Novel dipepetide core lipids, lipid carriers, and methods of using thereof

Abstract

The disclosure relates to novel lipids having a dipeptide core, lipid-based carriers containing such dipeptide-core containing lipids that can be used in the delivery of therapeutic agents, compositions, and methods thereof.

Description

[0001] NOVEL DIPEPETIDE CORE LIPIDS, LIPID CARRIERS,

[0002] AND METHODS OF USING THEREOF

[0003] FIELD OF THE INVENTION

[0004] The present disclosure generally relates to novel lipids, lipid-based carriers, pharmaceutical compositions, and methods of using thereof.

[0005] BACKGROUND

[0006] Lipid carriers such as lipid nanoparticles formed from ionizable amine-containing lipids can serve as therapeutic cargo vehicles for delivery of biologically active agents, such as coding RNAs (i.e., messenger RNAs (mRNAs), guide RNAs, circular RNA (circRNA)) and non-coding RNAs (i.e. antisense, siRNA), into cells.

[0007] Many biologically active agents including relatively large oligonucleotides, encoding protein-based drugs or nucleic acid-based drugs, are particularly difficult to be delivered into cells.

[0008] There thus continues to be a need in the art for novel lipid compounds for delivering therapeutic agents, such as nucleic acids, proteins, and small molecule drugs.

[0009] SUMMARY OF THE INVENTION

[0010] Disclosed herein are novel ionizable lipids that can be used in combination with at least one other lipid component, such as a sterol, a polymer conjugated lipid, and / or a neutral lipid, to form a lipid-based carrier, which may be used to facilitate the intracellular delivery of a therapeutic agent, such as a nucleic acid molecule, in vitro and / or in vivo. The novel ionizable lipids contain a core mimicking a peptide backbone structure (e.g., a dipeptide core), useful for formation of lipid-based carriers that may have properties advantageous for delivery of nucleic acid cargos, such as delivery of coding and non-coding RNAs, into cells.

[0011] One aspect of the invention relates to a compound of formula (IA- 1 ) or (IA-2):

[0012]

[0013] ^*aa? - *aa2* — W

[0014] D _ V _ T

[0015] (IA-2), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: aal and aa2 each are independently a natural or unnatural amino acid residue, wherein * represent the bonding to aal or aa 2 via its backbone N atom and • represents the bonding to aal or aa2 via its backbone carbonyl C atom;

[0016] each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;

[0017] each R, for each occurrence, is independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl;

[0018] each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;

[0019] j is 1, 2, or 3;

[0020] W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted with one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;

[0021] each A is independently absent, C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; and

[0022] each X is independently absent, X’,

[0023]

[0024] each R’ is independently Ci-Cs alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and

[0025] each X’ is independently a biodegradable moiety.

[0026] In some embodiments, in formula (IA-1) or (IA-2), A and X are not absent in at least one

[0027] or two of the

[0028]

[0029] tails.

[0030] In some embodiments, aal and aa2 each may be independently an a-amino acid, -amino acid, or y-amino acid. In some embodiments, aal and aa2 each may be independently a natural amino acid in D-stereoisomer or L-stereoisomer. In some embodiments, aal and aa2 each may be independently a non-canonical amino acid. In some embodiments, one of aal and aa2 is selected from the group consisting of Gly, Cys, Pro, and its variants thereof. In some embodiments, one of aal and aa2 has a hydrophobic side chain, and is selected from the group consisting of Ala, Vai, He, Leu, Met, Phe, Tyr, Trp, and its variants thereof. In some embodiments, one of aal and aa2 has a polar, uncharged side chain, and is selected from the group consisting of Ser, Thr, Asn, Gin, and its variants thereof. In some embodiments, one of aal and aa2 has an electrically charged side chain, and is selected from the group consisting of Arg, His, Lys, Asp, Glu, and its variants thereof.

[0031] In some embodiments, aal-aa2 is Gly-Gly, -Ala-Gly, Gly- -Ala,

[0032] His, Ala-Ala, Ala-Gly, Gly-Leu, Leu-Gly, Gly-Ile, Gly-Ser, Gly-SerHead

[0033]

[0034] Ser-Gly, Ser- -Ala, -Ala-Ser,

[0035] Glu, P-Ala-Glu, Gly-GluHead

[0036]

[0037] Gly-Asn, P-Ala-P-Ala, y-aminobutyric acid-Gly, or variants thereof.

[0038] In some embodiments, a variant of any one of the amino acid residue in aal and / or aa2 is a squaramide derivative of the amino acid.

[0039] Another aspect of the invention relates to compound of formula (IB-1) or (IB-2):

[0040]

[0041] a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein:

[0042] each of il and i2 is independently 0, 1, 2, or 3;

[0043] j is 1, 2, or 3; each of Rai, Ra2, Rbi, and Rb2 is, for each occurrence, independently H, Ci-Ce branched or unbranched alkyl, -(CRJR2)vR3, -(CR1R2)vSR4, -(CR1R2)vSeR4, -(CR1R2)vOR4, -(CR1R2)VCOR5, -(CR1R2)VC(O)OR4, -(CR1R2)VNR1R2, -(CR1R2)VN(R1)(CR1R2)VNR1R2, -(CR1R2)VC(O)NR1R2, -(CR1R2)VN(R1)C(O)NR1R2, -(CR1R2)VN(R1)C(O)R5, -(CR1R2)VC(S)NR1R2, -(CR1R2)VN(R1)C(S)NR1R2, or -(CR1R2)VN(R1)C(=NR1)NR1R2; or one Ra2or Rb2 adjacent to the N atom is taken together with its adjacent N atom and R variable to form a heterocyclic ring, optionally substituted with one or more alkyl, OR4, SR4, and / or halogen groups;

[0044] each R1is, for each occurrence, independently H, OH, or C1-C3 alkyl;

[0045] each R2is, for each occurrence, independently H or C1-C3 alkyl;

[0046] each R3is independently an aryl or heteroaryl, optionally substituted with one or more alkyl, C(O)OR4, OR4, SR4, oxo, and / or halogen groups;

[0047] each R4is independently H, C1-C3 alkyl, aryl,

[0048]

[0049] -(CR1R2)VC(O)(CR1R2)VNR1R2;

[0050] each R5is independently H, C1-C3 alkyl, -NR^CR^jvNR^2, or -(CR1R2)VC(O)(CR1R2)VNR1R2;

[0051] each v is independently an integer of 0-4;

[0052] each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;

[0053] each R is, for each occurrence, independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl;

[0054] each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;

[0055] W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;

[0056] each A is absent, independently C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; and

[0057] each X is independently absent, X’,

[0058]

[0059] each R’ is, for each occurrence, independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and

[0060] each X’ is independently a biodegradable moiety.

[0061] In some embodiments, in formula (IB-1) or (IB-2), A and X are not absent in at least one

[0062] or two of the

[0063]

[0064] tails.

[0065] In some embodiments, when il is not 0, Rai in both occurrences are H, and when i2 is not 0, both Ra2 in both occurrences are H. In some embodiments, il is 0, i2 is 0, or both il and i2 are 0. In some embodiments, il is 1, i2 is 1, or both il and i2 are 1. In some embodiments, il is 2, i2 is 2, or both il and i2 are 2.

[0066] In some embodiments, the core formula

[0067]

[0068] has the structure of:

[0069]

[0070]

[0071] Rqis H or OR4. In some embodiments, R2herein is H or CH3.

[0072] In some embodiments, j is 1 or 2.

[0073] In some embodiments, in any of the above formulas, each V is, for each occurrence, C2- ene.

[0074] In some embodiments, the compound has the structure of

[0075]

[0076] -,

[0077]

[0078] wherein each m is, for each occurrence, 2 or 3.

[0079] In some embodiments, the compound has the structure of

[0080]

[0081] -,

[0082]

[0083] (IIIA-12),

[0084]

[0085] (-),

[0086]

[0087] -,

[0088]

[0089] (-), wherein:

[0090] each m is, for each occurrence, 2 or 3;

[0091] Rpis H, C(O)OH, or OH;

[0092] RNis H, C(O)R5, or C1-C3 alkyl;

[0093]

[0094] R2and R5are each H or C1-C3 alkyl; and

[0095] v is 1-4.

[0096] In some embodiments, in any of the above formulas, each Y is, for each occurrence, -O-,

[0097]

[0098] In some embodiments, in any of the above formulas, at least two, at least three, or at least

[0099] four of the

[0100]

[0101] tails are the same.

[0102] In some embodiments, in any of the above formulas, at least one, at least two, at least A - X - B

[0103] three, or at least four of the

[0104]

[0105] tails are different.

[0106] A - X - B

[0107] In some embodiments, at least one or two of the

[0108]

[0109] tails has both A and B absent and has B being H or C1-C24 branched or unbranched alkyl.

[0110] In some embodiments, at least one or two of the

[0111]

[0112] tails has both A and B absent and has B being H or C1-C3 branched or unbranched alkyl.

[0113] In some embodiments, in any of the above formulas, in the

[0114]

[0115] in which A and X are not absent, each X’ is independently -OC(O)-, -C(O)O-, -OC(O)O-, -N(R10)C(O)-, -C(O)N(R10)-, -Y-V-OC(O), -Y-V-C(O)O-, -Y-V-N(R10)C(O)-, -Y-V-C(O)N(R10)-, -OC(O)-(C(R11)2)s-S-, -OC(O)-(C(R11)2)s-S-S-, -C(O)O-(C(R11)2)s-S-, -C(O)O-(C(R11)2)s-S-S-, -C(O)N(R10)-(C(R11)2)s-S-, -C(O)N(R10)-(C(R11)2)s-S-S-, -N(R10)C(O)-(C(R11)2)s-S-, -N(R10)C(O)-(C(R11)2)s-S-S-, or -S-S-; each R10is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each R11is independently H or C1-C3 alkyl; and each s is independently 1, 2, 3, 4, or 5.

[0116] In some embodiments, in any of the above formulas, each X’ is independently -OC(O)-,

[0117]

[0118] -N(R10)C(O)-(CHR11)s-S-, -N(R10)C(O)-(CH2)s-S-S-, or -S-S-; wherein R10and R11are each independently H or CH3.

[0119] In some embodiments, in any of the above formulas, each X is independently X’,

[0120]

[0121] A - X - B In some embodiments, in any of the above formulas, in the

[0122]

[0123] tail in which A and X are not absent, each A is independently C1-C9 branched or unbranched alkylene.

[0124] In some embodiments, in the

[0125]

[0126] tail where A is not absent, each A is independently C2-C7 alkylene.

[0127] In some embodiments, in any of the above formulas, each B is independently, for each occurrence, H or has the structure of:

[0128]

[0129]

[0130] each nl is independently an integer from 1 to 5;

[0131] each t is independently an integer from 0 to 5;

[0132] each u is independently an integer from 0 to 16,

[0133] each of ul, u2, u3, and u4, is independently, for each occurrence, an integer from 0 to 10; each of R21, R22, R23, and R24is independently, for each occurrence, H or C1-C3 alkyl; each R25is independently H, C1-C3 alkyl, or a saturated or unsaturated cyclic, optionally substituted with one or more alkyl groups.

[0134] In some embodiments, in any of the above formulas, each B is independently for each occurrence H or selected from the group consisting of:

[0135]

[0136]

[0137] wherein u is an integer from 7 to 16.

[0138] In some embodiments, the compound has the formula (IA-2) or (IB-2), wherein W is H, OR6, or NHR6, wherein R6is H or C1-C12 alkyl.

[0139] In some embodiments, the compound has the structure of formula (IIIA-1), and is one of the compounds listed in Table 1. In some embodiments, the compound has the structure of formula (IIIA-2), and is one of the compounds listed in Table 2. In some embodiments, the compound has the structure of formula (IIIA-3), and is one of the compounds listed in Table 3. In some embodiments, the compound has the structure of formula (IIIA-4), and is one of the compounds listed in Table 4. In some embodiments, the compound is one of the compounds listed in Table 5. In some embodiments, the compound is one of the compounds listed in Table 6. In some embodiments, the compound is one of the compounds listed in Table 7. In some embodiments, the compound is one of the compounds listed in Table 8.

[0140] Another aspect of the invention relates to a lipid-based carrier comprising a compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or formula (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein. In some embodiments, the lipid-based carrier is a lipid nanoparticle. In some embodiments, the lipid-based carrier further comprises a second lipid. In some embodiments, the second lipid is cationic, anionic, ionizable, or zwitterionic lipid. In some embodiments, the lipid-based carrier further comprises a sterol, a PEGylated lipid, a phospholipid, and / or a neutral lipid.

[0141] Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or formula (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable excipi ent. Certain aspect of the invention also relates to a pharmaceutical composition comprising the lipid-based carriers as described herein, and a pharmaceutically acceptable excipient.

[0142] The pharmaceutical composition may further comprise a therapeutic agent. In some embodiments, the therapeutic agent is a nucleic acid component. In some embodiments, the nucleic acid component is an RNA or DNA. In some embodiments, the DNA or RNA is linear, circular, single stranded, or double stranded. In some embodiments, the RNA is a mRNA or a circular RNA.

[0143] In some embodiments, the therapeutic agent is a protein or small molecule drug. In some embodiments, the pharmaceutical composition is a vaccine.

[0144] Another aspect of the invention relates to a method of delivering a therapeutic agent to a subject. The method comprises administering to the subject the pharmaceutical composition comprising a compound of formula (IA- 1 ), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or formula (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable excipient. Alternatively, the method comprises administering to the subject the pharmaceutical composition comprising the lipid-based carriers as described herein, and a pharmaceutically acceptabl e excipient.

[0145] These and other aspects of the disclosure will be apparent upon reference to the following detailed description.

[0146] DETAILED DESCRIPTION OF THE INVENTION

[0147] Definitions

[0148] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs.

[0149] As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0150] The term “lipids” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0151] The term “lipid component” refers to a component in the lipid-based carrier that includes one or more lipids. For example, the lipid component may include one or more of a cationic / anionic / ionizable / zwitterionic lipid, a neutral lipid, a PEGylated lipid, or other lipid, such as a phospholipid.

[0152] The terms “lipid-based carrier,” “lipid carrier,” and “lipid composition” herein can be used interchangeably to refer a composition comprising one or more lipids, and encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. These compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer.

[0153]

[0154] A “steroid” is a compound comprising a carbon skeleton of w- 'W,. A non-limiting example of a steroid is cholesterol.

[0155] As used herein, the term “compound,” is meant to include all the isomers and isotopes of the structure depicted, all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms (e.g., crystal polymorphs), crystal form mixtures, or anhydrides or hydrates thereof.

[0156] “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium (3H) and deuterium (2H). The compounds described herein or their pharmaceutically acceptable salts may include those being isotopically -labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as2H, TI, ”C,!3C,!4C,!3N,!5N,15O,170,18O,3!P,32P,35S,18F,3OC1,123I, and125I, respectively. These isotopically-labelled compounds could be useful to help determine or measure the effectiveness of the compounds, by characterizing, for example, the site or mode of action, or binding affinity to pharmacologically important site of action. Certain isotopically-labelled compounds are useful in drug and / or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.,JH, and carbon-14, i.e.,!4C, may be useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e.,2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be useful in some circumstances. Substitution with positron emitting isotopes, such asUC,18F,!5O and13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art.

[0157] A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0158] “Isomers.” The compounds described herein or their pharmaceutically acceptable salts may include all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like. For instance, the compounds can contain one or more stereocenters and may thus give rise to geometic isomers (e.g., double bond causing geometric E / Z isomers), enantiomers, diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers), and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- such as for sugar anomers, or as (D)- or (L)- such as for amino acids. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or deri vati ve) using, for example, chiral high pressure liquid chromatography (IIPLC). Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0159] The term “crystal polymorphs”, “polymorphs” or “crystal forms” means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

[0160] Crystall ization of the compounds di sclosed herein may produce a solvate. As used herein, the term “solvate” refers to an aggregate that comprises one or more molecules of an ionizable lipid of the disclosure with one or more molecules of solvent. The solvent may be water, m which case the solvate may be a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like. Alternatively, the solvent may be an organic solvent. Solvates of the compound may be true solvates, while in other cases, the compound may merely retain adventitious water or be a mixture of water plus some adventitious solvent.

[0161] As used herein, “ionizable lipid” refers to a lipid that can be ionized, e.g., dissociated to produce one or more electrically charged species, under a given condition (e.g., pH). In some embodiments, an ionizable lipid includes one or more positively charged amine groups. In some embodiments, ionizable lipids are ionizable such that they can dissociate to exist in a positively charged form depending on pH. The ionization of an ionizable lipid affects the surface charge of a lipid nanoparticle comprising the ionizable lipid under different pH conditions. The surface charge of the lipid nanoparticle in turn can influence its plasma protein absorption, blood clearance, and tissue distribution (Semple, S. C., et al.. Adv. Drug Deliv Rev 32:3-17 (1998)) as well as its ability to form endosomolytic non-bilayer structures (Hafez, I. M., et al., Gene Ther 8: 1188-1196 (2001)) that can influence the intracellular delivery of nucleic acids. In some embodiments, ionizable lipids are those that are generally neutral, e.g., at physiological pH (e.g., pH about 7), but can cany net charge(s) at an acidic pH or basic pH. In one embodiment, ionizable lipids are those that are generally neutral at pH about 7, but can carry net charge(s) at an acidi c pH. In one embodiment, ionizable lipids are those that are generally neutral at pH about 7, but can carry net charge(s) at a basic pH. In some embodiments, ionizable lipids do not include those cationic lipids or anionic lipids that generally carry net charge(s) at physiological pH (e.g., pH about 7).

[0162] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. A non-limiting example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include, for example, 1-(monomethoxy -poly ethyleneglycol)-, 3 -dimyristoylglycerol (PEG-DMG) and the like. As used herein, the terms “PEG-lipid” and “PEGylated lipid” are interchangeable and refer to a lipid comprising a polyethylene glycol component.

[0163] The term “neutral lipid” refers to any of a lipid that exists either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-5n-glycero-3 -phosphocholine (DPPC), l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, and steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0164] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty’ acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery' of the one or more elements to a cell.

[0165] The term “liposome” as used herein refers to a composition comprising an outer lipid layer membrane (e.g., a single lipid bi-layer known as unilamellar liposomes or multiple lipid bilayers known as multilamellar liposomes) surrounding an internal aqueous space which may contain a cargo. See, e.g., Cullis et ah, Biochim. Biophys Acta, 559: 399-420 (1987), which is incorporated herein by reference in its entirety. A unilamellar liposome generally has a diameter in the range of about 20 to about 400 nanometers (nm), about 50 to about 300 nm, about 100 to about 200 nm, or about 300 to about 400 nm. A multilamellar liposome usually has a diameter in the range of about 1 to about 10 pm and may comprise anywhere from 2 to hundreds of concentric lipid bilayers alternating with layers of an aqueous phase.

[0166] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) and comprising one or more lipids (e.g., the lipid compounds described herein, and a second lipid (e.g., cationic, anionic, ionizable, or zwitterionic lipid)) for encapsulation of a therapeutic agent. LNPs may also include neutral lipids such as phospholipid molecules belonging to the phosphatidylcholine (PC) class; sterols, such as cholesterol; and polyethylene glycol (PEG). LNPs may be taken up by cells via endocytosis and the ionizability of the lipids at low pH enables endosomal escape, which can allow release of cargo into the cytoplasm. LNPs are liposome-like structures. Exemplary lipid nanoparticle composition are formulations of ionizable lipids, sterols (or hydrophobic molecules), structural lipids such as phospholipids, polyethyleneglycol (PEG) lipids, and potentially additional components (see Nature Nanotechnology 15:313-320 (2020), which is incorporated herein by reference in its entirety), or single molecules containing combinations of ionizable lipid, sterol, structural phospholipid, and shielding groups (see Nature Materials 20:701-710 (2021), which is incorporated herein by reference in its entirety). These components may be mixed with a therapeutic agent (e.g., nucleic acid molecules such as mRNA or circRNA) to be formulated into LNP composition.

[0167] In some embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In some embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U. S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, 8,569,256, 5,965,542 and U. S. Patent Publication Nos.2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO 99 / 39741, WO 2017 / 117528, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO2011 / 141705, and WO 2001 / 07548, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0168] As used herein, the term “size” refers to the hydrodynamic diameter of a lipid nanoparticle population. The measurement of the size of a lipid-based carrier may be used to indicate the size and population distribution (polydispersity index, PDI) of the composition.

[0169] As used herein, the “polydispersity index” is a ratio between weight-average molar mass and Mn is the number-average molar mass that describes the homogeneity of the particle size distribution of a system. A small value, e.g., less than 0.3, indicates a narrow particle size distribution.

[0170] A polydispersity index may be used to indicate the homogeneity of a lipid-based carrier (e.g., liposome or LNP), e.g., the particle size distribution of the liposome or LNP. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A lipid-based carrier may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid-based carrier may be from about 0.10 to about 0.20.

[0171] As used herein, the term “apparent pKa” refers to the pH at which 50% of the lipid-based carrier (e.g., LNP) is protonated. This can be used as an indicator of the pH range that the lipid-based carrier (e.g., LNP) will be protonated, and thus initiate the endosomal escape process in a nucleotide delivery.

[0172] As used herein, the term “zeta potential” refers to the electrokinetic potential of lipid, e.g., in a lipid-based carrier (e.g., a LNP composition). The zeta potential may describe the surface charge of a LNP composition. Zeta potential is useful in predicting organ tropism and potential interaction with serum proteins.

[0173] The zeta potential of a lipid-based carrier (e.g., liposome or LNP) may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a liposome or LNP. Lipid-based carriers (e.g., liposomes or LNP) with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a liposome or LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0174] As used herein, “encapsulated” by a lipid refers a therapeutic agent, such as a nucleic acid (e.g., mRNA or circRNA), that is fully or partially encapsulated to by a lipid-based carrier (e.g., liposome or LNP). In some embodiments, nucleic acid (e.g., mRNA or circRNA) is fully encapsulated in a lipid-based carrier (e.g., liposome or LNP).

[0175] As used herein, “encapsulation efficiency” or “entrapment efficiency” refers to the percentage of an encapsulated cargo (e.g., a therapeutic agent) that is successfully incorporated into (e.g., encapsulated or otherwise associated with) the lipid-based carrier (e.g., a LNP or liposome), relative to the initial total amount of therapeutic agent provided. For example, if 97 mg of a therapeutic agent are encapsulated in a lipid-based carrier out of a total 100 mg of the therapeutic agent initially provided, the encapsulation efficiency may be given as 97%.

[0176] Encapsulation efficiency can be used to indicate the efficiency of an encapsulated cargo (e.g., a nucleic acid molecule) loading into the lipid composition using a particular formulation method and formulation recipe.

[0177] The efficiency of encapsulation of a cargo such as a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a lipid-based carrier (e.g., liposome or LNP) after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., at least 70%, 80%, 90%, 95%, close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the liposome or LNP described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0178] “Serum-stable” in relation to nucleic acid-lipid nanoparticles means that the nucleic acid is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay.

[0179] A “pharmaceutical composition” refers to a composition which may comprise an ionizable lipid of the disclosure and a medium generally accepted in the art for the delivery of the biologically active compound to mammals, e.g., humans. Such a medium includes pharmaceutically acceptable carriers, diluents or excipients therefor.

[0180] “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0181] “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0182] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-l,2-disulfonic acid, ethanesulfonic acid, 2 -hydroxyethanesulfonic acid, formic acid, fumaric acid, galactanc acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0183] “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Non-limiting examples of inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0184] An “effective amount” or “therapeutically effective amount” of a therapeutic agent such as a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g., an increase or inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the nucleic acid. An increase in expression of a target sequence is achieved when any measurable level is detected in the case of an expression product that is not present in the absence of the nucleic acid. In the case where the expression product is present at some level prior to contact with the nucleic acid, an increase in expression is achieved when the fold increase in value obtained with a nucleic acid such as mRNA or circRNA relative to control is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000 or greater. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with a nucleic acid such as antisense oligonucleotide relative to the control is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of suitable reporter proteins, as well as phenotypic assays known to those of skill in the art.

[0185] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are interchangeable and refer to RNA or DNA that is linear, branched, or circular; single or double stranded; or a hybrid thereof, regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also encompasses RNA / DNA hybrids. DNA may be in the form of antisense molecules, plasmid DNA, cDNA, deoxyribozyme (DNAzyme), PCR products, or vectors. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), circular RNA (circRNA), an siRNA or siRNA precursor, a microRNA (miRNA) or miRNA precursor, antisense RNA, micRNA, multivalent RNA, Dicer substrate RNA, Dicer substrate small interfering RNA (dsiRNA), viral RNA (vRNA), self-amplifying RNA, tRNA, asymmetric interfering RNA (aiRNA), pi wi -interacting RNA (piRNA), guide RNA (gRNA), ribozyme, and combinations thereof. The nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequence. A nucleic acid can contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases. Nucleotides typically contain a deoxyribose (DNA) or ribose (RNA) sugar and a base, and are typically linked together through phosphodiester bonds.

[0186] The term “nucleic acid” also encompasses nucleic acid analogs having other types of linkages or backbones (e g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others). Nucleic acids include those nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphorami dates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptidenucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolim et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0187] “Bases” or “nucleobases’ include purines and pyrimidines, which further include natural bases adenine, thymine, guanine, cytosine, uracil, and inosine; as well as natural analogs and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. Bases also include modified or non-canonical bases (e.g., hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5 -methylcytosine, and 5 hydroxymethylcytosine).

[0188] As used herein, the terms “linear polyribonucleotide,” “linear polyribonucleotide molecule,” “linear RNA,” and “linRNA” are used interchangeably and mean polyribonucleotide molecule having a 5’ and 3’ end. One or both of the 5’ and 3’ ends may be free ends or joined to another moiety. Linear RNA includes RNA that has not undergone circularization (e.g., is precircularized) and can be used as a starting material for circularization through, for example, splint ligation, or chemical, enzymatic, ribozyme- or splicing-catalyzed circularization methods.

[0189] As used herein, the terms “circular polyribonucleotide,” “circular polynucleotide molecule,” “circular RNA,” and “circRNA” are used interchangeably and mean a polyribonucleotide molecule that has a structure having no free ends (i.e., no free 3’ and / or 5’ ends), for example a polyribonucleotide molecule that forms a circular or end-less structure through covalent (e.g., covalently closed) or non-covalent bonds. The circular polyribonucleotide may be, e.g., a covalently closed polyribonucleotide.

[0190] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide. The term “gene product,” as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

[0191] As used herein, the term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.

[0192] As used herein, “methods of administration” may include both systemic delivery7and local delivery. “Systemic delivery” means that a useful, such as a therapeutic, amount of an agent is delivered to most parts of the body. Systemic delivery of a lipid-based carrier can be carried out by any means known in the art including, for example, intravenous, intraarterial, intramuscular, intradermal, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of a lipid-based carrier is by intravenous delivery. “Local delivery,” as used herein, refers to delivery of a therapeutic agent directly to a target site within an organism. For example, a therapeutic agent can be locally delivered by direct injection into a disease site such as a tumor, other target site such as a site of inflammation, or a target organ such as the liver, heart, pancreas, kidney, and the like. Local delivery can also include topical applications or localized injection techniques such as intramuscular, subcutaneous or intradermal injection. Local delivery does not preclude a systemic pharmacological effect. The present disclosure is also meant to encompass the in vivo metabolic products of the disclosed compounds. Such products may result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered compound, primarily due to enzymatic processes. Accordingly, the disclosure also encompasses the compounds produced by a process comprising administering the lipid compounds described herein to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled compound described herein in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples.

[0193] The phrase “induce expression of a desired protein” refers to the ability of a nucleic acid to increase expression of the desired protein. To examine the extent of protein expression, a test sample (e.g., a sample of cells in culture expressing the desired protein) or a test mammal (e.g., a mammal such as a human or an animal) model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model is contacted with a nucleic acid (e.g., nucleic acid in combination with a lipid of the present disclosure). Expression of the desired protein in the test sample or test animal is compared to expression of the desired protein in a control sample (e.g., a sample of cells in culture expressing the desired protein) or a control mammal (e.g., a mammal such as a human or an animal) model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model that is not contacted with or administered the nucleic acid. When the desired protein is present in a control sample or a control mammal, the expression of a desired protein in a control sample or a control mammal may be assigned a value of 1.0. In some embodiments, inducing expression of a desired protein is achieved when the ratio of desired protein expression in the test sample or the test mammal to the level of desired protein expression in the control sample or the control mammal is greater than 1, for example, about 1.1, 1.5, 2.0. 5.0 or 10.0. When adesired protein is not present in a control sample or a control mammal, inducing expression of a desired protein is achieved when any measurable level of the desired protein in the test sample or the test mammal is detected. One of ordinary skill in the art will understand appropriate assays to determine the level of protein expression in a sample, for example dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays, or assays based on reporter proteins that can produce fluorescence or luminescence under appropriate conditions.

