Novel ionizable lipids and lipid nanoparticles and methods of using them - Patents.com

Abstract

Novel ionizable lipids and lipid nanoparticles that can be used in the delivery of therapeutic cargo are disclosed.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 264,400, filed November 22, 2021, U.S. Provisional Patent Application No. 63 / 264,420, filed November 22, 2021, and U.S. Provisional Patent Application No. 63 / 322,952, filed March 23, 2022, all of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Lipid nanoparticles ("LNPs") formed from ionizable amine-containing lipids can function as therapeutic cargo vehicles for intracellular delivery of bioactive agents, such as coding RNA (i.e., messenger RNA (mRNA), guide RNA) and non-coding RNA (i.e., antisense, siRNA). LNPs can facilitate delivery of oligonucleotide agents across cell membranes and can be used to introduce components and compositions into living cells.

[0003] Bioactive agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs, and their derivatives, especially drugs containing relatively large oligonucleotides such as mRNA or guide RNA. Compositions for delivering promising mRNA therapeutics or editing technologies into cells, such as the delivery of components of the CRISPR / Cas9 system, are of particular interest.

[0004] With the recent emergence of pandemic diseases, messenger RNA therapy has become an increasingly important option for the treatment of various diseases, including viral infectious diseases and diseases associated with the deficiency of one or more proteins. Particular attention has also been paid to compositions that can stabilize and / or deliver RNA components and have properties that are useful for in vitro and in vivo delivery.

[0005] There continues to be a need in the art for novel lipid compounds for the development of lipid nanoparticles or other lipid delivery mechanisms for the delivery of therapeutic agents. The present invention addresses that need. Summary of the Invention

[0006] This specification discloses novel ionizable lipids that can be used in combination with at least one other lipid component, such as a neutral lipid, cholesterol, or a polymer-conjugated lipid, to form lipid nanoparticle compositions that can be used to promote intracellular delivery of therapeutic nucleic acids in vitro and / or in vivo.

[0007] This paper discloses ionizable amine-containing lipids that are useful for forming lipid nanoparticle compositions.Such LNP compositions can have advantageous properties for the delivery of nucleic acid cargo, such as the delivery of coding RNA and non-coding RNA to cells.Also provided is a method for treating various diseases or pathologies, such as those caused by infectious agents and / or protein deficiencies, using lipid nanoparticles of the present disclosure.

[0008] Ionizable lipids of formulas (I)-(XII) are disclosed below.

[0009] In some embodiments, an ionizable lipid of formula (I): [ka] , Disclosed is a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: Each A is independently C-C optionally substituted with a heteroatom or substituted with OH, SH, or halogen. 16 Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl; Each B is independently a C-C optionally substituted with a heteroatom or substituted with OH, SH, or halogen. 16Branched or unbranched alkyl or C1-C 16 branched or unbranched alkenyl; each X is independently a biodegradable moiety; and W is [ka] [ka] [ka] wherein: R5 is OH, SH, NR 10 R 11 and; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, NR 10 R 11 where R 10 and R 11 each independently is H, C1-C3 alkyl, or R 10 and R 11 are taken together to form a heterocyclic ring; R7 and R8 are taken together to form a ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, and Q is O, S, or NR 13 where each R 13 is H, C1-C5 alkyl.

[0010] In some embodiments, W is [ka] wherein: V is C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; and Each u is independently 2, 3, 4, or 5.

[0011] In some embodiments, when Z is absent, adjacent R and R are OH, NR 10 R 11 , or never be SH. In some embodiments, Q is O, S, or NH.

[0012] In some embodiments, B is C3-C 20 It is alkyl.

[0013] In some embodiments, an ionizable lipid of formula (II): [ka] , Disclosed is a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R10 and R 11 are grouped together to form a heterocyclic ring; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each X is independently a biodegradable moiety; Each R3 and each R4 independently represent H, C3-C 10 Branched or unbranched alkyl, or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; W is [ka] [ka] [ka] wherein: R5 is OH, SH, NR 10 R 11 and; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or each R 10 and each R 11 are taken together with the carbon atoms to which they are attached to form a heterocyclic ring; R7 and R8 are taken together to form a ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl; and Q is O, S, or NR 13 where each R 13 is H, C1-C5 alkyl.

[0014] In some embodiments, in any of the above formulas, X is selected from the group consisting of -OCO-, -COO-, -NHCO-, -CONH-, -C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(OR 13 )-O-(CH2) r -, wherein R 13 is a branched or unbranched C3-C10 alkyl, and r is 1, 2, 3, 4, or 5. In some embodiments, X is -OCO- or -COO-.

[0015] In some embodiments, Z is absent in any of the above formulas. In some embodiments, Z is O. In some embodiments, Z is S. In some embodiments, Z is NH.

[0016] In some embodiments, in any of the above formulas, at least one of R7 and R8 is H. In some embodiments, R7 and R8 are each H.

[0017] In some embodiments, s is 1 or 2 in any of the above formulas.

[0018] In some embodiments, u is 1 or 2 in any of the above formulas.

[0019] In some embodiments, m is 5, 6, 7, 8, or 9 in any of the above formulas.

[0020] In some embodiments, the pKa of the protonated form of the compound of any of the above formulas is from about 5.1 to about 8.0. In one embodiment, the pKa of the protonated form of the compound is from about 5.7 to about 6.4. In one embodiment, the pKa of the protonated form of the compound is from about 5.8 to about 6.2. In one embodiment, the pKa of the protonated form of the compound is from about 5.5 to about 6.0. In one embodiment, the pKa of the protonated form of the compound is from about 6.1 to about 6.3.

[0021] Also disclosed herein is a pharmaceutical composition comprising one or more compounds selected from the ionizable lipid compounds of the formula disclosed below and a therapeutic agent. In some embodiments, the pharmaceutical composition further comprises one or more components selected from neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. Such compositions can be useful for forming lipid nanoparticles for delivery of therapeutic agents.

[0022] In some embodiments, the present disclosure provides a method for delivering a therapeutic agent to a patient in need thereof, comprising administering to the patient a lipid nanoparticle composition comprising an ionizable lipid compound of the formula disclosed below, a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent. In some embodiments, the method further comprises preparing a lipid nanoparticle composition comprising an ionizable lipid compound of the formula disclosed below, a pharmaceutically acceptable salt thereof, and / or a stereoisomer of any of the foregoing, and a therapeutic agent.

[0023] These and other aspects of the present disclosure will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0026] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] Unless the context requires otherwise, throughout this specification and claims, the term "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in an open and inclusive sense, i.e., "including but not limited to."

[0028] The phrase "inducing the expression of a desired protein" refers to the ability of a nucleic acid to increase the expression of a desired protein.To determine the level of protein expression, a test sample (e.g., a sample of cells in culture that express the desired protein) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with a nucleic acid (e.g., a nucleic acid combined with a lipid of the present disclosure).The expression of the desired protein in the test sample or test animal is compared with the expression of the desired protein in a control sample (e.g., a sample of cells in culture that express the desired protein) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, which has not been contacted with or administered with a nucleic acid.If the desired protein is present in the control sample or control mammal, the expression of the desired protein in the control sample or control mammal can be assigned a value of 1.0. In some embodiments, induction of desired protein expression is achieved when the ratio of the desired protein expression level in a test sample or test mammal to the desired protein expression level in a control sample or control mammal is greater than 1, for example, about 1.1, 1.5, 2.0, 5.0, or 10.0. If the desired protein is not present in a control sample or control mammal, induction of desired protein expression is achieved when any measurable level of the desired protein is detected in the test sample or test mammal. Those skilled in the art will understand suitable assays for determining protein expression levels in a sample, such as 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.

[0029] The phrase "inhibit the 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 determine the degree of gene silencing, a test sample (e.g., a sample of cells in culture that express a target gene) or a test mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, is contacted with the nucleic acid that silences, reduces, or inhibits the expression of a target gene.The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a sample of cells in culture that express a target gene) or a control mammalian (e.g., a mammalian, such as a human or animal) model, such as a rodent (e.g., a mouse) or a non-human primate (e.g., a monkey) model, which is not contacted with or administered with nucleic acid.The expression of the target gene in the control sample or control mammalian can 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 a test sample or test mammal relative to the level of target gene expression in a control sample or 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 acid can silence, reduce or inhibit the expression of the target gene in the test sample or test mammal 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% compared to the level of target gene expression in the control sample or control mammal that is not contacted with or administered with the nucleic acid. Suitable assays for determining the level of target gene expression include, but are not limited to, testing protein or mRNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays known to those skilled in the art.

[0030] An "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to achieve the desired effect, e.g., increase or inhibit expression of a target sequence relative to the normal expression level detected in the absence of the nucleic acid. Increased expression of a target sequence is achieved when any measurable level of expression product not present in the absence of the nucleic acid is detected. When the expression product is present at a certain level before contact with the nucleic acid, increased expression is achieved when the fold increase over the value obtained using a nucleic acid, such as mRNA, relative to the 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 more. Inhibition of expression of a target gene or target sequence is achieved when the value obtained using a nucleic acid, such as an antisense oligonucleotide, relative to a 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, for example, examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence or luminescence of an appropriate reporter protein, and phenotypic assays known to those skilled in the art.

[0031] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA may be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA may be in the form of small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, polyvalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar binding properties to the reference nucleic acid. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, single-nucleotide polymorphisms, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with a mixed-base residue and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via a phosphate group.

[0032] "Bases" include purines and pyrimidines (which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs), and synthetic derivatives of purines and pyrimidines (which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides).

[0033] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

[0034] As used herein, "gene product" refers to the product of a gene, such as an RNA transcript or a polypeptide.

[0035] The term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, that are generally characterized by poor solubility in water but solubility in many organic solvents. They are typically divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0036] A "steroid" is a compound containing the following carbon skeleton: [ka] A non-limiting example of a steroid is cholesterol.

[0037] As used herein, "ionizable lipid" refers to a lipid that can be charged. In some embodiments, the ionizable lipid comprises one or more positively charged amine groups. In some embodiments, the ionizable lipid is ionizable so that it can exist in a positively charged form or a neutral form depending on the pH. The ionization of the ionizable lipid affects the surface charge of lipid nanoparticles comprising the ionizable lipid under different pH conditions. The surface charge of lipid nanoparticles can in turn affect their plasma protein absorption, blood clearance, and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17 (1998)), as well as their ability to form endosomolytic non-bilayer structures (Hafez, IM, et al., Gene Ther 8:1188-1196 (2001)), which can affect the intracellular delivery of nucleic acids. In some embodiments, the ionizable lipids include, for example, lipids that are generally neutral at physiological pH (e.g., about pH 7) but can carry a net charge at acidic or basic pH. In one embodiment, the ionizable lipids include lipids that are generally neutral at pH about 7 but can carry a net charge at acidic pH. In one embodiment, the ionizable lipids include lipids that are generally neutral at pH about 7 but can carry a net charge at basic pH. In some embodiments, the ionizable lipids do not include cationic or anionic lipids that generally carry a net charge at physiological pH (e.g., about pH 7).

[0038] The term "N:P ratio" refers to the molar ratio of ionizable (within the physiological pH range) nitrogen atoms in lipids to phosphate groups in nucleic acids (e.g., RNA), for example, in a lipid nanoparticle composition comprising a lipid component and nucleic acids (e.g., RNA).

[0039] The term "polymer-conjugated lipid" refers to a molecule that contains 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 that contains both a lipid portion and a polyethylene glycol portion.PEGylated lipids are known in the art and include, for example, l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG).

[0040] The term "neutral lipid" refers to any lipid that exists in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-5n-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramides, and steroids, such as sterols, and their derivatives. Neutral lipids may be synthetic or naturally occurring.

[0041] The term "PEG lipid" or "PEGylated lipid" refers to a lipid conjugate that includes a polyethylene glycol (PEG) moiety.

[0042] The term "phospholipid" refers to a lipid that contains a phosphate moiety and one or more carbon chains, such as an unsaturated fatty acid chain. A phospholipid may contain one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturated). Certain phospholipids can promote fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in membranes (e.g., cell membranes or intracellular membranes). The fusion of phospholipids with membranes can allow one or more components of lipid-containing compositions to pass through the membrane, for example, allowing one or more components to be delivered to cells.

[0043] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of a nanometer (e.g., 1-1,000 nm) and comprising one or more ionizable lipid compounds disclosed herein. In some embodiments, lipid nanoparticles comprising one or more ionizable lipid compounds disclosed herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing are included in compositions that can be used to deliver therapeutic agents, such as nucleic acids (e.g., mRNA), to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, lipid nanoparticles comprise one or more ionizable lipid compounds disclosed herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing, and nucleic acids. In some embodiments, lipid nanoparticles comprise one or more ionizable lipid compounds disclosed herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing, and nucleic acids. Such lipid nanoparticles typically contain one or more ionizable lipid compounds disclosed herein and one or more other lipids, such as neutral lipids, charged lipids, steroids, and polymer-conjugated lipids. In some embodiments, therapeutic agents such as nucleic acids can be encapsulated in the lipid portion of the lipid nanoparticle or within the aqueous space covered by some or all of the lipid portions of the lipid nanoparticle, thereby protecting them from enzymatic degradation or other undesirable effects induced by the host organism's or cell's machinery, such as harmful immune responses.

[0044] In some embodiments, the lipid nanoparticles have an average particle size of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, 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 acid is resistant to degradation by nuclease in aqueous solution when present in lipid nanoparticles.Nucleic acid-containing lipid nanoparticles and their preparation methods are described in, for example, U.S. Patent Publication Nos. 2004 / 0142025, 2007 / 0042031, and PCT Publication Nos. WO2013 / 016058 and WO2013 / 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 / 012 No. 3338, No. 2013 / 0022649, No. 2013 / 0017223, No. 2012 / 0295832, No. 2012 / 0183581, No. 2012 / 0172411, No. 2012 / 0027803, No. 2012 / 005818 No. 8, No. 2011 / 0311583, No. 2011 / 0311582, No. 2011 / 0262527, No. 2011 / 0216622, No. 2011 / 0117125, No. 2011 / 0091525, No. 2011 / 0076335, PCT Publication Nos. 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, and 1999 / 009076, as well as PCT Publication Nos. WO99 / 39741 and W and WO2017 / 117528, WO2017 / 004143, WO2017 / 075531, WO2015 / 199952, WO2014 / 008334, WO2013 / 086373, WO2013 / 086322, WO2013 / 016058, WO2013 / 086373, WO2011 / 141705, and WO2001 / 07548, the entire disclosures of which are incorporated herein by reference for all purposes.

[0045] The term "polydispersity index" or "PDI" refers to a ratio that describes the homogeneity of the particle size distribution of a system, such as a lipid nanoparticle composition. A small value, e.g., less than 0.3, indicates a narrow particle size distribution.

[0046] As used herein, "encapsulated" by a lipid refers to a therapeutic agent, such as a nucleic acid (e.g., mRNA), that is fully or partially encapsulated by a lipid nanoparticle. In some embodiments, the therapeutic agent, such as a nucleic acid (e.g., mRNA), is fully encapsulated in the lipid nanoparticle.

[0047] "Serum stability" in relation to nucleic acid-lipid nanoparticles means that the nucleic acid is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA. Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0048] Some administration techniques can result in systemic delivery of certain drugs but not others. "Systemic delivery" means that a useful (e.g., therapeutic) amount of the drug is delivered to most of the body. Systemic delivery of lipid nanoparticles can be achieved by any means known in the art, including, for example, intravenous, intraarterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is by intravenous delivery.

[0049] As used herein, "local delivery" refers to the direct delivery of a drug to a target site within an organism.For example, a drug can be delivered locally by direct injection into a disease site such as a tumor, other target site such as an inflammation site, or target organ such as the liver, heart, pancreas, or kidney.Local delivery can also include topical application or local injection techniques such as intramuscular, subcutaneous, or intradermal injection.Local delivery does not interfere with systemic pharmacological effects.

[0050] "Alkyl" refers to a group that is saturated or unsaturated (i.e., contains one or more double bonds (alkenyl) and / or triple bonds (alkynyl)), consists solely of carbon and hydrogen atoms, e.g., 1 to 24 carbon atoms (C1-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (C1-C 15 alkyl), 1 to 12 carbon atoms (C1-C 12 "C-C alkyl" refers to a straight or branched hydrocarbon chain radical having 1 to 8 carbon atoms (C-C alkyl), 1 to 8 carbon atoms (C-C alkyl), or 1 to 6 carbon atoms (C-C alkyl) and attached to the rest of the molecule by a single bond, for example, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Unless stated otherwise in the specification, alkyl groups are optionally substituted.

[0051] An "alkylene" or "alkylene chain" connects the rest of the molecule to a radical group and can be saturated or unsaturated (i.e., contain one or more double bonds (alkenylene) and / or triple bonds (alkynylene)), consist solely of carbon and hydrogen, and have from 1 to 24 carbon atoms (C1-C 24 Alkylene, 1 to 15 carbon atoms (C1-C 15 Alkylene, 1 to 12 carbon atoms (C1-C 12"Alkylene" refers to a straight or branched divalent hydrocarbon chain having 1 to 8 carbon atoms (C1-C8 alkylene), 1 to 6 carbon atoms (C1-C6 alkylene), 2 to 4 carbon atoms (C2-C4 alkylene), or 1 to 2 carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. 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 and to the radical group can be through one carbon or any two carbons within the chain.

[0052] As used herein, the term "substituted" refers to a group in which at least one hydrogen atom of any of the above groups (e.g., alkyl, alkylene, cycloalkyl, or cycloalkylene) has been replaced with, but is not limited to, a halogen atom such as F, CI, Br, or I; an oxo group (=O); a hydroxyl group (-OH); a C1-C 12 Alkyl group; Cycloalkyl group; -(C=O)OR; -O(C=O)R; -C(=O)R; -OR; -S(O) x R;-S-SR;-C(=O)SR;-SC(=O)R;-NRR';-R ’ C(=O)R;-C(=O)RR ’ ;-RC(=O)R'R”;-OC(=O)RR ’ ;-RC(=O)OR';-R ’ S(O) X R”R;-R ’ S(O) X R; and -S(O) x R-R′, where R, R′, and R″, in each occurrence, are independently selected from H, C-C 15 In some embodiments, the substituent is a C-C alkyl, or cycloalkyl, where x is 0, 1, or 2. 12In some embodiments, the substituent is an alkyl group. In some embodiments, the substituent is a cycloalkyl group. In some embodiments, the substituent is a halo group, such as fluoro. In some embodiments, the substituent is an oxo group. In some embodiments, the substituent is a hydroxyl group. In some embodiments, the substituent is an alkoxy group (-OR). In some embodiments, the substituent is a carboxyl group. In some embodiments, the substituent is an amine group (-NRR').

[0053] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstance may or may not occur, and that the specification includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the specification includes both substituted alkyl groups and alkyl groups that have no substituents.

