1305.9, observed = 1305.9.

[0445] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.9.

[0446] Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E7Ei3-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.9.

[0447] Diisopentyl 9,9'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate)) (GL-HEPES-E4-E7Es6-DS-3-E9Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0448] Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C58H110N4O14S2 [M+H] = 1151.6, observed = 1151.1.

[0449] Dibutyl 7,7'-((5-(2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7E4-DS-4-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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). APCI-MS analysis: calculated for C59H112N4O14S2 [M+H] = 1165.6, observed = 1165.2.

[0450] Dibutyl 7,7'-((4-((2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butanoyl)-oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.0.

[0451] Dibutyl 7,7'-((4-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C64H122N4O14S2 [M+H] = 1235.8, observed = 1235.0.

[0452] 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)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E7Ei3-DS-4-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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: calculated for C67H128N4O14S2 [M+H] = 1277.9, observed = 1277.0.

[0453] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E7Es6-DS-4-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1361.2.

[0454] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1319.0.

[0455] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1346.9.

[0456] Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)propyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-3-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.4.

[0457] Dibutyl 7,7'-((4-(2-(4-(2-((4-(bis(2-hydroxy-7-(isopentyloxy)-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-4-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.4.

[0458] Dibutyl 9,9'-((4-((2-(4-(2-((5-(bis(2-hydroxy-9-(isopentyloxy)-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1333.7.

[0459] Dibutyl 9,9'-((3-((2-(4-(2-((5-(bis(9-(2-ethylbutoxy)-2-hydroxy-9-oxononyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E9E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1362.0, observed = 1361.5.

[0460] Dibutyl 7,7'-((3-((2-(4-(2-((5-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7E4-DS-3-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.4.

[0461] Dibutyl 7,7'-((5-(2-(4-(2-((4-(bis(7-butoxy-2-hydroxy-7-oxoheptyl)amino)butyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7E4-DS-4-E7E4) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C61H116N4O14S2 [M+H] = 1193.7, observed = 1193.5.

[0462] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0463] Bis(2-ethylbutyl) 9,9'-((4-((2-(4-(2-((4-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-butanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)butyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E3-E7Ei3-DS-4-E9Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,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: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0464] Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: Calculated value C60H114N4O14S2[M+H]=1179.7, observed value=1179.8.

[0465] Diisopentyl 7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate)) (GL-HEPES-E4-E7Es6-DS-3-E7Ei5) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C66H126N4O14S2 [M+H] = 1263.8, observed = 1263.9.

[0466] Diisopentyl 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C70H134N4O14S2 [M+H] = 1319.9, observed = 1320.0.

[0467] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((3-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-propyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C72H138N4O14S2 [M+H] = 1348.0, observed = 1348.0.

[0468] Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((5-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)-pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Ei3-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,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). APCI-MS analysis: calculated for C62H118N4O14S2 [M+H] = 1207.7, observed = 1207.8.

[0469] Bis(2-ethylbutyl)7,7'-((3-((2-(4-(2-((5-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)-amino)pentanoyl)oxy)ethyl)piperazin-1-yl)ethyl)disulfanayl)propyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7Es6-DS-3-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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). APCI-MS analysis: calculated for C68H130N4O14S2 [M+H] = 1291.9, observed = 1291.9.

[0470] Diisopentyl 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Ei5-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.19(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: calculated for C71H136N4O14S2 [M+H] = 1334.0, observed = 1334.0.

[0471] Bis(2-ethylbutyl) 9,9'-((5-(2-(4-(2-((4-(bis(7-(2-ethylbutoxy)-2-hydroxy-7-oxoheptyl)amino)-butyl)disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(8-hydroxynonanoate) (GL-HEPES-E4-E9Es6-DS-4-E7Es6) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.19(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: calculated for C73H140N4O14S2 [M+H] = 1362.0, observed = 1362.0.

[0472] Dibutyl 7,7'-((4-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-4-oxobutyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E3-E7E4-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3)δ 4.99(hept,2H),4.19(t,2H),4.05(t,4H),3.63(m,4H),2.86-2.46(m,2 2H), 2.45-2.23(m, 16H), 1.88-1.30(m, 40H), 1.22(d, 12H), 0.92(t, 6H). APCI-MS analysis: calculated for C57H108N4O14S2 [M+H] = 1137.6, observed = 1137.7.

[0473] Dibutyl 7,7'-((5-(2-(4-(2-((3-(bis(2-hydroxy-7-isopropoxy-7-oxoheptyl)amino)propyl)-disulfanayl)ethyl)piperazin-1-yl)ethoxy)-5-oxopentyl)azanediyl)bis(6-hydroxyheptanoate) (GL-HEPES-E4-E7E4-DS-3-E7Ei3) [ka] 1 H NMR (300 MHz, CDCl 3 )δ 4.99(hept,2H),4.19(t,2H),4.05(t,4H),3.63(m,4H),2.86-2.46(m,2 2H),2.45-2.23(m,16H),1.88-1.30(m,42H),1.22(d,12H),0.92(t,6H). APCI-MS analysis: calculated for C58H110N4O14S2 [M+H] = 1151.6, observed = 1151.7.