[0194] The phrase “inhibiting expression of a target gene” refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To examine the extent of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) or a test mammal (e.g., a mammal such as a human or an animal) model such as a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model is contacted with a nucleic acid that silences, reduces, or inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) or a control mammal (e.g., a mammal such as a human or an animal) model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey) model that is not contacted with or administered the nucleic acid. The expression of the target gene in a control sample or a control mammal may be assigned a value of 100%. In some embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the level of target gene expression in the test sample or the test mammal relative to the level of target gene expression in the control sample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids are capable of silencing, reducing, or inhibiting the expression of a target gene by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relative to the level of target gene expression in a control sample or a control mammal not contacted with or administered the nucleic acid. Suitable assays for determining the level of target gene expression include, without limitation, examination of protein or mRNA l evels using techniques known to those of skill in the art, such as, e.g., dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0195] “Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest in a mammal, such as a human, having the disease or condition of interest, and includes:

[0196] (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it;

[0197] (ii) inhibiting the disease or condition, i.e., arresting its development;

[0198] (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been worked out) and it is therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians.

[0199] As used herein, the term “biodegradable moiety” refers to a moiety that includes one or more bonds that may undergo bond breaking reactions in a biological environment, e.g., in an organism, organ, tissue, cell, or organelle. For example, the biodegradable group may be metabolizable by the body of a mammal, such as a human (e.g., by hydrolysis). Some groups that contain a biodegradable bond include, for example, but are not limited to, esters, amides, disulfides, dithiols, and oximes.

[0200] The term “alkyl” refers to a straight or branched monovalent hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and attaching to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3 -methylhexyl, 2-methylhexyl, and the like. The alkyl group may have, for example, from one to twenty-four carbon atoms (C1-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (C6-C16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl), one to six carbon atoms (Ci-Cs alkyl), one to four carbon atoms (C1-C4 alkyl), one to three carbon atoms (C1-C3 alkyl), or one to two carbon atoms (C1-C2 alkyl). Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted.

[0201] The term “alkylene” or “alkylene chain” refers to a divalent form of an alkyl group, a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen and linking the rest of the molecule via a diradical group, e.g., methylene, ethylene, propylene, n-butylene, and the like. The alkylene group may have, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Cs alkylene), one to four carbon atoms (C1-C4 alkylene), one to three carbon atoms (C1-C3 alkylene), or one to two carbon atoms (C1-C2 alkylene). The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene group is optionally substituted.

[0202] The term “alkenyl” refers to a straight or branched hydrocarbon chain having one or more double bonds. Unless otherwise indicated, “alkenyl” generally refers to C2-C8 alkenyl (e.g., C2-Ce alkenyl, C2-C4 alkenyl, or C2-C3 alkenyl). Examples of alkenyl groups include, but not limited to, ethenyl, allyl, propenyl, 2-butenyl, 1 -pentenyl, penta-1, 4-dienyl, 3-hexenyl, 3-octenyl and the like.

[0203] The term “alkenylene” refers to a divalent form of an alkenyl group. The sp2and sp3carbons may optionally serve as the point of attachment of the alkenylene group. Examples of alkenylene groups include ethenylene, propenylene, n-butenylene, and the like. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Unless otherwise indicated, “alkynyl” generally refers to C2-C8 alkynyl (e.g., C2-C6 alkynyl, C2-C4 alkynyl, or C2-C3 alkynyl). Examples of alkynyl groups include ethynyl, propynyl, butynyl, and 3-methylbutynyl, pentynyl, hexynyl, and,the like.

[0204] The term “alkynylene” refers to a divalent form of an alkynyl group. The sp2and sp3carbons may optionally serve as the point of attachment of the alkynylene groups. Examples of alkynlene groups include ethynylene, propynylene, n-butynylene, and the like.

[0205] The term “cycloalkyl” or “cyclyl” as employed herein includes saturated and partially unsaturated, but not aromatic, cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0206] The term “cycloalkylene” refers to a divalent form of a cycloalkyl group.

[0207] The term “heterocyclyl,” “heterocycle,” “heterocyclic,” “heterocyclic radical,” or “heterocyclic ring” refers to non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, which can be completely saturated or which can contain one or more units of unsaturation, for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system. The heteroatoms may be selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). For each ring of the heterocycle, 0, 1, 2 or 3 atoms may be substituted by a substituent. The term also includes groups in which a heterocycle ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 1-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2J / -pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). Examples of heterocyclyl groups include indolinyl, isoindolinyl, piperazinyl, pyrrolidinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, quinuclidinyl, and the like.

[0208] The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0209] The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term “aryl” may be used interchangeably with the term “aryl ring.” Examples of aryl groups include phenyl, biphenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0210] The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl.

[0211] The term “arylalkoxy” refers to an alkoxy substituted with aryl.

[0212] The term “heteroaryl” or “heteroar-” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. The term also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloalkyl, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of heteroaryl groups include pyrrolyl, pyridyl, pyridazinyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, pyrazinyl, indolizinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, isothiazolyl, thiadiazolyl, purinyl, naphthyridinyl, pteridinyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4 / f-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3- / >]-l,4-oxazin-3(4 / / )-one and the like.

[0213] A divalent radical of an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, heterocyclyl is formed by removal of a hydrogen atom from an alkyl, alkenyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl radical, respectively (or by removal of two hydrogen atoms from an alkane, alkene, arene, heteroarene, cycloalkane, or heterocycle, respectively).

[0214] The term “alkoxy” refers to an -O-alkyl radical.

[0215] The term “alkoxyalkyl” refers to an alkyl substituted with an alkoxy group.

[0216] The term “hydroxyalkyl” refers to an alkyl substituted with a hydroxy group.

[0217] The term “alkylthio” refers to an S-alkyl radical.

[0218] The term “arylthio” refers to an S-aryl radical.

[0219] The term “aryloxy” refers to an O-aryl radical.

[0220] The term “arylamino” refers to an amino-aryl radical.

[0221] The term “alkylsulfonyl” refers to a -SO₂-alkyl radical. The term “haloalkyl” refers to an alkyl group in which one or more hydrogens are replaced by a halogen.

[0222] The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0223] The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

[0224] The term “aminoalkyl” refers to an alkyl substituted with an amino.

[0225] The term “alkylamino” refers to an amino substituted with an alkyl.

[0226] The term “aminocarbonyl” refers to an -C(O)-amino radical.

[0227] The term “halo” or “halogen” refers to any radical of fluorine, chlorine, bromine or iodine.

[0228] The term “optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0229] The term “substituted” used herein means that for any of the above groups (e.g., alkyl, hydroxyalkyl, alkylene, cycloalkyl, cycloalkylene, alkenyl, alkynyl, amino, aminocarbonyl, aryl, heterocyclyl, or heteroaryl), one or more hydrogen radicals in that group is replaced with the radical of a specified substituent including, but not limited to: halo (e.g., F, CI, Br, or I); oxo groups (=0); hydroxyl (-0H); alkoxy; alkoxyalkyl; arylalkoxy; alkyl (e.g., C1-C12 alkyl); cycloalkyl; alkenyl; alkynyl; aryl; arylalkyl heterocyclyl; heterocyclyl; heteroaryl; thiol; alkylthio; arylthio; alkylthioalkyl; arylthioalkyl; alkylsulfonyl; alkylsulfonylalkyl; arylsulfonylalkyl; aryloxy; carboxyalkyl; alkoxycarbonylalkyl; aminocarbonylalkyl; acyl; aminocarbonyl; alkylaminocarbonyl; arylaminocarbonyl; alkoxy carbonyl; aryloxycarbonyl; haloalkyl; amino; trifluoromethyl; cyano; nitro; alkylamino; arylamino; alkylaminoalkyl; arylaminoalkyl; aminoalkylamino; aralkoxycarbonyl; sulfonyl; alkylaminolactams; alkylaminoheteroaryls; alkylaminoheterocycyls; and aminosulfonamides. Exemplary substituents also include: -(C=0)0R‘, -0(C=0)R, -C(=0)R, -OR, -S(O)kR, -S-SR, -C(=O)SR, -SC(=O)R, -NRR’, -R”C(=0)R, -C(=0)RR’, -Rt”C(=0)RtRt’; -0C(=0)RR, -R”C(=0)0R, -RES(O)k RERE, - ”S(O)kRl, and -S(O)kRERE, wherein: REand REis each independently H, Ci-C15 alkyl or cycloalkyl, each R” is C1-C15 alkylene, and k is 0, 1 or 2. In some embodiments, the substituent is a C1-C12 alkyl group. In some embodiments, the substituent is a cycloalkyl group. In some embodiments, the substituent is a halo group, such as F, Cl, or Br. In some embodiments, the substituent is an oxo group. In some embodiments, the substituent is a hydroxyl group. In some embodiments, the substituent is a hydroxyalkylene group (-R‘ -OH). In some embodiments, the substituent is an alkoxy group (-OR1). In some embodiments, the substituent is a carboxyl group. In some embodiments, the substituent is an amino group (- RlRl’). Suitable substituents also include divalent substituents on a saturated carbon atom, including but are not limited to: =0, =S, =NNR*2, =NNHC(0)R*, =NNHC(0)0R*, =NNHS(0)2R*, =NR*, =N0R*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, substituted or unsubstituted Ci-6 alkyl, or an unsubstituted 5-6-membered saturated or partially unsaturated ring, or an aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0230] The novel dipeptide-core lipids

[0231] One aspect of the invention relates to a compound of formula (IA-1) or (IA-2):

[0232]

[0233] (IA-2), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein:

[0234] aal and aa2 each are independently a natural or unnatural amino acid residue, wherein * represents the bonding to aal or aa 2 via its backbone N atom and • represents the bonding to aal or aa2 via its backbone carbonyl C atom;

[0235] each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;

[0236] each R, for each occurrence, is independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl;

[0237] each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;

[0238] j is 1, 2, or 3;

[0239] W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each A is independently absent, C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; and

[0240] each X is independently absent, X’,

[0241]

[0242] each R’ is independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and

[0243] each X’ is independently a biodegradable moiety.

[0244] In some embodiments, in formula (IA-1) or (IA-2), A and X are not absent in at least one -I— A - X - B

[0245] of the *» tails. In some embodiments, in formula (IA-1) or (IA-2), A and X are

[0246]

[0247] A - X - B

[0248] not absent in at least two of the *> tails. In some embodiments, in formula (IA-1),

[0249]

[0250] A - X - B

[0251] A and X are not absent in at least three of the *» tails. In some embodiments, in -I— A - X - B

[0252] formula (IA-1), A and X are not absent in all of the *> tails.

[0253] In formula (IA-1) or (IA-2), the compound contains a dipeptide core of *aal* or *aa2«. Each aal and aa2 may be independently a natural or unnatural amino acid residue. *aal • or *aa2« indicates the direction of the amino acid backbone in aal or aa2, connecting to each other or connecting to the rest of the groups in formula (IA-1) or (IA-2). * represents the bonding to aal or aa 2 via its backbone N atom, and • represents the bonding to aal or aa2 via its backbone carbonyl C atom.

[0254] In some embodiments, aal and / or aa2 may be an a-amino acid (i.e., the amino group is located on the a carbon to the carbonyl group). For instance, aal may have the structure of

[0255]

[0256] and aa2 may have the structure of

[0257]

[0258] , wherein Rs represents the side chain of the amino acid. That is to say, the side chain in aal’s or aa2’s structure (e.g., Rs) is the side chain of the corresponding amino acid. In some embodiments, aal and / or aa2 may be one of the 20 common amino acids: Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai. In some embodiments, aal and / or aa2 may Sec (Selenocysteine) or Pyl (Pyrrolysine).

[0259] In some embodiments, aal and / or aa2 may be a P-amino acid (i.e., the amino group is located on the carbon to the carbonyl group). For instance, aal may have the structure of

[0260]

[0261] , wherein Rs represents the side chain of the amino acid. That is to say, the side chain in aal’s or aa2’s structure (e.g., Rs) is the side chain of the corresponding amino acid. In some embodiments, aal and / or aa2 may be P-Ala, P-Leu, P-Lys, P-Arg, P-Glu, P-Glu, P-Phe, or P-Tyr.

[0262] In some embodiments, aal and / or aa2 may be a y-amino acid (i.e., the amino group is located on the y carbon to the carbonyl group). For instance, aal may have the structure of

[0263]

[0264] in of the amino acid. That is to say, the side chain in aal’s or aa2’s structure (e.g., Rs) is the side chain of the corresponding amino acid. In some embodiments, aal and / or aa2 may be y-aminobutyric acid.

[0265] In some embodiments, aal and / or aa2 may be a 8-amino acid (i.e., the amino group is located on the 8 carbon to the carbonyl group).

[0266] In some embodiments, aal and / or aa2 may be a natural amino acid in L-stereoisomer. In some embodiments, aal and / or aa2 may be in D-stereoisomer.

[0267] In some embodiments, aal and / or aa2 may an unnatural amino acid or non-canonical amino acid. Non-canonical amino acids are typically non-proteinogenic amino acids, i.e., amino acids that cannot be incorporated into proteins during translation. Exemplary non-canonical

[0268]

[0269] amino acid are hydroxyproline ( -NH ), hydroxyarginine (e.g.,

[0270]

[0271] NH

[0272] dimethyl hydroxy arginine (e.g.,

[0273]

[0274] homocysteine (e.g.,

[0275]

[0276]

[0277] selenocysteine (N*L ), methyl tryptophan (

[0278]

[0279] , hydroxytryptophan

[0280]

[0281] , homoserine (e.g.,

[0282]

[0283] ylalanine (

[0284]

[0285] Because aal or aa2 can have a backbone taking the form of the backbone of a-, -, or y- amino acid, and because aal or aa2 can take the form of different stereoisomers (e.g., D- or L-), when referring to aal or aa2 being a specific amino acid, what is meant is that the side chain of aal or aa2 in aal’s or aa2’s structure (e.g., Rs) is the side chain of the corresponding amino acid. In some embodiments, aal and aa2 may be one of the 20 common amino acids: Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai.

[0286] In some embodiments, aal and aa2 may be a variant of any of the 20 common amino acids. By variant, it is meant that the side chain of aal or aa2 is derived from the side chain of the corresponding amino acid, with further functionalization of the side chain of the corresponding amino acid. For instance, when the side chain of an amino acid has a -OH group, its variant can refer to the functionalization of the -OH group by a functional group that is reactive to the -OH group. In some embodiments, the side chain of aal or aa2 may be a variant of Ser, Thr, or Tyr, by functionalizing respective -OH group to form a -O-C(O)-Rh, wherein Rhmay be optionally substituted alkyl or aminoalkyl. In one embodiment, the side chain of aal or aa2 may be a A)

[0287] RnJ ° variant of Ser, Thr, or Tyr, by functionalizing respective -OH group to form

[0288]

[0289] , wherein O is connected to the rest of the side chain of Ser, Thr, or Tyr, and each Rnmay be independently H or optionally substituted alkyl, or both Rntogether with N form a ring.

[0290] As another example, when the side chain of an amino acid has a -COOH group, its variant can refer to the functionalization of the -COOH group by a functional group that is reactive to the -COOH group. In some embodiments, the side chain of aal or aa2 may be a variant of Glu or Asp, by functionalizing respective -COOH group to form a -C(O)-NH-Rh, wherein Rhmay be H or optionally substituted alkyl or aminoalkyl. In one embodiment, the side chain of aal or aa2

[0291] may be a variant of Glu or Asp, by functionalizing respective -COOH group to form

[0292]

[0293] , wherein C(O) is connected to the rest of the side chain of Glu or Asp, and each Rnmay be independently H or optionally substituted alkyl, or both Rntogether with N form a ring.

[0294] As another example, when the side chain of an amino acid has a -NH2 group, its variant can refer to the functionalization of the NH2 group by a functional group that is reactive to the NH2 group. In some embodiments, the side chain of aal or aa2 may be a variant of Lys, Gin, Arg, or Asn, by functionalizing respective -NH2 group to form a -NH-C(O)-Rh, wherein Rhmay be H, or optionally substituted alkyl or aminoalkyl. In one embodiment, the side chain of aal or aa2 may be a variant of Lys, Gin, Arg, or Asn, by functionalizing respective -NH2 group to form a -NH-C(O)-CH3.

[0295] In some embodiments, one of aal and aa2 is selected from the group consisting of Gly, Cys, Pro, and its variants thereof. In some embodiments, one of aal and aa2 has a hydrophobic side chain, and is selected from the group consisting of Ala, Vai, He, Leu, Met, Phe, Tyr, Trp, and its variants thereof. In some embodiments, one of aal and aa2 has a polar, uncharged side chain, and is selected from the group consisting of Ser, Thr, Asn, Gin, and its variants thereof. In some embodiments, one of aal and aa2 has an electrically charged side chain, and is selected from the group consisting of Arg, His, Lys, Asp, Glu, and its variants thereof. In some embodiments, aal-aa2 is Gly-Gly, -Ala-Gly (or Gly- -Ala), Gly-Phe (or Phe- Gly), Gly-Ala (or Ala-Gly), Gly-His (or His- Leu (or Leu-Gly), Gly-Ile (or

[0296] Ile-Gly), Gly-Ser (or Ser-Gly), Gly-SerHead

[0297]

[0298] or SerHead-Gly, e.g.,

[0299]

[0300] -Ala (or -Ala-Ser), Gly-Arg (or Arg-Gly), Gly-Cys (or Cys-Gly), Gly-Gln (or Gln-Gly), Gln- -Ala (or -Ala-Gln), Glu- -Ala (or -Ala-Glu), Gly-Glu (or Glu-Gly), Gly-

[0301] GluHead (

[0302]

[0303] Lys (or Lys-Gly), Gly-Pro, Gly-Thr (or Thr-Gly), Gly-Asn (or Asn-Gly), P-Ala- -Ala, y-aminobutyric acid -Gly (or Gly-y-aminobutyric acid), or variants thereof. In these embodiments, SerHead is a variant of Ser by functionalizing -OH group as discussed above; and GluHead is a variant of Glu by functionalizing -COOH group as discussed above.

[0304] In some embodiments, a variant of any one of the amino acid residue in aal and / or aa2 is a squaramide or squaramide derivative of the amino acid. In some embodiment, the squaramide

[0305] or squaramide derivative of the amino acid residue has the structure

[0306]

[0307] wherein each R1is independently H, OH, or C1-C3 alkyl; each R2is, independently H or C1-C3 alkyl; each v is independently an integer of 0-4; and represents the connection point to the side chain of the amino acid residue.

[0308] Another aspect of the invention relates to compound of formula (IB-1) or (IB-2):

[0309]

[0310] thereof, or a stereoisomer thereof, wherein:

[0311] each of il and i2 is independently 0, 1, 2, or 3; and when il is not 0, Rai in both occurrences are H, and when i2 is not 0, both Ra2 in both occurrences are H;

[0312] j is 1, 2, or 3;

[0313] each of Rai, Ra2, Rbi, and Rb2 is, for each occurrence, independently H, Ci-Ce branched

[0314]

[0315] or Rb2 adjacent to the N atom is taken together with its adjacent N atom and R variable to form a heterocyclic ring, optionally substituted with one or more alkyl, OR4, SR4, and / or halogen groups;

[0316] each R1is, for each occurrence, independently H, OH, or C1-C3 alkyl;

[0317] each R2is, for each occurrence, independently H or C1-C3 alkyl;

[0318] each R3is independently an aryl or heteroaryl, optionally substituted with one or more alkyl, C(O)OR4, OR4, SR4, oxo, and / or halogen groups;

[0319] each R4is independently H, C1-C3 alkyl, aryl,

[0320]

[0321] -(CR1R2)VC(O)(CR1R2)VNR1R2;

[0322] each R5is independently H, C1-C3 alkyl, -NR^CR^jvNR^2, or -(CR1R2)VC(O)(CR1R2)VNR1R2;

[0323] each v is independently an integer of 0-4;

[0324] each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;

[0325] each R is, for each occurrence, independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl; each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;

[0326] W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;

[0327] each A is independently absent, C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; and

[0328] each X is independently absent, X’,

[0329]

[0330] each R’ is, for each occurrence, independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and

[0331] each X’ is independently a biodegradable moiety.

[0332] In some embodiments, in formula (IB-1) or (IB-2), A and X are not absent in at least one - — A - X - B

[0333] of the tails. In some embodiments, in formula (IB-1) or (IB-2), A and X are A - X - B

[0334] not absent in at least two of the

[0335]

[0336] tails. In some embodiments, in formula (IB-1),

[0337]

[0338] A - X - B

[0339] A and X are not absent in at least three of the *> tails. In some embodiments, in

[0340] formula (IB-1), A and X are not absent in all of the

[0341]

[0342] tails.

[0343] formula

[0344]

[0345] ula (IA-1) or (IA-2). Each of il and i2 is independently 0, 1, 2, or 3 (e.g., corresponding to a backbone of a,, y, or 5 amino acid, respectively). In some embodiments, il is 0. In some embodiments, i2 is 0. In some embodiments, both il and i2 are 0. In some embodiments, il is 1. In some embodiments, i2 is 1. In some embodiments, both il and i2 are 1. In some embodiments, il is 0, and i2 is 1. In some embodiments, il is 1, and i2 is 0. In some embodiments, il is 2. In some embodiments, i2 is 2. In some embodiments, both il and i2 are 2. In some embodiments, il is 3. In some embodiments, i2 is 3. In some embodiments, both il and i2 are 3.

[0346] Each of Rai, Ra2, Rbi, and Rb2 is, for each occurrence, independently H, Ci-Ce branched or unbranched alkyl, -(CRJR2)vR3, -(CR1R2)vSR4, -(CR1R2)vSeR4, -(CR1R2)vOR4, -

[0347]

[0348] hat is adjacent to the N atom may be taken together with its adjacent N atom and / or the R variable (that is connected to the N atom) to form a heterocyclic ring. The heterocyclic ring may be optionally substituted with one or more alkyl, OR4, SR4, and / or halogen groups.

[0349] In some embodiments, for each occurrence, at least one Rai is H, at least one Ra2is H, at least one Rbi is H, and at least one Rb2 is H.

[0350] In some embodiments, il is 0, one Rai is H. In some embodiments, i2 is 0, one Ra2 is H. In some embodiments, il is 0, each Rai is independently H or alkyl. In some embodiments, i2 is 0, each Ra2is independently H or alkyl.

[0351] In some embodiments, il is not 0, both Rai are H. In some embodiments, i2 is not 0, both Ra2 are H. In some embodiments, il is 1, both Rai are H, and one Rbi is H.

[0352] In some embodiments, il is 1, both Rbi are H, and at least one Rai is H. In some embodiments, i2 is 1, both Rb2 are H, and at least one Ra2is H. In some embodiments, i2 is 1, both Rb2 are H, and at least one Ra2 is H. In some embodiments, il is 2; both Rai are H; both Rbi on the C atom next to the C atom connecting to Rai are H; and at least one Rbi on the C atom next to the N atom is H. In some embodiments, il is 2; both Rai are H; both Rbi on the C atom next to the N atom are H; and at least one Rbi on the C atom next to the C atom connecting to Rai is H. In some embodiments, il is 2; all Rbi are H; and at least one Rai is H. In some embodiments, i2 is 2; both Ra2 are H; both Rb2 on the C atom next to the C atom connecting to Ra2 are H; and at least one Rb2 on the C atom next to the N atom is H. In some embodiments, i2 is 2; both Ra2 are H; both Rb2 on the C atom next to the N atom are H; and at least one Rb2 on the C atom next to the C atom connecting to Ra2 is H. In some embodiments, i2 is 2; all Rb2 are H; and at least one Ra2 is H.

[0353] In some embodiment, at least one Rai or Ra2 is H, Ci-Ce branched or unbranched alkyl, -(CRJR2)VR3, -(CR1R2)VSR4, -(CR1R2)vSeR4, -(CR1R2)vOR4, -(CR1R2)vCOR5, -(CR1R2)VC(O)OR4, -(CR1R2)VNR1R2, -(CR1R2)VC(O)NR1R2, -(CR1R2)VN(R1)C(O)R5, -(CR1R2)VC(S)NR1R2, or -(CR1R2)VNR1C(=NR1)NR1R2. In some embodiment, at least one Rbi or Rb2 is H, Ci-Ce branched or unbranched alkyl, -(CRJR2)vR3, -(CR1R2)vSR4, -(CR1R2)vSeR4, -(CR1R2)vOR4, -(CR1R2)VCOR5, -(CR1R2)VC(O)OR4, -(CR1R2)VNR1R2, -(CR1R2)VC(O)NR1R2, -(CR1R2)VN(R1)C(O)R5, -(CR1R2)VC(S)NR1R2, or -(CR1R2)vNR1C(=NR1)NR1R2. In some embodiment, one Ra2 or Rb2 that is adjacent to the N atom is taken together with its adjacent N atom and the R variable (connected to the N atom) to form a heterocyclic ring, which may be substituted with one or more alkyl, OR4, SR4, and / or halogen groups.

[0354] Each R1is, for each occurrence, independently H, OH, or C1-C3 alkyl. In some embodiments, each R1is, for each occurrence, independently H or C1-C3 alkyl (e.g., H or methyl). In some embodiments, R1is OH. Each R2is, for each occurrence, independently H or C1-C3 alkyl. In some embodiments, R2is C1-C3 alkyl (e.g., methyl). In some embodiments, R2is H.

[0355] Each R3is independently an aryl or heteroaryl, optionally substituted with one or more alkyl, C(O)OR4, OR4, SR4, and / or halogen groups. In some embodiments, each R3is independently an aryl (e.g., phenyl). In some embodiments, each R3is independently an aryl (e.g., phenyl) substituted with one or more alkyl, alkoxy, or hydroxy groups. In some embodiments, each R3is independently an aryl (e.g., phenyl) substituted with one or more halo and / or aralkoxy, wherein the aryl in the aralkoxy may be further substituted by one or more hydroxy and / or halo. In some embodiments, each R3is independently an aryl (e.g., phenyl) substituted with C(O)OR4. In some embodiments, each R3is independently a heteroaryl (e.g., indolyl, imidazolyl). In some embodiments, each R3is independently a heteroaryl (e.g., indolyl, imidazolyl), substituted with one or more alkyl, alkoxy, aralkoxy, hydroxy, C(O)OR4, or oxo groups.

[0356] E c

[0357] a h R4is independently H, C1-C3 alkyl, aryl,

[0358]

[0359] (CR1R2)VC(O)(CR1R2)VNR1R2. In some embodiments, each R4is independently H or C1-C3 alkyl (e.g., H or methyl). In some embodiments, each R4is independently an aryl (e.g., phenyl). In

[0360] some embodiments, each R4is independently

[0361]

[0362] In some embodiments, each R4is

[0363] independently

[0364]

[0365] In some embodiments, each R4is independently -(CR1R2)VC(O)(CR1R2)VNR1R2.

[0366] Each R5is independently H, C1-C3 alkyl, -NR^CR^jvNR^2, or -(CR1R2)VC(O)(CR1R2)VNR1R2. In some embodiments, each R5is independently H or C1-C3 alkyl (e.g., H or methyl). In some embodiments, each R5is independently NR^CR^jvNR^R2. In some embodiments, each R5is independently -(CR1R2)vC(O)(CR1R2)vNR1R2.

[0367] Each v is independently an integer of 0-4 (e.g., 0, 1, 2, 3, or 4).

[0368] Each R is, for each occurrence, independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl. In some embodiments, R is H. In some embodiments, R and N atom, together with one Ra2 or Rb2 adjacent to the N atom form a heterocyclic ring (e.g., a 5-member heterocyclic ring). In some embodiments, the heterocyclic ring may be substituted with one or more alkyl, OR4, SR4, and / or halogen groups (e.g., substituted with a C1-C3 alkyl, OH, SH, or Cl group).

[0369] In some embodiments, the dipeptide core moiety

[0370]

[0371] formula (IB-1) or (IB-2) has the structure of:

[0372]

[0373]

[0374]

[0375]

[0376] All the descriptions below are applicable to all the variables in all above aspects of the invention relating to the compounds of formula (IA-1), (IA-2), (IB-1), or (IB-2), or the compounds of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB-2).