[0054] The present disclosure also intends to encompass all pharmaceutically acceptable compounds of the ionizable lipid compounds of the formula disclosed herein, which are isotopically labeled by replacing one or more atoms with atoms having different atomic mass or mass number.Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, for example, 2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 C1, 123 I, and 125I. These isotope-labeled compounds can be useful for determining or measuring the effectiveness of compounds, for example, by characterizing the site or mode of action, or the binding affinity to pharmacologically important sites of action. Certain isotope-labeled lipid compounds, for example, those incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies. Radioisotopes such as tritium, i.e., 3 H, and carbon-14, i.e., 14 C may be useful for this purpose in view of its ease of incorporation and rapid means of detection.

[0055] Deuterium, i.e., 2 Substitution with heavy isotopes such as H may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be useful in some circumstances.

[0056] 11 C. 18 F, 15 O and 13 Substitution with positron-emitting isotopes, such as N, can be useful in positron emission topography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the Preparations and Examples set forth below, substituting an appropriate isotopically labeled reagent for the previously used non-labeled reagent.

[0057] The present disclosure is also intended to encompass in vivo metabolic products of the disclosed compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, esterification, etc., of the administered compound, primarily due to enzymatic processes. Accordingly, embodiments of the present disclosure include compounds produced by a process comprising administering an ionizable lipid of the present disclosure to a mammal for a period of time sufficient to yield its metabolic products. Such products are typically identified by administering a detectable dose of a radiolabeled compound of the present disclosure to an animal, such as a rat, mouse, guinea pig, monkey, or human, allowing sufficient time for metabolism to occur, and isolating the conversion products from urine, blood, or other biological sample.

[0058] A "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, flow agent, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that is approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine.

[0059] "Pharmaceutically acceptable salts" include both acid and base addition salts.

[0060] "Pharmaceutically acceptable acid addition salts" are salts that retain the biological effectiveness and properties of the free base, which are not biologically or otherwise undesirable, and include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and, 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-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid It refers to salts formed with organic acids such as 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, and undecylenic acid.

[0061] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and are not biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or 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, 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, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resin salts. Non-limiting examples of organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0062] The crystallization of the ionizable lipid disclosed herein can produce a solvate of the ionizable lipid.As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the ionizable lipid compound of the present disclosure and one or more solvent molecules.The solvent can be water, and in this case, the solvate can be a hydrate.Alternatively, the solvent can be an organic solvent.Therefore, the lipid compound of the present disclosure can exist as a hydrate, such as a monohydrate, a dihydrate, a hemihydrate, a sesquihydrate, a trihydrate, a tetrahydrate, etc., and corresponding solvate forms.The solvate of the lipid compound of the present disclosure can be a true solvate, while in other cases, the lipid compound of the present disclosure can simply retain extraneous water or be a mixture of water and some extraneous solvent.

[0063] A "pharmaceutical composition" refers to a composition that may include an ionizable lipid compound of the present disclosure and a vehicle generally accepted in the art for delivery of a biologically active compound to a mammal, e.g., a human. Such a vehicle includes a pharmaceutically acceptable carrier, diluent, or excipient therefor.

[0064] An "effective amount" or "therapeutically effective amount" refers to an amount of an ionizable lipid compound of the present disclosure that, when administered to a mammal, such as a human, is sufficient to effect treatment in the mammal, such as a human. The amount of an ionizable lipid compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the method of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art having regard to their own knowledge and this disclosure.

[0065] As used herein, "treating" or "treatment" includes treatment of a disease or condition of interest in a mammal, such as a human, having the disease or condition of interest; (i) preventing the onset of a disease or condition in a mammal, particularly where such mammal is predisposed to the condition but has not yet been diagnosed as having the condition; (ii) preventing the disease or condition, i.e., arresting its development; (iii) alleviating the disease or condition, i.e., causing regression of the disease or condition; or (iv) Relieving symptoms resulting from a disease or condition, i.e., alleviating pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may differ in that a particular illness or condition may not have a known causative agent (so that the etiology remains unknown) and therefore is not yet recognized as a disease, but only as an undesirable condition or syndrome, with some specific set of symptoms identified by a physician.

[0066] The ionizable lipid compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may contain one or more stereocenters and thus give rise to enantiomers, diastereomers, and other stereoisomers that can be defined in terms of absolute stereochemistry as (R)- or (S)-amino acids, or (D)- or (L)-. The present disclosure is intended to encompass all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) and (S), or (D) and (L) isomers can be prepared using chiral synthesis or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the ionizable lipid compounds described herein contain an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers.Similarly, all tautomers are also intended to be included.

[0067] "Stereoisomer" refers to a compound made up of the same atoms joined by the same bonds but having different, incompatible three-dimensional structures. 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.

[0068] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details.

[0069] Ionizable lipid compounds In some embodiments, an ionizable lipid of formula (I): [ka] a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, During the ceremony, Each A is independently a C-C group optionally interrupted by one or more heteroatoms or substituted by one or more OH, SH, or halogen groups. 16 Branched or unbranched alkylene or C1-C 16 branched or unbranched alkenylene; Each B is independently a C-C group optionally interrupted by one or more heteroatoms or substituted by one or more OH, SH, or halogen groups. 20 Branched or unbranched alkyl or C1-C 20 branched or unbranched alkenyl; each X is independently a biodegradable moiety; and W is [ka] [ka] [ka] wherein: R5 is (CH2) s OH, OH, SH, NR 10 R 11 and; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) s R 17 , or NR 10 R 11 where R 10 and R 11 each independently is H, C1-C3 alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, N(R 12 ), or a divalent heterocyclic group, wherein R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl; R 17 is OH, SH, or N(CH3)2; and Q is O, S, CH2, or NH.

[0070] In some embodiments, B is C3-C 20 It is alkyl.

[0071] In some embodiments, X is —OCO—, —COO—, —NHCO—, —CONH—, —C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(OR13 )-O-(CH2) r -, -O(CO)O-, where R 13 branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5. In some embodiments, X is —OCO— or —COO—.

[0072] In some embodiments, in Formula (I), W is alternatively: [ka] may be, wherein: V is a branched or unbranched C-C alkyl group optionally substituted with one or more OH, SH, and / or halogen groups. 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; each v is independently 0, 1, 2, 3, 4, or 5; R 17 is OH, SH, or N(CH3)2; and Each u is independently 1, 2, 3, 4, or 5.

[0073] In some embodiments, in Formula (I), W is alternatively: [ka] may be, wherein: V is C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; and Each u is independently 1, 2, 3, 4, or 5.

[0074] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: R 14 is a heterocyclic group; each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0075] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: Z is O, S, -C((CH2) v N(R 15 )2)-, or N(R 15 ) where R 15 is H, C1-C4 branched or unbranched alkyl, and v is 0, 1, 2, 3, 4, or 5; Each R 10 are independently H, or C1-C3 alkyl; and Each u is independently 0, 1, 2, 3, 4, or 5.

[0076] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: each Y is a divalent heterocyclic group; Q is O, S, or NH; and Each u is independently 1, 2, 3, 4, or 5.

[0077] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: R 14 is heterocyclic, NR 10 R 11 , C(O)NR 10 R 11 , or C(S)NR 10 R 11 and 、 In the formula, R 10 and R 11 each independently is H, C1-C3 alkyl, C3-C7 cycloalkyl, C3-C7 cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 are grouped together to form a heterocyclic ring; R 16 is H, ═O, ═S, or CN; each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0078] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: T is -NHC(O)O-, -OC(O)NH-, or optionally one or more -(CH) v OH, -(CH2) v SH, and / or -(CH2) v - a divalent heterocyclic group substituted with a halogen group; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; R 17 is OH, SH, or N(CH3)2; each v is independently 0, 1, 2, 3, 4, or 5; and Each u is independently 1, 2, 3, 4, or 5.

[0079] In some embodiments, in Formula (I), W is alternatively: [ka] , may be, wherein: T is —NHC(O)O—, —OC(O)NH—, or a divalent heterocyclic group; and Each u is independently 1, 2, 3, 4, or 5.

[0080] In some embodiments, when Z is absent, adjacent R and R are OH, NR 10 R 11 , or never be SH.

[0081] In some embodiments, the heterocyclic group is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic or heteroaromatic group.

[0082] In some embodiments, the present disclosure provides an ionizable lipid of formula (II): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, the formula: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each X is independently a biodegradable moiety; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; W is [ka] [ka] [ka] wherein: R5 is OH, SH, NR 10 R 11 and; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, NR 10 R 11 where R 10 and R 11 are each independently H, C1-C3 alkyl, or each R 10 and each R 11 are grouped together with the carbon atoms to which they are attached to form a heterocyclic ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , or SH; and Q is O, S, CH2, or NH.

[0083] In some embodiments, in formula (II), W may alternatively be any of the formulae above in alternative embodiments where W is as defined in formula (I). For example, in formula (II), W may alternatively be: [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] ,or [ka] The definitions of the variables in each of these formulas are the same as those defined in the same formula above in alternative embodiments where W is as defined in formula (I).

[0084] In some embodiments, X is —OCO—, —COO—, —NHCO—, —CONH—, —C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, -CON(R 13 )-, or -C(OR 13 )-O-(CH2) r -, -O(CO)O-, where R 13 is branched or unbranched C1-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0085] In some embodiments, W is [ka] wherein: V is C2-C 10 Alkenylene, C2-C10 Alkynylene, or C2-C 10 is heteroalkylene; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; and Each u is independently 2, 3, 4, or 5.

[0086] In some embodiments, the present disclosure provides an ionizable lipid of formula (III): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, the formula: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C3-C 10 Branched or unbranched alkyl, or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and Each s is independently 1, 2, 3, 4, or 5.

[0087] In some embodiments, the present disclosure provides an ionizable lipid of formula (IV): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, the formula: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C2-C 14 branched or unbranched alkyl (e.g., C1-C3 branched or unbranched alkyl), or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C3-C 10 Branched or unbranched alkyl, or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0088] In some embodiments, X is —OCO—, —COO—, —NHCO—, —CONH—, —C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(OR 13 )-O-(CH2) r -, wherein R 13 branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5.

[0089] In some embodiments, the present disclosure provides an ionizable lipid of formula (V): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0090] In some embodiments, the present disclosure provides an ionizable lipid of formula (VI): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, During the ceremony, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0091] In some embodiments, the present disclosure provides an ionizable lipid of formula (VII): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, the formula: Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C3-C 10 Branched or unbranched alkyl, or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0092] In some embodiments, the present disclosure provides an ionizable lipid of formula (VIII): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Each Z is absent, O, S, or NR 12 where R 12 is H, C1-C7 branched or unbranched alkyl, or C2-C7 branched or unbranched alkenyl, provided that when Z is absent, adjacent R1 and R2 are OH, NR 10 R 11 , or without being SH; Q is O, S, CH, or NH; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0093] In some embodiments, the present disclosure provides an ionizable lipid of formula (IX): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; V is a branched or unbranched C-C alkyl group optionally substituted with one or more OH, SH, and / or halogen groups. 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylene, or C2-C 10 is heteroalkylene; each R6 is independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, or cycloalkyl; Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2)vR 17 , or NR 10 R 11 wherein each v is independently 0, 1, 2, 3, 4, or 5; 17 is OH, SH, or N(CH3)2; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0094] In some embodiments, the present disclosure provides an ionizable lipid of formula (X): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C1-C3 branched or unbranched alkyl, or R10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; Z is O, S, -C((CH2) v N(R 15 )2)-, or -N(R 15 )-, wherein R 15 is H, C1-C4 branched or unbranched alkyl, and each v is independently 0, 1, 2, 3, 4, or 5; each R6 is independently H, or C1-C3 alkyl; each u is independently 0, 1, 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0095] In some embodiments, the present disclosure provides an ionizable lipid of formula (XI): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11are each independently H, C1-C3 branched or unbranched alkyl, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each s is independently 1, 2, 3, 4, or 5; T is -NHC(O)O-, -OC(O)NH-, or optionally one or more -(CH) v OH, -(CH2) v SH, -(CH2) v -a divalent heterocyclic group substituted with a halogen group, Each R7 and each R8 are independently H, C1-C3 branched or unbranched alkyl, C2-C3 branched or unbranched alkenyl, halogen, OH, SH, (CH2) v R 17 , or NR 10 R 11 where R 17 is OH, SH, or N(CH3)2; each v is independently 0, 1, 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0096] In some embodiments, the present disclosure provides an ionizable lipid of formula (XII): [ka] , With respect to pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, Each R1 and each R2 is independently H, C1-C3 branched or unbranched alkyl, OH, halogen, SH, or NR10 R 11 or each R1 and each R2 independently, taken together with the carbon atom to which they are attached, form a cyclic ring; R 10 and R 11 are each independently H, C-C branched or unbranched alkyl, C-C cycloalkyl, C-C cycloalkenyl, optionally substituted with one or more NH and / or oxo groups, or R 10 and R 11 are grouped together to form a heterocyclic ring; Each R3 and each R4 independently represent H, C2-C 14 Branched or unbranched alkyl (e.g., C3-C 10 branched or unbranched alkyl), or C3-C 10 branched or unbranched alkenyl, provided that at least one of R3 and R4 is not H; each X is independently a biodegradable moiety; each q is independently 2, 3, 4, or 5; R 14 is a heterocyclic group, NR 10 R 11 , C(O)NR 10 R 11 , or C(S)NR 10 R 11 and R 16 is H, ═O, ═S, or CN; each R6 is independently H or C1-C3 alkyl; each v is independently 0, 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; and Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0097] In some embodiments, in each of the above formulas (I)-(XII), X is —OC(O)—, —C(O)O—, —SS—, —N(R 18 )C(O)-, -C(O)N(R18 )-, -C(OR 13 )-O-, -C(O)O(CH2) a -, -OC(O)(CH2) a -, -C(O)N(R 18 )(CH2) a -, -N(R 18 )C(O)(CH2) a -, -C(OR 13 )-O-(CH2) a wherein each R 18 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and each R 13 independently, C3-C 10 alkyl, and each a is independently 0 to 16.

[0098] In some embodiments, in each of the above formulas, X is -OC(O)-, -C(O)O-, -C(O)O(CH) a - or -OC(O)(CH2) a In some embodiments, a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0099] In some embodiments, in each of the above formulas (I)-(XII), X is —C(O)N(R 18 )-, -N(R 18 )C(O)-, -C(O)N(R 18 )(CH2) a - or -N(R 18 )C(O)(CH2) a -, wherein R 18 is independently H, alkyl, alkenyl, or cycloalkyl. In some embodiments, a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0100] In some embodiments, in each of the above formulas (I)-(XII), X is —C(O)N(R 18 )-, -N(R 18 )C(O)-, -C(O)N(R 18 )(CH2) a- or -N(R 18 )C(O)(CH2) a -, wherein R 18 is independently H, alkyl, alkenyl, or cycloalkyl. In some embodiments, a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0101] In some embodiments, in each of the above formulas (I)-(XII), X is —C(OR 13 )-O-(acetal) or -C(OR 13 )-O-(CH2) s -, wherein R 13 is C3-C 10 It is alkyl.

[0102] In one embodiment, X is -SS-.

[0103] In some embodiments, in any of formulas (I) to (XII), X is -OCO-, -COO-, -NHCO-, -CONH-, -C(OR 13 )-O-, -COO(CH2) r -, -CONH(CH2) r -, or -C(OR 13 )-O-(CH2) r -, wherein R 13 branched or unbranched C3-C 10 alkyl, and r is 1, 2, 3, 4, or 5. In one embodiment, X is —OC(O)— or —C(O)O—.

[0104] In some embodiments, in any of the above formulas (I)-(XII), Z is absent. In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z is NH.

[0105] In some embodiments, in any of the above formulas (I)-(XII), each Z is absent. In some embodiments, each Z is S. In some embodiments, each Z is O. In some embodiments, each Z is NH.

[0106] In some embodiments, in any of the above formulas (I)-(XII), one Z is NH and the other Z is O.

[0107] In some embodiments, in any of the above formulas (I) to (XII), at least one of R7 and R8 is H. In some embodiments, each of R7 and R8 is H. In some embodiments, one of R7 and R8 is H and the other is methyl or (CH2) v R 17 wherein each v is independently 0, 1, or 2, and R 17 is OH or N(CH3)2.

[0108] In some embodiments, m is 5, 6, 7, 8, or 9 in any of the above formulas (I)-(XII).

[0109] In some embodiments, in any of the above formulas (I)-(XII), the heterocyclic group is piperazine, piperazinedione, piperazine-2,5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bis-piperazine, aromatic, or heteroaromatic group.

[0110] In some embodiments, in any of the above formulas (II) to (XII), R3 and R4 are each independently C5-C8 alkyl. In some embodiments, R3 is C4-C6 alkyl and R4 is C8 alkyl. In one embodiment, R3 is C6 alkyl and R4 is C8 alkyl. In one embodiment, each of R3 and R4 is C8 alkyl.

[0111] In some embodiments, in any of the above formulas (II)-(XII), R3 is H and R4 is C9-C 13 It is a branched or unbranched alkyl.

[0112] In some embodiments, in any of the above formulas (I)-(XII), each q is independently 2 or 3.

[0113] In some embodiments, in any of the above formulas (I)-(XII), each s is independently 1 or 2.

[0114] In some embodiments, in any of the above formulas (I)-(XII), each u is independently 0, 1, 2, or 3.

[0115] In some embodiments, in each of the above formulas (II)-(XII), R 1 and R 2 are each H.

[0116] In some embodiments, in any of the above formulas (I) to (XII), Q is O. In some embodiments, Q is CH.

[0117] In some embodiments, in any of the above formulas (I) to (XII), V is a branched or unbranched C2-C3 alkylene. In some embodiments, V is a C2-C3 alkylene substituted with OH. In some embodiments, V is a branched or unbranched C2-C3 alkenylene.

[0118] In some embodiments, in any of the above formulas (I)-(XII), Z is —C((CH) v N(R 15 )2)-, wherein each R 15 is H or methyl and v is 0, 1, 2, or 3.

[0119] In some embodiments, in any of the above formulas (I)-(XII), each R6 is independently H or methyl.

[0120] In some embodiments, in any of the above formulas (I)-(XII), T is optionally -(CH) vA divalent heterocyclic group substituted with OH (eg, a divalent piperazine or a divalent dioxopiperazine), where v is independently 0, 1, or 2.

[0121] In some embodiments, in any of the above formulas (I)-(XII), R 16 is H or =O.

[0122] In some embodiments, in any of the above formulas (I)-(XII), v is 0, 1, or 2.

[0123] In some embodiments, in any of the above formulas (I)-(XII), R 14 is a heterocyclic group (e.g., pyrrolidinyl). In some embodiments, R 14 is C(S)NR 10 R 11 where R 10 and R 11 are each C1-C3 alkyl. In some embodiments, R 14 is NR 10 R 11 where R 10 is H and R 11 is a C3-C5 cycloalkyl or C3-C5 cycloalkenyl optionally substituted with one or more NH and / or oxo groups.

[0124] In some embodiments, in each of the above formulas (II)-(XII), all four variables m in the same formula are the same.

[0125] In some embodiments, in each of the above formulas (II)-(XII), all four variables X in the same formula are the same.

[0126] In some embodiments, in each of the above formulas (II)-(XII), all four variables R3 in the same formula are the same.