[0474] The HEPBS-based cationic lipids described herein can also be prepared according to Scheme 3: 1. Scheme 3 [ka]

[0475] Intermediate [3]: [ka] To a solution of triphenylmethanethiol (5.0 g, 18.08 mmol) in EtOH (40 mL) and water (40 mL) was added a solution of NaOH (1.44 g, 36.16 mmol) in 40 mL of water. The reaction mixture was stirred for 10 min and a solution of 1,4-dibromobutane (3.65 g, 18.08 mmol) in EtOH (40 ml) 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 / Hexanes). The reaction mixture was diluted with dilute DCM and aqueous sodium bicarbonate and the organic layer was washed with brine. The organic layer was dried over sodium sulfate and concentrated under vacuum to give the crude compound. MeOH (15 mL) was added to the crude material and stirred at 0-10 °C for 15 min and the solid compound was filtered and dried under vacuum to give [3] (5.1 g, 69%) as a white solid.

[0476] result: 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),2.1 7(t,2H),1.82-1.77(m,2H),1.55-1.50(m,2H).LCMS: Purity 84.99% (low ionization).

[0477] Intermediate [5]: [ka] To a solution of [3] (5.0 g, 12.16 mmol) and [4] (3.16 g, 24.32 mmol) in ACN (75 mL) was added K2CO3 (6.72 g, 48.62 mmol). The reaction mixture was heated at 40 °C for 48 h. The progress of the reaction was monitored by TLC (2.5% MeOH in DCM). The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated in vacuo to give the crude product. The crude material was purified by flash chromatography (0-2.5% MeOH in DCM) to give [5] (2.6 g, 46%) as a white solid.

[0478] result: 1H NMR(400MHz,DMSO-d6):δ 7.41(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: Purity 98%. ESI-MS analysis: calculated for C29H37N2OS, [M+H] = 461.26, found = 461.29.

[0479] Intermediate [7]: [ka] 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), DIPEA (0.442 mL, 2.54 mmol) in DCM (8 mL) were added and stirred at room temperature for 14 h. After completion of the reaction monitored by MS. The reaction mixture was diluted with DCM and diluted with NaHCO 3 The organic layer was washed with water and brine. 2 SO 4 After drying and concentration, the crude compound was purified (eluent: 20% EtOAc in hexane) to give pure compound [7] as a colorless oil (0.77 g, 49%), which was confirmed by MS analysis.

[0480] result: ESI-MS analysis: Calculated value C 71 H 119 N 3 O 8 SSi 2、 [M+H]=1230.98, observed=1230.8.

[0481] Intermediate [8]: [ka] To a solution of [7] (0.77 g, 0.625 mmol) in DCM (3 mL) was slowly added TFA (3 mL) at room temperature and stirred for 0.5 h at room temperature. Triethylsilane (0.124 mL, 0.782 mmol) was slowly added thereto and stirred for 1 h. After completion of the reaction monitored by MS. The reaction mixture was concentrated to give the crude product [8] (quantitative), which was confirmed by MS analysis.

[0482] result: ESI-MS analysis: Calculated value C 52 H 105 N 3 O 8 SSi 2、 [M+H]=988.66, Observed=988.66.

[0483] Intermediate

[10] : [ka] To a solution of [8] (quantitative) in MeOH (4 mL), [9] (0.234 g, 1.06 mmol) was added at room temperature and stirred for 2 h. After completion of the reaction monitored by MS. The reaction mixture was concentrated and the crude compound was purified (eluent: 100% ethyl acetate, then 0-20% methanol in ethyl acetate) to obtain the pure product

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

[0484] result: ESI-MS analysis: Calculated value C 57 H 108 N 4 O 8 S 2 S 2、 [M+H]=1097.80; Observed=1097.8.

[0485] Intermediate

[12] : [ka] To a solution of

[10] (0.350 g, 0.319 mmol) and

[11] (0.322 g, 0.574 mmol) in chloroform, triethylamine (0.266 ml, 1.91 mmol) was added and reacted at room temperature for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated and carried to the next step without purification (0.800 g crude material).

[0486] ESI-MS analysis: Calculated value C82H 162 N 4 O 14 S 2 S 2、 [M+H]=1548.50; Observed=1548.8.