[0377] In the above formulas, each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups. Each R, for each occurrence, is independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl. In some embodiments, Y is -O- or -S-. In some embodiments, Y is -N(R)-. In some embodiments, R is H or C1-C3 alkyl (e.g., H or methyl). In some embodiments, Y is -NH- or -N(CHs)-. In some embodiments, Y is a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups. In one

[0378] embodiment,

[0379]

[0380] Each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene. In some embodiments, each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene (e.g., C2-C3 alkylene). In some embodiments, each V is, for each occurrence, independently C2-C6 branched or unbranched alkenylene (e.g., C2-C3 alkenylene). In some embodiments, each V is, for each occurrence, C3-C7 cycloalkylene (e.g., C3-C5 cycloalkylene).

[0381] The integer) represents the number of unit of-Y-V- in formula (IA-1) or (IB-1). Each j is 1, 2, or 3. In some embodiments, j is 1 or 2. Each unit of-Y-V- may be the same as or different than the other unit of -Y-V-.

[0382] R

[0383] -U-e.

[0384] In some embodiments, a unit of-Y-V- is s >. In some embodiments, an unit

[0385] of-Y-V- is

[0386]

[0387] . In some embodiments, a unit of-Y-V- is

[0388]

[0389] . In some

[0390] embodiments, a unit of-Y-V- is

[0391]

[0392] . In these embodiments, R is as defined above (e.g., R is H or methyl), and m is 1 or 2. In some embodiments, when j is 2 or 3, theY-V4Jmoiety represents any combination of the same or different unit of-Y-V- described herein.

[0393] In some embodiments, the compound described herein has the structure of

[0394]

[0395] (IIF-1). The definitions for the variables Rai, Ra2, Rbi, Rb2, Y, A, B, and X are the same as those described herein for formulas (IA-1) and / or formula (IB-1). Each m is, for each occurrence, 2 or

[0396] In some embodiments, the compounds have the formula (IA-2) or (IB-2).

[0397] In some embodiments, the compound has the structure of

[0398]

[0399]

[0400] The definitions for the variables Rai, Ra2, Rbi, Rb2, A, B, and X are the same as those described herein for formulas (IA-2) and / or formula (IB-2).

[0401] In some embodiments, the compound described herein has the structure of

[0402]

[0403]

[0404] (IIIA-14),

[0405]

[0406] -,

[0407]

[0408] -. he definitions for the variables Y, A, B, and X are the same as those described herein for formulas (IA-1) and / or formula (IB- 1). Each m is, for each occurrence, 2 or 3. Rpis H, C(O)OH, or OH. RNis H,

[0409] C(O)R5, or C1-C3 alkyl.

[0410]

[0411] are each H or Ci- C3 alkyl, vis 1-4.

[0412] In some embodiments, the compound described herein has the structure of

[0413]

[0414]

[0415]

[0416] The definitions for the variables W, A, B, and X are the same as those described herein for formulas (IA-2) and / or formula (IB-2). Rpis H, C(O)OH, or OH. RNis H, C(O)R5, or C1-C3

[0417] alkyl.

[0418]

[0419] are each H or C1-C3 alkyl, v is 1-4.

[0420] All the descriptions below are applicable to all the variables in all above aspects of the invention relating to the compounds of formula (IA-1), (IA-2), (IB-1), or (IB-2), or the compounds of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB-2).

[0421] In the above formulas (IA-2) or (IB-2), or the subgenus formulas of formula (IA-2) or (IB-2), W is R6, OR6, or NHR6. R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted with one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups. In some embodiments, W is R6. In some embodiments, W is OR6. In some embodiments, W is NHR6. In some embodiments, R6in the W variable is H or C1-C12 alkyl, such as C1-C3 alkyl. In some embodiments, W is hydroxy or alkoxy. -2— A - X - B

[0422] In the above formulas, each lipid compound has four tails of *» (formula

[0423] IA-1 or IB-1), or has two tails

[0424]

[0425] (formula IA-2 or IB-2).

[0426] -2— A - X - B

[0427] In some embodiments, at least two of the tails are the same. In some embodiments, in formula (IA-2) or (IB-2) or their subgenus formulas, both tails of

[0428]

[0429] are the same.

[0430] In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, at least -1— A - X - B

[0431] three of the tails are the same. In some embodiments formula (IA-1) or (IB- 1)

[0432] or their subgenus formulas, all four tails

[0433]

[0434] are the same.

[0435] -2— A - X - B

[0436] In some embodiments, at least one or at least two of the *» tails are different. In some embodiments, in formula (IA-2) or (IB-2) or their subgenus formulas, the two -2— A - X - B

[0437] tails of *> are different from each other.

[0438] In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, at least -1— A - X - B

[0439] three of the tails are different. In some embodiments, in formula (IA-1) or -I— A - X - B

[0440] (IB- 1) or their subgenus formulas, all four of the tails are different.

[0441] In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, among the -I— A - X - B

[0442] four tails of *>, three tails are the same and one tail is different.

[0443] In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, among the -i— A - X - B

[0444] four tails of *>, two tails are the same, but different from the other two tails that A - X - B are the same. That is to say, the compound contains two different sets of

[0445]

[0446] tails. In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, among the four -2— A - X - B

[0447] tails of *>, two tails are the same, and the other two tails are different from the two tails and also different from each other. That is to say, the compound contains three different

[0448] sets

[0449]

[0450] tails.

[0451] In some embodiments, in formula (IA-1) or (IB-1) or their subgenus formulas, all four

[0452] tails of

[0453]

[0454] are different from each other.

[0455] -1— A - X - B

[0456] In some embodiments, at least one or two of the *» tails has both A and B absent, in which B may be H or C1-C24 branched or unbranched alkyl. In some embodiments, at -I— A - X - B

[0457] least one or two of the has both A and B absent, in which B may be H or Ci- C3 branched or unbranched alkyl.

[0458] In the above formulas, in each tail of

[0459]

[0460] , X is independently absent, X’,

[0461]

[0462] each R’ is independently C1-C5 alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and X’ is independently a biodegradable moiety. In some embodiments, each R’ is independently C1-C5 alkyl (e.g., methyl).

[0463] In some embodiments, X is absent. In some embodiments, X is X’. In some

[0464]

[0465] X’ is a biodegradable moiety as defined herein. Non-limiting examples of biodegradable

[0466]

[0467] and — N(R10)C(O)-(C(R11)2)S-S-S —. The definitions for the variables Y and V are the same as those described herein for formulas (IA) and / or formula (IB). Each R10is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. In some embodiments, each R10is independently H or C1-C3 alkyl. Each R11is independently branched or unbranched C1-C15 branched or unbranched alkyl, C1-C15 branched or unbranched alkenyl, or cycloalkyl or substituted cycloalkyl. In some embodiments, each R11is independently H or C1-C3 alkyl. Each s is independently 1, 2, 3, 4, or 5.

[0468] In some embodiments, each X’ is independently -OC(O)-, -C(O)O-, -OC(O)O-, -N(R10)C(O)-, -C(O)N(R10)-, -Y-V-OC(O), -Y-V-C(O)O-, -Y-V-N(R10)C(O)-, -Y-V-C(O)N(R10)-, -OC(O)-(C(R11)2)S-S-, -OC(O)-(C(R11)2)S-S-S-, -C(O)O-(C(R11)2)s-S-, -C(O)O-(C(R11)2)s-S-S-, -C(O)N(R10)-(C(R11)2)s-S-, -C(O)N(R10)-(C(R11)2)s-S-S-, -N(R10)C(O)-(C(R11)2)s-S-, -N(R10)C(O)-(C(R11)2)s-S-S-, or -S-S-. The definitions for the variables Y and V are the same as those described herein for formulas (IA-1), (IA-2), (IB-1), and / or (IB-2). Each R10is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl. In some embodiments, each R10is independently H or C1-C3 alkyl. Each R11is independently H or C1-C3 alkyl. Each s is independently 1, 2, 3, 4, or 5.

[0469] In some embodiments, each X’ is independently -OC(O)-, -C(O)O-, -OC(O)O-,

[0470]

[0471] -N(R10)C(O)-(CH2)s-S-S-, or -S-S-. R10and R11are each independently H or CH3. Each s is independently 1, 2, 3, 4, or 5 (e.g., 1 or 2).

[0472] -2— A - X - B

[0473] In all the above formulas, in each tail of, A is independently absent, C1-C16 branched or unbranched alkylene, or C2-Ci6 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups. In some embodiments, in the case of optional heteroatom interruption (or substitution), the interruption (or substitution) may take place in the backbone chain, e.g., a heteroatom (e.g., O, N, or S) may replace the carbon atom in the backbone of the alkylene or alkenylene chain. In some embodiments, in the case of optional substitution with one or more OH, SH, and / or halogen groups, the substituent group (e.g., OH, SH, and / or halogen group) may replace a hydrogen atom of the alkylene or alkenylene group. In some -5— A - X - B

[0474] embodiments, in the *» tail where A is not absent, each A is independently C1-C9

[0475] -1— A - X - B branched or unbranched alkylene. In some embodiments, in the tail where A is not absent, each A is independently C2-C? alkylene. In some embodiments, in the tail where A and X are not absent, each tail is represented by

[0476]

[0477] , wherein each n is independently 2 to 7.

[0478] In some embodiments, the compound has the formula (IA-2) or (IB-2), wherein W is H, OR6, or NHR6, wherein R6is H or C1-C12 alkyl.

[0479] In some embodiments, the compound has the structure of

[0480]

[0481] -,

[0482]

[0483] wherein:

[0484] each n is independently 2 to 7;

[0485]

[0486] or -S-S-;

[0487] R10and R11are each independently H or CH3; each s is independently 1, 2, 3, 4, or 5 (e.g., 1 or 2). The definitions for all the other variables are the same as those described herein for formulas (IA-1), (IA-2), (IB-1), and / or (IB-2).

[0488] In all the above formulas, each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups. In some embodiments, in the case of optional heteroatom interruption (or substitution), the interruption (or substitution) may take place in the backbone chain, e.g., a heteroatom (e.g., O, N, or S) may replace the carbon atom in the backbone of the alkyl, alkenyl, alkynyl, or unsaturated monovalent hydrocarbon chain. In some embodiments, in the case of optional substitution with one or more OH, SH, and / or halogen groups, the substituent group (e.g., OH, SH, and / or halogen group) may replace a hydrogen atom of the alkyl, alkenyl, alkynyl, or unsaturated monovalent hydrocarbon chain.

[0489]

[0490] In some embodiments, B has the structure of, wherein t is an integer from 0 to 5;

[0491] and u is an integer from 0 to 16. In some embodiments, the sum of t and u ranges from 6 to 18. In some embodiments, the sum of t and u ranges from 6 to 10, or from 7 to 9. In some embodiments, the sum of t and u ranges from 14 to 18, or from 15 to 17, or is 16.

[0492]

[0493]

[0494] some embodiments, t is 1, 2, or 3.

[0495] In some embodiments, B has the structure

[0496]

[0497] wherein:

[0498] nl is an integer from 1 to 5;

[0499] each of ul, u2, u3, and u4 is independently, for each occurrence, an integer from 0 to 10; each of R21, R22, R23, and R24is independently, for each occurrence, H or C1-C3 alkyl; and each R25is independently H, C1-C3 alkyl, or a saturated or unsaturated cyclic, optionally substituted with one or more alkyl groups.

[0500] In some embodiments, nl is 1, 2, 3, or 4. In some embodiments, each of R21, R22, R23, R24, and R25is H. In some embodiments, each of R21, R22, R23, and R25is H; for at least one occurrence, two R24are both H; and for at least one occurrence, one of the two R24is H and the other is methyl. In some embodiments, each of R22, R23, and R25is H; and for at least one occurrence, one of the two R24is H and the other is methyl. In some embodiments, each of R21, R22, R23, and R24is H; and R25is a unsaturated 5-6 membered cyclic (e.g., unsaturated 6-membered cyclic), optionally substituted with one or more methyl. In some embodiments, each of R21, R22, and R23is H; for at least one occurrence, one of the two R24is H and the other is methyl; and R25is a unsaturated 5-6 membered cyclic (e.g., unsaturated 6-membered cyclic), optionally substituted with one or more methyl. In some embodiments, each of R21, R22, and R23is H; for at least one occurrence, two R24are both H; for at least one occurrence, one of the two R24is H and the other is methyl; and R25is a unsaturated 5-6 membered cyclic (e.g., unsaturated 6-membered cyclic), optionally substituted with one or more methyl. In some embodiments, u2 and u3 are each 0. In some embodiments, the sum of u2 and u3 is 1, 2, or 3. In some embodiments, the sum of t and ul is 0 or 1; and u4 is 0. In some embodiments, the sum of t and ul ranges from 2 to 10, or from 4 to 8; and u4 ranges from 2 to 10, or from 4 to 8.

[0501] In some embodiments, B has the structure

[0502]

[0503] wherein:

[0504] nl is an integer from 1 to 5;

[0505] each of ul, u2, u3, and u4 is independently, for each occurrence, an integer from 0 to 10; each of R21, R22, R23, and R24is independently, for each occurrence, H or C1-C3 alkyl; and each R25is independently H, C1-C3 alkyl, or a saturated or unsaturated cyclic, optionally substituted with one or more alkyl groups.

[0506] In some embodiments, nl is 1. In some embodiments, each of R21, R22, R23, R24, and R25is H. In some embodiments, u2 and u3 are each 0. In some embodiments, the sum of t and ul ranges from 2 to 10, or from 4 to 8; and u4 ranges from 2 to 10, or from 4 to 8.

[0507] In some embodiments, each B is independently selected from the group consisting of:

[0508]

[0509]

[0510] from 7 to 16.

[0511] In some embodiments, the compound has the structure of formula (IIIA-1). Non-limiting 5 examples of the lipid compounds of formula (IIIA-1) are set forth in Table 1 below.

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519] In some embodiments, the compound has the structure of formula (IIIA-2). Non-limiting examples of the lipid compounds of formula (IIIA-2) are set forth in Table 2 below.

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527] In some embodiments, the compound has the structure of formula (IIIA-3). Non-limiting examples of the lipid compounds of formula (IIIA-3) are set forth in Table 3 below.

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534] In some embodiments, the compound has the structure of formula (IIIA-4). Non-limiting examples of the lipid compounds of formula (IIIA-4) are set forth in Table 4 below.

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543] Additional non-limiting examples of the lipid compounds disclosed here are set forth in Table 5 below.

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553] Additional non-limiting examples of the lipid compounds disclosed here are set forth in Tables 6-8 below.

[0554] 5 Table 6. Exemplary lipid compounds.

[0555]

[0556]

[0557]

[0558]

[0559] Table 7. Exemplary lipid compounds.

[0560]

[0561]

[0562]

[0563]

[0564]

[0565] Table 8. Exemplary lipid compounds.

[0566]

[0567] Lipid-based carrier / Lipid Composition

[0568] The lipid compounds disclosed herein may be used to form a lipid-based carrier or lipid composition (e.g., liposome or lipid nanoparticle). Thus, another aspect of the invention relates to a lipid-based carrier comprising a compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein.

[0569] As described herein, suitable compounds to be used in the lipid-based carrier (or lipid composition) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.

[0570] In some embodiments, the lipid-based carrier contains one or more compounds described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid-based carrier is a liposome or a lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is a LNP.

[0571] In addition to one or more compounds described herein, the lipid-based carrier may further include a second lipid. In some embodiments, the disclosure relates to a lipid-based carrier comprising (i) one or more compound chosen from the compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), the compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing and (ii) a second lipid. In some embodiments, the lipid-based carrier comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the one or more lipid compounds disclosed herein.

[0572] In some embodiments, the second lipid is cationic, non-cationic (e.g., neutral, anionic, or zwitterionic), or ionizable. In some embodiments, the lipid-based carrier comprises about a 1: 1 ratio of the lipid compound and the second lipid (e.g., a helper lipid). In some embodiments, the second lipid is a cationic lipid, anionic lipid, ionizable lipid, or zwitterionic lipid.

[0573] In some embodiments, one or more lipid components different than the lipid compounds described herein comprise one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component(s) different than the lipid compounds described herein comprise(s) one or more helper lipids, one or more PEG lipids, and one or more neutral lipids.

[0574] In some embodiments, the lipid-based carrier may further comprise a sterol and a PEG lipid. In some embodiments, the lipid-based carrier may further comprise a sterol, a PEGylated lipid, a phospholipid, and / or a neutral lipid.

[0575] In some embodiments, one or more naturally occurring and / or synthetic lipid compounds may be used in the preparation of the lipid-based carrier / lipid composition. The lipid-based carrier / lipid composition may contain negatively charged lipids, positively charged lipids, or a combination thereof.

[0576] THE OTHER LIPID COMPONENTS

[0577] Charged and neutral Lipids

[0578] Examples of suitable negatively charged (anionic) lipids include, but are not limited to dimyrystoyl-, dipalmitoyl-, and distearoyl-phasphatidylglycerol; dimyrystoyl-, dipalmitoyl-, and dipalmitoyl-phosphatidic acid; dimyrystoyl-, dipalmitoyl-, and dipalmitoylphosphatidylethanolamine; and their unsaturated diacyl and mixed acyl chain counterparts as well as cardiolipin.

[0579] Examples of positively charged (cationic) lipids include, but are not limited to, N, N'-dimethyl-N, N'-dioctacyl ammonium bromide (DDAB) and chloride (DDAC), N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), 3P-[N-(N', N'-dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), l,2-dioleoyloxy-3-[trimethylammonio]-propane (DOTAP), l,2-dioctadecyloxy-3-[trimethylammonio] -propane (DSTAP), and 1,2-dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design, pages 1-394, which is herein incorporated by reference in its entirety.

[0580] Additional exemplary cationic lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), N-(l-(2,3 -dioleyloxy )propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-Dioleoyl-3-Dimethylammonium-propane (DODAP), l,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP), l,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan- 4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3 -dimethyl- 1 -(cis, cis-9',12'-octadecadienoxy)propane (CpLin DMA), N, N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA) and / or a mixture thereof. The neutral lipid can comprise dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), and / or a mixture thereof.

[0581] In some embodiments, the lipid components comprise one or more neutral lipids. The neutral lipids may be one or more phospholipids, such as one or more (poly)unsaturated lipids. A phospholipid moiety may be a phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. A fatty acid moiety may be a lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid.

[0582] In some embodiments, the neutral lipids may be phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine(LPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, phosphatidylcholine, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1.2-dimyristoyl -sn-glycero-phosphocholine (DMPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), 1 -stearoyl -2-oleoyl- phosphatidylcholine (SOPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn- glycero-3 -phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytaiioyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids may be acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0583] Steroids and other non-ionizable lipid components

[0584] In some embodiments, the lipid components comprise one or more steroids or analogues thereof. In some embodiments, the lipid components comprise sterols such as cholesterol, sitosterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.

[0585] In some embodiments, the lipid components comprise or consist of a mixture of one or more phospholipids and cholesterol or a derivative thereof. In some embodiments, the lipid components comprise or consist of one or more phospholipids, e.g., a cholesterol-free lipid formulation. In some embodiments, the lipid components comprise or consist of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid formulation.

[0586] In some embodiments, the lipid components comprise a phytosterol, or its combination with cholesterol. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol, stigmasterol, b-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175 and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S-160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175, and combinations thereof. In some embodiments, the phytosterol is a combination of Compound S-141, Compound S-140, Compound S-143 and Compound S-148. The detailed structures for Compounds S-140, S-141, S-143, S-148, S-151, S-156, S-157, S-159, S-160, S-164, S-165, S-170, S-173, and S-175 are those disclosed in Tables 1-2, 7, 13 and 15 in US 2019 / 0314291, the entire content of which is incorporated herein by reference in its entirety. In some embodiments, the phytosterol comprises a sitosterol, or a salt or ester thereof. In some embodiments, the phytosterol comprises a stigmasterol, or a salt or ester thereof. In some embodiments, the phytosterol is beta-sitosterol, or a salt or ester thereof.

[0587] In some embodiments, the lipid components of the lipid carrier comprises a phytosterol, or a salt or ester thereof; and / or cholesterol or a salt thereof.

[0588] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a splenic cell), and the phytosterol is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, the salt or ester thereof and combinations thereof. In some embodiments, the phytosterol is b-sitosterol. In some embodiments, the phytosterol is b-sitostanol. In some embodiments, the phytosterol is campesterol. In some embodiments, the phytosterol is brassicasterol.

[0589] In some embodiments, the target cell is a cell described herein (e.g., a liver cell or a splenic cell), and the phytosterol is selected from the group consisting of b-sitosterol, stigmasterol, the salt or ester thereof, and combinations thereof. In some embodiments, the phytosterol is b-sitosterol. In some embodiments, the phytosterol is stigmasterol.

[0590] Other examples of non-ionizable lipids include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, and sphingomyelin.

[0591] In some embodiments, these non-ionizable lipids comprise from 10 mol % to 60 mol %, from 20 mol % to 55 mol %, from 20 mol % to 45 mol %, 20 mol % to 40 mol %, from 25 mol % to 50 mol %, from 25 mol % to 45 mol %, from 30 mol % to 50 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 35 mol % to 45 mol %, from 37 mol % to 42 mol %, or 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0592] In embodiments where the lipid-based carrier / lipid composition contain a mixture of phospholipid and cholesterol or a cholesterol derivative, the mixture may comprise up to 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the lipid composition.

[0593] In some embodiments, the phospholipid component in the mixture may comprise from 2 mol % to 20 mol %, from 2 mol % to 15 mol %, from 2 mol % to 12 mol %, from 4 mol % to 15 mol %, or from 4 mol % to 10 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition. In some embodiments, the phospholipid component in the mixture comprises from 5 mol % to 10 mol %, from 5 mol % to 9 mol %, from 5 mol % to 8 mol %, from 6 mol % to 9 mol %, from 6 mol % to 8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0594] In some embodiments, the cholesterol component in the mixture may comprise from 25 mol % to 45 mol %, from 25 mol % to 40 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 27 mol % to 37 mol %, from 25 mol % to 30 mol %, or from 35 mol % to 40 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition. In some embodiments, the cholesterol component in the mixture comprises from 25 mol % to 35 mol %, from 27 mol % to 35 mol %, from 29 mol % to 35 mol %, from 30 mol % to 35 mol %, from 30 mol % to 34 mol %, from 31 mol % to 33 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0595] In embodiments where the lipid-based carrier / lipid compositions are phospholipid-free, the cholesterol or derivative thereof may comprise up to 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the lipid composition.

[0596] In some embodiments, the cholesterol or derivative thereof in the phospholipid-free lipid formulation may comprise from 25 mol % to 45 mol %, from 25 mol % to 40 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 31 mol % to 39 mol %, from 32 mol % to 38 mol %, from 33 mol % to 37 mol %, from 35 mol % to 45 mol %, from 30 mol % to 35 mol %, from 35 mol % to 40 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0597] In some embodiments, these non-ionizable lipids comprise from 5 mol % to 90 mol %, from 10 mol % to 85 mol %, from 20 mol % to 80 mol %, 10 mol % (e.g., phospholipid only), or 60 mol % (e.g., phospholipid and cholesterol or derivative thereof) (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0598] The percentage of these non-ionizable lipids present in the lipid composition is a target amount, and that the actual amount of non-ionizable lipid present in the lipid composition may vary, for example, by ± 5 mol %.

[0599] Lipid conjugates

[0600] The lipid-based carrier / lipid compositions may further comprise one or more lipid conjugates. Non-limiting examples of conjugated lipids include PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof.

[0601] In some embodiments, the lipid conjugate is a PEG-lipid or PEG-modified lipid (alternatively referred to as PEGylated lipid). A PEG lipid is a lipid modified with polyethylene glycol. Examples of PEG- lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DEG), PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides (PEG-CER), PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0602] In some embodiments, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and a PEG-modified di alkylglycerol.

[0603] In some embodiments, the PEG-lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-[amino(polyethy lene glycol)] (PEG-DSPE), PEG-di steryl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0604] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from 550 Daltons to 10,000 Daltons. In certain instances, the PEG moiety has an average molecular weight of from 750 Daltons to 5,000 Daltons (e.g., from 1,000 Daltons to 5,000 Daltons, from 1,500 Daltons to 3,000 Daltons, from 750 Daltons to 3,000 Daltons, from 750 Daltons to 2,000 Daltons). In some embodiments, the PEG moiety has an average molecular weight of 2,000 Daltons or 750 Daltons.

[0605] In certain instances, the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group. The PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester-containing linker moieties and ester-containing linker moieties. In some embodiments, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulphide (-S-S-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety). In some embodiments, a carbamate linker is used to couple the PEG to the lipid.

[0606] In some embodiments, an ester-containing linker moiety is used to couple the PEG to the lipid. Suitable ester-containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.

[0607] Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate. Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skill in the art.

[0608] In some embodiments, the PEG-lipid is phosphatidylethanolamine, e.g., phosphatidylethanolamines with mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids with carbon chain lengths in the range of CIO to C20. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0609] In some embodiments, the PEG-lipid is PEG-DAA conjugate, which is a PEG-didecyloxypropyl (CIO) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, aPEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxy propyl (C16) conjugate, or aPEG-distearyloxy propyl (Cl 8) conjugate. In some embodiments, the PEG has an average molecular weight of 750 or 2,000 Daltons. In some embodiments, the terminal hydroxyl group of the PEG is substituted with a methyl group.

[0610] In addition to the foregoing, other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxy ethylcellulose.

[0611] In some embodiments, the incorporation of any of the above-discussed PEG-lipids in the lipid composition can improve the pharmacokinetics and / or biodistribution of the lipid composition. For example, incorporation of any of the above-discussed PEG-lipids m the lipid composition can reduce the accelerated blood clearance (ABC) effect.

[0612] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from 0.1 mol % to 2 mol %, from 0.5 mol % to 2 mol %, from 1 mol % to 2 mol %, from 0.6 mol % to 1.9 mol %, from 0.7 mol % to 1.8 mol %, from 0.8 mol % to 1.7 mol %, from 0.9 mol % to 1.6 mol %, from 0.9 mol % to 1.8 mol %, from 1 mol % to 1.8 mol %, from 1 mol % to 1.7 mol %, from 1.2 mol % to 1.8 mol %, from 1.2 mol % to 1.7 mol %, from 1.3 mol % to 1.6 mol %, or from 1.4 mol % to 1.5 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition. In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from 0 mol % to 20 mol %, from 0.5 mol % to 20 mol %, from 2 mol % to 20 mol %, from 1.5 mol % to 18 mol %, from 2 mol % to 15 mol %, from 4 mol % to 15 mol %, from 2 mol % to 12 mol %, from 5 mol % to 12 mol %, or 2 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0613] In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprises from 4 mol % to 10 mol %, from 5 mol % to 10 mol %, from 5 mol % to 9 mol %, from 5 mol % to 8 mol %, from 6 mol % to 9 mol %, from 6 mol % to 8 mol %, or 5 mol %, 6 mol %, 7 mol%, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the lipid composition.

[0614] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid composition is a target amount, and the actual amount of lipid conjugate present in the composition may vary, for example, by ± 2 mol %. By controlling the composition and concentration of the lipid conjugate, one can control the rate at which the lipid conjugate exchanges out of the lipid-based carrier / lipid composition and, in turn, the rate at which the lipid-based carrier / lipid composition becomes fusogenic. In addition, other variables including, e.g., pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid-based carrier / lipid composition becomes fusogenic. Other methods which can be used to control the rate at which the lipid-based carrier / lipid composition becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, one can control the particle size of the lipid composition.

[0615] Other lipids

[0616] In some embodiments, the lipid-based carrier / lipid composition may comprise one or more other lipids, such as an ionizable lipid.

[0617] In one embodiment, the lipid-based carrier / lipid composition may comprise heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US Patent No. 9,867,888, which is incorporated by reference herein in its entirety.

[0618] In one embodiment, the lipid-based carrier / lipid composition may comprise 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-di enoate (LP01), e.g., as synthesized in Example 13 of WO 2015 / 095340, which is incorporated by reference herein in its entirety. In one embodiment, the lipid-based carrier / lipid composition may comprise Di((Z)-non-2-en-l-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Example 7, 8, or 9 of US 2012 / 0027803, which is incorporated by reference herein in its entirety.

[0619] In one embodiment, the lipid-based carrier / lipid composition may comprise l,l'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2 -hydroxy dodecyl) amino)ethyl)piperazin- 1 -yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO 2010 / 053572, which is incorporated by reference herein in its entirety.