[0127] In some embodiments, in each of the above formulas (II)-(XII), all four variables R4 in the same formula are the same.

[0128] In some embodiments, in each of the above formulas (II)-(XII), R 1 and R 2 are each H.

[0129] In each of the above formulas (II)-(XII), considering the four chains attached to the N atom and the variables in each of the four chains labeled as i, ii, iii, iv, respectively:

[0130] In some embodiments, mi and mii are the same, miii and miv are the same, and mi and miii are different;

[0131] In some embodiments, Xi and Xii are the same, Xiii and Xiv are the same, and Xi and Xiii are different;

[0132] In some embodiments, R3i and R3ii are the same, R3iii and R3iv are the same, and R3i and R3iii are different;

[0133] In some embodiments, R4i and R4ii are the same, R4iii and R4iv are the same, and R3i and R3iii are different.

[0134] In some embodiments, in each of the above formulas (II)-(XII), all four variables m in the same formula are different.

[0135] In some embodiments, in each of the above formulas (II)-(XII), all four variables X in the same formula are different.

[0136] In some embodiments, in each of the above formulas (II)-(XII), all four variables R3 in the same formula are different.

[0137] In some embodiments, in each of the above formulas (II)-(XII), all four variables R4 in the same formula are different.

[0138] In some embodiments, B or [ka] [ka] [ka] [ka] where t is 0, 1, 2, 3, 4, or 5.

[0139] In some embodiments, the pKa of the protonated form of an ionizable lipid compound described herein is about 5.1 to about 8.0, e.g., about 5.7 to about 6.5, about 5.7 to about 6.4, or about 5.8 to about 6.2. In some embodiments, the pKa of the protonated form of the compound is about 5.5 to about 6.0. In some embodiments, the pKa of the protonated form of the compound is about 6.1 to about 6.3.

[0140] Non-limiting examples of ionizable lipid compounds disclosed herein are listed below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

Table 1-38

Table 1-39

Table 1-40

[0141] Lipid Nanoparticle Composition The ionizable lipid disclosed herein can be used to form lipid nanoparticle compositions.In some embodiments, the lipid nanoparticle compositions further comprise one or more therapeutic agents.In some embodiments, the lipid nanoparticles in the composition encapsulate one or more therapeutic agents or associate with one or more therapeutic agents.

[0142] In some embodiments, the LNP composition has an N / P ratio of about 3 to about 10, e.g., an N / P ratio of about 6±1, or an N / P ratio of about 6±0.5. In some embodiments, the N / P ratio is about 6.

[0143] In some embodiments, the present disclosure relates to a combination comprising (i) one or more compounds selected from ionizable lipids of formulas (I)-(XII), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, and (ii) a lipid component. In some embodiments, the combination comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of one or more compounds of (i). In some embodiments, the combination comprises a ratio of about 1:1 between the compound of (i) and the lipid component (ii). In some embodiments, the combination is a lipid nanoparticle (LNP) composition.

[0144] In some embodiments, the present disclosure relates to lipid nanoparticle compositions comprising (i) one or more ionizable lipid compounds described herein and (ii) one or more lipid components.

[0145] In some embodiments, the one or more lipid components in the LNP composition comprise one or more helper lipids and one or more PEG lipids, hi some embodiments, the lipid components comprise one or more helper lipids, one or more PEG lipids, and one or more neutral lipids.

[0146] Non-ionizable lipid components neutral lipid In some embodiments, the lipid component comprises one or more neutral lipids.

[0147] The neutral lipid may be one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipid may be assembled into one or more lipid bilayers. Generally, the phospholipid may comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid may be a lipid of the formula: [ka] (In the formula, R p represents the phospholipid moiety, and R A and R Brepresent fatty acid moieties, which may be the same or different, with or without unsaturation. The phospholipid moiety may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, or sphingomyelin. The fatty acid moiety may be 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. Non-naturally occurring species, including naturally occurring species with modifications and substitutions such as branching, oxidation, cyclization, and alkynes, are also contemplated. For example, phospholipids may be functionalized with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds) or crosslinked to one or more alkynes. Under appropriate reaction conditions, when exposed to azide, the alkyne group can undergo copper-catalyzed cycloaddition. Such reactions can be useful for functionalizing the lipid bilayer of lipid nanoparticles to facilitate membrane permeation or cell recognition, or for conjugating lipid nanoparticles to useful components such as targeting or imaging moieties (e.g., dyes).

[0148] In some embodiments, the neutral lipid is a phospholipid, such as distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero- 3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-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, 1,2-diphytanoyl-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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl The phosphatidylethanolamine may be phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a mixture thereof.

[0149] Additional non-limiting examples of neutral lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG ... Other examples of phospholipids include diacylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are C 10 -C 24 The acyl group may be derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0150] Steroids and other non-ionizable lipid components In some embodiments, the lipid component comprises one or more steroids or their analogs.These lipid components can be considered as structural lipids, such as cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.In some embodiments, the structural lipid is cholesterol.In some embodiments, the structural lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0151] In some embodiments, the lipid component comprises sterol, such as cholesterol, sterol, and their derivatives.Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 5a-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestan, cholestenone, 5a-cholestanone, 5a-cholestanone, and cholesteryl decanoate; and mixtures thereof.In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0152] In some embodiments, non-ionizable lipid component comprises or consists of the mixture of one or more phospholipids and cholesterol or its derivatives.In some embodiments, non-ionizable lipid component comprises or consists of one or more phospholipids, for example, cholesterol-free lipid particle formulations.In some embodiments, non-ionizable lipid component comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid particle formulations.

[0153] In some embodiments, the LNP composition comprises a phytosterol or a combination of a phytosterol and cholesterol. In some embodiments, the phytosterol is selected from the group consisting of β-sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of β-sitosterol, β-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. In some embodiments, the phytosterol comprises sitosterol or a salt or ester thereof. In some embodiments, the phytosterol comprises stigmasterol or a salt or ester thereof. In some embodiments, the phytosterol comprises beta-sitosterol, [ka] a salt thereof, or an ester thereof.

[0154] In some embodiments, the LNP composition comprises a phytosterol, or a salt or ester thereof, and cholesterol, or a salt thereof.

[0155] In some embodiments, the target cell is a cell described herein (e.g., a hepatocyte or a splenocyte), and the phytosterol or salt or ester thereof is selected from the group consisting of β-sitosterol, β-sitostanol, campesterol, and brassicasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is β-sitostanol. In some embodiments, the phytosterol is campesterol. In some embodiments, the phytosterol is brassicasterol.

[0156] In some embodiments, the target cell is a cell described herein (e.g., a hepatocyte or a splenocyte), and the phytosterol or its salt or ester is selected from the group consisting of β-sitosterol, stigmasterol, and combinations thereof. In some embodiments, the phytosterol is β-sitosterol. In some embodiments, the phytosterol is stigmasterol.

[0157] Other examples of non-ionizable lipid components include non-phosphorus-containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0158] In some embodiments, the non-ionizable lipid component is present at 10 mol% to 60 mol%, 20 mol% to 55 mol%, 20 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 50 mol%, 25 mol% to 45 mol%, 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 35 mol% to 45 mol%, 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 or range therein) of the total lipid present in the lipid nanoparticle composition.

[0159] In embodiments where the lipid nanoparticle composition contains a mixture of phospholipids and cholesterol or a cholesterol derivative, the mixture may be present at up to 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipid present in the lipid nanoparticle composition.

[0160] In some embodiments, the phospholipid component in the mixture may be present at 2 mol% to 20 mol%, 2 mol% to 15 mol%, 2 mol% to 12 mol%, 4 mol% to 15 mol%, or 4 mol% to 10 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid nanoparticle composition. In some embodiments, the phospholipid component in the mixture may be present at 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 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 nanoparticle composition.

[0161] In some embodiments, the cholesterol component in the mixture may be present at 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 27 mol% to 37 mol%, 25 mol% to 30 mol%, or 35 mol% to 40 mol% (or any fraction thereof or range therein) of the total lipid present in the lipid nanoparticle composition. In some embodiments, the cholesterol component in the mixture may be present at 25 mol% to 35 mol%, 27 mol% to 35 mol%, 29 mol% to 35 mol%, 30 mol% to 35 mol%, 30 mol% to 34 mol%, 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 nanoparticle composition.

[0162] In embodiments in which the lipid nanoparticle composition does not contain phospholipids, cholesterol or a derivative thereof may be present at 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 nanoparticle composition.

[0163] In some embodiments, cholesterol or a derivative thereof in a phospholipid-free lipid particle formulation may be present at 25 mol% to 45 mol%, 25 mol% to 40 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 31 mol% to 39 mol%, 32 mol% to 38 mol%, 33 mol% to 37 mol%, 35 mol% to 45 mol%, 30 mol% to 35 mol%, 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 or range therein) of the total lipid present in the lipid nanoparticle composition.

[0164] In some embodiments, the non-ionizable lipid component may be present at 5 mol% to 90 mol%, 10 mol% to 85 mol%, 20 mol% to 80 mol%, 10 mol% (e.g., phospholipids only), or 60 mol% (e.g., phospholipids and cholesterol or a derivative thereof) (or any fraction or range therein) of the total lipid present in the lipid nanoparticle composition.

[0165] The percentage of non-ionizable lipid present in the lipid nanoparticle composition is a target amount, and the actual amount of non-ionizable lipid present can vary, for example, by ±5 mol %.

[0166] lipid conjugates The lipid nanoparticle composition described herein can further comprise one or more lipid conjugates.The conjugated lipid can prevent particle aggregation.Non-limiting examples of conjugated lipids include PEG-lipid conjugates, cationic polymer-lipid conjugates, and their mixtures.

[0167] In some embodiments, lipid conjugate is PEG lipid or PEG-modified lipid (also called PEGylated lipid).PEG lipid is lipid modified with polyethylene glycol.Examples of PEG lipid include but are not limited to PEG bound to dialkyloxypropyl (PEG-DAA), PEG bound to diacylglycerol (PEG-DAG), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DEG), PEG bound to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG-modified phosphatidic acid, PEG bound to ceramide (PEG-CER), PEG bound to cholesterol or its derivatives, and their mixtures.For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0168] In some embodiments, the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0169] In some embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxylpropyl-3-amine (PEG-c-DMA).

[0170] PEG is a linear water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified according to their molecular weight and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidylsuccinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), as well as compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0171] 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 examples, the PEG moiety has an average molecular weight of 750 daltons to 5,000 daltons (e.g., 1,000 daltons to 5,000 daltons, 1,500 daltons to 3,000 daltons, 750 daltons to 3,000 daltons, 750 daltons to 2,000 daltons). In some embodiments, the PEG moiety has an average molecular weight of 2,000 daltons or 750 daltons.

[0172] In certain examples, PEG can be optionally substituted with alkyl, alkoxy, acyl or aryl group.PEG can be directly conjugated to lipid, or can be linked to lipid via linker moiety.For example, any linker moiety suitable for connecting PEG to lipid can be used, including non-ester-containing linker moiety and ester-containing linker moiety.In some embodiments, linker moiety is non-ester-containing linker moiety. Suitable non-ester containing linker moieties include, but are not limited to, amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH2CH2C(O)-), succinamidyl (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In some embodiments, a carbamate linker is used to attach PEG to a lipid.

[0173] In some embodiments, PEG is attached to a lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof.

[0174] Phosphatidylethanolamines with various acyl chain groups of various chain lengths and degrees of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art.

[0175] In some embodiments, the phosphatidylethanolamine contains saturated or unsaturated fatty acids with carbon chain lengths ranging from C10 to C20. Phosphatidylethanolamines with monounsaturated or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0176] The term "diacylglycerol" or "DAG" includes compounds having two fatty acyl chains, R1 and R2, each having 2 to 30 carbon atoms, independently attached to the 1- and 2-positions of glycerol by an ester bond. The acyl groups may be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C12), myristoyl (CM), palmitoyl (C16), stearoyl (C18), and icosyl (C20). In some embodiments, R1 and R2 are the same, i.e., both R1 and R2 are myristoyl (i.e., dimyristoyl) and both R1 and R2 are stearoyl (i.e., distearoyl).

[0177] The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains, R and R', where R and R' both independently have 2 to 30 carbons. The alkyl groups may be saturated or have varying degrees of unsaturation.

[0178] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C10) conjugate, a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, or a PEG-distearyloxypropyl (C18) 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 replaced with a methyl group.

[0179] In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0180] In some embodiments, the PEG lipid is a compound of the formula [ka] , or a salt thereof, wherein: R 3PL1 -OR OPL1 and; R OPL1 is hydrogen, an optionally substituted alkyl, or an oxygen protecting group; r PL1 is an integer between 1 and 100, inclusive; L 1 is an optionally substituted C 1-10 alkylene and optionally substituted C 1-10 At least one methylene of the alkylene is independently selected from optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) and replaced with; D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; A is of the following formula: [ka] and; L 2 Each instance of is independently a bond or an optionally substituted C 1-6 alkylene, wherein optionally substituted C 1-6 One methylene unit of the alkylene is optionally O, N(R NPL1 ), S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), C(O)O, OC(O)O, -OC(O)N(R NPL1 ), NR NPL1 C(O)O, or NR NPL1 C(O)N(R NPL1 ) is replaced by; R 2SL Each instance of is independently an optionally substituted C 1-30 Alkyl, optionally substituted C 1-30 alkenyl, or optionally substituted C 1-30 alkynyl; optionally, R2SL one or more methylene units in the formula (I) are independently an optionally substituted carbocyclylene, an optionally substituted heterocyclylene, an optionally substituted arylene, an optionally substituted heteroarylene, N(R NPL1 ), O, S, C(O), C(O)N(R NPL1 ), NR NPL1 C(O), -NR NPL1 C(O)N(R NPL1 ), C(O)O, OC(O), OC(O)O, OC(O)N(R NPL1 ), NR NPL1 C(O)O, C(O)S, -SC(O), C(=NR NPL1 ), C(=NR NPL1 )N(R NPL1 ), NR NPL1 C(=NR NPL1 ), -NR NPL1 C(=NR NPL1 )N(R NPL1 ), C(S), C(S)N(R NPL1 ), NR NPL1 C(S), NR NPL1 C(S)N(R NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(R NPL1 )S(O), S(O)N(R NPL1 ), -N(R NPL1 )S(O)N(R NPL1 ), OS(O)N(R NPL1 ), N(R NPL1 )S(O)O, S(O)2, N(R NPL1 )S(O)2, -S(O)2N(R NPL1 ), N(R NPL1 )S(O)2N(R NPL1 ), OS(O)2N(R NPL1 ), or N(R NPL1 ) replaced by S(O)2O; R NPL1 each instance is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl; and p SL is either 1 or 2.

[0181] In some embodiments, the PEG lipid is a compound of the formula [ka] or a salt thereof, wherein r PL1 , L 1 ,D,m PL1 , and A is as defined above.

[0182] In some embodiments, the PEG lipid is a compound of the formula [ka] , or a salt or isomer thereof, wherein: R 3PEG -OR O and; R O is hydrogen, C 1-6 alkyl, or oxygen protecting groups; r PEG is an integer from 1 to 100 (e.g., 40 to 50, e.g., 45); R 5PEG is C 10-40 Alkyl (e.g., C 17 alkyl), C 10-40 Alkenyl, or C 10-40 alkynyl; optionally, R 5PEG One or more methylene groups in 3-10 Carbocyclylene, 4-10 membered heterocyclylene, C 6~10 Arylene, 4- to 10-membered heteroarylene, -N(R NPEG )-, -O-, -S-, -C(O)-, -C(O)N(R NPEG )-, -NR NPEG C(O)-, -NR NPEG C(O)N(RNPEG )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N(R NPEG )-, -NR NPEG ssC(O)O-, -C(O)S-, -SC(O)-, -C(=NR NPEG )-, -C(=NR NPEG )N(R NPEG )-, -NR NPEG C(=NR NPEG )-, -NR NPEG C(=)NR NPEG )N(R NPEG )-, -C(S)-, -C(S)N(R NPEG )-, -NR NPEG C(S)-, -NR NPEG C(S)N(R NPEG )-, -S(O)-, -OS(O)-, -S(O)O-, -OS(O)O-, -OS(O)2-, -S(O)2O-, -OS(O)2O-, -N(R NPEG )S(O)-, -S(O)N(R NPEG )-, -N(R NPEG )S(O)N(R NPEG )-, -OS(O)N(R NPEG )-, -N(R NPEG )S(O)O-, -S(O)2-, -N(R NPEG )S(O)2-, -S(O)2N(R NPEG )-, -N(R NPEG )S(O)2N(R NPEG )-, -OS(O)2N(R NPEG )-, or -N(R NPEG )S(O)2O-; and R NPEG Each instance of is independently hydrogen, C 1-6 alkyl, or nitrogen protecting groups.

[0183] In some embodiments, the PEG lipid is a compound of the formula [ka] where r PEG is an integer from 1 to 100 (for example, 40 to 50, such as 45).

[0184] In some embodiments, the PEG lipid is a compound of the formula [ka] or a salt or isomer thereof, wherein s PL1 is an integer from 1 to 100 (for example, 40 to 50, such as 45).

[0185] In some embodiments, the PEG lipid has the formula: [ka] or a pharmaceutically acceptable salt thereof, a tautomer or a stereoisomer thereof, wherein: R 8 and R 9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, the alkyl chain optionally being interrupted by one or more ester bonds (e.g., R 8 and R 9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms; and w has an average value in the range of 30 to 60 (eg, the average w is about 49).

[0186] In some embodiments, incorporating any of the above PEG-lipids into the lipid nanoparticle composition can improve the pharmacokinetics and / or biodistribution of the LNP composition. For example, incorporating any of the above PEG-lipids into the lipid nanoparticle composition can reduce the accelerated blood clearance (ABC) effect.

[0187] In some embodiments, the lipid conjugate (e.g., a PEG lipid) is present at 0.1 mol% to 2 mol%, 0.5 mol% to 2 mol%, 1 mol% to 2 mol%, 0.6 mol% to 1.9 mol%, 0.7 mol% to 1.8 mol%, 0.8 mol% to 1.7 mol%, 0.9 mol% to 1.6 mol%, 0.9 mol% to 1.8 mol%, 1 mol% to 1.8 mol%, 1 mol% to 1.7 mol%, 1.2 mol% to 1.8 mol%, 1.2 mol% to 1.7 mol%, 1.3 mol% to 1.6 mol%, or 1.4 mol% to 1.5 mol% (or any fraction or range therein) of the total lipid present in the lipid nanoparticle composition. In some embodiments, the lipid conjugate (e.g., a PEG lipid) is present at 0 mol% to 20 mol%, 0.5 mol% to 20 mol%, 2 mol% to 20 mol%, 1.5 mol% to 18 mol%, 2 mol% to 15 mol%, 4 mol% to 15 mol%, 2 mol% to 12 mol%, 5 mol% to 12 mol%, or 2 mol% (or any fraction or range therein) of the total lipid present in the lipid nanoparticle composition.