[0487] GL-HEPBS-E3(C6-Es-C1-3;5)-DS-4-(C6-Es-C1-3;5)

[13] : [ka] To a 20 mL polypropylene scintillation vial was added

[12] (crude material, 0.800 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 76.33 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was then cooled again to 0 °C, neutralized with solid sodium bicarbonate, diluted with ethyl acetate, and dissolved in NaHCO 3 The organic layer was washed with water and brine. 2 SO 4 The mixture was dried over 100 ml of water and concentrated. The crude product was purified to give compound

[13] (0.196 g, 46% over two steps). 1 Confirmed by 1 H NMR and MS analysis.

[0488] result: 1 H NMR (400 MHz, CDCl 3)4.19(t,2H),3.97(d,8H),3.64(br,4H),2.76-2.22(m,36H),1.86-1.74(m,2 H),1.73-1.56(m,15H),1.55-1.44(m,9H),1.43-1.26(m,28H),0.87(t,24H). ESI-MS analysis: Calculated value C 70 H 134 N 4 O 14 S 2、 [M+H]=1319.98; Observed=1319.8

[0489] The HEPBS-based cationic lipids described herein can also be prepared according to Scheme 4: Scheme 4 [ka]

[0490] Intermediate 5 was synthesized using the same procedure as in Scheme 3.

[0491] Intermediate [7]: [ka] 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), DIPEA (0.442 mL, 2.54 mmol) in DCM (8 mL) were added and stirred at room temperature for 14 h. After completion of the reaction monitored by MS. The reaction mixture was diluted with DCM and diluted with NaHCO 3 The organic layer was washed with water and brine. 2 SO 4 After drying and concentration, the crude compound was purified (eluent: 20% EtOAc in hexane) to give pure compound [7] as a colorless oil (0.77 g, 49%), which was confirmed by MS analysis.

[0492] result: ESI-MS analysis: Calculated value C71 H 119 N 3 O 8 SSi 2、 [M+H]=1230.98, observed=1230.8.

[0493] Intermediate [8]: [ka] To a solution of [7] (0.77 g, 0.625 mmol) in DCM (3 mL) was slowly added TFA (3 mL) at room temperature and stirred for 0.5 h at room temperature. Triethylsilane (0.124 mL, 0.782 mmol) was slowly added thereto and stirred for 1 h. After completion of the reaction monitored by MS. The reaction mixture was concentrated to give the crude product [8] (quantitative), which was confirmed by MS analysis.

[0494] result: ESI-MS analysis: Calculated value C 52 H 105 N 3 O 8 SSi 2、 [M+H]=988.66, Observed=988.66.

[0495] Intermediate

[10] : [ka] To a solution of [8] (quantitative) in MeOH (4 mL), [9] (0.234 g, 1.06 mmol) was added at room temperature and stirred for 2 h. After completion of the reaction monitored by MS. The reaction mixture was concentrated and the crude compound was purified (eluent: 100% ethyl acetate, then 0-20% methanol in ethyl acetate) to obtain the pure product

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

[0496] result: ESI-MS analysis: Calculated value C 57 H 108 N 4 O 8 S 2 S 2、[M+H]=1097.80; Observed=1097.8.

[0497] Intermediate

[12] : [ka] To a solution of

[10] (0.320 g, 0.291 mmol) and

[11] (0.287 g, 0.525 mmol) in chloroform, triethylamine (0.243 ml, 1.75 mmol) was added and reacted at room temperature for 2.5 hours. After the reaction was completed, the reaction mixture was concentrated and carried to the next step without purification (0.800 g crude material).

[0498] ESI-MS analysis: Calculated value C 81 H 160 N4O 14 S 2 S 2、 [M+H]=1534.48; Observed=1534.8.

[0499] GL-HEPBS-E3(C6-Es-C1-3;5)-DS-3-(C6-Es-C1-3;5)

[13] : [ka] To a 20 mL polypropylene scintillation vial was added

[12] (crude material, 0.800 g) along with 4 mL of dry tetrahydrofuran. The vial was cooled to 0-5 °C and HF / pyridine (2.0 mL, 77.03 mmol) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was then cooled again to 0 °C, neutralized with solid sodium bicarbonate, diluted with ethyl acetate, and diluted with NaHCO 3 The organic layer was washed with water and brine. 2 SO 4 The mixture was dried over 100 ml of water and concentrated. The crude product was purified to give compound

[13] (0.211 g, 55% over two steps). 1 Confirmed by 1 H NMR and MS analysis.

[0500] result: 1H NMR (400 MHz, CDCl 3 )4.19(t,2H),3.97(d,8H),3.64(br,4H),2.85-2.23(m,36H),1.89-1.74(m,4 H),1.73-1.55(m,12H),1.55-1.44(m,8H),1.43-1.28(m,30H),0.87(t,24H). ESI-MS analysis: Calculated value C 69 H 132 N 4 O 14 S 2、 [M+H]=1305.95; Observed=1305.8

[0501] Example 2 Lipid Nanoparticle Formulations The cationic lipids described herein can be used to prepare lipid nanoparticles by methods known in the art. For example, suitable methods include those described in WO 2018 / 089801, which is incorporated herein by reference in its entirety.