[0620] In one embodiment, the lipid-based carrier / lipid composition may comprise Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13 -dimethyl- 17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, e.g., Structure (I) from WO 2020 / 106946, which is incorporated by reference herein in its entirety.

[0621] In one embodiment, the lipid-based carrier / lipid composition may comprise MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO 2019 / 051289A9, which is incorporated by reference herein in its entirety.

[0622] In one embodiment, the lipid-based carrier / lipid composition may comprise ATX-002, e.g., as described in Example 10 of WO 2019 / 051289A9, which incorporated by reference herein in its entirety.

[0623] In one embodiment, the lipid-based carrier / lipid composition may comprise (13ZJ6Z)-A, A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO 2019 / 051289A9, which is incorporated by reference herein in its entirety.

[0624] In one embodiment, the lipid-based carrier / lipid composition may comprise Compound 6 or Compound 22, e.g., as described in Example 12 of WO 2019 / 051289A9, which is incorporated by reference herein in its entirety.

[0625] Examples of additional ionizable lipids that can be included in the lipid-based carrier / lipid composition include SM-86, ALC-0315, Lipid 10, Acuitas Lipid 9, and Acuitas Lipid 10 (see WO 2017 / 004143 Al, which is incorporated herein by reference in its entirety, as well as the compounds listed in Table 1 of WO 2019 / 051289, which is incorporated herein by reference.

[0626] The lipid-based carrier / lipid composition may contain 30-70% the lipid compound described herein, 0-60 % cholesterol, 0-30% phospholipid, and 1-10% polyethylene glycol (PEG). In some embodiments, the lipid-based carrier / lipid composition comprises 30-40% the lipid compound described herein, 40-50% cholesterol, and 10-20% PEG. In some embodiments, the lipid-based carrier / lipid composition comprises 50-75% the lipid compound described herein, 20-40% cholesterol, and 5-10% phospholipid, and 1-10% PEG. The lipid-based carrier / lipid composition may contain 60-70% the lipid compound described herein, 25-35% cholesterol, and 5-10% PEG.

[0627] In some embodiments, the lipid component of the lipid composition includes about 30 mol% to about 60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) the lipid compound described herein, about 0 mol% to about 30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15 mol% to about 50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0 mol% to about 10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid, provided that the total mol% of the lipid component does not exceed 100%.

[0628] In some embodiments, the lipid-based carrier / lipid composition may contain up to 90% lipid compound described herein, and 2-15% helper lipid.

[0629] The lipid-based carrier / lipid composition may be a lipid particle composition, for example containing 8-30% the lipid compound described herein, 5-30% helper lipid, and 0-20% cholesterol; 4-25% the lipid compound described herein, 4-25% helper lipid, 2- 25% cholesterol, 10- 35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% the lipid compound described herein, 2-30% helper lipid, 1- 15% cholesterol, 2- 35% cholesterol-PEG, and 1-20% cholesterolamine; or up to 90% the lipid compound described herein and 2-10% helper lipids, or even 100% the lipid compound described herein.

[0630] OTHER COMPONENTS FOR THE LIPID-BASED CARRIER / LIPID COMPOSITION

[0631] The lipid-based carrier / lipid composition may include one or more components in addition to those lipid components described above. For example, lipid-based carrier / lipid composition may include one or more small hydrophobic molecules such as a vitamin (e.g., vitamin A or vitamin E).

[0632] The lipid-based carrier / lipid composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents, or other components.

[0633] Suitable carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0634] A polymer may be used to encapsulate or partially encapsulate a nanoparticle composition. The polymer may be biodegradable and / or biocompatible. Suitable polymers include, but are not limited to, polyamines, polyethers,, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes,

[0635] poly acetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and poly arylates For example, a polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(gly colic acid) (PGA), poly(lactic acid-co-gly colic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D, L-lactide) (PDLA), poly(L-lactide) (PLEA), poly(D, L-lactide-co-caprolactone), poly(D, L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates,

[0636] poly alkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), poly siloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly (lauryl(meth)acrylate), poly (phenyl(meth)acry late), poly (methyl aery 1 ate), poly (isopropy 1 acrylate), poly(isobutyl acrylate), poly (octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholme) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and poly glycerol.

[0637] Suitable surface altering agents include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecylammoniumbromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carboci steine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, domase alfa, neltenexine, and erdosteine), and DNases (e.g., rhDNase). A surface altering agent may be disposed within a lipid nanoparticle and / or on the surface of a lipid nanoparticle (e.g., by coating, adsorption, covalent linkage, or other process).

[0638] The lipid-based carrier / lipid composition may also comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, cellular recognition, or imaging. The surface of a lipid nanoparticle may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging, and membrane permeation are well known in the art.

[0639] The lipid-based carrier / lipid composition may include any substance useful in pharmaceutical compositions. For example, the lipid nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrates, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included.

[0640] Suitable diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof

[0641] Suitable surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrus, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxy ethylene ethers, (e.g polyoxyethylene lauryl ether [BRU® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0642] Suitable binding agents may be starch (e.g cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethy lene oxide; polyethylene glycol; inorganic calcium salts, silicic acid; polymethacrylates, waxes, water; alcohol; and combinations thereof, or any other suitable binding agent.

[0643] Suitable preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothiogly cerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidme, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenyl ethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0644] Suitable lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0645] Suitable oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0646] In some embodiments, the lipid-based carrier / lipid composition further comprises one or more cryoprotectants. Suitable cryoprotective agents include, but are not limited to, a polyol (e.g., a diol or a triol such as propylene glycol (i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+ / - )-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol, 2,3 -butanediol, ethylene glycol, or diethylene glycol), a nondetergent sulfobetaine (e.g., NDSB-201 (3 -(l-pyridino)-l -propane sulfonate), an osmolyte (e.g., L-proline or trimethylamine N-oxide dihydrate), a polymer (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEGzk-DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG 20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG 5000, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K 15), pentaerythritol propoxylate, or polypropylene glycol P 400), an organic solvent (e.g., dimethyl sulfoxide (DMSO) or ethanol), a sugar (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, meso-erythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or a salt (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof), or any combination thereof.

[0647] In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the cryoprotectant and / or excipient is sucrose. In some embodiments, the cryoprotectant comprises sodium acetate. In some embodiments, the cryoprotectant and / or excipient is sodium acetate. In some embodiments, the cryoprotectant comprises sucrose and sodium acetate.

[0648] In some embodiments, the lipid-based carrier / lipid composition further comprises one or more buffers. Suitable buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-glucomc acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0649] In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, a tris buffer, or combinations thereof.

[0650] Therapeutic agent-loaded lipid compositions and pharmaceutical compositions

[0651] In some embodiments, provided herein are a therapeutic agent-loaded lipid-based carrier comprising:

[0652] a therapeutic agent;

[0653] one or more compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein; a helper lipid;

[0654] a sterol; and

[0655] a PEG-modified lipid.

[0656] Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or formula (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, and a pharmaceutically acceptable excipient. Certain aspect of the invention also relates to a pharmaceutical composition comprising the lipid-based carriers as described herein, and a pharmaceutically acceptable excipient.

[0657] The pharmaceutical composition may further comprise a therapeutic agent.

[0658] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, and a lipid composition comprising:

[0659] a therapeutic agent;

[0660] 35-65 mol % of one or more compound of formula (IA-1), (IA-2), (IB-1), or (IB-2), compound of the subgenus formulas of formula (IA-1), (IA-2), (IB-1), or (IB), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein;

[0661] 3-12 mol % of a helper lipid

[0662] 15-45 mol % of a sterol; and

[0663] 0.5-10 mol % of a PEG-modified lipid.

[0664] All above descriptions and all embodiments discussed in the above aspects relating to the lipid compounds, and the exemplary variables and compounds are all applicable to these aspects of the invention relating to the pharmaceutical composition.

[0665] All above descriptions and all embodiments discussed in the above aspects relating to the lipid-based carrier / lipid composition, including various other lipid components, are applicable to these aspects of the invention relating to the pharmaceutical composition.

[0666] In the lipid composition / pharmaceutical composition containing the therapeutic agent, the ratio of total lipid components to the therapeutic agent (e.g., an encapsulated therapeutic agent such as a nucleic acid) can be varied as desired. For example, the total lipid components to the therapeutic agent (mass or weight) ratio can be from about 10: 1 to about 30: 1. In some embodiments, the total lipid components to the therapeutic agent ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3: 1 to about 15:1, from about 4: 1 to about 10:1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9:1. The amounts of total lipid components and the therapeutic agent can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 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, or higher. Generally, the lipid composition’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0667] In some embodiments, the total therapeutic agent administered to the subject has a spleen to liver ratio of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the total therapeutic agent administered to the subject has a spleen to liver ratio of at least 1. In some embodiments, the total therapeutic agent administered to the subject has spleen to liver ratio of at least 5.

[0668] THERAPEUTIC AGENTS

[0669] Nucleic acid molecule

[0670] In some embodiments, the lipid composition / pharmaceutical composition further comprises one or more nucleic acid components. The nucleic acid component may be a plasmid, an immunostimulatory oligonucleotide, an antisense oligonucleotide, an antagomir, an aptamer, a deoxyribozyme (DNAzyme), or a ribozyme.

[0671] In some embodiments, the lipid composition / pharmaceutical composition further comprises one or more RNA and / or DNA components.

[0672] In some embodiments, the lipid composition / pharmaceutical composition further comprises one or more DNA components. In some embodiments, the DNA is linear DNA, circular DNA, single stranded DNA, or double stranded DNA. The DNA component may be in the form of antisense molecules, plasmid DNA, cDNA, DNAzyme, PCR products, or vectors.

[0673] In some embodiments, the lipid composition / pharmaceutical composition further comprises one or more RNA components. In some embodiments, the RNA is linear RNA, circular RNA, single stranded RNA, or double stranded RNA.

[0674] In some embodiments, the RNA is mRNA, microRNA (miRNA) or miRNA precursor, siRNA or siRNA precursor, RNA aptamer, linear RNA, circular RNA (circRNA), single stranded RNA, double stranded RNA, antisense RNA, tRNA, multivalent RNA, Dicer substrate RNA, Dicer substrate small interfering RNA, viral RNA, self-amplifying RNA, a short hairpin RNA, an asymmetric interfering RNA, a guide RNA, ribosomal RNA, long non-coding RNA, non-coding RNA, small noncoding RNA, small nuclear RNA, piwi-interacting RNA, small nucleolar RNA, small nuclear RNA, small cajal body-specific RNA, extracellular RNA, Y RNA, or heterogeneous nuclear RNA.

[0675] In one embodiment, the therapeutic agent is an mRNA (messenger RNA).

[0676] In one embodiment, the therapeutic agent is a miRNA (microRNA) or miRNA precursor. In one embodiment, the therapeutic agent is a siRNA (small interfering RNA) or siRNA precursor. In one embodiment, the therapeutic agent is a RNA aptamer.

[0677] In one embodiment, the therapeutic agent is a linear RNA.

[0678] In one embodiment, the therapeutic agent is a circular RNA (circRNA), e.g., a circular RNA encoding a therapeutic polypeptide, or a non-coding circular RNA.

[0679] In one embodiment, the therapeutic agent is a single stranded RNA or double stranded RNA.

[0680] In one embodiment, the therapeutic agent is an antisense RNA.

[0681] In one embodiment, the therapeutic agent is a tRNA (transfer RNA).

[0682] In one embodiment, the therapeutic agent is a multivalent RNA.

[0683] In one embodiment, the therapeutic agent is a Dicer substrate RNA or Dicer substrate small interfering RNA (dsiRNA).

[0684] In one embodiment, the therapeutic agent is a viral RNA (vRNA).

[0685] In one embodiment, the therapeutic agent is a self-amplifying RNA.

[0686] In one embodiment, the therapeutic agent is a short hairpin RNA (shRNA).

[0687] In one embodiment, the therapeutic agent is an asymmetric interfering RNA (aiRNA). In one embodiment, the therapeutic agent is a guide RNA (gRNA).

[0688] In one embodiment, the therapeutic agent is a rRNA (ribosomal RNA).

[0689] In one embodiment, the therapeutic agent is a IncRNA (long non-coding RNA).

[0690] In one embodiment, the therapeutic agent is a snRNA (small nuclear RNA).

[0691] In one embodiment, the therapeutic agent is a ncRNA (non-coding RNA).

[0692] In one embodiment, the therapeutic agent is a sncRNA (small noncoding RNA).

[0693] In one embodiment, the therapeutic agent is a snoRNA (small nucleolar RNA).

[0694] In one embodiment, the therapeutic agent is a piRNA (piwi-interacting RNA).

[0695] In one embodiment, the therapeutic agent is a scaRNA (small cajal body-specific RNA). In one embodiment, the therapeutic agent is an exRNA (extracellular RNA).

[0696] In one embodiment, the therapeutic agent is a Y RNA (small non-coding RNAs that are components of the R06O ribonucleoprotein particle).

[0697] In one embodiment, the therapeutic agent is a hnRNA (heterogeneous nuclear RNA). In some embodiments, the therapeutic agent is a circular polyribonucleotide (e.g., a circular RNA, circRNA), including one or more expression sequences encoding a polypeptide. The polyribonucleotide may include an IRES (Internal Ribosome Entry Site) operably linked to an expression sequence encoding a polypeptide. The circular polyribonucleotide may include a splice junction, e.g., joining a 5’ exon fragment and a 3’ exon fragment. The circular polyribonucleotide may include any one or more of the elements described herein. In some embodiments, the circular polyribonucleotide includes any feature, or any combination of features as disclosed in International Patent Publication Nos. WO2019 / 118919, WO 2020 / 023655, WO 2020 / 180751, WO 2020 / 180752, WO 2020 / 181013, WO 2020 / 198403, WO 2020 / 257730, WO 2020 / 257727, WO 2020 / 252436, each of which is hereby incorporated by reference in its entirety. Circular polyribonucleotide may be less susceptible to degradation by exonuclease as compared to linear RNA. As such, the circular polyribonucleotide can be more stable than a linear RNA, especially when incubated in the presence of an exonuclease. The increased stability of the circular polyribonucleotide compared with linear RNA makes circular polyribonucleotide more useful as a cell transforming reagent to produce polypeptides and can be stored more easily and for longer than linear RNA.

[0698] In some embodiments, the nucleic acid molecule is an enzymatic nucleic acid molecule. The term “enzymatic nucleic acid molecule” refers to a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.

[0699] In some embodiments, the nucleic acid molecule is an antisense nucleic acid. The term “antisense nucleic acid” refers to a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid) interactions and alters the activity of the target RNA.

[0700] In some embodiments, the nucleic acid molecule may be a 2-5A antisense chimera. The term “2-5 A antisense chimera” refers to an antisense oligonucleotide containing a 5'-phosphorylated 2'-5 '-linked adenylate residue.

[0701] In some embodiments, the nucleic acid molecule may be a triplex forming oligonucleotide. The term “triplex forming oligonucleotide” refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix.

[0702] In some embodiments, the nucleic acid molecule may be a decoy RNA. The term “decoy RNA” refers to a RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule.

[0703] In some embodiments, the nucleic acid molecule (e.g., RNA or DNA) encodes a therapeutic peptide or polypeptide, operably linked to a promoter for a DNA. The therapeutic peptide or polypeptide may be, e.g., a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e.g., a nuclease element of a CRISPR system, e.g., a Cas9, dCas9, aCas9-nickase, Cpf / Casl2a); a Crispr-linked enzyme, e.g., a base editor or prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a Gene Writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody; a receptor ligand; a receptor; a clotting factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder such as a centyrin, darpin, or adnectin.

[0704] In some embodiments, the one or more RNA components comprise a gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is a gRNA.

[0705] In some embodiments, the one or more RNA components comprise a Class 2 Cas nuclease mRNA and a gRNA. In some embodiments, the gRNA nucleic acid is or encodes a dual-guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is or encodes a singleguide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA. In some embodiments, the modified gRNA comprises a modification at one or more of the first five nucleotides at a 5’ end. In some embodiments, the modified gRNA comprises a modification at one or more of the last five nucleotides at a 3’ end.

[0706] In some embodiments, the one or more RNA components comprise an mRNA. In some embodiments, the one or more RNA components comprise an RNA-guided DNA-binding agent, for example a Cas nuclease mRNA (such as a Class 2 Cas nuclease mRNA) or a Cas9 nuclease mRNA.

[0707] All the nucleic acid molecules described herein can be chemically modified. The various modification strategy to the nucleic acid molecules are well known to one skilled in the art. In some embodiments, the nucleic acid molecule comprises one or more modifications selected from the group consisting of pseudouridine, 5 -bromouracil, 5-methylcytosine, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g. rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. In some embodiments, the antisense oligonucleotide may be a locked nucleic acid oligonucleotide (LNA). The term “locked nucleic acid (LNA)” refers to oligonucleotides that contain one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation (Grunweller A, Hartmann RK, BioDrugs, 21(4): 235-243 (2007)). In some embodiments, the lipid composition / pharmaceutical composition further comprises one or more template nucleic acids.

[0708] Additional examples of the nucleic acid molecules (including tumor suppressor genes, antisense oligonucleotides, siRNA, miRNA, or shRNA) may be found in U. S. Published Patent Application No. 2007 / 0065499 and U. S. Patent No. 7,780,882, which are incorporated by reference herein in their entireties.

[0709] In some embodiments, the lipid composition / pharmaceutical composition can include a plurality of nucleic acid molecules, which may be the same or different types.

[0710] Nuclei c acids for use with embodiments of this disclosure may be prepared according to any available technique. For mRNA, the primary methodology of preparation is, but not limited to, enzymatic synthesis (also termed in vitro transcription) which currently represents the most efficient method to produce long sequence-specific mRNA. In vitro transcription describes a process of template-directed synthesis of RNA molecules from an engineered DNA template comprised of an upstream bacteriophage promoter sequence (e.g., including but not limited to that from the T7, T3 and SP6 coliphage) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from a number of sources with appropriate techniques which are well known in the art including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, J. L and Conn, G. L., General protocols for preparation of plasmid DNA template and Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn GL. (ed), New York, N. Y. Humana Press, 2012).

[0711] Transcription of the RNA occurs in vitro using the linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine and cyti dine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resultant mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits including, but not limited to RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies) as well as with commercially available reagents including RNA polymerases and rNTPs. The methodology for in vitro transcription of mRNA is well known in the art. (see, e.g. Losick, R., 1972, In vitro transcription, Ann Rev Biochem v.41 409-46; Kamakaka, R. T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2: 11.6: 11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, N. Y. Humana Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0712] The desired in vitro transcribed mRNA may be purified from the undesired components of the transcription or associated reactions (including unincorporated rNTPs, protein enzyme, salts, short RNA oligos, etc.). Techniques for the isolation of the mRNA transcripts are well known in the art. Well known procedures include, for non-limiting examples, phenol / chloroform extraction or precipitation with either alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0713] Additional, non-limiting examples of purification procedures which can be used include size exclusion chromatography (Lukavsky, P. J. and Puglisi, J. D., 2004, Large-scale preparation and purification of polyacrylamide-free RN A oligonucleotides, RNA v.10, 889-893), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed), New York, N. Y. Humana Press, 2012). Purification can be performed using a variety of commercially available kits including, but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0714] Furthermore, while reverse transcription can yield large quantities of mRNA, the products can contain a number of aberrant RNA impurities associated with undesired polymerase activity which may need to be removed from the full-length mRNA preparation. These include short RNAs that result from abortive transcription initiation as well as double-stranded RNA (dsRNA) generated by RNA-dependent RNA polymerase activity, RNA-primed transcription from RNA templates and self-complementary 3' extension. It has been demonstrated that these contaminants with dsRNA structures can lead to undesired immunostimulatory activity through interaction with various innate immune sensors in eukaryotic cells that function to recognize specific nucleic acid structures and induce potent immune responses. This in turn, can dramatically reduce mRNA translation since protein synthesis is reduced during the innate cellular immune response.

[0715] Therefore, additional techniques to remove these dsRNA contaminants have been developed and are known in the art including but not limited to scaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H, Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 el42; Weissman, D., Pardi, N., Muramatsu, H, and Kariko, K., HPLC Purification of in vitro transcribed long RNA in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013). HPLC purified mRNA has been reported to be translated at much greater levels, particularly in primary cells and in vivo.

[0716] A significant variety of modifications have been described in the art which are used to alter specific properties of in vitro transcribed mRNA, and may improve its utility’. These include, but are not limited to modifications to the 5' and 3' termini of the mRNA. Endogenous eukaryotic mRNA typically contain a cap structure on the 5'-end of a mature molecule which plays an important role in medi ating binding of the mRNA Cap Binding Protein (CBP), which is in turn responsible for enhancing mRNA stability in the cell and efficiency of mRNA translation.

[0717] Therefore, highest levels of protein expression are achieved with capped mRNA transcripts. The 5 ’-cap contains a 5 ’-5 '-triphosphate linkage between the 5 '-most nucleotide and guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the ultimate and penultimate most 5 '-nucleotides on the 2'-hydroxyl group.

[0718] Multiple distinct cap structures can be used to generate the 5 '-cap of in vitro transcribed synthetic mRNA. 5 '-capping of synthetic mRNA can be performed co-transcriptionally with chemical cap analogs (i.e., capping during in vitro transcription). For example, the Anti -Reverse Cap Analog (ARC A) cap contains a 5 '-5 '-triphosphate guanine-guanine linkage where one guanine contains an N7 methyl group as well as a 3'-0-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcnptional process and the synthetic cap analog is not identical to the 5 '-cap structure of an authentic cellular mRNA, potentially reducing translatability’ and cellular stability. Alternatively, synthetic mRNA molecules may also be enzymatically capped post-transcriptionally. These may generate a more authentic 5 '-cap structure that more closely mimics, either structurally or functionally, the endogenous 5 '-cap which have enhanced binding of cap binding proteins, increased half-life and reduced susceptibility to 5' endonucleases and / or reduced 5’ decapping. Numerous synthetic 5'-cap analogs have been developed and are known in the art to enhance mRNA stability and translatability’ (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology’ v.969 (Rabinovich, P. H. Ed), 2013).

[0719] On the 3 '-terminus, a long chain of adenine nucleotides (poly-A tail) is normally added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved to free a 3' hydroxyl to which poly-A polymerase adds a chain of adenine nucleotides to the RNA in a process called polyadenylation. The poly- A tail has been extensively shown to enhance both translational efficiency and stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A), poly (A) binding protein and the regulation of mRNA stability, Trends Bio Sci v. 14373-377; Guhaniyogi, J. And Brewer, G, 2001, Regulation of mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M. And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard in eukaryotes, scavenger in bacteria, Cell, v. 11, 611-613).

[0720] Poly (A) tailing of in vitro transcribed mRNA can be achieved using various approaches including, but not limited to, cloning of a poly (T) tract into the DNA template or by post-transcriptional addition using Poly (A) polymerase. The first case allows in vitro transcription of mRNA with poly (A) tails of defined length, depending on the size of the poly (T) tract, but requires additional manipulation of the template. The latter case involves the enzymatic addition of a poly (A) tail to in vitro transcribed mRNA using poly (A) polymerase which catalyzes the incorporation of adenine residues onto the 3 'termini of RNA, requiring no additional manipulation of the DNA template, but results in mRNA with poly(A) tails of heterogeneous length. 5 '-capping and 3 '-poly (A) tailing can be performed using a variety of commercially available kits including, but not limited to Poly (A) Polymerase Tailing kit (EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit (Life Technologies) as well as with commercially available reagents, various ARCA caps, Poly (A) polymerase, etc.

[0721] In addition to 5' cap and 3' poly adenylation, other modifications of the in vitro transcripts have been reported to provide benefits as related to efficiency of translation and stability. It is well known in the art that pathogenic DNA and RNA can be recognized by a variety' of sensors within eukaryotes and trigger potent innate immune responses. The ability to discriminate between pathogenic and self DNA and RNA has been shown to be based, at least in part, on structure and nucleoside modifications since most nucleic acids from natural sources contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications, thus rendering it immunostimulatory which in turn can inhibit effective mRNA translation as outlined above. The introduction of modified nucleosides into in vitro transcribed m NA can be used to prevent recognition and activation of RNA sensors, thus mitigating this undesired immunostimulatory activity and enhancing translation capacity (see, e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: implication for therapeutic RNA development, Curr Opin Drug Discov Devel, v.lO 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko, K, In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013; Kariko, K, Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman, D., 2008, Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability, Mol Ther v.16, 1833-1840). The modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared monitored and utilized using general methods and procedures known in the art. A large variety of nucleoside modifications are available that may be incorporated alone or in combination with other modified nucleosides to some extent into the in vitro transcribed mRNA (see, e.g., US2012 / 0251618). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immune sensors with a concomitant enhanced translational capacity.

[0722] Other components of mRNA which can be modified to provide benefit in terms of translatability and stability include the 5' and 3' untranslated regions (UTR). Optimization of the UTRs (favorable 5' and 3' UTRs can be obtained from cellular or viral RNAs), either both or independently, have been shown to increase mRNA stability and translational efficiency of in vitro transcribed mRNA (see, e.g., Pardi, N., Muramatsu, II., Weissman, D., Kariko, K., In vitro transcription of long RNA containing modified nucleosides in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, P. H. Ed), 2013).

[0723] In addition to mRNA, other nucleic acid payloads may be used for this disclosure. For oligonucleoti des, methods of preparation include but are not limited to chemical synthesis and enzymatic, chemical cleavage of a longer precursor, in vitro transcription as described above, etc. Methods of synthesizing DNA and RN A nucleotides are widely used and well known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: apractical approach, Oxford [Oxfordshire], Ishington, D C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N. J.) Totowa, N. J.: Humana Press, 2005; both of which are incorporated herein by reference).

[0724] For plasmid DNA, preparation for use with embodiments of this disclosure commonly utilizes, but is not limited to, expansion and isolation of the plasmid DNA in vitro in a liquid culture of bacteria containing the plasmid of interest. The presence of a gene in the plasmid of interest that encodes resistance to a particular antibiotic (penicillin, kanamycin, etc.) allows those bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods of isolating plasmid DNA are widely used and well known in the art (see, e.g., Heilig, J., Elbing, K. L. and Brent, R., (2001), Large-Scale Preparation of Plasmid DNA, Current Protocols in Molecular Biology, 41:11: 1.7: 1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillstrom, S., Bjomestedt, R. and Schmidt, S. R., (2008), Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture, Biotechnol. Bioeng., 99: 557-566; and US 6, 197,553 Bl ). Plasmid isolation can be performed using a variety of commercially available kits including, but not limited to Plasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and Pure Yield MaxiPrep (Promega) kits as well as with commercially available reagents.

[0725] In some embodiments, the lipid compositions / pharmaceutical compositions are useful for expression of protein encoded by mRNA or circRNA. In some embodiments, provided herein are methods for expression of protein encoded by mRNA or circRNA.

[0726] In some embodiments, the lipid composition / pharmaceutical composition has an N / P ratio of from about 1:1 to about 30:1, for instance, from about 3:1 to about 20:1, from about 3:1 to about 15:1, from about 3: 1 to about 10: 1, or from about 3: 1 to about 6:1. For example, the N / P ratio of the nucleic acid molecule-encapsulated lipid composition may be about 6 ± 1, or the N / P ratio of the nucleic acid molecule-encapsulated lipid composition may be about 6 ± 0.5. In some embodiments, the N / P ratio of the nucleic acid molecule - encapsulated lipid composition ranges from about 3: 1 to about 15:1. In some embodiments, the N / P ratio of the nucleic acid molecule-encapsulated lipid composition is about 6. An N: P ratio refers to the molar ratio of the amines present in the lipid-based carrier (e.g., the amines in the ionizable lipids) to the phosphates present in the nucleic acid molecule. It is a factor for efficient packaging and potency.

[0727] Other Therapeutic Agents

[0728] The therapeutic agent can be a peptide or protein, a small molecule drug, encapsulated in the lipid composition. The lipid composition / pharmaceutical composition can contain two or more different therapeutic agents from the nucleic acid molecule, peptide or protein, and small molecule drug.

[0729] In some embodiments, the protein may be a peptide or polypeptide, e.g., a transcription factor; a chromatin remodeling factor; an antigen; a hormone; an enzyme (such as a nuclease, e.g., an endonuclease, e.g., a nuclease element of a CRISPR system, e.g., a Cas9, dCas9, aCas9-nickase, Cpf / Casl2a); a Crispr-linked enzyme, e.g., a base editor or prime editor; a mobile genetic element protein (e.g., a transposase, a retrotransposase, a recombinase, an integrase); a gene writer; a polymerase; a methylase; a demethylase; an acetylase; a deacetylase; a kinase; a phosphatase; a ligase; a deubiquitinase; an integrase; a recombinase; a topoisomerase; a gyrase; a helicase; a lysosomal acid hydrolase); an antibody; a receptor ligand; a receptor; a clotting factor; a membrane protein; a mitochondrial protein; a nuclear protein; an antibody or other protein scaffold binder such as a centyrin, darpin, or adnectin.