[0188] In some embodiments, the lipid conjugate (e.g., a PEG lipid) is present at 4 mol% to 10 mol%, 5 mol% to 10 mol%, 5 mol% to 9 mol%, 5 mol% to 8 mol%, 6 mol% to 9 mol%, 6 mol% to 8 mol%, or 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, or 10 mol% (or any fraction or range therein) of the total lipid present in the lipid nanoparticle composition.

[0189] The percentage of lipid conjugate (e.g., PEG lipid) present in the lipid nanoparticle composition is a target amount, and the actual amount of lipid conjugate present in the composition can vary, for example, by ±2 mol%. Those skilled in the art will understand that the concentration of lipid conjugate can vary depending on the lipid conjugate used and the rate at which the lipid particles become fusogenic.

[0190] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate is exchanged from lipid nanoparticles can be controlled, and thus the rate at which lipid nanoparticles become fusogenic.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which lipid nanoparticles become fusogenic.Other methods that can be used to control the rate at which lipid nanoparticles become fusogenic will be clear to those skilled in the art after reading this disclosure.In addition, by controlling the composition and concentration of lipid conjugate, the size of lipid nanoparticles can be controlled.

[0191] In some embodiments, lipid nanoparticle compositions may contain 30-70% ionizable lipid compounds, 0-60% cholesterol, 0-30% phospholipids, and 1-10% polyethylene glycol (PEG)-lipids. In some embodiments, LNP compositions may contain 30-40% ionizable lipid compounds, 40-50% cholesterol, and 10-20% PEG-lipids. In some embodiments, LNP compositions may contain 50-75% ionizable lipid compounds, 20-40% cholesterol, 5-10% phospholipids, and 1-10% PEG-lipids. In some embodiments, compositions may contain 60-70% ionizable lipid compounds, 25-35% cholesterol, and 5-10% PEG-lipids.

[0192] In some embodiments, the LNP composition may contain up to 90% ionizable lipid compounds and 2-15% helper lipids.

[0193] In some embodiments, the lipid nanoparticle composition may contain 8-30% ionizable lipid compounds, 5-30% helper lipids, and 0-20% cholesterol. In some embodiments, the lipid nanoparticle composition contains 4-25% ionizable lipid compounds, 4-25% helper lipids, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine. In some embodiments, the lipid nanoparticle composition contains 2-30% ionizable lipid compounds, 2-30% helper lipids, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine. In some embodiments, the lipid nanoparticle composition contains up to 90% ionizable lipid compounds and 2-10% helper lipids. In some embodiments, the lipid nanoparticle composition even contains 100% ionizable lipids.

[0194] Other components of the LNP composition The lipid nanoparticle composition may include one or more ingredients in addition to those described above. For example, the LNP composition may include one or more hydrophobic small molecules, such as vitamins (e.g., vitamin A or vitamin E) or sterols.

[0195] The lipid nanoparticle composition may also include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers, or other components.

[0196] Suitable carbohydrates can include monosaccharides (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

[0197] Polymers may be used to encapsulate or partially encapsulate the nanoparticle composition. The polymer may be biodegradable and / or biocompatible. Suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. For example, polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), 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), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl acetate), poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, and polymers of acrylic acid, 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)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). Examples of suitable polyols include polyols and copolymers thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarates, polyoxymethylene, poloxamers, polyoxamines, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

[0198] Suitable surface modifiers include, but are not limited to, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, artemisia, bromelain, papain, clerodendrum, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letostein, stepronin, tiopronin, gelsolin, thymosin β4, dornase alfa, neltenexin, and erdosteine), and DNase (e.g., rhDNase). Surface modifiers may be disposed within the lipid nanoparticles and / or on the surface of the lipid nanoparticles (e.g., by coating, adsorption, covalent bonding, or other process).

[0199] Lipid nanoparticle composition can also comprise one or more functionalized lipids.For example, lipid can be functionalized with alkyne group, which can undergo cycloaddition reaction when exposed to azide under suitable reaction conditions.In particular, lipid bilayer can be functionalized in this manner with one or more groups that are useful for promoting membrane permeation, cell recognition or imaging.The surface of lipid nanoparticle can also be conjugated with one or more useful antibodies.The functional groups and conjugates that are useful for target cell delivery, imaging and membrane permeation are well known in the art.

[0200] Lipid nanoparticle compositions may contain any substance useful in pharmaceutical compositions.For example, lipid nanoparticle compositions may contain one or more pharmaceutically acceptable excipients or auxiliary components, such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulation aids, disintegrants, fillers, glidants, liquid vehicles, binders, surfactants, isotonicity agents, thickeners or emulsifiers, buffers, lubricants, oils, preservatives, and other species.Excipients such as wax, butter, coloring agents, coating agents, flavoring agents, and fragrances may also be included.

[0201] Suitable diluents can 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, corn starch, 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, clay, 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), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0202] Suitable surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, 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., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl ... cellulose, 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®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone),Examples of suitable anti-inflammatory agents include 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.

[0203] Suitable binders may be starches (e.g., corn starch 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, breadwort gum, ghatti gum, mucilage of isopole shell, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binder.

[0204] Suitable preservatives may include, but are not limited to, antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, 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 antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, 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, hydroxybenzoates, 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, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (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®.

[0205] Suitable lubricants 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.

[0206] Suitable oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, juniper, chamomile, canola, caraway, carnauba, castor, cinnamon bark, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cucumber, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, and tawny. Ingredients include, but are not limited to, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, 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.

[0207] In some embodiments, the lipid nanoparticle composition further comprises one or more cryoprotectants. Suitable cryoprotectants include polyols (e.g., diols or triols, 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), non-surfactant sulfobetaines (e.g., NDSB-201 (3-(1-pyridino)-1-propanesulfonate)), osmolytes (e.g., L-proline or trimethylamine N-oxide dihydrate), polymers (e.g., polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG 2k-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 K15), pentaerythritol propoxylate, or polypropylene glycol P 400), organic solvents (e.g., dimethyl sulfoxide (DMSO) or ethanol), sugars (e.g., D-(+)-sucrose, D-sorbitol, trehalose, D-(+)-maltose monohydrate, mesoerythritol, xylitol, myo-inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate, or D-(+)-glucose monohydrate), or salts (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.

[0208] 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.

[0209] In some embodiments, the lipid nanoparticle composition further comprises one or more buffers.Suitable buffers include but are not limited to citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic 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 mixture, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixture, tromethamine, aminosulfonic acid buffer (for example, HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof.

[0210] In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, a Tris buffer, or a combination thereof.

[0211] In some embodiments, the lipid nanoparticle composition further comprises one or more nucleic acids, ionizable lipids, amphiphiles, phospholipids, cholesterol, and / or PEG-conjugated cholesterol.

[0212] therapeutic agent

[0213] In some embodiments, the lipid nanoparticle composition further comprises one or more therapeutic and / or prophylactic agents (eg, nucleic acid components).

[0214] In some embodiments, the therapeutic and / or prophylactic agent is a vaccine, a compound that elicits an immune response (e.g., a polynucleotide or nucleic acid molecule encoding a protein, polypeptide, or peptide, or a protein, polypeptide, or protein), and / or another therapeutic and / or prophylactic agent. Vaccines include compounds and formulations that can confer immunity against one or more pathologies associated with infectious diseases and can include mRNA encoding infectious disease-derived antigens and / or epitopes. Vaccines also include compounds and formulations that induce an immune response against cancer cells and can include mRNA encoding tumor cell-derived antigens, epitopes, and / or neoepitopes. In some embodiments, vaccines and / or compounds capable of eliciting an immune response are administered intramuscularly via a composition of the present disclosure.

[0215] In some embodiments, the therapeutic and / or prophylactic agent is a protein, for example, a protein required to enhance or replace a natural protein of interest. Such proteins or polypeptides may be naturally occurring or may be modified using methods known in the art, for example, to increase half-life. Exemplary proteins are intracellular, transmembrane, or secreted proteins, peptides, or polypeptides.

[0216] In some embodiments, the therapeutic and / or prophylactic agent comprises one or more RNA and / or DNA components. In some embodiments, the therapeutic and / or prophylactic agent comprises one or more DNA components. In some embodiments, the therapeutic and / or prophylactic agent comprises one or more RNA components.

[0217] In some embodiments, the one or more RNA components are selected from mRNA. In some embodiments, the mRNA is a modified mRNA.

[0218] In some embodiments, the one or more RNA components comprise a gRNA nucleic acid. In some embodiments, the gRNA nucleic acid is a gRNA.

[0219] 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 a dual guide RNA (dgRNA) or encodes a dual guide RNA (dgRNA). In some embodiments, the gRNA nucleic acid is a single guide RNA (sgRNA) or encodes a single guide RNA (sgRNA). In some embodiments, the gRNA is a modified gRNA. In some embodiments, the modified gRNA comprises a modification in one or more of the first five nucleotides at the 5' end. In some embodiments, the modified gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

[0220] 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, such as a Cas nuclease mRNA (such as a class 2 Cas nuclease mRNA) or a Cas9 nuclease mRNA.

[0221] In some embodiments, the therapeutic and / or prophylactic agent comprises one or more template nucleic acids.

[0222] In some embodiments, the therapeutic agent is selected from one or more nucleic acids, including, for example, mRNA, antisense oligonucleotides, plasmid DNA, microRNA (miRNA), miRNA inhibitors (antagomir / antimers), messenger RNA interference complementary RNA (micRNA), DNA, polyvalent RNA, Dicer substrate RNA, complementary DNA (cDNA), and the like.

[0223] Nucleic acids can be prepared according to any available technique. For mRNA, the primary preparation method, including but not limited to, is enzymatic synthesis (also known as in vitro transcription), which is currently the most efficient method for producing long sequence-specific mRNA. In vitro transcription describes the process of template-directed synthesis of RNA molecules from a modified DNA template consisting of a bacteriophage promoter upstream sequence (e.g., including but not limited to those derived from T7, T3, and SP6 coliphages) linked to a downstream sequence encoding the gene of interest. Template DNA can be prepared for in vitro transcription from many sources using suitable techniques well known in the art, such as, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDin 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, NY Humana Press, 2012, which are incorporated herein by reference in their entireties).

[0224] RNA transcription occurs in vitro using a linearized DNA template in the presence of the corresponding RNA polymerase and adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates (rNTPs) under conditions that support polymerase activity while minimizing potential degradation of the resulting mRNA transcript. In vitro transcription can be carried out using various commercially available kits, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), and commercially available reagents, including RNA polymerase and rNTPs. Methods for in vitro transcription of mRNA are well known in the art (see, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamaka, RT and Kraus, WL 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, NY Humana Press, 2010; Brunelle, JL 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)).

[0225] The desired in vitro transcribed mRNA can be purified from unwanted components of the transcription or related reaction, including unincorporated rNTPs, protein enzymes, salts, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include, by way of non-limiting example, phenol / chloroform extraction or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0226] Additional non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v.10,889-893 (incorporated herein in its entirety by reference)), silica-based affinity chromatography and polyacrylamide gel electrophoresis (Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LDin 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, NY Humana Press, 2012 (incorporated herein in its entirety by reference)).Purification can be carried out using various commercially available kits, including but not limited to SV Total Isolation System (Promega) and In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0227] Furthermore, while reverse transcription can produce large amounts of mRNA, the product may contain numerous aberrant RNA impurities associated with undesired polymerase activity, which may need to be removed from the full-length mRNA preparation. These include short RNAs resulting 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 impurities, which have dsRNA structures, can result in undesirable immunostimulatory activity through interactions with various innate immune sensors in eukaryotic cells that recognize specific nucleic acid structures and function to elicit strong immune responses. This, in turn, can dramatically reduce mRNA translation, as protein synthesis is reduced during the innate cellular immune response. Therefore, additional techniques have been developed to remove these dsRNA impurities, including but not limited to scalable HPLC purification, and are known in the art (see, for example, 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, PHEd), 2013 (incorporated herein in their entirety by reference)). It has been reported that HPLC-purified mRNA is translated at a much higher level, especially in primary cells and in vivo.

[0228] A wide variety of modifications have been described in the art that can be used to alter specific properties of in vitro transcribed mRNA and improve its utility. These include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap structure at the 5' end of the mature molecule, which plays an important role in mediating the binding of mRNA cap-binding protein (CBP), which in turn contributes to enhancing intracellular mRNA stability and the efficiency of mRNA translation. Therefore, the highest level of protein expression is achieved with capped mRNA transcripts. The 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and a guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Additional modifications include methylation of the very end and penultimate positions of the 5'-most nucleotide on the 2'-hydroxyl group.

[0229] 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 by co-transcription (i.e., capping during in vitro transcription) with chemical cap analogs. 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 and a 3'-0-methyl group. However, up to 20% of transcripts remain uncapped during this co-transcription process, and synthetic cap analogs are not identical to the 5'-cap structure of authentic cellular mRNA, potentially resulting in reduced translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped after transcription. These can generate more authentic 5'-cap structures that structurally or functionally mimic endogenous 5'-caps with enhanced cap-binding protein binding, increased half-life, 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, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology v.969 (Rabinovich, PHEd), 2013, which is incorporated herein by reference in its entirety).

[0230] At the 3'-end, a long chain of adenine nucleotides (poly-A tail) is usually added to mRNA molecules during RNA processing. Immediately after transcription, the 3' end of the transcript is cleaved, freeing the 3' hydroxyl for poly-A polymerase to add a chain of adenine nucleotides to the RNA in a process called polyadenylation. Poly(A) tails have been widely shown to enhance both the translation 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.14 373-377; 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, v1 1, 611-613, which are incorporated herein by reference in their entireties).

[0231] Poly(A) tailing of in vitro transcribed mRNA can be achieved using various techniques, including, but not limited to, cloning a poly(T) tract into a DNA template or post-transcriptional addition using poly(A) polymerase. The former allows for in vitro transcription of mRNA with a poly(A) tail of a defined length depending on the size of the poly(T) tract, but requires additional template manipulation. The latter requires enzymatic addition of a poly(A) tail to in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of RNA. This does not require additional DNA template manipulation, but results in mRNAs with poly(A) tails of heterogeneous lengths. 5'-capping and 3'-poly(A) tailing can be performed using various 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 commercially available reagents, various ARCA caps, poly(A) polymerases, etc.

[0232] In addition to 5' cap and 3' polyadenylation, other modifications of in vitro transcription products have been reported to bring about benefits related to translation efficiency and stability.It is well known in the art that pathogenic DNA and RNA can be recognized by various sensors in eukaryotes and induce strong innate immune responses.Since most nucleic acids from natural sources contain modified nucleosides, it has been shown that the ability to distinguish pathogenic DNA and RNA from self-DNA and RNA is at least partly based on structure and nucleoside modification.In contrast, in vitro synthesized RNA lacks these modifications, and therefore can cause immune stimulation, which in turn can inhibit the above-mentioned effective mRNA translation.Introduction of modified nucleosides into in vitro transcribed mRNA can be used to prevent recognition and activation of RNA sensors, thus alleviating this undesirable immunostimulatory activity and enhancing translational competence (e.g., Kariko, K. And Weissman, D. 2007, Naturally occurring nucleoside modifications suppress the immunostimulatory activity of RNA: Implications for therapeutic RNA development, Curr Opin Drug Discov Devel, v.10 523-532; 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, PHEd), 2013; Kariko, K., Muramatsu, H., Welsh, FA, 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 (incorporated herein by reference in their entirety). Modified nucleosides and nucleotides used in the synthesis of modified RNAs can be prepared, monitored, and utilized using common methods and procedures known in the art.A variety of nucleoside modifications are available that can be incorporated into in vitro transcribed mRNA to some extent, either alone or in combination with other modified nucleosides (see, e.g., US2012 / 0251618, incorporated herein by reference in its entirety). In vitro synthesis of nucleoside-modified mRNA has been reported to have reduced ability to activate immunosensors while simultaneously enhancing translational capacity.

[0233] Other components of mRNA that can be modified to benefit translatability and stability include 5' and 3' untranslated region (UTR).Optimizing UTR (advantageous 5' and 3'UTR can be obtained from cellular or viral RNA) has been shown to increase mRNA stability and the translation efficiency of in vitro transcribed mRNA, either together or independently (see, for example, 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, PHEd), 2013 (these are incorporated herein by reference in their entirety)).

[0234] In addition to mRNA, other nucleic acid payloads may be used in the present disclosure. For oligonucleotides, preparation methods include, but are not limited to, chemical synthesis, enzymatic and chemical cleavage of long-chain precursors, in vitro transcription as described above, etc. The synthesis method of DNA and RNA nucleotides is widely used and well known in the art (see, for example, Gait, MJ (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Ishington, DC: IRL Press, 1984, and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v.288 (Clifton, NJ) Totowa, NJ: Humana Press, 2005 (both are incorporated herein by reference)).

[0235] For plasmid DNA, preparation for use in embodiments of the present disclosure generally utilizes, but is not limited to, in vitro propagation and isolation of the plasmid DNA in liquid cultures 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 bacteria containing the plasmid of interest to selectively grow in antibiotic-containing cultures. Methods for 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., Bjornestedt, 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 U.S. Pat. No. 6,197,553 B1, which are incorporated herein by reference in their entireties). 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 commercially available reagents.

[0236] The amount of therapeutic and / or prophylactic agent in a lipid nanoparticle composition can depend on the size, composition, desired target and / or use, or other characteristics of the LNP composition, as well as the characteristics of the therapeutic and / or prophylactic agent. For example, the amount of RNA useful in an LNP composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and / or prophylactic agent and other components (e.g., lipids) in the LNP composition can also vary. In some embodiments, the wt / wt ratio of lipid component to therapeutic and / or prophylactic agent in the LNP composition can be from about 5:1 to about 60:1, e.g., 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of lipid component to therapeutic and / or prophylactic agent can be from about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1.

[0237] In some embodiments, the lipid nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and their amounts can be selected to achieve a specific N:P ratio. The N:P ratio of an LNP composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. Generally, lower N:P ratios are preferred. The one or more RNAs, lipids, and their amounts can be selected to achieve an N:P ratio of about 2:1 to about 30:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio can be about 5.67:1.

[0238] Generation of lipid nanoparticle compositions In some embodiments, lipid nanoparticle compositions can be prepared by first combining an ionizable lipid compound described herein with or without a helper lipid and / or other lipid components (e.g., a phospholipid (e.g., DOPE or DSPC), a PEG lipid (e.g., 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol, also known as PEG-DMG), a structural lipid (e.g., cholesterol)) in a buffer solution, and then forming lipid nanoparticles, e.g., via nanoprecipitation.

[0239] In some embodiments, the lipid nanoparticle composition may be made, for example, according to the methods described in WO2020 / 160397, the entire contents of which are incorporated herein by reference.

[0240] Characterization of nanoparticle compositions The properties of lipid nanoparticle composition can depend on its components.For example, lipid nanoparticles that contain cholesterol as structural lipid can have different properties from lipid nanoparticles that contain different structural lipids.Similarly, the properties of lipid nanoparticles can depend on the absolute or relative amounts of its components.For example, lipid nanoparticles that contain a higher molar fraction of phospholipids can have different properties from lipid nanoparticles that contain a lower molar fraction of phospholipids.Properties can also vary depending on the method and conditions of nanoparticle composition preparation.