[0502] The lipid nanoparticles in the examples of the present invention were formulated using Process A of WO 2018 / 089801 (see, for example, Example 1 and Figure 1 of WO 2018 / 089801). Process A ("A") refers to a conventional method of encapsulating mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles. In the exemplary process, an ethanol solution of a mixture of lipids (cationic lipids, phosphatidylethanolamine, cholesterol and polyethylene glycol-lipids) at a fixed lipid-to-mRNA ratio was combined with an aqueous buffer solution of target mRNA at acidic pH under controlled conditions to obtain a homogenous suspension of LNPs. After ultrafiltration and diafiltration into a suitable dilution system, the resulting nanoparticle suspension was diluted to a final concentration, filtered and stored frozen at -80°C until use.

[0503] The lipid nanoparticle formulations in Table 3 were prepared by Process A. All of the lipid nanoparticle formulations were composed of hEPO mRNA and different lipids (cationic lipid: DMG-PEG2000: cholesterol: DOPE / DSPC) in the molar % ratios specified in Table 3.

[0504] [Table 22]

[0505] The cationic lipids of the present invention were evaluated in lipid nanoparticle formulation 1. MC3 was evaluated in lipid nanoparticle formulation 2, which is an exemplary MC3 formulation.

[0506] Example 3 Delivery of hEPO mRNA by Intramuscular Administration Mouse studies In summary, lipid screening studies were performed in 6-8 week old female BALB / cJ mice. Mice were dosed with 0.1 μg in 30 μL of LNP by a single intramuscular (IM) injection into the gastrocnemius leg muscle. Blood samples were taken 6 and 24 hours after injection and hEPO levels were measured in mouse serum using an ELISA assay according to the manufacture's protocol. WO 2022 / 099003A1 also describes an in vivo assay for intramuscular administration (e.g., page 46, paragraph

[0206] ).

[0507] Further details of the intramuscular experiments carried out in this application are provided below.

[0508] [Table 23]

[0509] Test materials and treatment regimens Test materials were kept RNase-free during loading into the syringes (where applicable).

[0510] Compound test class: Oligonucleotides ABSL-1 Treatment regimen: On day 1, animals in groups 1-13 were administered intramuscular injection under light 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 MC3 control. The cationic lipid MC3 is the current gold standard for in vivo delivery of, for example, siRNA (see WO 2010 / 144740).

[0511] Test animals animal:

[0512] [Table 24]

[0513] Acclimatization: Animals were allowed to acclimate to the testing facility for at least 24 hours.

[0514] Housing: All animals were socially housed in polycarbonate cages with contact bedding in the animal room.

[0515] Food and water: Food (Envigo irradiated 2918 diet) and filtered tap water were provided ad libitum to the animals.

[0516] In-life observations and measurements Animal Health Checks: Animals underwent cage-side health check observations at least once daily.

[0517] Clinical Observations: Clinical observations were performed on all animals on Day 1 prior to dose administration and prior to euthanasia. Clinical observations were performed more frequently if any abnormal clinical signs were displayed by the animals on study.

[0518] Body weight: Body weight was recorded before administration of test material and was rounded to the nearest 0.1 g.

[0519] Interim Sample Collection: Interim whole blood (approximately 50 μL) was collected by tail snip or saphenous vein at 6 and 24 hours (±5%) post dose administration. Blood samples were collected into serum separator tubes, allowed to clot for at least 10 minutes at room temperature, and serum was extracted by centrifugation at a minimum of 1000 g for 10 minutes at ambient temperature. All serum samples were stored at nominal -70°C until analysis hEPO by the testing laboratory. Results of EPO analysis were included in the data submission.

[0520] [Table 25]

[0521] Final Steps Euthanasia: On day 2, 24 h after dosing, all animals were euthanized by CO 2 Euthanasia was performed by asphyxiation followed by thoracotomy and terminal blood collection.

[0522] Terminal blood draw: Whole blood was collected by cardiac puncture into serum separator tubes, allowed to clot for at least 10 minutes at room temperature, and serum extracted by centrifugation at a minimum of 1000 g for 10 minutes at ambient temperature. Serum samples were stored at nominal -70°C until analyzed for hEPO by the testing laboratory.

[0523] [Table 26]

[0524] In vitro assay: ELISA assay: Human erythropoietin (hEPO) levels in serum samples were determined by an ELISA kit (R&D systems, catalogue no. DEP-00) according to the manufacture's instructions and the results were included in the data submission. The "shaker" protocol was used. Serum samples were diluted 1:40 to 1:100.