[0730] In some embodiments, the lipid composition / pharmaceutical composition can include a plurality of protein molecules, which may be the same or different types.

[0731] In some embodiments, the therapeutic agent is a small molecule drug, for instance, a small molecule drug approved for use in humans by an appropriate regulatory authority.

[0732] In some embodiments, the lipid composition / pharmaceutical composition can include a plurality of small molecule drugs, which may be the same or different types.

[0733] In some embodiments, the therapeutic agent is a vaccine. In some embodiments, the vaccine is a RNA vaccine, such as a RNA cancer vaccine or RNA vaccine for infectious disease (e.g., an influenza virus vaccine or a coronavirus vaccine (e.g., COVID-19 vaccine).

[0734] Other Ingredients

[0735] The pharmaceutical compositions may contain one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers or excipients for use in pharmaceutical formulations are described in Remington: The Science and Practice of Pharmacy, 21stEd., Gennaro, Ed., Lippencott Williams & Wilkins (2005); Handbook of Pharmaceutical Excipients, 6thEdition, Rowe et al., Eds., Pharmaceutical Press (2009); and the USP / NF (United States Pharmacopeia and the National Formulary), which are herein incorporated by reference in their entirety.

[0736] In some embodiments, the pharmaceutically acceptable excipient includes one or more of an antioxidant, binder, antiadherent, buffer, coloring agent, diluent (e.g., solid or liquid), disintegrant (e.g., coatings disintegrate), dispersing agent, dyestuff, filler, emulsifier, flavoring agent, lubricant, pH adjuster, pigment, preservative, stabilizer, solubilizing agent, solvent, suspending agent, sweetener, or wetting agent, or combination thereof.

[0737] Examples of suitable excipients include, without limitation, acacia, alginate, calcium phosphate, calcium carbonate, calcium silicate, carbopol gel, carboxymethyl cellulose, carnauba wax, cellulose, crospovidone, dextrose, diacetylated monoglycerides, ethylcellulose, gelatin, glyceryl monostearate 40-50, gum acacia, gum arabic, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hypromellose phthalate, hypromellose, lactose, lecithin, magnesium stearate, kaolin, methacrylic acid copolymer type C, mannitol, methyl cellulose, methylhydroxybenzoate, microcrystalline cellulose, povidone, polyethylene glycol, polysorbate 80, polyvinylpyrrolidone, propylhydroxybenzoate, sodium carboxymethyl cellulose sodium hydroxide, sodium stearyl fumarate, sodium starch glycolate, starch, sorbitan monooleate sorbitol, sorbic acid, sucrose, talc, tragacanth, talc, triethyl citrate, titanium dioxide, yellow ferric oxide, talc, oil medium (e.g., peanut oil, liquid paraffin, mineral oil, olive oil, almond oil, glycerin, propylene glycol), or water,

[0738] When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. As is known in the art, the type of diluent can vary depending upon the intended route of administration.

[0739] The pharmaceutical compositions can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl chloride, hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans, chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt- forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0740] Suitable carriers or excipients for the pharmaceutical compositions may also include a substance that enhances the ability of the body of an individual to absorb the LNP or liposome. Suitable carriers and / or excipients also include any substance that can be used to bulk up formulations with a LNP or liposome, to allow for convenient and accurate dosage. In addition, carriers and / or excipients may be used in the manufacturing process to aid in the handling of a LNP or liposome. Depending on the route of administration, a d form of medication, different carriers and / or excipients may be used.

[0741] Carriers and / or excipients may also include vehicles and / or diluents. “Vehicles” indicates any of various media acting usually as solvents or carriers; “diluent” indicates a diluting agent which is issued to dilute an active ingredient of a composition, suitable diluent include any substance that can decrease the viscosity of a medicine. The type and amounts of carriers and / or excipients are chosen in function of the chosen pharmaceutical form; suitable pharmaceutical forms are liquid systems like solutions, infusions, suspensions; semisolid systems like colloids, gels, pastes or creams; solid systems like powders, granulates, tablets, capsules, pellets, microgranulates, mini tablets, microcapsules, micropellets, suppositories; etc.

[0742] Each of the above systems can be suitably formulated for normal, delayed or accelerated release, using techniques well-known in the art.

[0743] FORMULATIONS, DOSAGES, AND ROUTES OF ADMINISTRATION

[0744] The pharmaceutical compositions described herein can be prepared according to standard techniques, as well as those techniques described herein. For instance, the pharmaceutical compositions can be manufactured in a conventional manner, e.g., by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Methods well known in the art for making formulations are known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21stEd., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and Encyclopedia of Pharmaceutical Technology, eds. J. S warbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York.

[0745] The therapeutic agent may be encapsulated in the lipid composition, for instance, the therapeutic agent may be completely or partially located in the interior space of the LNPs, within the lipid layer / membrane, or associated with the exterior surface of the lipid layer / membrane. One purpose of incorporating therapeutic agents into LNPs is to protect the therapeutic agents from environments which may contain enzymes or chemicals or conditions that degrade the therapeutic agents and / or systems or receptors that cause the rapid excretion of the therapeutic agents. Moreover, incorporating therapeutic agents into LNPs may promote uptake of the therapeutic agent, and hence, may enhance the therapeutic effect.

[0746] In some embodiments, in the pharmaceutical composition, the lipid components to therapeutic agent ratio (mass / mass ratio; w / w ratio) can range from about 1:1 to about 25:1, 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0747] The lipid composition or pharmaceutical composition may contain about 5 to about 95% by weight the therapeutic agent, based on the weight of the lipid composition or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical composition contains about 5%, about 10%, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 95% by weight, based on the weight of the lipid composition or pharmaceutical composition, of the therapeutic agent. In some embodiments, the lipid composition or pharmaceutical composition contains the therapeutic agent in an amount about 5-95%, about 5-90%, about 5-80 %, about 5-70 %, about 5-60%, about 5-50%, about 5-40%, about 5-30%, about 5-20%, about 5-10%, about 10-95%, about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-95%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-95%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%, based on the weight of the lipid composition or pharmaceutical composition.

[0748] The lipid composition or pharmaceutical compositions can contain total lipids at an amount of about 5 to about 95% by weight, based on the weight of the lipid composition or pharmaceutical composition. In some embodiments, the lipid composition or pharmaceutical compositions contain total lipids at an amount of about 5-95%, about 5-90%, about 5-80 %, about 5-70 %, about 5-60%, about 5-50%, about 5-40%, about 5-30%, about 5-20%, about 5-10%, about 10-95%, about 10-90%, about 10-80%, about 10-70%, about 10-60%, about 10-50%, about 10-40%, about 10-30%, about 10-20%, about 20-95%, about 20-90%, about 20-80%, about 20-70%, about 20-60%, about 20-50%, about 20-40%, about 20-30%, about 30-95%, about 30-90%, about 30-80%, about 30-70%, about 30-60%, about 30-50%, about 30-40%, about 40-95%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-95%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 60-95%, about 60-90%, about 60-80%, about 60-70%, about 70-95%, about 70-90%, about 70-80%, about 80-95%, about 80-90%, or about 90-95%, based on the weight of the lipid composition or pharmaceutical composition.

[0749] The compositions of this disclosure may be administered by various routes, for example, to effect systemic delivery via intravenous, parenteral, intraperitoneal, or topical routes. In some embodiments, a siRNA may be delivered intracellularly, for example, in cells of a target tissue such as lung or liver, or in inflamed tissues. In some embodiments, this disclosure provides a method for delivery of siRNA in vivo. A nucleic acid-lipid composition may be administered intravenously, subcutaneously, or intraperitoneally to a subject.

[0750] The compositions and methods of the disclosure may be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or dermal delivery, or by topical delivery to the eyes, ears, skin, or other mucosal surfaces. In some aspects of this disclosure, the mucosal tissue layer includes an epithelial cell layer. The epithelial cell can be pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal, or gastrointestinal. The compositions of this disclosure can be administered using conventional actuators such as mechanical spray devices, as well as pressurized, electrically activated, or other types of actuators.

[0751] The compositions of this disclosure may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Pulmonary delivery of a composition of this disclosure is achieved by administering the composition in the form of drops, particles, or spray, which can be, for example, aerosolized, atomized, or nebulized. Particles of the composition, spray, or aerosol can be in either a liquid or solid form. Non-limiting examples of systems for dispensing liquids as a nasal spray are disclosed in U. S. Pat. No. 4,511,069. Such formulations may be conveniently prepared by dissolving compositions according to the present disclosure in water to produce an aqueous solution, and rendering said solution sterile. The formulations may be presented in multidose containers, for example in the sealed dispensing system disclosed in U. S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems have been described in TRANSDERMAL SYSTEMIC MEDICATION, Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in U. S. Pat. No. 4,778,810. Additional aerosol delivery forms may include, e.g., compressed air-Jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or mixtures thereof.

[0752] Nasal and pulmonary spray solutions of the present disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be from pH 6.8 to 7.2. The pharmaceutical solvents employed can also be a slightly acidic aqueous buffer of pH 4-6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.

[0753] In some embodiments, provided herein is a pharmaceutical product which includes a solution containing a composition of this disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0754] A dosage form of the composition of this disclosure can be liquid, in the form of droplets or an emulsion, or in the form of an aerosol.

[0755] A dosage form of the composition of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0756] To prepare compositions for pulmonary delivery within the present disclosure, the biologically active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the active agent(s).

[0757] Examples of additives include pH control agents such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is a liquid, the tonicity of the composition, as measured with reference to the tonicity of 0.9% (w / v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0758] The biologically active agent may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid-gly colic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-gly colic acid) copolymer, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as poly glycerin fatty acid esters, sucrose fatty acid esters, etc., can be employed as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking, and the like. The carrier can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the biologically active agent.

[0759] Compositions for mucosal, nasal, or pulmonary delivery may contain a hydrophilic low molecular weight compound as a base or excipient. Such hydrophilic low molecular weight compounds may provide a passage medium through which a water-soluble active agent, such as a physiologically active peptide or protein, may diffuse through the base to the body surface where the active agent is absorbed. The hydrophilic low molecular weight compound may optionally absorb moisture from the mucosa or the administration atmosphere and may dissolve the water-soluble active peptide. In some embodiments, the molecular weight of the hydrophilic low molecular weight compound is less than or equal to 10,000, such as not more than 3,000.

[0760] Examples of hydrophilic low molecular weight compounds include polyol compounds, such as oligo-, di- and monosaccharides including sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, polyethylene glycol, and mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[0761] The compositions of this disclosure may alternatively contain as pharmaceutically acceptable carriers substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0762] In some embodiments, the biologically active agent may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agent can be prepared with carriers that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system, or bioadhesive gel. Prolonged delivery of the active agent, in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monosterate hydrogels and gelatin.

[0763] In some embodiments, the lipid composition, pharmaceutical compositions, or dosage units contain about 0.01 to about 1000 mg of one or more lipid compounds described herein. In some embodiments, the lipid composition, pharmaceutical compositions, or dosage units contain about 0.01, about 0.1, about 0.5, about 1, about 5, about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, 250, about 275, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 mg of one or more lipid compounds described herein. In some embodiments, the lipid composition, pharmaceutical compositions, or dosage units contain about 0.01 to about 750 mg, about 0.01 to about 500 mg, about 0.01 to about 250 mg, about 0.01 to about 100 mg, about 0.01 to about 50 mg, about 0.01 to about 25 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 0.1 mg, about 0.1 to about 1000 mg, about 0.1 to about 750 mg, about 0.1 to about 500 mg, about 0.1 to about 250 mg, about 0.1 to about 100 mg, about 0.1 to about 50 mg, about 0.1 to about 25, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 1 mg, about 1 to about 1000 mg, about 1 to about 750 mg, about 1 to about 500 mg, about 1 to about 250 mg, about 1 to about 100 mg, about 1 to about 50 mg, about 1 to about 25 mg, about 1 to about 10 mg, about 1 to about 5 mg, about 5 to about 1000 mg, about 5 to about 750 mg, about 5 to about 500 mg, about 5 to about 250 mg, about 5 to about 100 mg, about 5 to about 50 mg, about 5 to about 25 mg, about 5 to about 10 mg, about 10 to about 1000 mg, about 10 to about 750 mg, about 10 to about 500, about 10 to about 250 mg, about 10 to about 100 mg, about 10 to about 50 mg, about 10 to about 25 mg, about 25 to about 1000 mg, about 25 to about 750 mg, about 25 to about 500 mg, about 25 to about 250 mg, about 25 to about 100 mg, about 25 to about 50 mg, about 50 to about 1000, mg about 50 to about 750 mg, about 50 to about 500 mg, about 50 to about 250 mg, about 50 to about 100 mg, about 100 to about 1000 mg, about 100 to about 750 mg, about 100 to about 500 mg, about 100 to about 250 mg, about 250 to about 1000 mg, about 250 to about 750 mg, about 250 to about 500 mg, about 500 to about 1000 mg, about 500 to about 750 mg, or about 750 to about 1000 mg of one or more lipid compounds described herein.

[0764] Methods of Using the Lipid Composition

[0765] Another aspect of the present disclosure provides methods for delivering a therapeutic agent to a subject (e.g., a patient) in need thereof, comprising administering to said subject (e.g., patient) the pharmaceutical composition comprising a compound of formula (IA- 1 ), (IA-2), (IB-1), or (IB-2), a compound of the subgenus formulas of formula (IA- 1 ), (IA-2), (IB-1), or (IB-2), or any of the compounds belonging to any subgenus or species of these formulas disclosed herein, a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and the therapeutic agent. Certain aspect of the invention provides methods for delivering a therapeutic agent to a subject (e.g., a patient) in need thereof, comprising administering to said subject (e.g., patient) the pharmaceutical composition comprising the lipid-based carrier / lipid composition as described herein, and a pharmaceutically acceptable excipient.

[0766] In some embodiments, provided herein is a method of delivering the therapeutic agent to at least one organ chosen from the pancreas, one or both lungs, and the spleen of a subject in need thereof with a minimum amount delivered elsewhere in body, such as in the liver, of the subject. In some embodiments, the method delivers the therapeutic agent to the pancreas and / or one or both lungs a subject in need thereof with a minimum amount delivered elsewhere in body, such as in the liver, of the subject.

[0767] In some embodiments, less than 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the total therapeutic agent administered to the subject is delivered to the liver of the subject. In some embodiments, less than 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total therapeutic agent administered to the subject is delivered to the liver of the subject.

[0768] In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to the subject is delivered to the pancreas, spleen, and / or one or both lungs of the subject. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to the subject is delivered to the pancreas of the subject. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to the subject is delivered to the lungs of the subject. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the total therapeutic agent administered to the subject is delivered to the spleen of the subject.

[0769] In some embodiments, the total therapeutic agent administered to the subject has a spleen to liver ratio of at least 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the total therapeutic agent administered to the subject has a spleen to liver ratio of at least 1. In some embodiments, the total therapeutic agent administered to the subject has spleen to liver ratio of at least 5.

[0770] As used herein, the percent amount of the total therapeutic agent administered to the subject and delivered to a location in the subject is measured by the level of protein expression, or mRNA knockdown level.

[0771] In some embodiments, the lipid composition or pharmaceutical composition disclosed herein may be used for a variety of purposes, including delivery of encapsulated or associated (e.g., complexed) therapeutic agents such as nucleic acids to cells, in vitro and / or in vivo.

[0772] Accordingly, in some embodiments, provided are methods of treating or preventing diseases or disorders in a subject in need thereof comprising administering to the subject the lipid composition or pharmaceutical composition described herein. In some embodiments, the lipid composition encapsulates or is associated with a suitable therapeutic agent, wherein the lipid composition comprises one or more of the novel lipid compounds described herein, a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing.

[0773] In some embodiments, the lipid composition described herein are useful for delivery of therapeutic agent. In some embodiments, the therapeutic agent is chosen from one or more nucleic acids, including, e.g., mRNA, circular RNA (circRNA), antisense oligonucleotide, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomirs / antimirs), messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalent RNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, in some embodiments, disclosed herein are methods of inducing expression of a desired protein in vitro and / or in vivo by contacting cells with a lipid composition comprising one or more novel lipid compounds described herein, wherein the lipid composition encapsulates or is associated with a nucleic acid that is expressed to produce a desired protein (e.g., a messenger RNA or plasmid encoding the desired protein) or inhibit processes that terminate expression of mRNA (e.g., miRNA inhibitors). In some embodiments, disclosed herein are methods of decreasing expression of target genes and proteins in vitro and / or in vivo by contacting cells with a lipid composition comprising one or more novel lipid compounds described herein, wherein the lipid composition encapsulates or is associated with a nucleic acid that reduces target gene expression (e.g., an antisense oligonucleotide or small interfering RNA (siRNA)). In some embodiments, disclosed herein are methods for co-delivery of one or more nucleic acid (e.g. mRNA and plasmid DNA). separately or in combination, such as may be useful to provide an effect requiring colocalization of different nucleic acids (e.g. mRNA encoding for a suitable gene modifying enzyme and DNA segment(s) for incorporation into the host genome).

[0774] In some embodiments, the lipid compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA. In some embodiments, provided herein are methods for upregulating endogenous protein expression comprising delivering miRNA inhibitors targeting one or more miRNA regulating one or more mRNA.

[0775] In some embodiments, the lipid compositions are useful for down-regulating (e.g., silencing) the protein levels and / or mRNA levels of target genes. In some embodiments, provided herein are methods for down-regulating (e.g., silencing) protein and / or mRN A levels of target genes.

[0776] In some embodiments, the lipid compositions are useful for delivery of mRNA and plasmids for expression of transgenes. In some embodiments, provided herein are methods for delivering mRNA and plasmids for expression of transgenes.

[0777] In some embodiments, the lipid compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody. In some embodiments, provided herein are methods for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antigen or antibody.

[0778] Non-limiting exemplary embodiments of the lipid compounds of the present disclosure, lipid compositions comprising the same, and their use to deliver agents (e.g., therapeutic agents, such as nucleic acids) and / or to modulate gene and / or protein expression are described in further detail below.

[0779] In some embodiments, the disclosure relates to a method of gene editing, comprising contacting a cell with the lipid composition described herein. In some embodiments, the disclosure relates to any method of gene editing described herein, comprising cleaving DNA.

[0780] In some embodiments, the disclosure relates to a method of cleaving DNA, comprising contacting a cell with the lipid composition described herein.

[0781] In some embodiments, the disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a single stranded DNA nick. In some embodiments, the disclosure relates to any method of cleaving DNA described herein, wherein the cleaving step comprises introducing a double-stranded DNA break. In some embodiments, the disclosure relates to any method of cleaving DNA described herein, wherein the LNP composition comprises a Class 2 Cas mRNA and a guide RNA nucleic acid. In some embodiments, the disclosure relates to any method of cleaving DNA described herein, further comprising introducing at least one template nucleic acid into the cell. In some embodiments, the disclosure relates to any method of cleaving DNA described herein, comprising contacting the cell with the lipid composition described herein comprising a template nucleic acid.

[0782] In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the method comprises administering the lipid composition described herein to an animal, for example a human. In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the method comprises administering the lipid composition described herein to a cell, such as a eukaryotic cell.

[0783] In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the method comprises administering the mRNA formulated in a first lipid composition and a second lipid composition comprising one or more of an mRNA, a gRNA, a gRNA nucleic acid, and a template nucleic acid. In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the first and second lipid compositions are administered simultaneously. In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the first and second lipid compositions are administered sequentially.

[0784] In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the method comprises administering the mRNA and the guide RNA nucleic acid formulated in a single lipid composition described herein.

[0785] In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the gene editing results in a gene knockout.

[0786] In some embodiments, the disclosure relates to any method of gene editing described herein, wherein the gene editing results in a gene correction.

[0787] In some embodiments, the disclosure relates to methods for in vivo delivery of interfering RNA to the lung of a mammalian subject.

[0788] In some embodiments, relates to methods of treating a disease or disorder in a mammalian subject. In some embodiments, these methods comprise administering a therapeutically effective amount of the lipid composition described herein to a subject having a disease or disorder associated with expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition. EXAMPLES

[0789] The following examples are for illustrative purposes only and are not intended to limit, in any way, the scope of the present invention.

[0790] Example 1. Synthesis of compound 2538

[0791]

[0792] To a solution of 2-(tert-butoxycarbonylamino)acetic acid (296.43 mg, 1.69 mmol, 1.5 eq), 2-(tert-butoxycarbonylamino)acetic acid (296.43 mg, 1.69 mmol, 1.5 eq) in dichloromethane (10 mL) was added EDCI (324.38 mg, 1.69 mmol, 1.5 eq), and DMAP (27.56 mg, 225.61 pmol, 0.2 eq). The mixture was stirred at 25 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 20 mL) and brine (2 x 20 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 1 / 1, 3% NH3 H2O) to afford 7-[2-[[2-(tert-butoxycarbonylamino)acetyl]amino] ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.45 g, 519.42 pmol, 46.05% yield).JH NMR (400 MHz, CDCh), 4.81-4.93 (m, 1H), 4.01-4.11 (m, 2H), 3.74-3.83 (m, 2H), 3.24-3.45 (m, 2H), 2.22-2.62 (m, 10H), 1.58-1.68 (m, 10H), 1.44-1.48 (m, 9H), 1.24-1.38 (m, 52H), 0.89 (t, J=6.4 Hz, 9H).

[0793] Step 2:

[0794] To a solution of 7-[2-[[2-(tert-butoxycarbonylamino)acetyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (0.35 g, 404.00 pmol, 1 eq) in dichloromethane (1.5 mL) was added TFA (767.50 mg, 6.73 mmol, 0.5 mL, 16.66 eq). The mixture was stirred at 25 °C for 3 hours. The reaction mixture was adjusted to pH=7 with saturated NaHCO₃ aqueous and extracted with ethyl acetate (3 x 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1, 3% NH3 H2O) to afford 7-[2-[(2-aminoacetyl)amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.24 g, 313.22 pmol, 77.53% yield).

[0795]

[0796] NMR (400 MHz, CDCh), 4.82-4.94 (m, 1H), 4.03-4.09 (m, 2H), 3.29-3.47 (m, 4H), 2.33-2.83 (m, 6H), 2.25-2.32 (m, 4H), 1.58-1.67 (m, 8H), 1.44-1.52 (m, 6H), 1.24-1.35 (m, 48H), 0.89 (t, J=6.8 Hz, 9H).

[0797] Step 3:

[0798] To a solution of 7-[2-[(2-aminoacetyl)amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.24 g, 313.22 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (226.82 mg, 313.22 pmol, 1 eq) in di chloromethane (5 mL) was added dropwise EDCI (120.09 mg, 626.45 pmol, 2 eq), DMAP (7.65 mg, 62.64 pmol, 0.2 eq). The mixture was stirred at 25 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, ethyl acetate / MeOH=l / 0 to 10 / 1, 5% NH3 THF) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient:40%-90% B over 12.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of 10: 1 ACN: TEA (3 x 10 mL). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]acetyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (12 mg, 8.07 pmol, 2.6% yield, 99% purity). 'H NMR (400 MHz, CDCh), 7.89 (brs, 1H), 6.37 (brs, 1H), 4.82-4.92 (m, 2H), 4.01-4.10 (m, 4H), 3.81-3.92 (m, 2H), 3.05-3.36 (m, 4H), 2.11-2.68 (m, 18H), 1.58-1.65 (m, 14H), 1.41-1.49 (m, 8H), 1.20-1.35 (m, 102H), 0.89 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 737.0. LCMS-CAD: (1 / 2M+H+): 737.0 @ 12.918 min. LCMS-ELSD:

[0799] (1 / 2M+H+): 736.8 @ 12.679 min.

[0800] Example 2. Synthesis of compound 2539

[0801]

[0802] A mixture of 7-[[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1 g, 1.50 mmol, 1 eq) in benzyl prop-2-enoate (15 mL) was degassed and purged with N23 times, and then the mixture was stirred at 50 °C for 8 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleumether / ethyl acetate=5 / l to 1 / 1) to afford 7-[(3-benzyloxy-3-oxo-propyl)-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1.24 g, crude).

[0803] Step 2:

[0804] To a solution of 7-[(3-benzyloxy-3-oxo-propyl)-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.9 g, 1.09mmol, 1 eq) in ethyl acetate (9 mL) was added to Pd / C (900.00 mg, 845.71 pmol, 10% purity) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 8 hours. The mixture was fdtered through celite and the solvent removed under reduced pressure to afford 3-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (0.8 g, 1.01 mmol,

[0805]

[0806] 4.94 (m, 1H), 4.01-4.12 (m, 2H), 2.97-3.04 (m, 2H), 2.72-2.82(m, 4H), 2.58-2.67 (m, 2H), 2.26-2.34 (m, 4H), 1.24-1.65 (m, 62H), 0.89 (t, J=6.4 Hz, 9H).

[0807] Step 3:

[0808] A mixture of 7-[2-[(2-aminoacetyl)amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.75 g, 978.82 pmol, 1 eq), 3-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (722.54 mg, 978.82 pmol, 1 eq), EDCI (375.28 mg, 1.96 mmol, 2 eq), and DMAP (23.92 mg, 195.76 pmol, 0.2 eq) in dichloromethane (8mL) was degassed and purged with N23 times, and then the mixture was stirred at 25°C for 8 hours under N2 atmosphere. The reaction mixture was diluted with ethyl acetate (30 mL) and washed with H2O (3 x 10 mL). The combined organic layers were dried over Na2SO4, fdtered, and concentrated under reduced pressure.

[0809] The residue was purified by column chromatography (SiCh, ethyl acetate / MeOH=l / 0 to 10 / 1, 5% NH3 THF) and prep-HPLC (column: Xselect CSH C18100*30mm*5um; mobile phase:

[0810] [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-90% B over 12.0 min). The mixture was diluted with brine (50 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with a mixture of 10: 1 ACN / TEA (3 x 10 mL). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[[3-[[2-[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-2-oxo-ethyl]amino]-3-oxo-propyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.228 g, 153.39 pmol, 15.6% yield).

[0811]

[0812] NMR (400 MHz, CDCh), 8.81 (brs, 1H), 4.78-4.94 (m, 2H), 4.03-4.11 (m, 4H), 3.78-4.01 (m, 2H), 3.18-3.46 (m, 2H), 2.12-3.05 (m, 22H), 1.58-1.64 (m, 14H), 1.42-1.49 (m, 8H), 1.21-1.38 (m, 102H), 0.88 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 743.9.

[0813] LCMS-CAD: (1 / 2M+H+): 743.9 @ 14.591 mm. LCMS-ELSD: (1 / 2M+H+): 744.3 @ 14.716 min. Example 3. Synthesis of compound 2540

[0814]

[0815] To a solution of (2S)-2-(benzyloxy carbonylamino) propanoic acid (409.20 mg, 1.83 mmol, 1.3 eq) in di chloromethane (5 mL) was added EDCI (540.63 mg, 2.82 mmol, 2 eq) and DMAP (34.45 mg, 282.02 pmol, 0.2 eq). Then 7-[2-aminoethyl- [8-(l-octylnonoxy)-8-oxo-octyl] amino] heptyl decanoate (1 g, 1.41 mmol, 1 eq) was added to the mixture. The mixture was stirred at 25 °C for 8 hours. The reaction mixture was quenched by addition water (10 mL) at 25 °C, and then extracted with di chloromethane (2 x 10 mL). The combined organic layers were washed with brine, dried over NazSCh, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 0 / 1) to afford 7-[2-[[(2S)-2-(benzyloxycarbonylamino)propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxooctyl] amino]heptyl decanoate (0.76 g, 831.16 pmol, 58.91% yield).

[0816] Step 2:

[0817] To a solution of Pd / C (640.11 mg, 601.50 pmol, 10% purity, 1 eq) in ethyl acetate (10 mL) was added 7-[2-[[(2S)-2-(benzyloxycarbonylamino)propanoyl]amino]ethyl-[8-(l- octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (0.55 g, 601.50 pmol, 1 eq). The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 at 15 Psi, 25 °C for 8 hours. The mixture was filtered through celite and the solvent removed under reduced pressure to afford 7-[2-[[(2S)-2-aminopropanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (0.938 g, 1.20 mmol, 99.93% yield).