[0241] Lipid nanoparticles can be characterized by various methods. For example, microscopy (e.g., transmission electron microscope or scanning electron microscope) can be used to examine the morphology and size distribution of nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (e.g., manufactured by Malvern Instruments Ltd., Malvern, Worcestershire, UK) can also be used to measure multiple properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0242] In some embodiments, the particle size, polydispersity index (PDI) and zeta potential of lipid nanoparticle compositions can be determined by a zeta potential analyzer. An exemplary zeta potential analyzer is the Zetasizer Nano ZS (e.g., manufactured by Malvern Instruments Ltd. (Malvern, Worcestershire, UK)). For such determination, the lipid nanoparticle composition can be dispersed in a buffer solution, for example, in 1x PBS when determining particle size and in 15mM PBS when determining zeta potential.

[0243] In some embodiments, the mean diameter of the lipid nanoparticle composition (e.g., empty LNP or therapeutic-agent-loaded LNP) is tens to hundreds of nanometers as measured by dynamic light scattering (DLS). In some embodiments, the mean diameter of the LNP composition is about 40 nm to about 150 nm. In some embodiments, the mean diameter of the LNP composition is about 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. In some embodiments, the mean diameter of the LNP composition is about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 150 nm, about 70 nm to about 130 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 150 nm, about 80 nm to about 130 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, about 90 nm to about 150 nm, about 90 nm to about 130 nm, or about 90 nm to about 100 nm. In certain embodiments, the mean diameter of the LNP compositions is about 70 nm to about 130 nm, or about 70 nm to about 100 nm. In some embodiments, the mean diameter of the LNP compositions is about 80 nm. In some embodiments, the mean diameter of the LNP compositions is about 100 nm. In some embodiments, the mean diameter of the LNP compositions is about 110 nm. In some embodiments, the mean diameter of the LNP compositions is about 120 nm.

[0244] In some embodiments, the polydispersity index ("PDI") of lipid nanoparticles (e.g., empty LNPs or therapeutic agent-loaded LNPs) formulated with the ionizable lipid compounds of the present disclosure is less than 0.3. In some embodiments, the lipid nanoparticles formulated with the ionizable lipid compounds of the present disclosure have a PDI of about 0 to about 0.25. In some embodiments, the lipid nanoparticles formulated with the ionizable lipid compounds of the present disclosure have a PDI of about 0.10 to about 0.20.

[0245] The surface hydrophobicity of lipid nanoparticles can be measured by normalized generalized polarization (N-GP) using Laurdan (GPL). In this method, the fluorescent aminonaphthalene ketone lipid Laurdan is post-inserted onto the nanoparticle surface, and the fluorescence spectrum of Laurdan is collected to determine the normalized generalized polarization (N-GP). In some embodiments, the surface hydrophobicity, expressed as N-GP, is between about 0.5 and about 1.5. For example, in some embodiments, the surface hydrophobicity, expressed as N-GP, of lipid nanoparticles formulated with ionizable lipid compounds of the present disclosure is about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, or about 1.5. In some embodiments, the surface hydrophobicity, expressed as N-GP, of lipid nanoparticles formulated with ionizable lipid compounds of the present disclosure is about 1.0 or about 1.1.

[0246] The zeta potential of lipid nanoparticles can be used to indicate the electrokinetic potential of the composition.For example, zeta potential can describe the surface charge of lipid nanoparticle compositions.Lipid nanoparticles with relatively low positive or negative charge are generally desirable, because species with higher charge may have undesirable interactions with cells, tissues and other elements in the body. In some embodiments, the zeta potential of the lipid nanoparticles may be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0247] The concentration of a therapeutic and / or prophylactic agent (e.g., RNA) in a lipid nanoparticle composition can be determined by UV-visible spectroscopy. The lipid nanoparticle composition can be dispersed in a buffer solution, and 100 μL of the diluted formulation in a solvent for such determination, e.g., 1x PBS, can be added to 900 μL of a 4:1 (v / v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution can be recorded at 230 nm to 330 nm, for example, on a DU800 spectrophotometer (e.g., Beckman Coulter, Inc., Brea, CA). The concentration of the therapeutic and / or prophylactic agent in the nanoparticle composition can be calculated based on the extinction coefficient of the therapeutic and / or prophylactic agent used in the composition and the difference between the absorbance at a wavelength of, e.g., 260 nm and the baseline value at a wavelength of, e.g., 330 nm.

[0248] The encapsulation efficiency of a therapeutic agent and / or prophylactic agent in a lipid nanoparticle composition describes the amount of the therapeutic agent and / or prophylactic agent that is encapsulated in or otherwise associated with the lipid nanoparticle after preparation, relative to the initial amount provided.It is desirable that the encapsulation efficiency is high (e.g., close to 100%).The encapsulation efficiency can be measured, for example, by comparing the amount of the therapeutic agent and / or prophylactic agent in a solution containing loaded LNPs before and after the loaded LNPs are disrupted by one or more organic solvents or surfactants.Fluorescence can also be used to measure the amount of free therapeutic agent and / or prophylactic agent (e.g., RNA) in the solution.

[0249] For example, encapsulation efficiency may be assessed using assays known to those skilled in the art. In one embodiment, the QUANT-IT™ RIBOGREEN® RNA assay (e.g., from Invitrogen Corporation, Carlsbad, CA) may be used. In one embodiment, the sample may be diluted to a concentration of approximately 5 μg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted sample may be transferred to a polystyrene 96-well plate, and either 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution may be added to the well. The plate may be incubated at 37° C. for 15 minutes. RIBOGREEN® reagent may be diluted 1:100 in TE buffer, and 100 μL of this solution may be added to each well. The fluorescence intensity can be measured using a fluorescence plate reader (e.g., Wallac Victor 1420 Multilabel Counter (Perkin Elmer, Waltham, MA)) at an excitation wavelength of, for example, about 480 nm and an emission wavelength of, for example, about 520 nm. The fluorescence value of the reagent blank can be subtracted from the fluorescence value of each sample, and the percentage of free RNA can be determined by dividing the fluorescence intensity of the untreated sample (without the addition of Triton X-100) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0250] In some embodiments, for loaded LNPs formulated with ionizable lipid compounds of the present disclosure, the encapsulation efficiency of the therapeutic and / or prophylactic agent is at least 50%, e.g., 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 is at least 80%. In some embodiments, the encapsulation efficiency is at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and / or prophylactic agent is 80%-100%.

[0251] Additional Exemplary LNP Formulations

[0252] Lipid nanoparticles may contain a lipid component and one or more additional components, such as a therapeutic agent and / or a prophylactic agent. Lipid nanoparticle compositions may be designed for one or more specific applications or targets. The components of the lipid nanoparticles may be selected based on the specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a specific formulation of the lipid nanoparticle composition may be selected for a specific application or target, for example, depending on the efficacy and toxicity of a specific combination of components.

[0253] In some embodiments, the lipid component of the lipid nanoparticle composition comprises one or more ionizable lipid compounds described herein, phospholipids (e.g., unsaturated lipids, e.g., DOPE or DSPC), PEG lipids, and structured lipids.

[0254] In some embodiments, the lipid component of the lipid nanoparticle composition comprises one or more of the ionizable lipid compounds, phospholipids, PEG lipids, and structured lipids described herein.

[0255] In some embodiments, the LNP compositions comprise one or more ionizable lipid compounds, phospholipids, structured lipids, PEG-lipids, and one or more therapeutic and / or prophylactic agents described herein.

[0256] In some embodiments, the LNP composition comprises one or more ionizable lipid compounds described herein in an amount of about 40% to about 60%.

[0257] In some embodiments, the LNP composition comprises phospholipids in an amount of about 0% to about 20%. For example, in some embodiments, the LNP composition comprises DSPC in an amount of about 0% to about 20%.

[0258] In some embodiments, the LNP composition comprises about 30% to about 50% structured lipids. For example, in some embodiments, the LNP composition comprises about 30% to about 50% cholesterol.

[0259] In some embodiments, the LNP composition comprises PEG lipids in an amount of about 0% to about 5%. For example, in some embodiments, the LNP composition comprises PEG-1 or PEG-2 lipids. 2k -Contains DMG in an amount of about 0% to about 5%.

[0260] In some embodiments, the lipid component of the nanoparticle composition comprises about 30 mol% to about 60 mol% of one or more ionizable lipid compounds described herein, about 0 mol% to about 30 mol% of phospholipids, about 18.5 mol% to about 48.5 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids, provided that the total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises about 35 mol% to about 55 mol% of one or more ionizable lipid compounds described herein, about 5 mol% to about 25 mol% of phospholipids, about 30 mol% to about 40 mol% of structural lipids, and about 0 mol% to about 10 mol% of PEG lipids. In one embodiment, the lipid component comprises about 50 mol% of one or more ionizable lipid compounds described herein, about 10 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In one embodiment, the lipid component comprises about 40 mol% of one or more ionizable lipid compounds described herein, about 20 mol% of phospholipids, about 38.5 mol% of structural lipids, and about 1.5 mol% of PEG lipids. In some embodiments, the phospholipids may be DOPE or DSPC. In some embodiments, the PEG lipids may be PEG-1 or PEG 2k -DMG and / or the structural lipid may be cholesterol.

[0261] In some embodiments, the LNP composition comprises about 40 mol% to about 60 mol% of one or more ionizable lipid compounds described herein, about 0 mol% to about 20 mol% of phospholipids, about 30 mol% to about 50 mol% of structural lipids, and about 0 mol% to about 5 mol% of PEG-1 or PEG-2 lipids. In some embodiments, the LNP composition comprises about 40 mol% to about 60 mol% of one or more ionizable lipid compounds described herein, about 0 mol% to about 20 mol% of DSPC, about 30 mol% to about 50 mol% of cholesterol, and about 0 mol% to about 5 mol% of PEG-1 or PEG-2 lipids. 2k -Includes DMG.

[0262] Lipid nanoparticles may be designed for one or more specific applications or targets. For example, nanoparticle compositions may be designed to deliver therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems within a mammalian body. The physiochemical properties of lipid nanoparticles may be altered to increase selectivity for specific bodily targets. For example, particle size may be adjusted based on the fenestration size of different organs. The therapeutic and / or prophylactic agents included in LNP compositions may also be selected based on the desired delivery target. For example, therapeutic and / or prophylactic agents may be selected for a specific indication, condition, disease, or disorder and / or for delivery (e.g., localized or specific delivery) to specific cells, tissues, organs, or systems within a mammalian body. In certain embodiments, lipid nanoparticle compositions may contain mRNA encoding a polypeptide of interest that can be translated intracellularly to produce the polypeptide of interest. Such compositions may be designed for specific delivery to a specific organ. In some embodiments, compositions may be designed for specific delivery to the mammalian liver.

[0263] In vivo formulation testing To monitor the effectiveness of lipid nanoparticle compositions in delivering therapeutic and / or prophylactic agents to target cells, different nanoparticle compositions containing specific therapeutic and / or prophylactic agents (e.g., modified or natural RNA such as mRNA) can be prepared and administered to a population of animals. Animals (e.g., mice, rats, or non-human primates) can be administered a single dose containing the LNP compositions described herein and mRNA expressing a protein, such as human erythropoietin (hEPO) or luciferase, intravenously, intramuscularly, intra-arterially, or intratumorally. A control composition containing PBS can also be used.

[0264] Upon administration of the LNP composition to an animal, the dose delivery profile, dose response, and toxicity of a particular formulation and dose can be measured by enzyme-linked immunosorbent assay (ELISA), bioluminescence imaging, or other methods. For LNP compositions containing mRNA, the time course of protein expression can also be assessed. Samples collected from the animal for evaluation may include blood, serum, and tissues (e.g., muscle tissue and internal tissues at the site of intramuscular injection), and sample collection may involve sacrificing the animal.

[0265] In some embodiments, hEPO concentrations can be determined using the Simple Plex Assay (ProteinSimple) enzyme-linked lectin assay (ELLA) using a human erythroprotein cartridge. The assay standards can be calibrated according to the 2.IRP WHO preparation.

[0266] LNP compositions containing mRNA are useful in evaluating the efficacy and usefulness of various formulations for the delivery of therapeutic and / or prophylactic agents. High levels of protein expression induced by the administration of a composition containing mRNA indicate higher mRNA translation and / or mRNA delivery efficiency of the nanoparticle composition. Because non-RNA components are not thought to affect the translation mechanism itself, higher levels of protein expression likely indicate higher delivery efficiency of therapeutic and / or prophylactic agents by a given nanoparticle composition compared to other nanoparticle compositions or their absence.

[0267] In some embodiments, an in vivo expression assay can be used to assess the efficacy of expression of the ionizable lipids of the present disclosure.

[0268] In some embodiments, protein expression (e.g., hEPO) can be measured in mice after administration of the loaded LNP composition. In some embodiments, the concentration of hEPO in serum can be tested after administration (e.g., about 6 hours after injection).

[0269] In some embodiments, the LNP composition can be administered intravenously to mice (eg, CD-1 mice).

[0270] In some embodiments, residual levels of lipids in the organs or tissues of a subject may be measured after administration (e.g., 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours after administration). In some embodiments, residual levels of lipids of the present disclosure may be measured in the liver.

[0271] In some embodiments, in vitro expression assays may be used to evaluate lipid and LNP compositions.

[0272] In some embodiments, cells (e.g., HeLa) may be seeded onto imaging plates (e.g., poly-D-lysene coated) and cultured in serum (e.g., human serum, mouse serum, cynomolgus monkey serum, or fetal bovine serum).

[0273] In some embodiments, an LNP composition comprising mRNA expressing a fluorescent protein (e.g., green fluorescent protein (GFP)) and a fluorescent lipid (e.g., rhodamine-DOPE) may be added to a plate, and the plate may be imaged for uptake and expression. In some embodiments, expression may be assessed by measuring fluorescence (e.g., from GFP). In some embodiments, uptake (accumulation) may be assessed by measuring the fluorescent signal from the fluorescent lipid (e.g., rhodamine-DOPE).

[0274] Methods of Using LNP Compositions

[0275] In some embodiments, provided herein are methods for delivering a therapeutic agent (i.e., cargo) to at least one organ selected from the pancreas, one or both lungs, and the spleen of a subject in need thereof, comprising administering to the subject a lipid nanoparticle composition comprising one or more ionizable lipid compounds disclosed herein (e.g., compounds of Formulae (I)-(XII)), while minimizing delivery to other parts of the subject's body, such as the liver.

[0276] In some embodiments, the method delivers a therapeutic agent (i.e., cargo) to the pancreas and / or one or both lungs of a subject in need thereof, with minimal delivery to other parts of the body, such as the subject's liver.

[0277] In some embodiments, less than 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% of the total therapeutic cargo administered to a subject is delivered to the subject's liver, hi some embodiments, less than 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total therapeutic cargo administered to a subject is delivered to the subject's liver.

[0278] In some embodiments, greater 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 cargo administered to a subject is delivered to the subject's pancreas and / or one or both lungs. In some embodiments, greater 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 cargo administered to a subject is delivered to the subject's pancreas. In some embodiments, greater 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 cargo administered to a subject is delivered to the subject's lungs.

[0279] As used herein, the percentage of the total therapeutic cargo administered to a subject and delivered to a site in the subject is measured by protein expression levels or mRNA knockdown levels.

[0280] In some embodiments, the method of delivering a therapeutic agent cargo disclosed above comprises administering to a subject a lipid nanoparticle composition comprising one or more ionizable lipid compounds disclosed herein encapsulating a therapeutic agent cargo. In some embodiments, the lipid nanoparticles in the lipid nanoparticle composition are formed from one or more compounds selected from ionizable lipids of formulas (I)-(XII), pharmaceutically acceptable salts thereof, and any stereoisomers thereof. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (I), pharmaceutically acceptable salts thereof, and any stereoisomers thereof. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (II), pharmaceutically acceptable salts thereof, and any stereoisomers thereof. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from ionizable lipids of formula (III), pharmaceutically acceptable salts thereof, and any stereoisomers thereof. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (IV), a pharmaceutically acceptable salt thereof, and any of the aforementioned stereoisomers. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (V), a pharmaceutically acceptable salt thereof, and any of the aforementioned stereoisomers. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VI), a pharmaceutically acceptable salt thereof, and any of the aforementioned stereoisomers. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VII), a pharmaceutically acceptable salt thereof, and any of the aforementioned stereoisomers. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VIII), a pharmaceutically acceptable salt thereof, and any of the aforementioned stereoisomers. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (IX), a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing.In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (X), a pharmaceutically acceptable salt thereof, and any stereoisomers of the foregoing. In some embodiments, the lipid nanoparticles are formed from one or more compounds selected from an ionizable lipid of formula (VII), a pharmaceutically acceptable salt thereof, and any stereoisomers of the foregoing.

[0281] Non-limiting exemplary embodiments of the disclosed ionizable lipids, lipid nanoparticles, and compositions comprising them, and their uses for delivering pharmaceutical agents (e.g., therapeutic agents such as nucleic acids) and / or modulating gene and / or protein expression, are described in further detail below.

[0282] In some embodiments, the ionizable lipid and lipid nanoparticle compositions disclosed herein can be used for a variety of purposes, including the delivery of encapsulated or associated (e.g., complexed) therapeutic agents, such as nucleic acids, to cells in vitro and / or in vivo. Accordingly, in some embodiments, methods are provided for treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a lipid nanoparticle composition described herein. In some embodiments, the lipid nanoparticles encapsulate or are associated with a suitable therapeutic agent, and the lipid nanoparticles comprise one or more of the novel ionizable lipids described herein, pharmaceutically acceptable salts thereof, and / or stereoisomers of any of the foregoing.

[0283] In some embodiments, the lipid nanoparticles of the present disclosure are useful for delivery of therapeutic cargo.

[0284] In some embodiments, disclosed herein are methods for inducing expression of a desired protein in vitro and / or in vivo by contacting cells with lipid nanoparticles comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid that is expressed to produce the desired protein (e.g., a messenger RNA or a plasmid encoding the desired protein) or a nucleic acid that blocks a process that terminates mRNA expression (e.g., an miRNA inhibitor).

[0285] In some embodiments, disclosed herein are methods for reducing target gene and protein expression in vitro and / or in vivo by contacting cells with lipid nanoparticle compositions comprising one or more of the novel ionizable lipids described herein, wherein the lipid nanoparticles encapsulate or are associated with a nucleic acid (e.g., an antisense oligonucleotide or a small interfering RNA (siRNA)) that reduces target gene expression.

[0286] Disclosed herein in some embodiments are methods for co-delivery of one or more nucleic acids (e.g., mRNA and plasmid DNA), individually or in combination, which may be useful, for example, to produce an effect requiring co-localization of different nucleic acids (e.g., mRNA encoding a suitable genetic modification enzyme and a DNA segment for integration into the host genome).

[0287] In some embodiments, the lipid nanoparticle compositions are useful for expressing proteins encoded by mRNA. In some embodiments, provided herein are methods for expressing proteins encoded by mRNA.