[0525] Reporting and Data Retention Data Submission: Tabular data summaries of animal allocation, individual and group means (where applicable) of dose administration and euthanasia times, body weights, in vitro analyses of clinical observations, and mortality (where applicable) were provided for this study.

[0526] [Table 27]

[0527] Example 4: Laurdan assay for determining generalized polarization (GP) values Using a Laudan probe, the lipid packing of lipid nanoparticles containing second generation cationic lipids derived from the "Good" buffer of the present invention was compared to lipid nanoparticles containing other cationic lipids derived from "Good" buffer.

[0528] The formulations were diluted in buffer solutions of pH 4.5, 5.5, 6.5 or 7.5 and laurdan molecules were added to give a final laurdan concentration of 1 μm. The solutions were incubated at room temperature for 3 hours and protected from light. GP values ​​were calculated based on the fluorescence values ​​to give an idea of ​​the formulation lipid membrane packing. Samples were analyzed using a Spectra Max M5 Multi-Mode microplate reader. A fluorescence excitation wavelength of 340 nm was used with emission wavelengths of 440 and 490 nm. GP values ​​were calculated using the following formula: GP = (AUC 440 -AUC 490 ) / (AUC 440 +AUC 490 ).

[0529] Further details of the Laurdan assay 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 (both incorporated herein by reference).

[0530] The Laudan probe inserts itself uniformly into the hydrophilic / hydrophobic interface of lipid bilayers and is used to measure polarity changes in the bilayer environment that may be related to lipid membrane packing and order. Generalized polarization (GP) values ​​were calculated from the shift in fluorescence intensity from 440 nm to 490 nm when the Laudan probe interacts with water molecules in the lipid membrane. Lower GP values ​​are associated with hydrated and fluid membranes, while higher GP values ​​typically imply fewer water molecules and more ordered lipid packing. GP values ​​of lipid nanoparticles (LNPs) were measured in pH 7.5, 6.5, 5.5, and 4.5 buffers to simulate the endosomal pH shift that occurs when particles are taken up into cells. It is believed that lower pH levels (4.5 and 5.5) may result in lower GP values ​​for all formulations tested compared to pH 6.5 and 7.5. This suggests that the lipid nanoparticles (LNPs) become more fluid and less ordered as the pH environment decreases. It is believed that the lipid nanoparticles containing the second generation cationic lipids derived from "Good" buffer have an overall higher GP value compared to the lipid nanoparticles containing other cationic lipids derived from "Good" buffer. It is believed that the additional ester and / or carbon branching in the lipid tail of the second generation cationic lipids derived from "Good" buffer results in a more densely packed membrane compared to other cationic lipids derived from "Good" buffer. It is believed that a positive trend is observed between the amount of hEPO produced in mice at pH 6.5 and the GP value for the lipid nanoparticles containing the second generation cationic lipids derived from "Good" buffer. One hypothesis of the possible correlation between GP value and protein production is that particles with tighter bilayer packing may perform better in vivo by increasing lipid nanoparticle (LNP) stability under physiological pH conditions.

[0531] In summary, the lipid nanoparticles containing the second generation cationic lipids derived from the "Good" buffer of the present invention are believed to have higher overall generalized polarization (GP) values ​​compared to other cationic lipids derived from "Good" buffer.There is a positive linear correlation between the Laudan GP value and the amount of EPO produced in 6 hours in mice.The increase in GP value is believed to correlate with the increase in EPO protein in pH 6.5 solution.

[0532] Example 5: In vitro degradation test In vitro lipolysis by mouse / human lung S9. Assay format - Includes 4 or 5 time points in triplicate. I. Assay Procedure: 1) Plan the experiment, compounds, and reagents. 2) Each lipid is dissolved in DMSO or IPA to make a 5 mM stock, then diluted to a 200 μM working solution with IPA. 3) Thaw mouse and human lung S9. 4) Prepare a pooled incubation mixture on ice with the following reaction recipe: 5) Dispense 495 μL of the incubation mixture prepared in step 4 into each well of a 2 mL 96-well plate. 6) Add 5 μL of compound to each well to start the reaction. Take t0 sample (as in step 8). 7) Cover the plate with two layers of breathable seal and incubate the plate in CO at 37°C. 2 Incubate in an incubator on an orbital shaker at 150 rpm. 8) At each time point, mix the incubation mixture 5 times with a pipette, then transfer 70 μL of the incubation mixture to a new plate. Immediately store in a -20°C freezer. 9) Add 210 μL (3x volume) of ice-cold stop solution to each well of the collected sample plate. Mix on an orbital shaker at 600 rpm for 15 minutes. 10) Centrifuge the quenched plate at 3800 rpm for 10 minutes at 4° C. and transfer the supernatant to a new plate. 11) Load the supernatant onto the filtration plate and centrifuge again at 3800 rpm for 5 minutes at 4° C. Collect the final sample onto a new plate for LC / MS. II. Time Course and Stop Solution: 4-5 time points (hours): e.g., 0, 4, 8, 24, 48 hours Stop solution: 1:1:1 ACN / MeOH / IPA (v / v / v), containing propranolol and MC3 as internal standards. Store at 4°C. III. Reactant components and recipe: Mouse / human lung S9