[0818]

[0819] NMR (400 MHz, CDCh), 4.84-4.90 (m, 1H), 4.06 (t, J=6.8 Hz, 2H), 3.36-3.53 (m, 3H), 2.31-2.62 (m, 4H), 2.28-2.30 (m, 4H), 1.44-1.64 (m, 20H), 1.26-1.35 (m, 47H), 0.89 (t, J=6.8 Hz, 9H).

[0820] Step 3:

[0821] To a solution of 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (407.62 mg, 562.89 umol, 1.2 eq) in di chloromethane (5 mL) was added EDCI (179.85 mg, 938.16 umol, 2 eq) andDMAP (11.46 mg, 93.82 umol, 0.2 eq). Then 7-[2-[[(2S)-2aminopropanoyl] amino] ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (0.366 g, 469.08 umol, 1 eq) was added to the mixture. The mixture was stirred at 25 °C for 5 hours. The reaction mixture was quenched by addition water (10 mL), and then extracted with

[0822] di chloromethane (2 x 10 mL). The combined organic layers were washed with brine, dried over Na2SO4, fdtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 55%-90% B over 12.0 min). The reaction mixture was adjusted pH=7 with NaHCOs and extracted with ethyl acetate (3 x 5 mL). The combined organic layers were washed with brine (20 mL) and dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate =10 / 1 to 1 / 1) to afford 7-[2-[[(2S)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]propanoyl] amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (85 mg, 55.47 pmol, 82.45% yield, 97% purity).

[0823]

[0824] NMR (400 MHz, CDCh), 7.82 (d, J=6.0 Hz, 1H), 6.46 (brs, 1H), 4.84-4.90 (m, 2H), 4.40-4.44 (m, 1H), 4.04-4.08 (m, 4H), 3.03-3.32 (m, 4H), 2.28-2.47 (m, 18H), 1.60-1.64 (m, 12H), 1.50-1.52 (m, 7H), 1.26-1.38 (m, 108H), 0.89 (t, J=7.2Hz, 18H). (1 / 2M+H+): 743.9. LCMS-CAD: (1 / 2M+H+): 743.9 @ 13.248 mm. LCMS-ELSD: (1 / 2M+H+): 743.8 @ 13.281 mm. Example 4. Synthesis of compound 2541

[0825]

[0826] Step 1:

[0827] To a solution of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1 g, 1.41 mmol, 1 eq), (2S)-2-(benzyloxycarbonylamino)-3-phenyl-propanoic acid (633.10 mg, 2.12 mmol, 1.5 eq) in dichloromethane (10 mL) was added dropwise EDCI (405.47 mg, 2.12 mmol, 1.5 eq), DMAP (34.45 mg, 282.02 pmol, 0.2 eq). The mixture was stirred at 25 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 10 mL) and brine (2 x 10 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate= 1 / 0 to 3 / 13% NEL THF ) to afford 7-[2-[[(2S)-2-(benzyloxycarbonylamino)-3-phenyl-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1 g, 969.22 pmol, 68.74% yield, 96% purity).

[0828]

[0829] NMR (400 MHz, CDCh), 7.28-7.39 (m, 6H), 7.16-7.27 (m, 4H), 6.07-6.24 (m, 1H), 5.37-5.49 (m, 1H), 5.04-5.14 (m, 2H), 4.81-4.94 (m, 1H), 4.31-4.44 (m, 1H), 3.98-4.10 (m, 2H), 3.01-3.31 (m, 4H), 2.06-2.65 (m, 10H), 1.58-1.65 (m, 12H), 1.47-1.51 (m, 2H), 1.24-1.33 (m, 48H), 0.88 (t, J=6.4 Hz, 9H). Step 2:

[0830] To a solution of Pd / C (0.1 g, 93.97 pmol, 10% purity, 1.86e-l eq) in ethyl acetate (5 mL) was added 7-[2-[[(2S)-2-(benzyloxycarbonylamino)-3-phenyl-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.5 g, 504.80 pmol, 1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 5 hours. The mixture was fdtered through celite and the solvent was removed under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate= 1 / 0 to 0 / 1 3% NH3 THF) to afford 7-[2-[[(2S)-2-amino-3-phenyl-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.3 g, 343.32 pmol, 68.01% yield).

[0831]

[0832] NMR (400 MHz, CDCh), 7.31-7.39 (m, 2H), 7.16-7.24 (m, 3H), 4.78-4.94 (m, 1H), 3.98-4.12 (m, 2H), 3.56-3.68 (m, 1H), 3.16-3.46 (m, 3H), 2.15-2.83 (m, 10H), 1.96-2.12 (m, 1H), 1.57-1.67 (m, 10H), 1.47-1.52 (m, 4H), 1.21-1.40 (m, 48H), 0.89 (t, J=6.4 Hz, 9H).

[0833] Step 3:

[0834] To a solution of 7-[2-[[(2S)-2-amino-3-phenyl-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.3 g, 350.32 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l -octyl nonoxy)-8-oxo-octyl]amino]acetic acid (279.05 mg, 385.36 pmol, 1.1 eq) in dichloromethane (5 mL) was added dropwise EDCI (134.31 mg, 700.65 pmol, 2 eq), DMAP (8.56 mg, 70.06 pmol, 0.2 eq). The mixture was stirred at 25 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate= 1 / 0 to 0 / 1, 3% NH3 THF ) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 65%-95% B over 15.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with a mixture of acetonitrile and TEA (3 x 10 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN / MeOH(3 / l) (2 x 10 mL), and then the hexane layers were concentrated under reduced pressure to afford 7-[2-[[(2S)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-3-phenyl-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (25 mg, 15.52 pmol, 4.4% yield, 97% purity). iH NMR (400 MHz, CDCh), 7.83 (brs, 1H), 7.31-7.36 (m, 1H), 7.11-7.22 (m, 4H), 6.19 (brs, 1H), 4.81-4.95 (m, 2H), 4.49-4.62 (m, 1H), 4.01-4.12 (m, 4H), 2.95-3.28 (m, 6H), 2.20-2.45 (m, 18H), 1.56-1.68 (m, 18H), 1.46-1.48 (m, 4H), 1.22-1.37 (m, 102H), 0.88 (t, J=6.4 Hz, 18H). (1 / 2M+H+): 782.0.

[0835] LCMS-CAD: (1 / 2M+H+): 782.0 @ 9.736 min. LCMS-ELSD: (1 / 2M+H+): 782.2 @ 9.318 min.

[0836]

[0837]

[0838]

[0839] Step 1:

[0840] To a solution of heptadecan A-ol (10.45 g, 40.75 mmol, 1 eq), EDCI (15.62 g, 81.49 mmol, 2 eq), DMAP (2.49 g, 20.37 mmol, 0.5 eq) in di chloromethane (200 mL) was added 8- bromooctanoic acid (10 g, 44.82 mmol, 1.1 eq) at 25 °C. The mixture was stirred at 25 °C for 8 hours. The reaction mixture was diluted with H2O (1000 mL) and extracted with ethyl acetate (3 x 600 mL). The combined organic layers were dried over Na2S()4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 0) to afford 1 -octylnonyl 8-bromooctanoate (50 g, crude).

[0841] Step 2:

[0842] A mixture of benzyl (2S)-2-aminopropanoate;hydrochloride (23.36 g, 108.33 mmol, 5 eq), KI (3.60 g, 21.67 mmol, 1 eq), TEA (13.15 g, 129.99 mmol, 18.09 mL, 6 eq) in acetonitrile (30 mL) was stirred at 25 °C for 1 hour under N2 atmosphere. To the mixture was added 1-octylnonyl 8-bromooctanoate (10 g, 21.67 mmol, 1 eq) and stirred at 80 °C for 8 hours under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 10 / 1) to afford 1-octylnonyl 8-[[(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl]amino] octanoate (5 g, 8.93 mmol, 50.00% yield, 100% purity). (M+H+): 560.5. LCMS:

[0843] (M+H+): 560.5 @ 1.783 mm.

[0844] Step 3:

[0845] To a solution of decanoic acid (61.81 g, 358.80 mmol, 69.21 mL, 1 eq), DMAP (8.77 g, 71.76 mmol, 0.2 eq), EDCI (82.54 g, 430.56 mmol, 1.2 eq) in dichloromethane (1000 mL) was added 7-bromoheptan-l-ol (70 g, 358.80 mmol, 1 eq) at 25 °C. The mixture was stirred at 25 °C for 8 hours. The reaction mixture was diluted with H2O (100 mL) and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 0) to afford 7-bromoheptyl decanoate (92 g, 263.35 mmol, 73.39% yield).

[0846] Step 4:

[0847] A mixture of 1 -octylnonyl 8-[[(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl]amino]octanoate (3.5 g, 6.25 mmol, 1 eq), TEA (2.53 g, 25.01 mmol, 3.48 mL, 4 eq) in acetonitrile (20 mL) was stirred at 25 °C for 1 hour under N2 atmosphere. To the mixture was added KI (1.04 g, 6.25 mmol, 1 eq), 7-bromoheptyl decanoate (8.74 g, 25.01 mmol, 4 eq) and stirred at 80 °C for 7 hours under N2 atmosphere. The reaction mixture was diluted with H2O (20 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over Na2SCh, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=30 / l to 20 / 1) to afford 7-[[(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.6 g, 673.67 pmol, 10.77% yield). *H NMR (400 MHz, CDCh), 7.32-7.39 (m, 5H), 5.14 (s, 2H), 4.86-4.89 (m, 1H), 4.05 (t, J=6.8 Hz, 2H), 3.55-3.57 (m, 1H), 2.26-2.56 (m, 8H), 1.27-1.62 (m, 65H), 0.89 (t, J=7.2 Hz, 9H). Step 5:

[0848] To a solution of Pd / C (37.50 mg, 35.24 pmol, 10% purity, 9.73e-2 eq) in ethyl acetate (20 mL) was added 7-[[(lS)-2-benzyloxy-l-methyl-2-oxo-ethyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.3 g, 362.19 pmol, 1 eq) under N2 atmosphere. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 3 hours. The mixture was filtered through celite and the solvent removed under reduced pressure to afford (2S)-2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (0.21 g, 284.49 pmol, 78.55% yield).

[0849] Step 6:

[0850] To a solution of (2S)-2-(benzyloxycarbonylamino)propanoic acid (440.67 mg, 1.97 mmol, 1 eq), EDCI (567.66 mg, 2.96 mmol, 1.5 eq), DMAP (48.23 mg, 394.82 pmol, 0.2 eq) in dichloromethane (20 mL) was added 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1.4 g, 1.97 mmol, 1 eq) at 25 °C. The mixture was stirred at 25 °C for 8 hours. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1) to afford 7-[2-[[(2S)-2-(benzyloxycarbonylamino)propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1.5 g, 1.26 mmol, 63.99% yield). (M+H+): 914.7

[0851] Step 7:

[0852] To a solution of Pd / C (0.1 g, 93.97 pmol, 10% purity, 8.59e-2 eq) in ethyl acetate (30 mL) was added 7-[2-[[(2S)-2-(benzyloxycarbonylamino)propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (1 g, 1.09 mmol, 1 eq) under N2 atmosphere. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 3 hours. The mixture was filtered through celite and the solvent removed under reduced pressure to afford 7-[2-[[(2S)-2-aminopropanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.7 g, 861.26 pmol, 78.75% yield).

[0853] Step 8:

[0854] To a solution of (2S)-2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (0.21 g, 284.49 pmol, 1 eq), EDCI (109.07 mg, 568.97 pmol, 2 eq), DMAP (17.38 mg, 142.24 pmol, 0.5 eq) in dichloromethane (10 mL) was added 7-[2-[[(2S)-2-aminopropanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (221.97 mg, 284.49 pmol, 1 eq) at 25 °C. The mixture was stirred at 25 °C for 8 hours. The reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-95% B over 15.0 min). The mixture was diluted with brine (100 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with a mixture of acetonitrile and TEA (3 x 20 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with acetonitrile (2 x 20 mL). The hexane phase was concentrated under N2 atmosphere. The residue was purified by prep-SEC (column: REGIS (s,s) WHELK-01

[0855] (250mm* 30mm, 5 um); mobile phase: [CO2-IPA(0.1%NH3H2O)]; B%:45%, isocratic elution mode) to afford 7-[2-[[(2S)-2-[[(2S)-2-[7-decanoyloxy heptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoyl]amino]propanoyl]amino]ethyl -[8-(l-octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (80 mg, 53.32 pmol, 18.74% yield, 100% purity). 'H NMR (400 MHz, CDCh), 7.84 (brs, 1H), 6.49 (brs, 1H), 4.85-4.88 (m, 2H), 4.39 (brs, 1H), 4.06 (t, J=6.4 Hz, 4H), 3.03-3.35 (m, 3H), 2.26-2.51 (m, 18H), 1.62-1.64 (m, 8H), 1.50-1.60 (m, 6H), 1.26-1.30 (m, 116H), 0.89 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 751.0. LCMS-CAD: (1 / 2M+H+): 751.0 @ 13.167 mm. LCMS-ELSD: (1 / 2M+H+): 750.9 @ 13.172 mm.

[0856] Example 6. Synthesis of compound 2620

[0857]

[0858] Step 1:

[0859] A mixture of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1.3 g, 1.83 mmol, 1 eq), (2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (376.17 mg, 1.83 mmol, 1 eq) and EDCI (702.82 mg, 3.67 mmol, 2 eq), DMAP (44.79 mg, 366.62 pmol, 0.2 eq) in dichloromethane (10 mL) was degassed and purged with N23 times, and then the mixture was stirred at 20 °C for 8 hours under N2 atmosphere. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 20 mL) and brine (2 x 20 mL), dried with anhydrous Na2SO4, fdtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 2 / 1, 3% NEP THF) and prep-HPLC (column: X-Select CSH Phenyl-Hexyl 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 30%-70% B over 16.0 min). The reaction mixture was adjusted pH=7 with aqueous saturated NaHCO₃ and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford 7-[2-[[(2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (0.7 g, 687.22 pmol, 61.60% yield).

[0860]

[0861] NMR (400 MHz, CDCh), 5.58 (brs, 1H), 4.74-5.05 (m, 1H), 3.85-4.25 (m, 4H), 3.55-3.65 (m, 1H), 3.32-3.52 (m, 2H), 2.27-3.01 (m, 10H), 1.51-1.68 (m, 12H), 1.45-1.48 (m, 9H), 1.21-1.42 (m, 50H), 0.87 (t, J=6.8 Hz, 9H).

[0862] Step 2:

[0863] To a solution of 7-[2-[[(2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.1 g, 111.56 pmol, 1 eq) in dioxane (1 mL) was added HCl / dioxane (4 M, 1 mL, 35.85 eq). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was adjusted pH=7 with aqueous saturated NaHCO₃ and extracted with ethyl acetate (3 x 5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1, 3% NH3 H2O) to afford 7-[2-[[(2S)-2-amino-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.07 g, 80.88 pmol, 72.50% yield).

[0864]

[0865] NMR (400 MHz, CDCh), 7.62 (brs, 1H), 4.78-4.94 (m, 1H), 4.03-4.12 (m, 2H), 3.78-3.88 (m, 1H), 3.64-3.71 (m, 1H), 3.18-3.52 (m, 3H), 2.25-2.62 (m, 10H), 1.58-1.67 (m, 6H), 1.41-1.52 (m, 6H), 1.22-1.37 (m, 50H), 0.89 (t, J=6.8 Hz, 9H).

[0866] Step 3:

[0867] To a solution of 7-[2-[[(2S)-2-amino-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (0.070 g, 87.91 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (63.66 mg, 87.91 pmol, 1 eq) in di chloromethane (5 mL) was added dropwise EDCI (33.71 mg, 175.82 pmol, 2 eq), DMAP (2.15 mg, 17.58 pmol, 0.2 eq). The mixture was stirred at 20 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, ethyl acetate / MeOH=l / 0 to 10 / 1, 5% NH3 THF) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase:

[0868] [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-95% B over 18.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of acetonitrile and TEA (3 x 10 mL, 10:1).

[0869] The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[(2S)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] acetyl]amino]-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.05 g, 31.62 pmol, 67.86% yield, 95% purity).

[0870]

[0871] NMR (400 MHz, CDCh), 8.12-8.27 (m, 1H), 6.71 (brs, 1H), 4.80-4.92 (m, 2H), 4.32-4.48 (m, 1H), 4.01-4.12 (m, 5H), 3.54-3.68 (m, 1H), 3.38-3.51 (m, 1H), 3.18-3.34 (m, 1H), 3.01-3.15 (m, 2H), 2.23-2.72 (m, 18H), 1.58-1.68 (m, 14H), 1.39-1.52 (m, 12H), 1.20-1.34 (m, 98H), 0.89 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 751.8. LCMS-CAD: (1 / 2M+H+): 751.8 @ 12.875 mm. LCMS-ELSD: (1 / 2M+H+): 751.8 @ 15.002 mm.

[0872]

[0873] To a solution of7-[2-[[(2S)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-3-hydroxy-propanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.5 g, 332.80 pmol, 1 eq), 3-(dimethylamino)propanoic acid;hydrochloride (126.51 mg, 665.61 pmol, 2 eq, HC1), DMAP (4.07 mg, 33.28 pmol, 0.1 eq), EDCI (95.70 mg, 499.21 pmol, 1.5 eq) in dichloromethane (5 mL). The mixture was stirred at 20 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 10 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 3 / 1, 3% NH3 H2O) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobilephase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient:45%-95% B over 16.0 mm). The mixture was diluted witli brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SOi, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of acetonitrile and TEA (3 x 10 mL, 10: 1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[(2S)-2-[[2-[7- decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-3-[3- (dimethylamino)propanoyloxy]propanoyl]amino]ethyl-[8-(l -octylnonoxy)-8-oxo- octyl] ammo] heptyl decanoate (0.095 g, 59.32 pmol, 38.00% yield, 100% purity). *11 NMR (400 MHz, CDCI3), 8.06 (brs, 1H), 4.81-4.94 (m, 2H), 4.60-4.72 (m, 1H), 4.25-4.59 (m, 2H), 3.95-4.15 (m, 4H), 2.77-3.75 (m, 10H), 2.19-2.74 (m, 22H), 1.58-1.74 (m, 14H), 1.41-1.55 (m, 12H), 1.20- 1.39 (m, 98H), 0.88 (t, J-6.8 Hz, 18H). (1 / 2M+H+): 801.2. LCMS-CAD: (1 / 2MH-L): 801.2 @ 11.739 mm. LCMS-ELSD: (1 / 2M+H+): 801.2 @ 11.836 min.

[0874]

[0875] Step 1:

[0876] A solution of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (2 g, 2.82 mmol, 1 eq), (2S)-2-(tert-butoxycarbonylamino)-5-guanidino-pentanoic acid (773.62 mg, 2.82 mmol, 1 eq), HOBt (381.07 nig, 2.82 mmol, 1 eq), BOP (1.25 g, 2.82 mmol, 1 eq), DIEA (728.97 mg, 5.64 mmol, 982,44 pL, 2 eq) in DMF (20 ml) was stirred at 20 °C for 8 hours. The reaction mixture was diluted with H2O (100 mL) and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate / MeOH=l / l to 0 / 1) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient:32%-75% B over 20.0 min). The mixture was adjusted pH=7 with saturated aqueous NaHCOs and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford heptadecan-9-yl (S)-ll-(7-(decanoyloxy)heptyl)-6-(3-guanidinopropyl)-2,2-dimethyl-4,7-dioxo-3-oxa-5,8,ll-triazanonadecan-19-oate (1 g, 973.61 pmol, 34.52% yield).

[0877]

[0878] NMR (400 MHz, CDCh), 8.09 (brs, 1H), 6.89-7.07 (m, 3H), 5.72-5.74 (m, 1H), 4.84-4.87 (m, 1H), 4.04-4.16 (m, 3H), 3.26-3.28 (m, 4H), 2.28-2.43 (m, 10H), 1.95-2.05 (m, 2H), 1.43-1.61 (m, 4H), 1.25-1.32 (m, 69H), 0.88 (t, J=6.8 Hz, 9H).

[0879] Step 2:

[0880] To a solution of heptadecan-9-yl (S)-ll-(7-(decanoyloxy)heptyl)-6-(3-guanidinopropyl)-2,2-dimethyl-4,7-dioxo-3-oxa-5,8,ll-triazanonadecan-19-oate (0.3 g, 310.73 pmol, 1 eq) in dioxane (2 mL) was added HCl / dioxane (4 M, 1.20 mL, 15.45 eq). The mixture was stirred at 20 °C for 3 hours. The crude reaction mixture was adjusted to pH=7 with saturated aqueous NaHCOs and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford (S)-7-((2-(2-amino-5-guanidinopentanamido)ethyl)(8-(heptadecan-9-yloxy)-8-oxooctyl)amino)heptyl decanoate (210 mg, 242.67 pmol, 78.10% yield).

[0881] Step 3:

[0882] A solution of (S)-7-((2-(2-amino-5-guanidinopentanamido)ethyl)(8-(heptadecan-9-yloxy)-8-oxooctyl)amino) heptyl decanoate (0.21 g, 242.67 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]acetic acid (175.73 mg, 242.67 pmol, 1 eq), HOBt (32.79 mg, 242.67 pmol, 1 eq), BOP (107.33 mg, 242.67 pmol, 1 eq), DIEA (31.36 mg, 242.67 pmol, 42.27 pL, 1 eq) in DMF (5 mL) was stirred at 20 °C for 8 hours. The reaction mixture was diluted with H2O 100 mL and extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3. MeOH=l / l / 0 to 0 / 1 / 0.1) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase:

[0883] [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 40%-90% B over 15.0 min). The mixture was diluted with ethyl acetate (60 mL) and washed with brine (4 x 500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of acetonitrile and TEA (3 x 20 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with acetonitrile (2 x 20 mL). The hexane phase was concentrated under N2 atmosphere to afford di(heptadecan-9-yl) (S)-9,18-bis(7-(decanoyloxy)heptyl)-13-(3-guanidinopropyl)-ll,14-dioxo-9,12,15,18-tetraazahexacosanedioate (0.15 g, 93.54 pmol, 38.54% yield, 98% purity).

[0884]

[0885] NMR (400 MHz, CDCh), 8.07-8.41 (m, 1H), 7.04 (brs, 3H), 4.82-4.89 (m, 2H), 4.39-4.53 (m, 1H), 3.12-4.07 (m, 17H), 2.26-2.47 (m, 11H), 1.27-1.67 (m, 128H), 0.89 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 785.9.

[0886] LCMS-CAD: (1 / 2M+H+): 785.9 @ 11.769 min. LCMS-ELSD: (1 / 2M+H+): 786.2 @ 11.851 mm.

[0887]

[0888] A mixture of (4S)-4-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-5-[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-5-oxo-pentanoic acid (0.1 g, 64.75 pmol, 1 eq), NH4CI (34.64 mg, 647.49 pmol, 10 eq) andHOBt (13.12 mg, 97.12 pmol, 1.5 eq), EDCI (18.62 mg, 97.12 pmol, 1.5 eq), TEA (19.66 mg, 194.25 pmol, 27.04 pL, 3 eq) in DMF (5 mL) was degassed and purged with N23 times, and then the mixture was stirred at 20 °C for 8 hours under N2 atmosphere. The combined organic phase was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 3 / 1, 3% NH3 H2O) and diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL). The hexane layers were concentrated under reduced pressure, diluted with hexane (10 mL) and washed with acetonitrile (2 x 10 mL), and then the hexane layers were concentrated under reduced pressure. The residue was purified by prep-HPLC (column: Xselect CSH Cl 8 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-90% B over 16.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with a mixture of acetonitrile and TEA (3 x 10 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[(2S)-5-amino-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl] amino]-5-oxo-pentanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (10 mg, 6.35 pmol, 9.8% yield, 98% purity).

[0889]

[0890] MHz, CDCh), 8.53 (brs, 1H), 8.06 (brs, 1H), 6.51 (brs, 1H), 5.35 (brs, 1H), 4.80-4.94 (m, 2H), 4.33-4.48 (m, 1H), 3.98-4.13 (m, 4H), 2.88-3.74 (m, 7H), 2.07-2.61 (m, 18H), 1.86-2.01 (m, 1H), 1.71-1.79 (m, 14H), 1.53-1.59 (m, 8H), 1.36-1.39 (m, 4H), 1.08-1.26 (m, 98H), 0.89 (t, J=6.4 Hz, 18H). (1 / 2M+H+): 772.3. LCMS-CAD:

[0891] (1 / 2M+H+): 772.3 @ 12.878 mm. LCMS-ELSD: (1 / 2M+H+): 772.3 @ 12.931 mm.

[0892] Example 10. Synthesis of compound 2625

[0893]

[0894]

[0895] A mixture of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (4 g, 5.64 mmol, 1 eq), (2S)-5-benzyloxy-2-(tert-butoxycarbonylamino)-5-oxo-pentanoic acid (2.28 g, 6.77 mmol, 1.2 eq) and EDCI (1.30 g, 6.77 mmol, 1.2 eq), DMAP (137.81 mg, 1.13 mmol, 0.2 eq) in dichloromethane (40 mL) was degassed and purged with N23 times, and then the mixture was stirred at 20 °C for 8 hours under N2 atmosphere. The combined organic phase was diluted with ethyl acetate (40 mL) and washed with water (3 x 20 mL) and brine (2 x 20 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 2 / 1, 3% NH3 THF) to afford 7-[2-[[(2S)-5-benzyloxy-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (4.5 g, 4.29 mmol, 76.02% yield). 'H NMR (400 MHz, CDCh), 7.28-7.43 (m, 5H), 5.05-5.22 (m, 2H), 4.79-4.93 (m, 1H), 3.41-4.41 (m, 4H), 2.94-3.33 (m, 3H), 1.81-2.68 (m, 12H), 1.68-1.75 (m, 8H), 1.53-1.59 (m, 4H), 1.38-1.42 (m, 9H), 1.15-1.35 (m, 50H), 0.89 (t, J=6.4 Hz, 9H).

[0896] Step 2:

[0897] To a solution of 7-[2-[[(2S)-5-benzyloxy-2-(tert-butoxycarbonylamino)-5-oxo-pentanoyl] amino] ethyl-[8-(l -octyl nonoxy)-8-oxo-octyl]amino]heptyl decanoate (4.5 g, 4.38 mmol, 1 eq) in dioxane (20 mL) was added HCl / dioxane (4 M, 20 mL, 18.28 eq). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was adjusted to pH=7 with saturated aqueous NaHCCh and extracted with ethyl acetate (3 x 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1, 3% NH3 H2O) to afford 7-[2-[[(2S)-2-amino-5- benzyloxy-5-oxo-pentanoyl]ammo]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (3 g, 775.52 pmol, 17.73% yield).

[0898] Step 3:

[0899] To a solution of 7-[2-[[(2S)-2-amino-5-benzyloxy-5-oxo-pentanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (0.68 g, 732.43 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (530.39 mg, 732.43 pmol, 1 eq) in dichloromethane (10 mL) was added dropwise EDCI (280.82 mg, 1.46 mmol, 2 eq), DMAP (17.90 mg, 146.49 pmol, 0.2 eq). The mixture was stirred at 20 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 10 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, ethyl acetate / MeOH=l / 0 to 10 / 1, 5% NIL THF) to afford 7-[2-[[(2S)-5-benzyloxy-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl] amino]-5-oxo-pentanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1 g, crude).

[0900] Step 4:

[0901] To a solution of Pd / C (0.3 g, 281.90 pmol, 10% purity, 5.12e-l eq) in ethyl acetate (10 mL) was added 7-[2-[[(2S)-5-benzyl oxy-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino] acetyl] amino]-5-oxo-pentanoyl]amino] ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.9 g, 550.61 pmol, 1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 8 hours. The mixture was filtered through celite and the solvent was removed under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 3 / 1, with 3% NIL. THF) and prep-HPLC (column: Xselect CSH Cl 8 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient:45%-95% B over 16.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA 30 mL (10 mL*3, 10: 1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford (4S)-4-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-5-[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-5-oxo-pentanoic acid (0.36 g, 233.10 pmol, 42% yield, 100% purity). (400 MHz, CDCL), 8.74 (brs, 1H), 4.82-4.90 (m, 2H), 4.63-4.79 (m, 1H), 4.02-4.09 (m, 4H), 3.82-3.97 (s, 1H), 2.95-3.58 (m, 12H), 2.44-2.53 (m, 2H), 2.24-2.39 (m, 10H), 2.02-2.20 (m, 2H), 1.71-1.78 (m, 4H), 1.56-1.68 (m, 14H), 1.42-1.52 (m, 8H), 1.16-1.34 (m, 98H), 0.89 (t, J=6.4 Hz, 18H). (1 / 2M+H+): 772.8. LCMS-CAD: (1 / 2M+H+): 772.8 @ 12.827 min. LCMS-ELSD: (1 / 2M+H+): 773.2 @ 12.960 min.