[0288] In some embodiments, lipid nanoparticle compositions are useful for up-regulating endogenous protein expression by delivering miRNA inhibitors that target a specific miRNA or a group of miRNAs that regulate one target mRNA or several mRNAs.In some embodiments, provided herein is a method for up-regulating endogenous protein expression, comprising delivering miRNA inhibitors that target one or more miRNAs that regulate one or more mRNAs.

[0289] In some embodiments, the lipid nanoparticle compositions are useful for down-regulating (e.g., silencing) the protein level and / or mRNA level of a target gene. In some embodiments, provided herein are methods for down-regulating (e.g., silencing) the protein and / or mRNA level of a target gene.

[0290] In some embodiments, lipid nanoparticles are useful for delivering mRNA and plasmids for transgene expression. In some embodiments, methods are provided herein for delivering mRNA and plasmids for transgene expression.

[0291] In some embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from the expression of a protein, such as increasing the production of red blood cells by delivery of a suitable erythropoietin mRNA, or protecting against infection by delivery of an mRNA encoding a suitable antigen or antibody. In some embodiments, methods are provided herein for inducing a pharmacological effect resulting from the expression of a protein, such as increasing the production of red blood cells by delivery of a suitable erythropoietin mRNA, or protecting against infection by delivery of an mRNA encoding a suitable antigen or antibody.

[0292] In some embodiments, the present disclosure relates to methods of gene editing comprising contacting a cell with an LNP, hi some embodiments, the present disclosure relates to any method of gene editing described herein comprising cleaving DNA.

[0293] In some embodiments, the present disclosure relates to methods of cleaving DNA comprising contacting a cell with an LNP composition.

[0294] In some embodiments, the present 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 present 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 present 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 present disclosure relates to any method of cleaving DNA described herein, further comprising introducing at least one template nucleic acid into a cell. In some embodiments, the present disclosure relates to any method of cleaving DNA described herein, comprising contacting a cell with an LNP composition comprising a template nucleic acid.

[0295] In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering an LNP composition to an animal, e.g., a human. In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering an LNP composition to a cell, such as a eukaryotic cell.

[0296] In some embodiments, the present disclosure relates to any method of gene editing described herein, comprising administering mRNA formulated in a first LNP composition and a second LNP composition, the first LNP composition comprising one or more of mRNA, gRNA, gRNA nucleic acid, and template nucleic acid. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered simultaneously. In some embodiments, the present disclosure relates to any method of gene editing described herein, wherein the first and second LNP compositions are administered sequentially.

[0297] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein comprising administering mRNA and guide RNA nucleic acids formulated in a single LNP composition.

[0298] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein, wherein the gene editing results in a gene knockout.

[0299] In some embodiments, the present disclosure relates to any of the methods of gene editing described herein, wherein the gene editing results in a gene correction.

[0300] In some embodiments, the present disclosure relates to methods for in vivo delivery of interfering RNA to the lungs of a mammalian subject.

[0301] In some embodiments, the methods relate to methods of treating a disease or disorder in a mammalian subject, hi some embodiments, the methods comprise administering a therapeutically effective amount of a composition of the present disclosure 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.

[0302] The composition of the present disclosure can be administered by various routes, for example, to achieve systemic delivery via intravenous, parenteral, intraperitoneal or local route.In some embodiments, siRNA can be delivered intracellularly, for example, in the cells of target tissue such as lung or liver, or in inflammatory tissue.In some embodiments, the present disclosure provides a method for delivering siRNA in vivo.Nucleic acid-lipid composition can be administered to subject intravenously, subcutaneously or intraperitoneally.

[0303] The compositions and methods of the present disclosure can be administered to a subject by various mucosal administration methods, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal or cutaneous delivery, or topical delivery to the eye, ear, skin, or other mucosal surfaces. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, oral, epidermal, or gastrointestinal. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices and pressurized, electrically operated, or other types of actuators.

[0304] The compositions of the present disclosure may be administered in aqueous solution as nasal or pulmonary sprays, or may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved, for example, by administering the composition in the form of drops, particles, or sprays, which may be aerosolized, atomized, or nebulized. The particles, spray, or aerosol of the composition may be in either liquid or solid form. A non-limiting example of a system for dispensing a liquid as a nasal spray is disclosed in U.S. Pat. No. 4,511,069. Such formulations may be conveniently prepared by dissolving a composition according to the present disclosure in water to form an aqueous solution and sterilizing the solution. The formulation may be placed in a multi-dose container in a sealed dispensing system, for example, as disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, Y.W. Chien ed., Elsevier Publishers, New York, 1985, and U.S. Pat. No. 4,778,810. Additional aerosol delivery forms can include, for example, compressed air jet nebulizers, ultrasonic nebulizers, and piezoelectric nebulizers that deliver bioactive agents dissolved or suspended in a pharmaceutical solvent, such as water, ethanol, or mixtures thereof.

[0305] The nasal and pulmonary spray solutions of the present disclosure typically contain a drug to be delivered and are optionally formulated with a surfactant, such as a non-ionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present disclosure, the nasal spray solution further contains a propellant. The pH of the nasal spray solution may be between pH 6.8 and 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer solution with a pH of between 4 and 6. Other ingredients, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0306] In some embodiments, the present disclosure is a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or nasal spray or aerosol.

[0307] The dosage form of the composition of the present disclosure may be a liquid in the form of drops or an emulsion, or in the form of an aerosol.

[0308] The dosage form of the composition of the present disclosure may be a solid that can be reconstituted in a liquid before administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0309] To prepare compositions for pulmonary delivery within the present disclosure, bioactive agents can be combined with various pharmaceutically acceptable additives, as well as bases or carriers for dispersion of the active agent.

[0310] 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), tonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), 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, measured relative to the tonicity of 0.9% (w / v) saline taken as a single unit, is typically adjusted to a value that does not induce substantial irreversible tissue damage in the mucosa at the administration site. Generally, the tonicity of the solution is adjusted to 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0311] The bioactive agent may be dispersed in a base or vehicle, which may include a hydrophilic compound capable of dispersing the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, polycarboxylic acids or their salts, copolymers of carboxylic acid 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, natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and their non-toxic metal salts. Biodegradable polymers, such as polylactic acid, poly(lactic acid-glycolic acid) copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof, are often selected as the base or carrier. Alternatively or additionally, synthetic fatty acid esters, such as polyglycerin fatty acid esters and sucrose fatty acid esters, can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and can impart enhanced structural integrity to the carrier by partial crystallization, ionic bonding, crosslinking, etc. Carriers can be supplied 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 selected carriers in this context can result in enhanced absorption of the bioactive agent.

[0312] Compositions for mucosal, nasal, or pulmonary delivery may contain hydrophilic low-molecular-weight compounds as bases or excipients. Such hydrophilic low-molecular-weight compounds can provide a passageway for water-soluble active agents, such as physiologically active peptides or proteins, to diffuse through the base to the body surface where they are absorbed. The hydrophilic low-molecular-weight compounds can optionally absorb moisture from the mucous membrane or the administration atmosphere, dissolving the water-soluble active peptide. In some embodiments, the molecular weight of the hydrophilic low-molecular-weight compounds is 10,000 or less, for example, 3,000 or less. Examples of hydrophilic low-molecular-weight compounds include polyol compounds, such as sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentiose, glycerin, polyethylene glycol, and oligosaccharides, disaccharides, and monosaccharides, including mixtures thereof. Further examples of hydrophilic low molecular weight compounds include N-methylpyrrolidone, alcohols (eg, oligovinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.), and mixtures thereof.

[0313] Alternatively, the compositions of the present disclosure may contain pharmaceutically acceptable carriers required for approximate physiological conditions, such as pH adjusting agents and buffering agents, isotonicity adjusting agents, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof.For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, including, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0314] In certain embodiments of the present disclosure, the bioactive agent may be administered in a sustained-release formulation, for example, in a composition containing a slow-release polymer. The active agent may be prepared with a carrier that protects it from rapid release, for example, a release-controlled vehicle, such as a polymeric microencapsulated delivery system or a bioadhesive gel. The long-term delivery of the active agent in various compositions of the present disclosure may be achieved by including an agent that delays absorption, for example, aluminum monostearate hydrogel and gelatin in the composition. [Example]

[0315] Example 1. Synthesis of various ionizable lipids

[0316] 1.1: Synthesis of Intermediate 8 (Int. 8) (Intermediate of Compound 2211) [ka]

[0317] Step 1: To a mixture of 8-(tert-butoxycarbonylamino)octanoic acid (25.0 g, 96.40 mmol, 1.2 equiv.) in DCM (1000 mL) was added DMAP (4.91 g, 40.17 mmol, 0.5 equiv.), heptadecan-9-ol (20.60 g, 80.33 mmol, 1 equiv.), and EDCI (46.20 g, 241.00 mmol, 3.0 equiv.). The mixture was heated under a N atmosphere. 、 The mixture was stirred at 25° C. for 12 hours. The reaction mixture was diluted with EtOAc and washed with H2O. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography to give 1-octylnonyl 8-(tert-butoxycarbonylaminooctanoate) (24.0 g, crude) as a yellow oil. The crude product was used in the next step.

[0318] Step 2: To a solution of 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (12.0 g, 24.11 mmol, 1.0 equiv.) in DCM (100 mL) was added TFA (46.20 g, 405.18 mmol, 30 mL, 16.81 equiv.). The mixture was stirred at 25 °C for 5 h. The reaction mixture was adjusted to pH = 7.0 with saturated aqueous NaHCO3, extracted with EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography to give 1-octylnonyl 8-aminooctanoate (15.0 g, 37.72 mmol, 78% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.64(brs,2H),4.84-4.88(m,1H),2.84(t,J=7.6Hz,2H),2. 28(t,J=7.6Hz,2H),1.50-1.61(m,8H),1.26-1.33(m,30H),0.88(t,J=6.8Hz,6H). LCMS: [M+H] + :398.6

[0319] Step 3: To a mixture of 6-bromohexanoic acid (22.64 g, 116.07 mmol, 1 equiv.) in DCM (1 mL) was added DMAP (2.84 g, 23.21 mmol, 0.2 equiv.), undecan-1-ol (20.0 g, 116.07 mmol, 1.0 equiv.), and EDCI (22.25 g, 116.07 mmol, 1.0 equiv.). The mixture was heated under a N atmosphere. 、 The mixture was stirred at 25° C. for 12 hours. The reaction mixture was diluted with HO and extracted with EtOAc. The combined organic layers were dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography to give undecyl 6-bromohexanoate (36.0 g, 103.05 mmol, 89% yield) as a yellow oil. 1H NMR(400MHz,CDCl3),4.07(t,J=6.8Hz,2H),3.41(t,J=6.8Hz,2H),2.33(t,J=7.2Hz,2H),1.87 -1.91(m,2H),1.63-1.68(m,4H),1.48-1.50(m,2H),1.27-1.32(m,16H),0.89(t,J=6.4Hz,3H).

[0320] Step 4: To a solution of 1-octylnonyl 8-aminooctanoate (1.0 g, 2.51 mmol, 1.0 equiv.) and undecyl 6-bromohexanoate (878.47 mg, 2.51 mmol, 1.0 equiv.) in DMF (20 mL) was added K2CO3 (1.04 g, 7.54 mmol, 3.0 equiv.). The mixture was stirred at 80 °C for 5 h. The reaction mixture was diluted with HO and extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography to give 1-octylnonyl 8-[(6-oxo-6-undecoxy-hexyl)amino]octanoate (0.5 g, 750.63 μmol, 30% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),4.06(t,J=6.8Hz,2H),2.59-2.60(m,4H),2.28-2.31(m, 4H),1.60-1.65(m,6H),1.50-1.52(m,8H),1.27-1.36(m,48H),0.89(t,J=6.4Hz,9H).LCMS:[M+H] + :666.8

[0321] 1.2: Synthesis of Compound 1 (Compound 2217) [ka]

[0322] Step 1: To a solution of intermediate 8 (1.0 mmol) in DMF (0.5 M) is added K2CO3 (1.5 mmol) and KI (1.5 mmol). To the above mixture is added a solution of intermediate 9 (2.0 mmol) in DMF (0.5 M) and stirred at 80 °C for 12 h. The reaction mixture is filtered and concentrated under reduced pressure to give a residue that is purified by silica gel chromatography to give intermediate 10.

[0323] Step 2: To a solution of intermediate 8 (1.0 mmol) in THF (0.5 M) is added MeNH (2.0 mmol), and the reaction mixture is stirred for 12 h at 100° C. under microwave conditions. The mixture is then concentrated under reduced pressure and purified by silica gel chromatography to give compound 1.

[0324] 1.3: Synthesis of Compound 2 (Compound 2218) [ka]

[0325] Step 1: To a solution of intermediate 8 (1.0 mmol) in DMF (0.5 M) is added DIEA (1.5 mmol) and intermediate 11 (1.5 mmol). The mixture is stirred at 80° C. for 12 hours. The reaction mixture is filtered and concentrated under reduced pressure to give a residue that is purified by silica gel chromatography to give intermediate 12.

[0326] Step 2: To a solution of intermediate 12 (2.5 mmol) in DCM (0.5 M) was added TEA (3.0 mmol) and triphosgene (1.0 mmol). The mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and concentrated under reduced pressure to give a residue that was purified by silica gel chromatography to give intermediate compound 2.

[0327] 1.4: Synthesis of Compound 3 (Compound 2219) [ka]

[0328] Step 1: To a solution of intermediate 8 (1.0 mmol) in DCM (0.5 M) is added intermediate 13 (1.5 mmol) at 0 °C, and the reaction mixture is warmed to room temperature and stirred for 12 h. The reaction is then quenched by adding 1 M HCl, diluted with HO, and extracted with EtOAc. The organic layer is dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that is purified by silica gel chromatography to give intermediate 14.

[0329] Step 2: To a solution of intermediate 14 (1.0 mmol) in THF (0.5 M) is added TFA (2.0 mmol) and the reaction is stirred at 25 °C for 5 h. The reaction mixture is then neutralized with saturated NaHCO and extracted with EtOAc. The organic layer is dried over NaSO, filtered, and concentrated under reduced pressure to give a residue that is purified by silica gel chromatography to give intermediate 15.

[0330] Step 3: To a solution of intermediate 16 (1.0 mmol) in MeOH (0.25 M) at 0 °C, SOCl (1.2 mmol) is added and the reaction mixture is stirred at room temperature for 3 h. The reaction is neutralized with saturated NaHCO and concentrated under reduced pressure. The residue is resuspended in H O and extracted with EtOAc. The organic layer is dried over Na SO , filtered, and concentrated under reduced pressure to give a residue that is purified by silica gel chromatography to give intermediate 17.

[0331] Step 4: To a solution of intermediate 15 (1.0 mmol) in DMF (0.5 M) is added intermediate 17 (2.0 mmol) and the reaction is stirred for 12 h at 110° C. The reaction mixture is concentrated to give a residue which is purified by silica gel chromatography to give compound 3.

[0332] 1.5: Synthesis of Compound 4 (Compound 2220) [ka] To a solution of intermediate 15 (2.5 mmol) in DCM (0.5 M) is added intermediate 18 (1.0 mmol) and TEA (2.5 mmol) at 0 °C. The mixture is stirred at 25 °C for 12 h. The reaction mixture is diluted with HO and extracted with EtOAc. The combined organic layers are dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue is purified by silica gel chromatography to give compound 4.

[0333] 1.6: Synthesis of Compound 5 (Compound 2221) [ka] To a solution of intermediate 15 (2.5 mmol) in DCM (0.5 M) is added intermediate 19 (1.0 mmol) and TEA (2.5 mmol) at 0 °C. The mixture is stirred at 25 °C for 12 h. The reaction mixture is diluted with HO and extracted with EtOAc. The combined organic layers are dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue is purified by silica gel chromatography to give compound 5.

[0334] 1.7: Key Intermediate 23 (Intermediate 23) [ka]

[0335] Intermediate 23 is synthesized according to the procedure reported for intermediate 8.

[0336] 1.8: Synthesis of Compound 6 (Compound 2209) [ka]

[0337] Compound 6 is synthesized according to the procedure reported for compound 1.

[0338] 1.9: Synthesis of Compound 7 (Compound 2210) [ka]

[0339] Compound 7 is synthesized according to the procedure reported for compound 2.

[0340] 1.10: Synthesis of Compound 8 (Compound 2211) [ka]

[0341] Compound 8 is synthesized according to the procedure reported for compound 3.

[0342] 1.11: Synthesis of Compound 9 (Compound 2212) [ka]

[0343] Compound 9 is synthesized according to the procedure reported for compound 4.

[0344] 1.12: Synthesis of Compound 10 (Compound 2213) [ka]

[0345] Compound 10 is synthesized according to the procedure reported for compound 5.

[0346] 1.13: Synthesis of Compound 2252 [ka]

[0347] Step 1: To a suspension of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (500 mg, 705.04 μmol, 2 equiv.), DMAP (4.31 mg, 35.25 μmol, 0.1 equiv.), TEA (71.34 mg, 705.04 μmol, 98.13 μL, 2 equiv.), and 4A molecular sieves (500 mg) in DCM (15 mL) was added (E)-but-2-enedioyl dichloride (53.92 mg, 352.52 μmol, 38.24 μL, 1 equiv.) in DCM (5 mL) dropwise under a N atmosphere at 15 °C for 0.5 h, and the mixture was then stirred at 15 °C for 8 h. The reaction mixture was filtered, and the filtrate was diluted with 20 mL of H2O and then extracted with 100 mL (50 mL x 2) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 1 / 1, 5% NH3 H2O) and preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1) to give compound 2252, i.e., 1-octylnonyl 8-[2-[[(E)-4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-4-oxo-but-2-enoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (250 mg, 166.51 μmol, 47.23% yield, 100% purity), as a colorless oil. 1 H NMR(400MHz,CDCl3),6.87(s,2H),6.50(brs,2H),4.84-4.90(m,2H),4.07(t,J=6.8Hz,4H),3.39-3.36(m,4H),2.58(t,J=6.0Hz,4H),2.4 1(m,8H),2.30(m,8H),1.60-1.67(m,12H),1.48-1.55(m,8H),1.38-1.45(m,8H),1.23-1.35(m,96H),0.88(t,J=6.8Hz,18H).LCMS:(M+2H + ):749.8@3.831 minutes.

[0348] 1.14: Synthesis of compound 2275 [ka]

[0349] Step 1: To a solution of heptadecan-9-ol (10 g, 7.80 mmol, 1 equiv.) and 8-bromooctanoic acid (9.55 g, 8.58 mmol, 1.1 equiv.) in DCM (200 mL) was added EDCI (1.79 g, 9.36 mmol, 1.2 equiv.) and DMAP (2.38 g, 3.90 mmol, 0.5 equiv.). The mixture was stirred at 15 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 15 °C and then extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were washed with 400 mL (200 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 5 / 1) to obtain the compound 1-octylnonyl 8-bromooctanoate (17.75 g, 38.46 mmol, yield 98.63%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.85-4.89(m,1H),3.40(t,J=6.8,2H),2.29(t,J=7.6Hz,2H),1.84-1 .86(m,2H),1.58-1.69(m,2H),1.39-1.57(m,6H),1.25-1.35(m,28H),0.88(t,J=6.8,6H).