[0533] [Table 28]

[0534] Example 6: RiboGreen Assay The encapsulation efficiency of mRNA in lipid nanoparticles can be determined using the Invitrgen RiboGreen assay kit. Unencapsulated mRNA was directly detected. Total mRNA was measured after dissolving lipid nanoparticles in the presence of Triton X-100. Encapsulation efficiency was calculated as (total mRNA-unencapsulated mRNA) / total mRNA x 100%. The RiboGreen assay is a fluorescence-based method for determining mRNA concentration (total and free) and % encapsulation using Quant-iT™ RiboGreen® RNA reagent in mRNA containing lipid nanoparticles.

[0535] Materials / Reagents Triton-X, 98%, for molecular biology, DNAse, RNase and protease free, Acros Organics, Cat. AC327371000 Ultra-pure DNase / RNase-free distilled water, Life Technologies, catalog 10977-023 RNaseZap® RNase Decontamination Solution Life Technologies, Catalog AM9784 Quant-iT™ RiboGreen® RNA Reagent Life Technologies, Catalog R11491 or Quant-iT™ RiboGreen® RNA Assay Kit Life Technologies, Catalog R11490 RNase-free 20X TE buffer Life Technologies, catalog T11493 RNaseZap® RNase Decontamination Solution LifeTechnologies, Catalog AM9784

[0536] device Molecular Devices Gemini EM Microplate Reader RNase-free microcentrifuge tube (2.0 mL) RNase-free Flacon tubes (15 and 50 mL) Vortex mixer Corning® 96-well specialty optical microplates with clear background (Cat. No. 3615).

[0537] Preparation of mRNA standards

[0538] [Table 29]

[0539] [Table 30]

[0540] Sample preparation

[0541] [Table 31]

[0542] [Table 32]

[0543] 200x RiboGreen dye preparation

[0544] [Table 33]

[0545] procedure To each of the standards (blank, mRNA-1, mRNA-2, mRNA-3, mRNA-4, mRNA-5) and samples (free and total mRNA), add 1.0 mL of 200x Ribogreen reagent solution and mix gently by inversion. This is a 2x dilution. Using the reverse pipetting technique, add 200 μL of each standard and sample in triplicate to a 96-well Costar Black plate with a clear background. Ensure there are no air bubbles in the plate before the fluorescence reading. Read the fluorescent signal using the following instrument parameters: Reading type: Fluorescence, bottom read Excitation: 485nm; Cutoff: 515nm; Emission: 530nm Plate type: 96-well Costar Black with clear background

[0546] Data analysis Plot the mean fluorescence from each calibration standard versus concentration to generate a linear calibration curve using MS Excel software. Calculate the coefficient of determination (R 2 ) is R 2 Must be >0.99.

[0547] The linear equations generated can be interpreted as follows: y=mx+c During the ceremony, Y = Mean fluorescence value m: Tilt x: Concentration (μg / mL) c:y intercept · Using the linear equation, calculate the free and total mRNA concentrations in the test samples by replacing the y values ​​in the equation with the respective mean fluorescence values ​​of each sample. Once the concentration has been determined, the actual concentration in the sample can be back-calculated by multiplying the concentration in the test sample by the dilution factor (DF) as follows: Free mRNA concentration = Free mRNA concentration in test sample x 800 (DF) Total mRNA concentration = total mRNA concentration in test sample x 4000 (DF) The concentration of encapsulated mRNA can be determined by subtracting the concentration of free mRNA from the total mRNA. The encapsulation percentage can then be calculated by taking the ratio of encapsulated mRNA to total mRNA and multiplying the result by 100.

[0548] Example 7 Delivery of human erythropoietin (hEPO) mRNA by intramuscular (IM) administration Lipid nanoparticle (LNP) formulations encapsulating hEPO mRNA were prepared by process A as described above for IM administration. The administered LNP composition contained 1.5% PEG, 40% cationic lipid, 28.5% cholesterol, and 30% DOPE (N / P ratio 4). After LNP formulation, the nanoparticles were first buffer exchanged with 20% EtOH, then a final buffer exchange with 10% trehalose. The LNPs were characterized for size, PDI, encapsulation, and mRNA concentration. For hEPO animal administration studies, the LNPs were diluted to 3.33 g / mL in 10% trehalose. Mice were administered intramuscularly into the right gastric muscle at 0.1 g in a volume of 30 μL. Blood samples were taken 6 and 24 hours after injection to measure the amount of hEPO protein produced in the serum. EPO protein amount was detected using an ELISA assay from a commercially available kit. FIG. 1 shows that lipid nanoparticles comprising lipids described herein are highly effective in delivering hEPO mRNA, demonstrating high levels of hEPO protein expression 6 hours after an IM injection dose.