[0902] Example 11. Synthesis of compound 2626

[0903]

[0904] A mixture of (4S)-4-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-5-[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-5-oxo-pentanoic acid (0.1 g, 64.75 pmol, 1 eq), N', N'-dimethylethane-1,2-diamine (19.98 mg, 226.62 pmol, 24.75 pL, 3.5 eq) and EDCI (24.82 mg, 129.50 pmol, 2 eq), DMAP (1.58 mg, 12.95 pmol, 0.2 eq) in dichloromethane (5 mL) was degassed and purged with N23 times, and then the mixture was stirred at 20 °C for 8 hours under N2 atmosphere. The combined organic phase was diluted with ethyl acetate (10 mL)and washed with water (3 x 5 mL) and brine (2x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-SFC (column: (s,s) WHELK-01

[0905] (250mm* 30mm, 5 um); mobile phase: [CO2-EtOH(0.1%NH3H2O)]; B%:50%, isocratic elution mode). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[(2S)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]-5-[2-(dimethylamino)ethylamino]-5-oxo-pentanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (15 mg, 9.29 pmol, 14.3% yield, 100% purity). 'H NMR (400 MHz, CDCh), 8.15 (brs, 1H), 4.80-4.93 (m, 2H), 4.68 (brs, 1H), 3.99-4.12 (m, 4H), 3.61 (brs, 4H), 2.62-3.38 (m, 15H), 2.24-2.52 (m, 15H), 1.82-1.93 (m, 2H), 1.55-1.68 (m, 14H), 1.42-1.53 (m, 12H), 1.18-1.40 (m, 98H), 0.89 (t, J=6.4Hz, 18H). (1 / 2M+H+): 808.0. LCMS-CAD:

[0906] (1 / 2M+H+): 808.0 @ 12.861 mm. LCMS-ELSD: (1 / 2M+H+): 808.2 @ 11.649 mm.

[0907]

[0908] A mixture of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (2 g, 2.82 mmol, 1 eq), (2S)-2-(benzyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoic acid (1.07 g, 2.82 mmol, 1 eq) and EDCI (1.08 g, 5.64 mmol, 2 eq), DMAP (68.91 mg, 564.03 pmol, 0.2 eq) in dichloromethane (20 mL) was degassed and purged with N23 times, and then the mixture was stirred at 20 °C for 8 hours under N2 atmosphere. The combined organic phase was diluted with ethyl acetate (20 mL) and washed with water (3 x 10 mL) and brine (2 x 10 mL), dried with anhydrous Na2SO4, fdtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 2 / 1, 3%

[0909] NH3 THF) to afford 7-[2-[[(2S)-2-(benzyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1.9 g, 1.54 mmol, 54.70% yield, 87% purity). (400 MHz, CDCh), 7.26-7.39 (m, 5H), 6.46 (brs, 1H), 5.48 (brs, 1H), 5.04-5.17 (m, 2H), 4.82-4.93 (m, 1H), 4.55-4.69 (m, 1H), 3.98-4.16 (m, 3H), 3.02-3.33 (m, 4H), 1.97-2.67 (m, 10H), 1.80-1.90 (m, 1H), 1.65-1.74 (m, 12H), 1.52-1.58 (m, 4H), 1.38-1.42 (m, 9H), 1.18-1.28 (m, 51H), 0.89 (t, J=6.4 Hz, 9H).

[0910] Step 2:

[0911] A solution of Pd / C (0.1 g, 93.97 pmol, 10% purity, 1.26e-l eq) in ethyl acetate (5 mL) was added 7-[2-[[(2S)-2-(benzyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.8 g, 746.55 pmol, 1 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 3 hours. The mixture was fdtered through celite and the solvent was removed under reduced pressure to afford 7-[2-[[(2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl] amino]heptyl decanoate (0.6 g, 537.62 pmol, 72.01% yield). 'H NMR (400 MHz, CDCh), 4.80-4.94 (m, 1H), 4.61 (brs, 1H), 4.02-4.12 (m, 2H), 3.05-3.42 (m, 5H), 2.17-2.68 (m, 10H), 1.70-1.90 (m, 2H), 1.62-1.69 (m, 8H), 1.48-1.53 (m, 6H), 1.32-1.37 (m, 9H), 1.09-1.29 (m, 52H), 0.88 (t, J=6.4 Hz, 9H).

[0912] Step 3:

[0913] To a solution of 7-[2-[[(2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.6 g, 640.02 pmol, 1 eq), 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (463.47 mg, 640.02 pmol, 1 eq) in dichloromethane (5 mL) was added dropwise EDCI (245.39 mg, 1.28 mmol, 2 eq), DMAP (15.64 mg, 128.00 pmol, 0.2 eq). The mixture was stirred at 20 °C for 8 hours. The combined organic phase was diluted with ethyl acetate (10 mL) and washed with water (3 x 5 mL) and brine (2 x 5 mL), dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 3 / 1, 3% NH3 H2O) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient:45%-95% B over 15.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA (3 x 10 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 7-[2-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.4 g, 209.30 pmol, 49.14% yield). NMR (400 MHz, CDCh), 7.80 (brs, 1H), 6.40 (brs, 1H), 4.81-4.94 (m, 2H), 4.64-4.77 (m, 1H), 4.28-4.39 (m, 1H), 4.01-4.09 (m, 4H), 2.92-3.44 (m, 7H), 2.25-2.52 (m, 16H), 1.49-1.69 (m, 26H), 1.41-1.45 (m, 9H), 1.20-1.38 (m, 104H), 0.89 (t, J=6.4 Hz, 18H).

[0914] Step 4:

[0915] To a solution of7-[2-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]hexanoyl]amino]ethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.3 g, 182.53 pmol, 1 eq) in dioxane (1 mL) was added HCl / di oxane (4 M, 1 mL, 21.91 eq). The mixture was stirred at 20 °C for 2 hours. The reaction mixture was adjusted pH=7 with saturated NaHCOs aqueous and extracted with ethyl acetate (3 x 5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1, 3% NH3 H2O) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase:

[0916] [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 52%-92% B over 15.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA 30 mL (10 mL*3, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 1-octylnonyl 10-[[(2S)-6-amino-2-[[2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]hexanoyl]amino]-9-(7-decanoyloxyheptylamino)decanoate (0.07 g, 43.99 pmol, 33.95% yield, 97% purity). 'H NMR (400 MHz, CDCh), 8.56 (brs, 1H), 7.80 (brs, 1H), 5.70 (brs, 1H), 4.78-4.99 (m, 2H), 4.23-4.49 (m, 1H), 3.94-4.12 (m, 4H), 2.83-3.85 (m, 12H), 2.14-2.68 (m, 12H), 1.95-2.07 (m, 2H), 1.76-1.87 (m, 4H), 1.57-1.68 (m, 14H), 1.36-1.49 (m, 14H), 1.15-1.32 (m, 96H), 0.89 (t, J=6.8 Hz, 18H). (1 / 2M+H+): 772.5. LCMS-CAD: (1 / 2M+H+): 772.5 @ 10.619 mm. LCMS-ELSD: (1 / 2M+H+): 772.6 @ 11.753 mm. Example 13. Synthesis of compound 2628

[0917]

[0918]

[0919] Step 1:

[0920] To a solution of heptadecan-9-ol (30 g, 116.97 mmol, 1 eq) in dichloromethane (300 mL) was added EDCI (26.91 g, 140.37 mmol, 1.2 eq) and DMAP (7.15 g, 58.49 mmol, 0.5 eq) and 8-bromooctanoic acid (26.10 g, 116.97 mmol, 1 eq). The mixture was stirred at 20 °C for 8 hours. The reaction mixture was quenched by addition H2O (500 mL) at 0 °C, and then extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=100 / l to 10 / 1) to afford 1-octylnonyl 8-bromooctanoate (40 g, 86.66 mmol, 74.09% yield).

[0921] Step 2:

[0922] To a solution of 1-octylnonyl 8-bromooctanoate (30 g, 65.00 mmol, 1 eq) was

[0923] added phenylmethanamine (34.82 g, 324.99 mmol, 35.43 mL, 5 eq). The mixture was stirred at 20 °C for 8 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 0 / 1) to afford 1-octylnonyl 8-(benzylamino)octanoate (19 g, 37.78 mmol, 73.72% yield).

[0924]

[0925] NMR (400 MHz, CDCh), 7.27-7.59 (m, 5H), 4.83-4.86 (m, 1H), 4.00 (s, 2H), 2.71-2.75 (m, 2H), 2.21-2.25 (m, 2H), 1.82 (s, 2H), 1.49-1.58 (m, 6H), 1.21-1.27 (m, 31H), 0.88 (t, J=6.4 Hz, 6H).

[0926] Step 3

[0927] To a solution of 7-bromoheptan-l-ol (10 g, 51.26 mmol, 1 eq) in dichloromethane (100 mL) was added EDCI (49.13 g, 256.28 mmol, 5 eq), DMAP (1.88 g, 15.38 mmol,

[0928] 0.3 eq) and decanoic acid (9.71 g, 56.38 mmol, 10.88 mL, 1.1 eq). The mixture was stirred at 20 °C for 8 hours. The reaction mixture was quenched by addition H2O (500 mL) at 0 °C, and then extracted with ethyl acetate (3 x 300 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 0 / 1) to afford 7-bromoheptyl decanoate (60 g, 171.75 mmol, 67.02% yield). Step 4:

[0929] To a solution of 1 -octylnonyl 8-(benzylamino)octanoate (10 g, 20.50 mmol, 1 eq) in DMF (100 mL) was added K2CO3 (8.50 g, 61.50 mmol, 3 eq), KI (6.81 g, 41.00 mmol, 2 eq) and 7-bromoheptyl decanoate (8.59 g, 24.60 mmol, 1.2 eq). The mixture was stirred at 50 °C for 8 hours. The reaction mixture was quenched by addition H2O (100 mL) at 0 °C, and then extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=100 / l to 0 / 1) to afford 7-[benzyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (12 g, 15.23 mmol, 74.31% yield).

[0930] Step 5:

[0931] To a solution of 7-[benzyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (18 g, 23.80 mmol, 1 eq) in ethyl acetate (180 mL) was added to Pd / C (5 g, 4.70 mmol, 10% purity, 1.97e-l eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (50 Psi) at 20 °C for 8 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=50 / l to 0 / 1) to afford 7-[[8-(l -octylnonoxy)-8-oxo-octyl]amino] heptyl decanoate (10 g, 15.01 mmol, 63.07% yield).

[0932]

[0933] NMR (400 MHz, CDCh), 4.85-4.88 (m, 1H), 4.04-4.08 (m, 2H), 2.58-2.62 (m, 4H), 2.26-2.31 (m,4H), 1.60-1.63 (m 8H), 1.41-1.51 (m, 8H), 1.26-1.36 (m, 47H), 0.88 (t, J=6.4 Hz, 9H).

[0934] Step 6:

[0935] To a solution of 7-[[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (6 g, 9.01 mmol, 1 eq) in DMF (60 mL) was added K2CO3 (3.73 g, 27.02 mmol, 3 eq), KI (2.99 g, 18.02 mmol, 2 eq) and benzyl 2-bromoacetate (2.27 g, 9.91 mmol, 1.55 mL, 1.1 eq). The mixture was stirred at 80 °C for 8 hours. The reaction mixture was quenched by addition H2O 60 mL at 0 °C, and then extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na2SO4, fdtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 0 / 1) to afford 7-[(2-benzyloxy-2-oxo-ethyl)-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (3.6 g, 3.14 mmol, 34.85% yield, 71% purity).

[0936] Step 7:

[0937] A solution of 7-[(2-benzyloxy-2-oxo-ethyl )-[8-(l-octylnonoxy) -8-oxo-octyl] amino] heptyl decanoate (3.6 g, 4.42 mmol, 1 eq) in ethyl acetate (50 mL) was added to Pd / C (360.00 mg, 338.28 pmol, 10% purity, 7.65e-2 eq) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 Psi) at 20 °C for 8 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 0 / 1) to afford 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (2.8 g, 3.87 mmol, 87.46% yield).

[0938]

[0939] NMR (400 MHz, CDCh), 4.84-4.87 (m, 1H), 4.03-4.15 (m, 3H), 3.49 (s, 2H), 3.01-3.05 (m,4H), 2.26-2.31 (m, 4H), 1.60-1.63 (m 6H), 1.50-1.51 (m, 4H), 1.30-1.35 (m, 54H), 0.88 (t, J=6.8Hz, 9H).

[0940] Step 8:

[0941] To a solution of 7-[2-aminoethyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (2 g, 2.82 mmol, 1 eq in dichloromethane (20 mL) was added EDCI (648.76 mg, 3.38 mmol, 1.2 eq) DMAP (172.27 mg, 1.41 mmol, 0.5 eq and ((benzyloxy)carbonyl)-D-proline (773.26 mg, 3.10 mmol, 1.1 eq. The mixture was stirred at 20 °C for 8 hours. The reaction mixture was quenched by addition H2O (20 mL) at 0 °C, and then extracted with ethyl acetate (3 x 40 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 0 / 1) to afford benzyl (R)-2-((2-((7-(decanoyloxy)heptyl)(8-(heptadecan-9-yloxy) -8-oxooctyl) amino) ethyl)carbamoyl)pyrrolidine-l -carboxylate (2 g, 2.13 mmol, 75.41% yield).

[0942] Step 9:

[0943] To a solution of benzyl (R)-2-((2-((7-(decanoyloxy)heptyl)(8-(heptadecan-9-yloxy)-8-oxooctyl) amino)ethyl) carbamoyl)pyrrolidine-l -carboxylate (700 mg, 744.35 pmol, 1 eq in ethyl acetate (7 mL) was added to Pd / C (200 mg, 187.93 pmol, 10% purity, 2.52e-l eq under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 Psi) at 20 °C for 8 hours. The mixture was filtered and the filtrate

[0944] was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=20 / l to 0 / 1) to afford (R)-7-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-(pyrrolidine-2-carboxamido)ethyl)amino)heptyl decanoate (450 mg, 558.11 pmol, 74.98% yield)

[0945]

[0946] 4.87 (m, 1H), 4.03-4.06 (m, 2H), 3.71-3.82 (m, 1H), 3.36-3.40 (m, 2H), 2.91-3.11 (m, 2H), 2.42-2.76 (m, 6H), 2.26-2.31 (m, 4H), 2.11-2.18 (m, 2H), 1.75-1.99 (m, 8H), 1.60-1.63 (m, 6H), 1.50-1.51 (m, 8H), 1.25-1.31 (m, 45H), 0.87 (t, J=6.4 Hz, 9H).

[0947] Step 10:

[0948] To a solution of (R)-7-((8-(heptadecan-9-yloxy)-8-oxooctyl)(2-(pyrrolidine-2-carboxamido) ethyl) amino)heptyl decanoate (450 mg, 558.11 pmol, 1 eq in dichloromethane (5 mL) was added EDCI (128.39 mg, 669.73 pmol, 1.2 eq) and DMAP (34.09 mg, 279.06 pmol, 0.5 eq and 2-[7-decanoyloxyheptyl-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (444.57 mg, 613.92 pmol, 1.1 eq. The mixture was stirred at 20 °C for 8 hours. The reaction mixture was quenched by addition H2O (10 mL) at 0 °C, and then extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=10 / l to 0 / 1). The residue was purified by prep-HPLC (column: Xselect CSH C18 100*30 mm*5um; mobile phase: [H2O(0.1% TFA)-ACN: THF=1:1]; gradient:45%-95% B over 15.0 min). The mixture was diluted with brine (100 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA 60 mL (20 mL*3, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with ACN (2 x 20 mL). The hexane phase was concentrated under N2 atmosphere to afford (R)-7-((2-(2-((2-((7-(decanoyloxy)heptyl)(8-(heptadecan-9-yloxy)-8-oxooctyl)amino)ethyl)carbamoyl)pyrrolidin-l-yl)-2-oxoethyl)(8-(heptadecan-9-yloxy)-8-oxooctyl)amino)heptyl decanoate (150 mg, 98.19 pmol, 17.00% yield,

[0949]

[0950]

[0951] Step 1:

[0952] To a solution of propanal (12.5 g, 215.22 mmol, 15.66 mL, 1 eq) in THF (500 mL) was added bromo(decyl)magnesium (1 M, 250.00 mL, 1.16 eq) at -78 °C. The mixture was stirred at 20 °C under N2 for 8 hours. The reaction mixture was quenched by addition saturated NH4Q (500 mL) at 0 °C under N2 atmosphere, and stirred at 0 °C for 0.5 hours. The mixture was extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with brine (500 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / 0 to 10 / 1) to afford tndecan-3-ol (46.5 g, 232.08 mmol, 53.92% yield).

[0953]

[0954] NMR (400 MHz, CDCk), 3.51-3.59 (m, 1H), 1.40-1.49 (m, 4H), 1.28 (brs, 16H), 0.96 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 6.8 Hz, 3H).

[0955] Step 2:

[0956] To a mixture of tridecan-3-ol (10 g, 49.91 mmol, 1 eq), 6-bromohexanoic acid (11.68 g, 59.89 mmol, 1.2 eq) and DMAP (1.22 g, 9.98 mmol, 0.2 eq) in dichloromethane (1000 mL) was added EDCI (19.14 g, 99.82 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C under N2 for 8 hours. The mixture was added H2O (1000 mL), extracted with di chloromethane (500 mL*3), combined organic layers were washed with brine (800 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 10 / 1) to afford 1 -ethylundecyl 6-bromohexanoate (40 g, 105.99 mmol, 70.79% yield).

[0957]

[0958] NMR (400 MHz, CDCh), 4.77-4.88 (m, 1H), 3.42 (t, J = 6.8 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.89 (q, J = 7.2 Hz, 2H), 1.67 (q, J = 7.6 Hz, 2H), 1.49-1.54 (m, 4H), 1.26-1.30 (m, 18H), 0.86-0.91 (m, 6H).

[0959] Step 3:

[0960] To a mixture of 1 -ethylundecyl 6-bromohexanoate (3.74 g, 9.92 mmol, 1.1 eq), 1-octylnonyl 8-(benzylamino)octanoate (4.4 g, 9.02 mmol, 1 eq), K2CO3 (3.74 g, 27.06 mmol, 3 eq) and KI (748.68 mg, 4.51 mmol, 0.5 eq) in DMF (100 mL) was stirred at 80 °C under N2 for 8 hours. To the mixture was added H2O (150 mL), extracted with ethyl acetate (3 x 100 mL), and the combined organic layers were washed with brine (150 mL*2), dried over Na2SC>4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 10 / 1) to afford 1-octylnonyl 8-[benzyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (10 g, crude).

[0961] Step 4:

[0962] To a solution of Pd / C (2.04 g, 1.91 mmol, 10% purity, 0.3 eq in ethyl acetate (100 mL) was added 1-octylnonyl 8-[benzyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (5 g, 6.38 mmol, 1 eq) under N2. The mixture was stirred at 30 °C for 12 hours under H.(50 Psi) The mixture was filtered under N2, and filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1) to afford 1-octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (5.3 g, 7.64 mmol, 59.88% yield)

[0963]

[0964] 4.91 (m, 2H), 2.56-2.64 (m, 4H), 2.29-2.32 (m, 10.2 Hz, 4H), 1.52-1.70 (m, 13H), 1.23-1.36 (m, 52H), 0.85-0.92 (m, 12H).

[0965] Step 5:

[0966] To a mixture of 1-octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (5.3 g, 7.64 mmol, 1 eq), K2CO3 (3.17 g, 22.91 mmol, 3 eq and KI (633.71 mg, 3.82 mmol, 0.5 eq in DMF (30 mL) was added benzyl 2-bromoacetate (2.62 g, 11.45 mmol, 1.80 mL, 1.5 eq. The mixture was stirred at 80 °C under N2 for 8 hours. The mixture was added H2O (50 mL), extracted with ethyl acetate (3 x 30 mL), combined organic layers were washed with brine (50 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 10 / 1) to afford 1-octylnonyl 8-[(2-benzyloxy-2-oxo-ethyl)-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (4.8 g, crude).

[0967] Step 6:

[0968] To a solution of Pd / C (2 g, 1.88 mmol, 10% purity, 0.66 eq in ethyl acetate (50 mL) was added 1-octylnonyl 8-[(2-benzyloxy-2-oxo-ethyl)-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (2.4 g, 2.85 mmol, 1 eq under N2. The mixture was stirred at 20 °C for 8 hours under H2 (15 Psi). The mixture was filtered under N2, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to ethyl acetate / methanol = 10 / 1) to afford 2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (3 g, 3.99 mmol, 69.99% yield, 100% purity).

[0969]

[0970] 4.91 (m, 2H), 3.45 (s, 2H), 2.95-3.06 (m, 4H), 2.30-2.33 (m, 4H), 1.60-1.74 (m, 8H), 1.48-1.54 (m, 6H), 1.21-1.37 (m, 50H), 0.84-0.92 (m, 12H). Step 7:

[0971] To a mixture of 1 -octylnonyl 8-[2-aminoethyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (4.15 g, 5.63 mmol, 1 eq), 2-(tert-butoxycarbonylamino)acetic acid (1.08 g, 6.19 mmol, 1.1 eq) and DMAP (343.85 mg, 2.81 mmol, 0.5 eq) in dichloromethane (100 mL) was added EDCI (3.24 g, 16.89 mmol, 3 eq) at 0 °C. The mixture was stirred at 20 °C under N2 for 8 hours. The mixture was added H2O (150 mL), extracted with di chloromethane (3 x 100 mL), and the combined organic layers were washed with brine (2 x 150 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1) to afford 1-octylnonyl 8-[2-[[2-(tert-butoxycarbonylamino)acetyl]amino]ethyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (6.5 g, 7.27 mmol, 64.55% yield).

[0972]

[0973] NMR (400 MHz, CDCh), 4.78-4.91 (m, 2H), 3.75-3.86 (m, 2H), 3.27-3.45 (m, 2H), 2.41 (brs, 4H), 2.30 (td, J = 7.4, 12.4 Hz, 4H), 1.59-1.69 (m, 8H), 1.48-1.55 (m, 8H), 1.46 (s, 9H), 1.23-1.34 (m, 50H), 0.85-0.91 (m, 12H). Step 8:

[0974] To a solution of 1-octylnonyl 8-[2-[[2-(tert-butoxycarbonylamino)acetyl]amino]ethyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.5 g, 1.68 mmol, 1 eq) in dichloromethane (15 mL) was added TFA (7.68 g, 67.31 mmol, 5.00 mL, 40.14 eq). The mixture was stirred at 20 °C under N2 for 2 hours. The mixture reaction was concentrated under reduced pressure. Then the mixture was added sodium bicarbonate solution (10 mL) and H2O (5 mL), extracted with dichloromethane (10 mL*3), and the combined organic layers were washed with brine (10 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to afford 1-octylnonyl 8-[2-[(2-aminoacetyl)amino] ethyl- [6-(l -ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.3 g, crude).

[0975] Step 9:

[0976] To a mixture of 2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino] acetic acid (1.08 g, 1.44 mmol, 1.2 eq), 1-octylnonyl 8-[2-[(2-aminoacetyl)amino]ethyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (950 mg, 1.20 mmol, 1 eq) and DMAP (73.06 mg, 598.02 pmol, 0.5 eq) in dichloromethane (15 mL) was added EDCI (458.57 mg, 2.39 mmol, 2 eq) at 0 °C. The mixture was stirred at 20 °C under N2 for 8 hours. The mixture was added H2O (10 mL), extracted with ethyl acetate (20 mL*3), combined organic layers were washed with brine (10 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 1) and prep-HPLC (column: Xselect CSH Cl 8 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-95% B over 16.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA 30 mL (10 mL*3, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 1 -octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[2-[[2-[[2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]acetyl] amino]ethyl]amino]octanoate (20 mg, 13.09 pmol, 0.90%

[0977]

[0978] To a solution of 1-octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (4.1 g, 5.91 mmol, 1 eq) and tert-butyl 3-bromopropanoate (8.64 g, 41.34 mmol, 6.90 mL, 7 eq) in DMF (100 mL) was added K2CO3 (2.45 g, 17.72 mmol, 3 eq) and KI (490.23 mg, 2.95 mmol, 0.5 eq). The mixture was stirred at 80 °C for 8 hours. The mixture was concentrated under reduced pressure. The mixture was added H2O (100 mL), extracted with ethyl acetate (3 x 100 mL), combined organic layers were washed with brine (3 x 100 mL), dried over Na2SO4, fdtered and the fdtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 9 / 1) to afford 1-octylnonyl 8-[(3-tert-butoxy-3-oxo-propyl)-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (5 g, 6.08 mmol, 51.47% yield)

[0979]

[0980] 4.92 (m, 2H), 2.59-2.77 (m, 2H), 2.23-2.46 (m, 10H), 1.49-1.67 (m, 14H), 1.45 (s, 9H), 1.18-1.39 (m, 50H), 0.83-0.93 (m, 12H).

[0981] Step 2:

[0982] To a solution of 1-octylnonyl 8-[(3-tert-butoxy-3-oxo-propyl)-[6-(l-ethylundecoxy)-6-oxo-hexyl] amino]octanoate (5 g, 6.08 mmol, 1 eq) in dichloromethane (50 mL) was added TFA (23.03 g, 201.93 mmol, 15 mL, 33.21 eq). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure. The reaction mixture was adjusted to pH=7 with saturated aqueous NaHCCh and the mixture was added H2O (20 mL), extracted with ethyl acetate (3 x 30 mL), and the combined organic layer was washed with brine (3 x 30 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to afford 3-[[6-(l -ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (4.4 g, crude).

[0983]

[0984] 4.93 (m, 2H), 2.85-2.94 (m, 2H), 2.64-2.77 (m, 4H), 2.51 (t, J = 6.4 Hz, 2H), 2.30 (m, 4H), 1.49-1.70 (m, 16H), 1.23-1.36 (m, 48H), 0.83-0.94 (m, 12H).

[0985] Step 3:

[0986] To a solution of 1-octylnonyl 8-[2-[(2-aminoacetyl)amino]ethyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.7 g, 2.14 mmol, 1 eq) and 3-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoic acid (1.97 g, 2.57 mmol, 1.2 eq) in dichloromethane (20 mL) was added EDCI (1.23 g, 6.42 mmol, 3 eq) and DMAP (130.74 mg, 1.07 mmol, 0.5 eq) at 0 °C. The mixture was stirred at 25 °C for 8 hours. The mixture was added H2O (30 mL), extracted with di chloromethane (3 x 30 mL), combined organic layers were washed with brine (3 x 30 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 4) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H20(0.05%TFA)-ACN: THF=1:1]; gradient: 50%-90% B over 18.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA (3 x 10 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 1-octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[3-[[2-[2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-2-oxo-ethyl] amino]-3-oxo-propyl]amino]octanoate (23 mg, 16.21 pmol, 0.75% yield, 100% purity). NMR (400 MHz, CDCh), 8.71 (brs, 1H), 6.48 (brs, 1H), 4.80-4.88 (m, 4H), 3.89-4.10 (m, 2H), 3.28-3.88 (m, 6H), 3.11 (s, 1H), 2.28-2.47 (m, 17H), 1.55-1.64 (m, 14H), 1.51-1.53 (m, 16H), 1.27-1.31 (m, 98H), 0.86-0.90 (m, 24H). (1 / 2M+H+): 771.8. LCMS-CAD: (1 / 2M+H+): 771.8 @ 13.769 mm. LCMS-ELSD: (1 / 2M+H+): 771.8 @ 13.852 min.

[0987]

[0988] Step 1:

[0989] To a solution of 1 -octylnonyl 8-bromooctanoate (5 g, 10.83 mmol, 1 eq) and tert-butyl N- (3 -amino propyl)carbamate (7.55 g, 43.33 mmol, 7.57 mL, 4 eq) in DMF (200 mL) was added K2CO3 (4.49 g, 32.50 mmol, 3 eq) and KI (1.80 g, 10.83 mmol, 1 eq). The mixture was stirred at 80 °C for 8 hours. The mixture was concentrated under reduced pressure. The mixture was added H2O (30 mL), extracted with ethyl acetate (3 x 100 mL), combined organic layers were washed with brine (2 x 100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 3 / 1) to afford 1-octylnonyl 8-[3-(tert-butoxycarbonylamino)propylamino] octanoate (21 g, 37.85 mmol, 87.34% yield).