[0350] Step 2: To a solution of phenylmethanamine (552.75 mg, 1.03 mmol, 562.30 μL, 1 equiv) in DMF (50 mL) was added KCO (3.56 g, 5.16 mmol, 5 equiv) and KI (2.14 g, 2.58 mmol, 2.5 equiv), followed by the addition of 1-octylnonyl 8-bromooctanoate (5 g, 2.17 mmol, 2.1 equiv) in DMF (20 mL). The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched at 15 °C by the addition of 100 mL of HO and then extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were washed with 100 mL (50 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to obtain the compound 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3.25 g, 3.74 mmol, yield 72.55%) as a colorless oil.

[0351] Step 3: A solution of 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (2.5 g, 575.74 μmol, 1 equiv.) and Pd / C (1.25 g, 575.74 μmol, 10% purity, 1.00 equiv.) in 50 mL of EtOAc was stirred under H2 (50 Psi) at 15 °C for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 0) to give the compound 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.5 g, 1.93 mmol, 66.95% yield) as a colorless oil.

[0352] Step 4: To a solution of 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1 g, 1.28 mmol, 1 equiv.) in DCM (10 mL) was added tert-butyl N-(2-oxoethyl)carbamate (306.78 mg, 1.93 mmol, 1.5 equiv.). The mixture was stirred at 15 °C for 30 min, and then NaBH(OAc) (544.61 mg, 2.57 mmol, 2 equiv.) was added to the mixture at 15 °C and stirred for 8 h. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain the compound 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (360 mg, 390.67 μmol, yield 30.41%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.99(s,1H),4.84-4.90(m,2H),3.14(brs,2H),2.49-2.55(m,2H),2.39(t,J=6.8Hz,3H ),2.28(t,J=7.6Hz,4H),1.56-1.70(m,4H),1.35-1.52(m,26H),1.20-1.32(m,63H),0.89(t,J=6.4Hz,12H).

[0353] Step 5: To a solution of 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (360 mg, 390.67 μmol, 1 equiv.) in DCM (10 mL) was added TFA (3.64 g, 35.92 mmol, 5 mL, 91.95 equiv.). The mixture was stirred at 15 °C for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 0) to give the compound 1-octylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (71 mg, 86.44 μmol, 22.13% yield) as a yellow oil.

[0354] Step 6: To a suspension of 1-octylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (71 mg, 86.44 μmol, 2 equiv.), TEA (13.12 mg, 129.66 μmol, 18.05 μL, 3 equiv.), DMAP (528.00 μg, 4.32 μmol, 0.1 equiv.), and 4A molecular sieves (50 mg, 1.00 equiv.) in DCM (3 mL) was added butanedioyl dichloride (6.70 mg, 43.22 μmol, 4.75 μL, 1 equiv.) in DCM (1 mL) dropwise over 30 min at 15 °C. The mixture was stirred at 15 °C for 4 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, ethyl acetate:¥MeOH = 10:1) to give compound 2275, i.e., 1-octylnonyl 8-[2-[[4-[2-[bis[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (20 mg, 11.36 μmol, 26.29% yield, 99% purity), as a colorless oil. 1 H NMR(400MHz,CDCl3),6.25(s,2H),4.83-4.90(m,4H),3.27(s,4H),2.26-2.52(m,24H),1.59-1.64(m,1 0H),1.48-1.52(m,12H),1.38-1.42(m,6H),1.20-1.32(m,124H),0.89(t,J=6.4Hz,24H).LCMS:(M / 2+H + ):863.0@12.517 minutes.

[0355] 1.15: Synthesis of compounds 2277 and 2213 [ka]

[0356] Step 1: To a solution of 8-bromooctanoic acid (10.21 g, 45.75 mmol, 1.1 equiv.) and nonan-1-ol (6 g, 41.59 mmol, 1 equiv.) in DCM (100 mL) was added DMAP (1.02 g, 8.32 mmol, 0.2 equiv.) and EDCI (9.57 g, 49.91 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (50 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine (50 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give nonyl 8-bromooctanoate (10 g, 27.19 mmol, yield 65.38%, purity 95%) as a colorless oil.

[0357] Step 2: To a solution of 8-(tert-butoxycarbonylamino)octanoic acid (10 g, 38.56 mmol, 1 equiv.) and heptadecan-9-ol (10 g, 38.99 mmol, 1.01 equiv.) in DCM (100 mL) was added DMAP (942.16 mg, 7.71 mmol, 0.2 equiv.) and EDCI (8.87 g, 46.27 mmol, 1.2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was added to HO (100 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine (50 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) to give 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (8 g, 16.07 mmol, 41.68% yield) as a colorless oil.

[0358] Step 3: A solution of 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (2 g, 4.02 mmol, 1 equiv) in HCl / EtOAc (4 M, 20 mL, 19.91 equiv) was stirred for 8 h at 20° C. The mixture was concentrated under reduced pressure to give 1-octylnonyl 8-aminooctanoate (5 g, crude) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.85-4.89(m,1H),2.72(t,J=7.2Hz,2H),2.28(t,J=7.6Hz,2H),2.2 0(s,2H),1.61-1.65(m,2H),1.45-1.55(m,6H),1.25-1.35(m,30H),0.89(t,J=6.8Hz,6H).

[0359] Step 4: To a solution of 1-octylnonyl 8-aminooctanoate (5.01 g, 12.59 mmol, 1.1 equiv.) in DMF (100 mL) was added KI (2.28 g, 13.74 mmol, 1.2 equiv.) and K2CO3 (4.75 g, 34.35 mmol, 3 equiv.), followed by a solution of nonyl 8-bromooctanoate (4 g, 11.45 mmol, 1 equiv.) in DMF (20 mL). The mixture was then stirred at 80 °C for 12 h. The mixture was filtered, and the filtrate was added to HO (50 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give nonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (4 g, 5.40 mmol, 47.20% yield, 90% purity) as a yellow oil.

[0360] Step 5: To a solution of nonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1 g, 1.50 mmol, 1 equiv.) in DMF (5 mL) was added K2CO3 (1.04 g, 7.51 mmol, 5 equiv.) and KI (249.21 mg, 1.50 mmol, 1 equiv.), followed by the addition of tert-butyl N-(2-bromoethyl)carbamate (1.51 g, 6.76 mmol, 4.5 equiv.). The mixture was stirred at 80 °C for 12 h. The mixture was filtered, and the filtrate was added to HO (10 mL) and extracted with EtOAc (5 mL × 3). The organic layer was washed with brine (5 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to give 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1 g, 1.24 mmol, 41.15% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.91(s,1H),4.76-4.85(m,1H),3.98(t,J=6.8Hz,2H),3.06-3.07(m,2H),2.41-2.50(m,2H),2. 25-2.35(m,4H),2.15-2.25(m,4H),1.50-1.65(m,7H),1.25-1.45(m,18H),1.17-1.25(m,50H),0.81(t,J=6.8Hz,9H).

[0361] Step 6: A solution of 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (1 g, 1.24 mmol, 1 equiv) in TFA (10.78 g, 94.54 mmol, 7 mL, 76.51 equiv) and DCM (14 mL) was stirred for 2 h at 20° C. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and further purified by preparative TLC (SiO2, ethyl acetate:MeOH = 3:1, with 3% NH3·H2O added) to give compound 2277, i.e., nonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (500 mg, 705.04 μmol, 57.06% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.85-4.89(m,1H),4.06(t,J=6.8Hz,2H),2.90-2.95(m,2H),2.65-2.75(m,2H),2.50-2.60(m, 4H),2.20-2.30(m,4H),1.55-1.70(m,6H),1.40-1.55(m,8H),1.20-1.40(m,48H),0.89(t,J=6.8Hz,9H).LCMS:(M+H + ):709.4@10.079 minutes.

[0362] Step 7: To a solution of 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (100 mg, 141.01 μmol, 2.1 equiv.) and TEA (40.00 mg, 395.30 μmol, 55.02 μL, 5.89 equiv.) in DCM (5 mL), butanedioyl dichloride (10.41 mg, 67.15 μmol, 7.38 μL, 1 equiv.) was added at 0° C. under N2, and the mixture was stirred at 20° C. for 1 h. The mixture was added to saturated NaHCO3 (20 mL) and extracted with EtOAc (10 mL × 3). The organic layer was 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 (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and further purified by preparative TLC (SiO2, ethyl acetate:MeOH = 5:1, with 3% NH3·H2O) to give compound 2213, i.e., 8-[2-[[4-[2-[(8-nonoxy-8-oxo-octyl)-[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (26 mg, 17.33 μmol, 25.81% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),6.27(s,2H),4.85-4.89(m,2H),4.06(t,J=6.8Hz,4H),3.26(s,4H),2.51(brs,8H),2.26-2.41(m ,16H),1.58-1.65(m,12H),1.45-1.55(m,8H),1.35-1.40(m,8H),1.20-1.35(m,96H),0.86-0.91(m,18H).LCMS:(M / 2+H + ):750.5@12.146 minutes.

[0363] Example 2. Preparation of lipid nanoparticle compositions C12-200 is a commercially available ionizable lipid with the chemical name 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol). The composition consisted of ionizable lipid: structural lipid: sterol: PEG-lipid (C12-200: DOPE: cholesterol: 14:0 PEG2000 PE) in a molar ratio of 35:16:46.5:2.5, respectively. The lipids were solubilized in ethanol. These lipids were mixed in the above molar ratio and diluted to a total lipid concentration of 5.5 mM in ethanol (organic phase). The mRNA solution (aqueous phase) was prepared with RNAse-free water and 100 mM citrate buffer pH 3 to obtain a final concentration of 50 mM citrate buffer. The N:P ratio of ionizable lipid to mRNA was maintained at 15:1.

[0364] All other compositions, namely, MC3 (a commercially available ionizable lipid with the chemical name (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) and the novel ionizable lipids 7669, 7671, 7668, 767, and 7650, are composed of ionizable lipid:structural lipid:sterol:PEG-lipid (SDA lipid number: DSPC:cholesterol:14:0 PEG2000 PE) in a molar ratio of 50:38.5:10:1.5, respectively. The lipids are solubilized in ethanol. The compositions are then processed as described above, except that the N:P ratio of ionizable lipid to mRNA in the formulation is maintained at 6:1. The lipid mixture and mRNA solution are mixed in a volume ratio of 1:3 on a NanoAssemblr Ignite (Precision Nanosystems) at a total flow rate of 9 mL / min. The resulting composition is then loaded into a Slide-A-Lyzer G2 dialysis cassette (10k MWCO) and dialyzed in 200x the sample volume of 1x PBS at room temperature for 4 hours with gentle agitation. The PBS is refreshed, and the composition is further dialyzed for at least 14 hours at 4°C with gentle agitation. The dialyzed composition is then collected and concentrated by centrifugation at 2000 x g using an Amicon Ultra centrifugal filter (100k MWCO). The concentrated particles are characterized for size, polydispersity, and particle concentration using a Zetasizer Ultra (Malvern Panalytical), and for mRNA encapsulation efficiency using a Quant-iT RiboGreen RNA Assay Kit (ThermoFisher Scientific).

[0365] NP formulation Lipids were solubilized in ethanol. These lipids were mixed at the indicated molar ratio and diluted in ethanol (organic phase) to a total lipid concentration of 5.5 mM. The mRNA solution (aqueous phase) was prepared in RNAse-free water and 100 mM citrate buffer, pH 3, to give a final concentration of 50 mM citrate buffer and an mRNA concentration of 0.167 mg / mL (1:1 FLuc:EPO).

[0366] The recLemon formulation consisted of ionizable lipids, structural lipids, sterols, and PEG-lipids (C12-200: lemon lipids: cholesterol: 14:0 PEG2000 PE) in molar ratios of 35:50:12.5:2.5, respectively. Lipids were solubilized in ethanol, except for lemon lipids, which were solubilized in 4:1 DMF:methanol. The formulations were then processed as described above.

[0367] The lipid mixture and mRNA solution were mixed at a volume ratio of 1:3 on a NanoAssemblr Ignite (Precision Nanosystems) at a total flow rate of 9 mL / min. The resulting formulation was then loaded into a Slide-A-Lyzer G2 dialysis cassette (10k MWCO) and dialyzed in 200x the sample volume of 1x PBS at room temperature for 2 hours. The PBS was refreshed, and the formulation was further dialyzed for at least 14 hours at 4°C with gentle agitation. The dialyzed formulation was then collected and concentrated by centrifugation at 3000 x g using an Amicon Ultra centrifugal filter (100k MWCO). The concentrated formulation was characterized for size, polydispersity, and particle concentration using a Zetasizer Ultra (Malvern Panalytical) and for mRNA encapsulation efficiency using a Quant-iT RiboGreen RNA Assay Kit (ThermoFisher Scientific).

[0368] TNS(pKa) assay (Protocol adapted from "A Novel Amino Lipid Series for mRNA Delivery: Impeded Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates," Sabnis, Staci et al., Molecular Therapy, Volume 26, Issue 6, pp. 1509-1519)

[0369] Twenty buffer solutions (10 mM sodium phosphate, 10 mM sodium borate, 10 mM sodium citrate, and 150 mM sodium chloride in distilled water) with unique pH values ​​ranging from 3.0 to 12.0 were prepared using 1 M sodium hydroxide and 1 M hydrochloric acid. 3.25 μL of LNP (0.04 mg / mL mRNA in PBS) was incubated with 2 μL of TNS reagent (0.3 mM in DMSO) and 90 μL of buffer solution of each pH value (described above) in a 96-well black-walled plate. Each pH condition was performed in triplicate wells. TNS fluorescence was measured using a Biotek Cytation Plate reader at excitation / emission wavelengths of 321 / 445 nm. Fluorescence values ​​were then plotted and fitted using a four-parameter sigmoidal curve. From the fit, the pH value resulting in the full-width at half-maximum fluorescence was calculated and reported as the apparent LNP pKa value.

[0370] Example 3. In vivo bioluminescence imaging Eight- to nine-week-old female Balb / c mice were used for the bioluminescence-based ionizable lipid screening approach. Mice were obtained from Jackson Laboratories (JAX Stock: 000651) and allowed to acclimate for one week before treatment. After placing the animals under a heat lamp for several minutes, they were introduced into a restraining chamber. The tail was wiped with an alcohol pad (Fisher Scientific), and 100 μL of the above lipid nanoparticle composition containing 10 μg of total mRNA (5 μg Fluc + 5 μg EPO) was injected intravenously using a 29G insulin syringe (Covidien). Any bleeding that occurred was stopped using a sterile gauze pad (Fisher Scientific), and the animals were returned to their home cage. Four to six hours after administration, the animals were injected with 200 μL of 15 mg / mL D-luciferin (GoldBio) and placed in an isoflurane induction chamber set up to deliver 2.5% isoflurane at an oxygen flow rate of 1 to 2 liters per minute. After 5 minutes of isoflurane exposure, the mice were placed in the nose cone set up in an IVIS Lumina LT imaging system (PerkinElmer). LivingImage software was used for imaging. Whole-body bioluminescence was captured with automatic exposure, after which the animals were removed from the IVIS and placed in a CO2 chamber for euthanasia. After placing the animals in the dorsal recumbent position, cardiac puncture was performed on each animal, and blood was collected using a 25G insulin syringe (BD). Blood was collected into lithium-heparin-coated tubes (Fisher Scientific) and immediately placed on ice. Once all blood samples have been collected, the tubes are spun at 2000 G for 10 minutes using a tabletop centrifuge, and the plasma is aliquoted into individual Eppendorf tubes (Fisher Scientific) and stored at -80°C for subsequent EPO quantification. EPO levels in plasma are determined using an EPO MSD kit (Meso Scale Diagnostics).

[0371] The molar ratios of the components of each LNP composition, the results of the characterization of each LNP composition according to the method described in Example 2, and the bioluminescence results of each LNP composition according to the method described in Example 3 are summarized in Table 1 below. [Table 2]

[0372] Thus, ionizable lipid scaffolds demonstrate selective delivery of therapeutic cargo outside the liver and are expected to have low hepatotoxicity due to low hepatic lipid levels.

[0373] Example 4: Synthesis of exemplary ionizable lipid compounds 4.1: Synthesis of compound 2213 [ka]

[0374] Step 1: To a mixture of 8-(tert-butoxycarbonylamino)octanoic acid (25 g, 96.40 mmol, 1.2 equiv.) in DCM (1000 mL) was added DMAP (4.91 g, 40.17 mmol, 0.5 equiv.), heptadecan-9-ol (20.60 g, 80.33 mmol, 1 equiv.), and EDCI (46.20 g, 241.00 mmol, 3 equiv.). The mixture was stirred at 25 °C under a N atmosphere for 12 h. LCMS showed 48% of the desired product. The reaction mixture was diluted with EtOAc (200 mL × 3) and washed with 200 mL of H2O. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 1 / 0) to obtain the compound 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (24 g, crude) as a yellow oil.

[0375] Step 2: To a solution of 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (12 g, 24.11 mmol, 1 equiv.) in DCM (100 mL) was added TFA (46.20 g, 405.18 mmol, 30 mL, 16.81 equiv.). The mixture was stirred at 25 °C for 5 h. The reaction mixture was adjusted to pH 7 with saturated aqueous NaHCO3, extracted with EtOAc (200 mL × 3), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 0 / 1 to ethyl acetate / MeOH = 3 / 1) to give compound 1-octylnonyl 8-aminooctanoate (15 g, 37.72 mmol, 78.23% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.64(brs,2H),4.84-4.88(m,1H),2.84(t,J=7.6Hz,2H),2. 28(t,J=7.6Hz,2H),1.50-1.61(m,8H),1.26-1.33(m,30H),0.88(t,J=6.8Hz,6H). [ka]

[0376] Step 3: To a solution of 8-bromooctanoic acid (10.21 g, 45.75 mmol, 1.1 equiv) and nonan-1-ol (6 g, 41.59 mmol, 1 equiv) in DCM (100 mL) was added DMAP (1.02 g, 8.32 mmol, 0.2 equiv) and EDCI (9.57 g, 49.91 mmol, 1.2 equiv) and stirred at 20 °C for 8 h. The mixture was added to HO (50 mL) and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine (50 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give nonyl 8-bromooctanoate (10 g, 27.19 mmol, yield 65.38%, purity 95%) as a colorless oil.

[0377] Step 4: To a solution of 1-octylnonyl 8-aminooctanoate (5 g, 12.57 mmol, 1.1 equiv.) in DMF (100 mL) was added KI (2.28 g, 13.74 mmol, 1.2 equiv.) and K2CO3 (4.75 g, 34.35 mmol, 3 equiv.), followed by addition of a solution of nonyl 8-bromooctanoate (4 g, 11.45 mmol, 1 equiv.) in DMF (20 mL), followed by stirring at 80 °C for 12 h. The mixture was filtered, and the filtrate was added to HO (50 mL) and extracted with EtOAc (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to obtain the compound 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (4 g, 5.40 mmol, yield 47.20%, purity 90%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.85-4.90(m,1H),4.06(t,J=7.2Hz,2H),2.59(t,J=6.8Hz,3H),2.26 -2.31(m,4H),1.60-1.70(m,6H),1.40-1.55(m,8H),1.20-1.35(m,49H),0.86-0.91(m,9H).