[0549] The polydispersity index (PDI) of the lipid nanoparticles can be determined by diluting the formulation in 10% trehalose at an mRNA concentration of approximately 0.1 mg / ml and then measuring the size with a Malvern Zetasizer.

[0550] The lipid nanoparticle size can be obtained using a Malvern Zetasizer Nano-ZS.

[0551] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope thereof.

[0552] All references, patents, or applications (U.S. or foreign) cited in this application are hereby incorporated by reference as if written in their entirety herein. In the event of any conflict, the material literally disclosed herein will control.

[0553] Numbered embodiments 1. A compound having a structure according to formula (I): [ka] or a pharma- ceutically acceptable salt thereof, A 1 teeth, [ka] and -SS-, and the left side of each structure shown is selected from -(CH 2 ) bound to a-; Z 1 teeth, [ka] and -SS-, and the right side of each structure shown is selected from -(CH 2 ) bound to 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; Each R 1A , R 1B , R 1C and R 1D is optionally substituted (C 3 ~C 6 ) alkyl.

[0554] 2. The compound of numbered embodiment 1 having a structure according to formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each c and d is independently selected from 3, 4, or 6.

[0555] 3. The compound of numbered embodiment 1 having a structure according to formula (Ib): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each e and f is independently selected from 3, 4, or 6.

[0556] 4. The compound of numbered embodiment 1 having a structure according to formula (Ic): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each c and d is independently selected from 3, 4, or 6.

[0557] 5. The compound of numbered embodiment 1 having a structure according to formula (Id): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each e and f is independently selected from 3, 4, or 6.

[0558] 6. The compound of numbered embodiment 1 having a structure according to formula (Ie): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each c and d is independently selected from 3, 4, or 6.

[0559] 7. The compound of numbered embodiment 1 having a structure according to formula (If): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each e and f is independently selected from 3, 4, or 6.

[0560] 8. The compound of numbered embodiment 1 having a structure according to formula (Ig): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH2 ) a- and Z 1 is -SS-, and each c and d is independently selected from 3, 4, or 6.

[0561] 9. The compound of numbered embodiment 1 having a structure according to formula (Ih): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (d) b is 2; (e) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-; or (f) b is 2 and A 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-, and each e and f is independently selected from 3, 4, or 6.

[0562] 10. The compound of numbered embodiment 1 having a structure according to formula (Ii): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0563] 11. The compound of numbered embodiment 1 having a structure according to formula (Ij): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0564] 12. The compound of numbered embodiment 1 having a structure according to formula (Ik): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0565] 13. The compound of numbered embodiment 1 having a structure according to formula (Im): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0566] 14. The compound of numbered embodiment 1 having a structure according to formula (In): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0567] 15. The compound of numbered embodiment 1 having a structure according to formula (Io): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0568] 16. The compound of numbered embodiment 1 having a structure according to formula (Ip): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0569] 17. The compound of numbered embodiment 1 having a structure according to formula (Iq): [ka] or a pharma- ceutically acceptable salt thereof (optionally: (c) b is 2; or (d) b is 2 and A is 1 teeth [ka] The left side of the illustrated structure is -(CH 2 ) a- and Z 1 is -SS-).

[0570] 18.A 1 and Z 1 or a pharma- ceutically acceptable salt thereof.

[0571] 193.A 1 and Z 1 The compound of any one of numbered embodiments 1-17, or a pharma- ceutically acceptable salt thereof, wherein:

[0572] 20.A 1 but [ka] The left side of the illustrated structure is -(CH 2 20. The compound of any one of numbered embodiments 1-19, or a pharma- ceutically acceptable salt thereof, which is bound to a-.

[0573] 21.A 1 but [ka] The left side of the illustrated structure is -(CH 2 20. The compound of any one of numbered embodiments 1-19, or a pharma- ceutically acceptable salt thereof, which is bound to a-.

[0574] 22.A 1 or a pharma- ceutically acceptable salt thereof.

[0575] 23.Z 1 but [ka] and on the right side of the illustrated structure is -(CH 2 ) a-, or a pharma- ceutically acceptable salt thereof.

[0576] 24.Z 1 but [ka] and on the right side of the illustrated structure is -(CH 2 ) a-, or a pharma- ceutically acceptable salt thereof.

[0577] 25.Z 1 The compound of any one of numbered embodiments 1-22, or a pharma- ceutically acceptable salt thereof, wherein:

[0578] 26. The compound of any one of numbered embodiments 1-25, wherein b is 2; or a pharma- ceutically acceptable salt thereof.