[0990]

[0991] NMR (400 MHz, CDCh), 5.15 (s, 1H), 4.80-4.93 (m, 1H), 3.22 (d, J= 5.4 Hz, 2H), 2.52-2.76 (m, 4H), 2.28 (t, J = 7.4 Hz, 2H), 1.85-2.07 (m, 2H), 1.47-1.76 (m, 11H), 1.45 (s, 9H), 1.24-1.33 (m, 28H), 0.88 (t, J = 6.8 Hz, 6H).

[0992] Step 2:

[0993] To a solution of 1-octylnonyl 8-[3-(tert-butoxycarbonylamino)propylamino]octanoate (10 g, 18.02 mmol, 1 eq) and 1 -ethylundecyl 6-bromohexanoate (7.48 g, 19.82 mmol, 1.1 eq) in DMF (200 mL) was added K2CO3 (7.47 g, 54.07 mmol, 3 eq) and KI (2.99 g, 18.02 mmol, 1 eq). The mixture was stirred at 80 °C for 8 hours. The mixture was concentrated under reduced pressure. The mixture was added H2O (50 mL), extracted with ethyl acetate (3 x 100 mL), combined organic layers were washed with brine (2 x 100 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 4 / 1) to afford 1-octylnonyl 8-[3-(tert-butoxycarbonylamino)propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (13 g, 15.27 mmol, 42.36% yield)

[0994]

[0995] 4.90 (m, 1H), 4.78-4.84 (m, 1H), 2.82-3.43 (m, 4H), 2.23-2.52 (m, 6H), 1.47-1.73 (m, 28H), 1.18-1.40 (m, 50H), 0.80-0.96 (m, 12H).

[0996] Step 3:

[0997] To a solution of 1-octylnonyl 8-[3-(tert-butoxycarbonylamino)propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino] octanoate (8 g, 9.40 mmol, 1 eq) and in dichloromethane (70 mL) was added TFA (35.09 g, 307.71 mmol, 22.86 mL, 32.75 eq). The mixture was stirred at 25 °C for 2 hours. The crude reaction mixture was adjusted to pH=7 with saturated aqueous NaHCCh and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to ethyl acetate / Methanol= 15 / 1) to afford 1-octylnonyl 8-[3-aminopropyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (6.8 g, 9.05 mmol, 96.33% yield). 'H NMR (400 MHz, CDCh), 4.84-4.89 (m, 1H), 4.78-4.83 (m, 1H), 2.96-3.19 (m, 6H), 2.76 (s, 2H), 2.24-2.36 (m, 4H), 1.89-3.19 (m, 2H), 1.47-1.67 (m, 16H), 1.21-1.39 (m, 50H), 0.80-0.97 (m, 12H)

[0998] Step 4:

[0999] To a solution of 1-octylnonyl 8-[3-aminopropyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino] octanoate (6.3 g, 8.39 mmol, 1 eq) and 2-(tert-butoxycarbonylamino)acetic acid (1.62 g, 9.22 mmol, 1.1 eq) in di chloromethane (100 mL) was added EDCI (4.82 g, 25.16 mmol, 3 eq) andDMAP (512.24 mg, 4.19 mmol, 0.5 eq) at 0 °C. The mixture was stirred at 25 °C for 8 hours. The mixture was concentrated under reduced pressure. The mixture was added H2O (30 mL), extracted with di chloromethane (60 mL*3), combined organic layers were washed with brine (3 x 30 mL), dried over Na2SO4, fdtered and the fdtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to ethyl acetate / methanol= 15 / 1) to afford 1-octylnonyl 8-[3-[[2-(tert-butoxycarbonylamino)acetyl]amino] propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (5.76 g, 6.34 mmol, 75.61% yield).

[1000]

[1001] NMR (400 MHz, CDCh), 7.52 (d, J = 4.8 Hz, 1H), 5.19- 5.29 (m, 1H), 4.76-4.93 (m, 2H), 3.77 (d, J = 5.2 Hz, 2H), 3.30-3.45 (m, 2H), 2.41-2.60 (m, 4H), 2.26-2.35 (m, 4H), 1.48-1.70 (m, 20H), 1.46 (s, 9H), 1.23-1.35 (m, 48H), 0.83-0.93 (m, 12H). Step 5:

[1002] To a solution of 1-octylnonyl 8-[3-[[2-(tert-butoxycarbonylamino)acetyl]amino]propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.5 g, 1.65 mmol, 1 eq) in dichloromethane (15 mL) was added TFA (7.68 g, 67.31 mmol, 5 mL, 40.76 eq). The mixture was stirred at 25 °C for 2 hours. The mixture was concentrated under reduced pressure. The mixture was added H2O (10 mL), extracted with ethyl acetate (3 x 20 mL), combined organic layers were washed with brine (20 mL*2), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure to afford 1-octylnonyl 8-[3-[(2-aminoacetyl)amino]propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl] amino] octanoate (1.2 g, 1.48 mmol, 89.91% yield). 'H NMR (400 MHz, CDCh), 10.53-10.83 (m, 1H), 9.05 (s, 1H), 8.32-8.78 (m, 1H), 4.71-5.04 (m, 2H), 3.88 (s, 2H), 3.41 (s, 2H), 2.91-3.30(m, 6H), 2.23-2.40(m, 4H), 1.97-2.05 (m, 2H), 1.55-1.82 (m, 14H), 1.22-1.43 (m, 50H), 0.83-0.94 (m, 12H)

[1003] Step 6:

[1004] To a solution of 1-octylnonyl 8-[3-[(2-aminoacetyl)amino]propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.2 g, 1.48 mmol, 1 eq) and 2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetic acid (1.34 g, 1.78 mmol, 1.2 eq) in dichloromethane (20 mL) was added EDCI (853.79 mg, 4.45 mmol, 3 eq) and DMAP (90.68 mg, 742.29 pmol, 0.5 eq) at 0 °C. The mixture was stirred at 25 °C for 8 hours. The mixture was added H2O (20 mL), extracted with dichloromethane (3 x 20 mL), combined organic layers were washed with brine (20 mL*3), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 4) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-95% B over 16.0 min). The mixture was diluted with brine (100 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA (3 x 20 mL, 10:1). The hexane phase was concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with ACN (2 x 20 mL). The hexane phase was concentrated under N2 atmosphere to afford 1 -octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[3-[[2-[[2-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]acetyl]amino]acetyl] amino]propyl]amino]octanoate (125 mg, 81.04 pmol, 18.66% yield, 100% purity).

[1005]

[1006] MHZ, CDC13), 8.03 (s, 1H), 4.79 - 4.88 (m, 4H), 3.97-4.17 (m, 4H), 2.97-3.44 (m, 10H), 2.26-2.50 (m, 10H), 1.57-1.99 (m, 12H), 1.41-1.51 (m, 13H), 1.27- 1.37 (m, 105H), 0.86-0.90 (m, 24H). (l / 2M+H+): 772.0. LCMS-CAD: (l / 2M+H+): 772.0@ 13.563 min.

[1007] LCMS-ELSD: (l / 2M+H+): 772.2@ 13.640 mm.

[1008] Example 17. Synthesis of compound 2632

[1009]

[1010] Step 1:

[1011] To a solution of l-octylnonyl 8-[3-[[2-(tert-butoxycarbonylamino)acetyl]amino]propyl- [6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (2 g, 2.20 mmol, 1 eq) in dichloromethane (20 mL) was added TEA (10.75 g, 94.24 mmol, 7 mL, 42.80 eq). The mixture was stirred at 25 °C for 2 hours. The crude reaction mixture was adjusted to pH=7 with saturated aqueous NaHCCh and extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to afford l-octylnonyl 8-[3-[(2-aminoacetyl)amino]propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl]amino]octanoate (1.9 g, crude). NMR (400 MHz, CDCl₃), 8.42 (s, 1H), 4.75-4.91 (m, 2H), 3.33-3.68 (m, 4H), 2.92-3.19 (m, 6H), 2.23-2.40 (m, 4H), 2.00 (s, 2H), 1.46-1.78 (m, 16H), 1.17-1.43 (m, 50H), 0.76-0.97 (m, 12H). Step 2:

[1012] To a solution of 1-octylnonyl 8-[3-[(2-aminoacetyl)amino]propyl-[6-(l-ethylundecoxy)-6-oxo-hexyl] amino] octanoate (1.9 g, 2.35 mmol, 1 eq) in dichloromethane (20 mL) was added EDCI (1.35 g, 7.05 mmol, 3 eq) and 3-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl] amino] propanoic acid (2.16 g, 2.82 mmol, 1.2 eq) and DMAP (143.58 mg, 1.18 mmol, 0.5 eq) at 0 °C. The mixture was stirred at 25 °C for 8 hours. The mixture was added H2O (30 mL), extracted with di chloromethane (3 x 30 mL), combined organic layers were washed with brine (3 x 30 mL), dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=l / O to 1 / 4) and prep-HPLC (column: Xselect CSH C18 100*30mm*5um; mobile phase: [H2O(0.1%TFA)-ACN: THF=1:1]; gradient: 50%-90% B over 16.0 min). The mixture was diluted with brine (60 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under N2 atmosphere. The residue was diluted with hexane (20 mL) and washed with the mixture of ACN and TEA (3 x 10 mL, 10:1). The hexane phase wasconcentrated under N2 atmosphere. The residue was diluted with hexane (10 mL) and washed with ACN (2 x 10 mL). The hexane phase was concentrated under N2 atmosphere to afford 1-octylnonyl 8-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[3-[[2-[3-[[6-(l-ethylundecoxy)-6-oxo-hexyl]-[8-(l-octylnonoxy)-8-oxo-octyl]amino]propanoylamino]acetyl] amino]propyl]amino]octanoate (0.762 g, 479.76 pmol, 46.67% yield, 98% purity). ¹H NMR (400 MHz, CDCl₃), 7.82-9.20 (m, 1H), 4.80-4.88 (m, 4H), 3.84 (s, 2H), 3.31-3.44 (m, 2H), 2.81-3.04 (m, 5H), 2.28 - 2.49 (m, 16H), 2.03 (s, 1H), 1.55-1.63 (m, 10H), 1.51-1.53 (m, 20H), 1.26-1.30 (m, 100H), 0.86-0.90 (m, 24H). (1 / 2M+H+): 779.0.

[1013] LCMS-CAD: (1 / 2M+H+): 779.0 @ 13.593 min. LCMS-ELSD: (1 / 2M+H+): 779.0 @ 13.720 min.

[1014] Example 18. Synthesis of compound 2633

[1015]

[1016]

[1017] Step 1:

[1018] To a solution of heptadecan-9-amine (6.87 g, 26.89 mmol, 1 eq) and 8-bromooctanoic acid (6 g, 26.89 mmol, 1 eq) in dichloromethane (500 mL) was added DMAP (657.10 mg, 5.38 mmol, 0.2 eq) and EDCI (10.31 g, 53.79 mmol, 2 eq) at 0 °C and the mixture was stirred at 20 °C for 12 hours. The reaction mixture was diluted with H2O (500 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic layers were washed with brine (600 mL*3) and dried over Na2SO4, filtered and the filtrate was concentrated. The residue was purified byflash silica gel chromatography (SiCh, petroleum ether / ethyl acetate=10 / l) to afford 8-bromo-N-(l-octylnonyl)octanamide (33 g, 71.65 mmol, 53.29% yield).

[1019]

[1020] NMR (400 MHz, CDCl₃), 5.16 (brs, 1H), 3.82 - 3.99 (m, 1H), 3.40 (t, J=6.82 Hz, 2H), 2.17 (t, J=7.50 Hz, 2H), 1.72-1.92 (m, 2H), 1.57-1.69 (m, 2H), 1.38-1.51 (m, 4H), 1.22-1.35 (m, 29H), 0.88 (brt, J=6.75 Hz, 6H).

[1021] Step 2:

[1022] To a solution of phenylmethanamine (3.07 g, 28.66 mmol, 3.12 mL, 2 eq) in DMF (500 mL) were added K2CO3 (5.94 g, 42.99 mmol, 3 eq), KI (4.76 g, 28.66 mmol, 2 eq) and 8-bromo-N-(l -octylnonyl) octanamide (6.6 g, 14.33 mmol, 1 eq), and the mixture was stirred at 80 °C for 12 hours under N2 atmosphere. The reaction mixture was diluted with H2O (150 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (SiCh, petroleum ether / ethyl acetate=O / l) to afford 8-(benzylamino)-N-(l-octylnonyl) octanamide (25 g, 51.35 mmol, 71.67% yield). ¹H NMR (400 MHz, CDCl₃), 7.19-7.29 (m, 5H), 5.18 (brd, J=9.13 Hz, 1H), 3.82-3.92 (m, 1H), 3.74 (s, 2H), 2.58 (t, J=7.19 Hz, 2H), 2.09 (t, J=7.57Hz, 2H), 1.70 (brs, 2H), 1.52-1.63 (m, 2H), 1.36-1.51 (m, 4H), 1.18-1.30 (m, 3 OH), 0.83 (brt, J=6.75 Hz, 6H).

[1023] Step 3:

[1024] To a solution of undecyl 6-bromohexanoate (2.94 g, 8.40 mmol, leq) in DMF (100 mL) was added K2CO3 (3.48 g, 25.20 mmol, 3 eq) and KI (697.33 mg, 4.20 mmol, 0.5 eq), then 8-(benzylamino)-N-(heptadecan-9-yl)octanamide (4.09 g, 8.40 mmol, 1 eq) was added to the mixture, then stirred at 80 °C for 6 hours. The residue was diluted with H2O (200 mL) and extracted with ethyl acetate (3 x 200 mL). The combined organic phase was washed with brine (200 mL), dried over Na2SO4, fdtered and the fdtrate was concentrated. The residue was purified by flash silica gel chromatography (SiCh, petroleum ether / ethyl acetate=20 / l) to afford undecyl 6-(benzyl(8-(heptadecan-9-ylamino)-8-oxooctyl)amino)hexanoate (10.4 g, 13.77 mmol, 81.95% yield). NMR (400 MHz, CDCl₃), 7.27 (s, 6H), 5.12 (brd, J=9.13 Hz, 1H), 4.01-4.20 (m, 3H), 3.90 (brd, J=7.88 Hz, 1H), 3.54 (brs, 1H), 2.32-2.45 (m, 3H), 2.28 (brt, J=7.57 Hz, 2H), 2.14 (t, J=7.57 Hz, 2H), 1.56-1.68 (m, 7H), 1.47 (brs, 5H), 1.26 (brd, J=4.13 Hz, 50H), 0.83-0.92 (m, 9H). Step 4:

[1025] To a solution of Pd / C (7.33 g, 6.89 mmol, 10% purity, 0.5 eq) in ethyl acetate (150 mL) was added undecyl 6-[benzyl-[8-(l-octylnonylamino)-8-oxo-octyl]amino]hexanoate (10.4 g, 13.77 mmol, 1 eq) at 20 °C under An, and the reaction mixture was stirred at 20 °C for 12 hours under H2(50 Psi). The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (SiCh, petroleum ether / ethyl acetate=O / l) to afford undecyl 6-[[8-(l-octylnonylamino)-8-oxo-octyl]amino]hexanoate (6 g, 9.02 mmol, 65.51% yield). ¹H NMR (400 MHz, CDCl₃), 5.16 (brd, J=9.13 Hz, 1H), 4.04 (t, J=6.69 Hz, 2H), 3.87 (brd, J=8.00 Hz, 1H), 2.57 (q, J=6.80 Hz, 4H), 2.29 (t, J=7.50 Hz, 2H), 2.13 (t, J=7.50 Hz, 2H), 1.55-1.68 (m, 7H), 1.40-1.54 (m, 7H), 1.21-1.33 (m, 49H), 0.79-0.94 (m, 9H).

[1026] Step 5:

[1027] To a solution of undecyl 6-[[8-(l-octylnonylamino)-8-oxo-octyl]amino]hexanoate (6 g, 9.02 mmol, 1 eq) in DMF (100 mL) were added K2CO3 (3.74 g, 27.06 mmol, 3 eq), KI (2.99 g, 18.04 mmol, 2 eq) and benzyl 2-bromoacetate (3.10 g, 13.53 mmol, 2.12 mL, 1.5 eq), and the mixture was stirred at 80 °C for 12 hours under N2 atmosphere. The reaction mixture was diluted with H2O (150 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (SiCh, petroleum ether / ethyl acetate=20 / l) to afford undecyl 6-[(2-benzyloxy-2-oxo-ethyl)-[8-(l-octylnonylamino)-8-oxo-octyl]amino]hexanoate (6 g, 7.38 mmol, 81.78% yield).

[1028]

[1029] NMR (400 MHz, CDCl₃), 7.28 (d, J=4.75 Hz, 5H), 5.06 (s, 2H), 3.97 (t, J=6.69 Hz, 2H), 3.82 (brs, 1H), 3.28 (s, 2H), 2.39-2.55 (m, 4H), 2.15-2.28 (m, 3H), 2.06 (brt, J=7.57Hz, 2H), 1.31-1.61 (m, 12H), 1.08-1.28 (m, 50H), 0.69-0.86 (m, 9H).

[1030] Step 6:

[1031] To a solution of Pd / C (7.85 g, 7.38 mmol, 10% purity, 1 eq) in ethyl acetate (100 mL) was added undecyl 6-[(2-benzyloxy-2-oxo-ethyl)-[8-(l-octylnonylamino)-8-oxo-octyl]amino]hexanoate (6 g, 7.38 mmol, 1 eq) at 20 °C under A and the reaction mixture was stirred at 20 °C for 12 hours under H2 (50 Psi)...

Claims

1. WHAT IS CLAIMED IS:

1. A compound of formula (IA-1) or (IA-2):

7.

11.

12. (IA-2), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein:13.aal and aa2 each are independently a natural or unnatural amino acid residue, wherein * represents the bonding to aal or aa 2 via its backbone N atom and • represents the bonding to aal or aa2 via its backbone carbonyl C atom;14.each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;15.each R, for each occurrence, is independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl;16.each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;17.j is 1, 2, or 3;18.W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;19.each A is independently absent, C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;21.each X is independently absent, X’,22. 24.each R’ is independently Ci-Cs alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and each X’ is independently a biodegradable moiety,26.provided that in at least one or two of the27.

28. tails, A and X are not absent.

2. The compound of claim 1, wherein aal and aa2 each are independently:30.an a-amino acid, P-amino acid, or y-amino acid, a natural amino acid in L-stereoisomer or D-stereoisomer, or a non-canonical amino acid.

3. The compound of claim 1, wherein one of aal and aa2 is selected from the group consisting of Gly, Cys, Pro, and its variants thereof.

4. The compound of claim 1, wherein one of aal and aa2 has a hydrophobic side chain, and is selected from the group consisting of Ala, Vai, He, Leu, Met, Phe, Tyr, Trp, and its variants thereof.

5. The compound of claim 1, wherein one of aal and aa2 has a polar, uncharged side chain, and is selected from the group consisting of Ser, Thr, Asn, Gin, and its variants thereof.

6. The compound of claim 1, wherein one of aal and aa2 has an electrically charged side chain, and is selected from the group consisting of Arg, His, Lys, Asp, Glu, and its variants thereof.

7. The compound of claim 1, wherein aal-aa2 is Gly-Gly, P-Ala-Gly, Gly-P-Ala, Gly -Phe, Gly-38.

39. Pro, P-Ala-Pro, Gly-Thr, Gly-Asn, P-Ala-P-Ala, y- aminobutyric acid-Gly, or variants thereof.

8. The compound of one of claims 3-7, wherein the variant of the amino acid is a squaramide or squaramide derivative.

9. A compound of formula (IB-1) or (IB-2):

42.

43. salt thereof, or a stereoisomer thereof, wherein:44.each of il and i2 is independently 0, 1, 2, or 3;45.j is 1, 2, or 3;46.each of Rai, Ra2, Rbi, and Rb2 is, for each occurrence, independently H, Ci-Ce branched or unbranched alkyl, -(CRJR2)vR3, -(CR1R2)vSR4, -(CR1R2)vSeR4, -(CR1R2)vOR4, -(CR1R2)VCOR5, -(CR1R2)VC(O)OR4, -(CR1R2)VNR1R2, -(CR1R2)VN(R1)(CR1R2)VNR1R2, -(CR1R2)VC(O)NR1R2, -(CR1R2)VN(R1)C(O)NR1R2, -(CR1R2)VN(R1)C(O)R5, -(CR1R2)VC(S)NR1R2, -(CR1R2)VN(R1)C(S)NR1R2, or -(CR1R2)VN(R1)C(=NR1)NR1R2; or one Ra2or Rb2 adjacent to the N atom is taken together with its adjacent N atom and R variable to form a heterocyclic ring, optionally substituted with one or more alkyl, OR4, SR4, and / or halogen groups;47.each R1is, for each occurrence, independently H, OH, or C1-C3 alkyl;48.each R2is, for each occurrence, independently H or C1-C3 alkyl;49.each R3is independently an aryl or heteroaryl, optionally substituted with one or more alkyl, C(O)OR4, OR4, SR4, oxo, and / or halogen groups;52.each R4is independently H, C1-C3 alkyl, aryl,53.

54. -(CR1R2)VC(O)(CR1R2)VNR1R2;55.each R5is independently H, C1-C3 alkyl, -NR^CR^jvNR^2, or -(CR1R2)VC(O)(CR1R2)VNR1R2;56.each v is independently an integer of 0-4; each Y is, for each occurrence, independently -O-, -S-, -N(R)-, a divalent heterocyclic, optionally substituted with one or more alkyl, OH, SH, and / or halogen groups;57.each R is, for each occurrence, independently H, C1-C3 branched or unbranched alkyl, or C2-C3 branched or unbranched alkenyl;58.each V is, for each occurrence, independently Ci-Ce branched or unbranched alkylene, C3-C7 cycloalkylene, or C2-C6 branched or unbranched alkenylene;59.W is R6, OR6, or NHR6, wherein R6is H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;60.each A is independently absent, C1-C16 branched or unbranched alkylene, or C2-C16 branched or unbranched alkenylene, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups; each B is independently H, C1-C24 branched or unbranched alkyl, C2-C24 branched or unbranched alkenyl, C2-C24 branched or unbranched alkynyl, or a C2-C24 branched or unbranched, unsaturated monovalent hydrocarbon chain containing two or more double bonds or triple bonds, each of these groups being optionally interrupted by one or more heteroatoms or optionally substituted with one or more OH, SH, and / or halogen groups;62.each X is independently absent, X’,63. 65.each R’ is, for each occurrence, independently Ci-Cs alkyl, C2-C5 alkenyl, or C2-C5 alkynyl; and66.each X’ is independently a biodegradable moiety,67.-f— A - X - B68.provided that in at least one or two of the tails, A and X are not absent.

10. The compound of claim 9, wherein il is 0, i2 is 0, or both il and i2 are 0.

11. The compound of claim 9, wherein il is 1, i2 is 1, or both il and i2 are 1.

12. The compound of claim 9, wherein il is 2, i2 is 2, or both il and i2 are 2.

3. The compound of claim 9, wherein the core formula73.

74. RNis H, R2, or C(O)R5; and75.Rqis H or OR414. The compound of claim 1 or 9, wherein j is 1 or 2.

15. The compound of claim 1 or 9, wherein each V is, for each occurrence, C2-C3 alkylene.

16. The compound of claim 9, wherein the compound has the structure of83.

84. wherein each m is, for each occurrence, 2 or 3.

7. The compound of claim 9, wherein the compound has the structure of92.

98. 100.(IIIA-14),105.

106. ,116.

117. (-), or118.

119. (IIIA-30), wherein:120.each m is, for each occurrence, 2 or 3;121.Rpis H, C(O)OH, or OH;122.RNis H, C(O)R5, or C1-C3 alkyl;124. 126.R2and R5are each H or C1-C3 alkyl; and127.v is 1-4.

18. The compound of any one of claims 1, 9, and 14-17, wherein each Y is, for each occurrence,130.

19. The compound of any one of claims 1-18, wherein at least two, at least three, or at least four -i— A - X - B133.of the tails are the same.

20. The compound of any one of claims 1-18, wherein at least one, at least two, at least three, or135.at least four of the136.

137. tails are different.

21. The compound of any one of claims 1-18, wherein at least one or two of the141.

142. tails has both A and X absent, and has B being H or C1-C24 branched or unbranched alkyl.

22. The compound of any one of claims 1-18, wherein at least one or two of the -2— A - X - B143.tails has both A and X absent, and has B being H or C1-C3 branched or unbranched alkyl.144.-I— A - X - B23. The compound of any one of claims 1-18, wherein in the *> tail in which X is not absent, each X’ is independently -OC(O)-, -C(O)O-, -OC(O)O-, -N(R10)C(O)-, -C(O)N(R10)-, -Y-V-OC(O), -Y-V-C(O)O-, -Y-V-N(R10)C(O)-, -Y-V-C(O)N(R10)-, -OC(O)-(C(Rn)2)s-S-, -OC(O)-(C(R11)2)s-S-S-, -C(O)O-(C(Rn)2)s-S-, -C(O)O-(C(R11)2)s-S-S-, -C(O)N(R10)-(C(R11)2)s-S-, -C(O)N(R10)-(C(R11)2)s-S-S-, -N(R10)C(O)-(C(R11)2)s-S-, -N(R10)C(O)-(C(RH)2)8-S-S-, or -S-S-;146.wherein:147.each R10is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; each R11is independently H or C1-C3 alkyl; and148.each s is independently 1, 2, 3, 4, or 5.

24. The compound of claim 23, wherein each X’ is independently -OC(O)-, -C(O)O-, -OC(O)O-,153.

154. N(R10)C(O)-(CH2)S-S-S-, or -S-S-, wherein R10and R11are each independently H or CH3.

25. The compound of claim 23 or 24, wherein each X is independently X’,156.

157. , or159. 161.-I— A - X - B26. The compound of any one of claims 1-18, wherein in the *> tail in which A and X are not absent, each A is independently C1-C9 branched or unbranched alkylene.

27. The compound of claim 26, wherein each A is independently C2-C? alkylene.

28. The compound of any one of claims 1-18, wherein each B is independently, for each occurrence, H or has the structure of:

165.

167.

168. wherein:169.each nl is independently an integer from 1 to 5;170.each t is independently an integer from 0 to 5;171.each u is independently an integer from 0 to 16,172.each of ul, u2, u3, and u4, is independently, for each occurrence, an integer from 0 to 10; each of R21, R22, R23, and R24is independently, for each occurrence, H or C1-C3 alkyl; each R25is independently H, C1-C3 alkyl, or a saturated or unsaturated cyclic, optionally substituted with one or more alkyl groups.

29. The compound of claim 28, wherein, each B is independently H or selected from the group consisting of:

175.

176. from 7 to 16.

30. The compound of any one of claims 1-29, wherein the compound has the formula (IA-2) or (IB-2), wherein W is H, OR6, or NHR6, wherein R6is H or C1-C12 alkyl.

31. A lipid-based carrier comprising a compound of any one of the preceding claims, wherein the lipid-based carrier is a lipid nanoparticle.

32. The lipid-based carrier of claim 31, further comprising a second lipid.

33. The lipid-based carrier of claim 32, wherein the second lipid is cationic, anionic, ionizable, or zwitterionic lipid.

34. The lipid-based carrier of claim 31, further comprising a sterol, aPEGylated lipid, a phospholipid, and / or a neutral lipid.

35. A pharmaceutical composition comprising the compound of any one of claims 1-30, or the lipid-based carrier of any one of claims 31-34; and a pharmaceutically acceptable excipient.

36. The pharmaceutical composition of claim 35, further comprising a therapeutic agent.

37. The pharmaceutical composition of claim 36, wherein the therapeutic agent is a nucleic acid component.

38. The pharmaceutical composition of claim 37, wherein the nucleic acid component is an RNA or DNA.

39. The pharmaceutical composition of claim 38, wherein the DNA or RNA is linear, circular, single stranded, or double stranded.

40. The pharmaceutical composition of claim 38, wherein the RNA is a mRNA or a circular RNA.

41. The pharmaceutical composition of claim 36, wherein the therapeutic agent is a protein or small molecule drug.

42. The pharmaceutical composition of claim 38, wherein the pharmaceutical composition is a vaccine.

43. A method of delivering a therapeutic agent to a subject, the method comprising administering to the subject the pharmaceutical composition of any one of claims 35-42.

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