[0579] 27. The compound of any one of numbered embodiments 1-25, wherein b is 3; or a pharma- ceutically acceptable salt thereof.

[0580] 28. The compound of any one of numbered embodiments 1-25, wherein b is 4; or a pharma- ceutically acceptable salt thereof.

[0581] 29. The compound of numbered embodiment 1 having a structure according to formula (Ir): [ka] or a pharma- ceutically acceptable salt thereof (optionally, each of c, d, e and f is independently selected from 3, 4, or 6).

[0582] 30. The compound of any one of numbered embodiments 1-29, wherein each a is 3; or a pharma- ceutically acceptable salt thereof.

[0583] 31. The compound of any one of numbered embodiments 1-29, wherein each a is 4; or a pharma- ceutically acceptable salt thereof.

[0584] 32. The compound of any one of numbered embodiments 1-29, wherein the value of a on the left side of the depicted formula is 3 and the value of a on the right side of the depicted formula is 4; or a pharma- ceutically acceptable salt thereof.

[0585] 33. The compound of any one of numbered embodiments 1-29, wherein the value of a on the left side of the depicted formula is 4 and the value of a on the right side of the depicted formula is 3; or a pharma- ceutically acceptable salt thereof.

[0586] 34. The compound of any one of numbered embodiments 1 or 18-33, wherein in the compound of formula (I) or (Ir), c, d, e and f are the same; or a pharma- ceutically acceptable salt thereof.

[0587] 35. The compound of any one of numbered embodiments 1 or 18-34, wherein in the compound of formula (I) or (Ir), c, d, e and f are 3; or a pharma- ceutically acceptable salt thereof.

[0588] 36. The compound of any one of numbered embodiments 1 or 18-34, wherein in the compound of formula (I) or (Ir), c, d, e and f are 4; or a pharma- ceutically acceptable salt thereof.

[0589] 37. The compound of any one of numbered embodiments 1 or 18-34, wherein in the compound ...

Claims

1. Equation (I): 【Chemistry 1】 A compound having the structure of , or a pharmaceutically acceptable salt thereof (wherein formula: A 1 teeth, 【Chemistry 2】 And selected from -S-S-, the left side of each illustrated structure is -(CH 2 ) to be joined to a-; Z 1 teeth, 【Transformation 3】 And selected from -S-S-, the right side of each illustrated structure is -(CH 2 ) to be joined to 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; Each R 1A , R 1B , R 1C and R 1D is independently selected from optionally substituted (C 3 ~C 6 ) alkyl).

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

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

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein each a is 3.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein each a is 4.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the value of a on the left side of the illustrated formula is 3, and the value of a on the right side of the illustrated formula is 4.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the value of a on the left side of the illustrated formula is 4, and the value of a on the right side of the illustrated formula is 3.

8. Each R 1A , R 1B , R 1C and R 1D However, the compound according to claim 1 or a pharmaceutically acceptable salt thereof, independently selected from the following: 【Transformation 5】 。

9. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein each a is 3.

10. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein each a is 4.

11. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the value of a on the left side of the illustrated formula is 3, and the value of a on the right side of the illustrated formula is 4.

12. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the value of a on the left side of the illustrated formula is 4, and the value of a on the right side of the illustrated formula is 3.

13. Each R 1A , R 1B , R 1C and R 1D However, the compound according to claim 3 or a pharmaceutically acceptable salt thereof, independently selected from the following: 【Transformation 6】 。

14. A composition comprising a cationic lipid according to any one of claims 1 to 13, and further comprising the following: (i) One or more noncationic lipids, (ii) One or more cholesterol-based lipids, and (iii) One or more PEG-modified lipids.

15. The composition according to claim 14, wherein the composition is lipid nanoparticles, and optionally liposomes.

16. The composition according to claim 15, wherein the lipid nanoparticles encapsulate mRNA encoding nucleic acids, optionally peptides or proteins.

17. The composition according to claim 15, wherein the lipid nanoparticles optionally contain mRNA encoding a peptide or protein for use in a vaccine.

18. The composition according to claim 17 for use in therapeutic purposes.

19. A composition according to claim 17 for use in a method for treating or preventing a disease suitable for treatment or prevention by the peptide or protein encoded by the mRNA, wherein the mRNA optionally encodes an antigen and / or the disease is (a) a protein deficiency (which optionally affects the liver, lungs, brain or muscles), (b) an autoimmune disease, (c) an infection, or (d) cancer.

20. The composition for use according to claim 18, administered intravenously, intrathecally, or intramuscularly, or optionally by pulmonary delivery via spray.

21. The composition for use according to claim 19, administered intravenously, intrathecally, or intramuscularly, or optionally by pulmonary delivery via spray.

Images