[0378] Step 5: To a solution of nonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (250 mg, 375.31 μmol, 1 equiv.) in DMF (5 mL) was added KCO (259.36 mg, 1.88 mmol, 5 equiv.) and KI (62.30 mg, 375.31 μmol, 1 equiv.), followed by the addition of tert-butyl N-(2-bromoethyl)carbamate (336.42 mg, 1.50 mmol, 4 equiv.). The mixture was stirred at 80 °C for 12 h. The mixture was filtered, and the filtrate was added to HO (5 mL) and extracted with EtOAc (5 mL × 3). The organic layer was washed with brine (5 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) to obtain the compound 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (200 mg, 247.13 μmol, yield 65.85%) as a colorless oil.

[0379] Step 6: A solution of nonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (160 mg, 197.70 μmol, 1 equiv.) in TFA (3.08 g, 27.01 mmol, 2 mL, 136.63 equiv.) and DCM (4 mL) was stirred at 20 °C for 2 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, ethyl acetate:methanol = 1 / 0 to 5 / 1) to give the compound nonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (45 mg, 63.45 μmol, 32.10% yield, purity) as a colorless oil. 1H NMR(400MHz,CDCl3),4.85-4.90(m,1H),4.06(t,J=6.4Hz,2H),2.93(t,J=5.6Hz,2H),2.69(t,J=6.0Hz,2H),2.59(t ,J=7.6Hz,4H),2.26-2.32(m,4H),1.60-1.70(m,6H),1.40-1.55(m,8H),1.20-1.35(m,48H),0.89(t,J=6.4Hz,9H). LCMS: (M+H + ):709.4@10.079 minutes.

[0380] Step 7: To a solution of 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (100 mg, 141.01 μmol, 2.1 equiv.) and TEA (40.00 mg, 395.30 μmol, 55.02 μL, 5.89 equiv.) in DCM (5 mL) was added butanedioyl dichloride (10.41 mg, 67.15 μmol, 7.38 μL, 1 equiv.) under N at 0° C., and the mixture was stirred at 20° C. for 1 h. The mixture was added to saturated NaHCO (20 mL) and extracted with EtOAc (10 mL × 3). The organic layer was washed with brine (10 mL × 2), dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1) and further purified by preparative TLC (SiO, ethyl acetate / MeOH = 5:1 with 3% NH.H.sub.2O) to give compound 8-[2-[[4-[2-[(8-nonoxy-8-oxo-octyl)-[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]nonyl octanoate (26 mg, 17.33 μmol, 25.81% yield) as a yellow oil. 1H NMR(400MHz,CDCl3),6.27(brs,2H),4.85-4.90(m,2H),4.06(t,J=6.4Hz,4H),3.26(s,4H),2.51(s,8H),2.26-2.40( m,16H),1.58-1.64(m,12H),1.40-1.55(m,8H),1.35-1.40(m,8H),1.20-1.35(m,96H),0.86-0.91(m,18H).LCMS:(M+H + ):1499.7@12.146 minutes.

[0381] 4.2: Synthesis of compound 2218 [ka]

[0382] Step 1: To a mixture of 8-(tert-butoxycarbonylamino)octanoic acid (25 g, 96.40 mmol, 1.2 equiv.) in DCM (1000 mL) was added DMAP (4.91 g, 40.17 mmol, 0.5 equiv.), heptadecan-9-ol (20.60 g, 80.33 mmol, 1 equiv.), and EDCI (46.20 g, 241.00 mmol, 3 equiv.). The mixture was stirred at 25 °C under a N atmosphere for 12 h. LCMS showed 48% of the desired product. The reaction mixture was diluted with 600 mL (200 mL × 3) of EtOAc and washed with 200 mL of HO. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 1 / 0) to give the compound 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (24 g, crude) as a yellow oil. The crude product was used in the next step without detection by H NMR.

[0383] Step 2: To a solution of 1-octylnonyl 8-(tert-butoxycarbonylamino)octanoate (12 g, 24.11 mmol, 1 equiv.) in DCM (100 mL) was added TFA (46.20 g, 405.18 mmol, 30 mL, 16.81 equiv.). The mixture was stirred at 25 °C for 5 h. The reaction mixture was adjusted to pH 7 with saturated aqueous NaHCO3, extracted with 600 mL (200 mL × 3) of EtOAc, dried over Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 0 / 1 to ethyl acetate / MeOH = 3 / 1) to give compound 1-octylnonyl 8-aminooctanoate (15 g, 37.72 mmol, 78.23% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),5.64(brs,2H),4.84-4.88(m,1H),2.84(t,J=7.6Hz,2H),2. 28(t,J=7.6Hz,2H),1.50-1.61(m,8H),1.26-1.33(m,30H),0.88(t,J=6.8Hz,6H). LCMS: (M+H + ):398.6@1.010 minutes.

[0384] Step 3: To a mixture of 6-bromohexanoic acid (22.64 g, 116.07 mmol, 1 equiv.) in DCM (1 mL) was added DMAP (2.84 g, 23.21 mmol, 0.2 equiv.), undecan-1-ol (20 g, 116.07 mmol, 1 equiv.), and EDCI (22.25 g, 116.07 mmol, 1 equiv.). The mixture was stirred at 25 °C under a N atmosphere for 12 h. The reaction mixture was diluted with 200 mL of HO and extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 40 / 1) to obtain the compound undecyl 6-bromohexanoate (36 g, 103.05 mmol, yield 88.78%) as a yellow oil. 1H NMR(400MHz,CDCl3),4.07(t,J=6.8Hz,2H),3.41(t,J=6.8Hz,2H),2.33(t,J=7.2Hz,2H),1.87 -1.91(m,2H),1.63-1.68(m,4H),1.48-1.50(m,2H),1.27-1.32(m,16H),0.89(t,J=6.4Hz,3H).

[0385] Step 4: To a solution of 1-octylnonyl 8-aminooctanoate (1 g, 2.51 mmol, 1 equiv.) and undecyl 6-bromohexanoate (878.47 mg, 2.51 mmol, 1 equiv.) in DMF (20 mL) was added K2CO3 (1.04 g, 7.54 mmol, 3 equiv.). The mixture was stirred at 80 °C for 5 h. LCMS showed 56% of the desired product. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, ethyl acetate:MeOH = 1 / 0 to 10 / 1) to obtain the compound 1-octylnonyl 8-[(6-oxo-6-undecoxy-hexyl)amino]octanoate (0.5 g, 750.63 μmol, yield 29.85%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),4.06(t,J=6.8Hz,2H),2.59-2.60(m,4H),2.28-2.31(m, 4H),1.60-1.65(m,6H),1.50-1.52(m,8H),1.27-1.36(m,48H),0.89(t,J=6.4Hz,9H).LCMS:(M+H + ):666.8@1.168 minutes.

[0386] Step 5: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (1 g, 1.50 mmol, 1 equiv.) in ACN (3 mL) was added DIEA (388.04 mg, 3.00 mmol, 522.97 μL, 2 equiv.) and 2-iodoethanol (387.24 mg, 2.25 mmol, 176.02 μL, 1.5 equiv.). The mixture was stirred at 80 °C for 8 h. TLC showed that 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate remained and one new major spot had formed. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate:MeOH=10 / 1 to 1 / 1) to obtain the compound 1-octylnonyl 8-[2-hydroxyethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (0.6 g, 844.88 μmol, yield 56.28%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,1H),4.06(t,J=6.8Hz,2H),3.55(brs,2H),2.27-2.62(m,10 H),1.62-1.63(m,6H),1.50-1.52(m,8H),1.27-1.31(m,48H),0.89(t,J=6.8Hz,9H).LCMS:(M+H + ):710.9@1.187 minutes.

[0387] Step 6: A mixture of bis(trichloromethyl)carbonate (16.71 mg, 56.33 μmol, 0.2 equiv.) in DCM (2 mL) was added over 1 h at 0 °C to a mixture of TEA (28.50 mg, 281.63 μmol, 39.20 μL, 1 equiv.) and 1-octylnonyl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (200 mg, 281.63 μmol, 1 equiv.) in DCM (5 mL), and the mixture was stirred under N2 at 0 °C for 0.5 h. TLC showed that 1-octylnonyl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate remained and two new spots had formed. The reaction mixture was quenched by adding 10 mL of HO at 0 °C and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give the compound 1-octylnonyl 8-[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethoxycarbonyloxy]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (20 mg, 13.83 μmol, 4.91% yield, 100% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.86-4.89(m,2H),4.15(t,J=6.4Hz,4H),4.06(t,J=6.8Hz,4H),2.71(t,J=6.4Hz,4H),2.42-2.47(m,8H) ),2.26-2.32(m,8H),1.60-1.65(m,12H),1.50-1.52(m,6H),1.39-1.46(m,10H),1.27-1.31(m,96H),0.89(t,J=6.4Hz,18H). LCMS: (1 / 2M+H + ):723.5@2.752 minutes.

[0388] 4.3: Synthesis of Compound 2220 [ka]

[0389] Step 1: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (7 g, 10.51 mmol, 1 equiv.) and tert-butyl N-(2-oxoethyl)carbamate (3.35 g, 21.02 mmol, 2 equiv.) in DCM (200 mL) was added sodium triacetoxyboranide (6.68 g, 31.53 mmol, 3 equiv.) at 15 °C. The mixture was degassed and purged with N three times, then stirred under N atmosphere at 15 °C for 8 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was diluted with 100 mL of HO and extracted with 600 mL (300 mL × 2) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1, 5% NH3·H2O) to obtain the compound 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (5.0 g, 6.18 mmol, 58.79% yield) as a colorless oil. LCMS: (M+H + ):809.7@1.083 minutes.

[0390] Step 2: To a solution of 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (5 g, 6.18 mmol, 1 equiv.) in DCM (45 mL) was added TFA (16.50 g, 144.71 mmol, 10.71 mL, 23.42 equiv.) dropwise at 15 °C. The mixture was stirred at 15 °C for 10 h under a N atmosphere. The reaction mixture was adjusted to pH = 7.0 with 80 mL of saturated aqueous NaHCO and extracted with 450 mL (150 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1, 5% NH3·H2O) to obtain the compound 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (3.1 g, 4.37 mmol, yield 70.75%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.06(t,J=6.8Hz,2H),2.71(t,J=6.4Hz,2H),2.37-2.46(m,6H), 2.29(q,J=7.6Hz,4H),1.59-1.68(m,6H),1.38-1.52(m,10H),1.27-1.31(m,48H),0.88(t,J=6.8Hz,9H).

[0391] Step 3: To a suspension of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (100 mg, 141.01 μmol, 2 equiv.), TEA (14.27 mg, 141.01 μmol, 19.63 μL, 2 equiv.), DMAP (861.34 μg, 7.05 μmol, 0.1 equiv.), and 4A molecular sieves (200 mg) in DCM (3 mL) was added a solution of propanedioyl dichloride (9.94 mg, 70.50 μmol, 6.85 μL, 1 equiv.) in DCM (0.5 mL) dropwise over 40 min at 0 °C. The mixture was stirred at 15 °C under a N atmosphere for 4 h. The reaction mixture was filtered, diluted with 10 mL of H2O, and then extracted with 100 mL (50 mL x 2) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 1 / 1, 5% NH3·H2O) to give the crude product, which was then purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 1:1) followed by preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1) to give the compound 1-octylnonyl 8-[2-[[3-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-3-oxo-propanoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (27 mg, 17.98 μmol, 25.51% yield, 99% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),7.14(brs,2H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),3.30(q,J=5.6Hz,4H),3.14(s,2H),2.53(t,J=6.0Hz,4H),2.40(q,J=6 .0Hz,8H),2.30(q,J=7.6Hz,8H),1.60-1.65(m,12H),1.48-1.55(m,8H),1 .39-1.46(m,8H),1.26-1.35(m,96H),0.88(t,J=6.8Hz,18H).LCMS:(M / 2+H + ):743.7@3.869 minutes.

[0392] 4.4: Synthesis of Compound 2221 [ka]

[0393] To a suspension of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (100 mg, 141.01 μmol, 2 equiv.), TEA (21.40 mg, 211.51 μmol, 29.44 μL, 3 equiv.), DMAP (861.34 μg, 7.05 μmol, 0.1 equiv.), and 4A molecular sieves (100 mg) in DCM (4 mL) was added butanedioyl dichloride (10.93 mg, 70.50 μmol, 7.75 μL, 1 equiv.) in DCM (0.5 mL) dropwise over 40 min at 15 °C. The mixture was stirred at 15 °C for 4 h under a N atmosphere. The reaction mixture was filtered, diluted with 15 mL of H2O, and then extracted with 150 mL (50 mL x 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1, 5% NH3·H2O) to give the crude product, which was then purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 1:1) followed by preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1) to give the compound 1-octylnonyl 8-[2-[[4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (30 mg, 19.99 μmol, yield 28.36%, purity 100%) as a pale yellow oil. 1H NMR(400MHz, CDCl3),6.33(brs,2H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),3.27(q,J=5.6Hz,4H),2.51(s,8H),2.40(q,J=6.0Hz ,8H),2.30(q,J=7.6Hz,8H),1.59-1.69(m,12H),1.48-1.53(m,8H),1.38-1.45(m,8H),1.26-1.35(m,96H),0.88(t,J=6.8Hz,18H). LCMS: (M+H + ):750.5@3.601 points.

[0394] 4.5: Synthesis of compound 2246

change

[0395] A mixture of bis(trichloromethyl)carbonate (16.74 mg, 56.40 μmol, 0.2 equiv.) in DCM (2 mL) was added to a mixture of 4A molecular sieves (50 mg), TEA (28.54 mg, 282.02 μmol, 39.25 μL, 1 equiv.), and 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (0.2 g, 282.02 μmol, 1 equiv.) in DCM (5 mL) at 0 °C over 1 h. The mixture was then stirred under N2 atmosphere at 0 °C for 3 h. TLC showed complete consumption of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate and the formation of one new major spot. The reaction mixture was quenched by adding 10 mL of HO at 0 °C. The mixture was extracted with 30 mL (10 mL × 3) of EtOAc, and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified twice by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 1 to 0 / 1, with 10% NH.HO) to give the compound 1-octylnonyl 8-[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylcarbamoylamino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (50 mg, 34.62 μmol, 12.28% yield, 100% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.09(brs,1H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),3.22(s,4H),2.52(s,4H),2.40-2.41(m,8H) ,2.26-2.31(m,8H),1.62-1.63(m,12H),1.51-1.52(m,8H),1.41-1.44(m,8H),1.27-1.39(m,96H),0.89(t,J=5.2Hz,18H). LCMS: (1 / 2M+H + ):722.6@2.681 minutes.

[0396] 4.6: Synthesis of Compound 2248 [ka]

[0397] Step 1: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxy-hexyl)amino]octanoate (1 g, 1.50 mmol, 1 equiv.) in ACN (15.0 mL) was added DIEA (388.05 mg, 3.00 mmol, 522.98 μL, 2 equiv.) and 2-iodoethanol (387.24 mg, 2.25 mmol, 176.02 μL, 1.5 equiv.) dropwise at 15 °C. The mixture was degassed and purged with N 3 times, then stirred at 80 °C under N 3 for 12 h. The reaction mixture was diluted with 20 mL of HO and extracted with 100 mL (50 mL × 2) of EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 1 / 1, 5% NH3·H2O) to obtain the compound 1-octylnonyl 8-[2-hydroxyethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (800 mg, 1.13 mmol, yield 75.04%) as a colorless oil.

[0398] Step 2: To a suspension of triphosgene (300 mg, 1.01 mmol, 8.97 e-1 equiv.) and 4A molecular sieves (50 mg) in DCM (10 mL), 1-octylnonyl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (800 mg, 1.13 mmol, 1 equiv.) and TEA (113.99 mg, 1.13 mmol, 156.80 μL, 1 equiv.) in DCM (10 mL) were added dropwise under a N atmosphere at 0 °C for 3.5 h. The reaction mixture was quenched by adding 20 mL of HO at 0 °C and extracted with 120 mL (40 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 5 / 1, 5% NH3·H2O) to obtain the compound 1-octylnonyl 8-[2-chloroethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (560 mg, 768.59 μmol, yield 68.23%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.06(t,J=6.8Hz,2H),3.48(t,J=7.6Hz,2H),2.76(t,J=6.8Hz,2H),2.45(q, J=5.6Hz,4H),2.30(q,J=8.0Hz,4H),1.56-1.66(m,6H),1.40-1.52(m,8H),1.27-1.35(m,48H),0.88(t,J=6.8Hz,9H).

[0399] Step 3: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (1 g, 1.50 mmol, 1 equiv.) and tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (933.59 mg, 3.75 mmol, 2.5 equiv.) in DCM (15 mL) was added KI (49.84 mg, 300.25 μmol, 0.2 equiv.) and KCO (414.96 mg, 3.00 mmol, 2 equiv.) at 20 °C. The mixture was degassed and purged with N three times, then stirred under N at 80 °C for 8 h. The reaction mixture was diluted with 30 mL of HO and extracted with 200 mL (2 x 100 mL) of EtOAc. The combined organic layers were washed with 80 mL (40 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 3 / 1, 5% NH3 HO) to give the compound tert-butyl 4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]piperazine-1-carboxylate (300 mg, 341.53 μmol, 22.75% yield) as a colorless oil.

[0400] Step 4: To a solution of tert-butyl 4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]piperazine-1-carboxylate (300 mg, 341.53 μmol, 1 equiv.) in DCM (10 mL) was added TFA (7.70 g, 67.53 mmol, 5 mL, 197.73 equiv.) dropwise at 20 °C. The mixture was degassed and purged with N 3 times, then stirred under N 3 atmosphere at 20 °C for 4 h. The reaction mixture was adjusted to pH = 7.0 with saturated aqueous NaHCO 3 and extracted with 120 mL (40 mL × 3) of EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1, 5% NH3·H2O) to give the compound 1-octylnonyl 8-[(6-oxo-6-undecoxy-hexyl)-(2-piperazin-1-ylethyl)amino]octanoate (140 mg, 179.88 μmol, 52.67% yield) as a colorless oil. LCMS: (M / 2+H + ):778.7@2.702 minutes.

[0401] Step 5: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)-(2-piperazin-1-ylethyl)amino]octanoate (120 mg, 154.19 μmol, 1 equiv.) and 1-octylnonyl 8-[2-chloroethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (280.85 mg, 385.46 μmol, 2.5 equiv.) in DMF (6 mL) was added KI (12.80 mg, 77.09 μmol, 0.5 equiv.) at 20 °C. The mixture was degassed and purged with N 3 times, then stirred under N 3 at 50 °C for 4 h. The reaction mixture was diluted with 20 mL of HO and extracted with 120 mL (60 mL × 2) of EtOAc. The combined organic layers were washed with 80 mL (40 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 8 / 1 to 3 / 1, 5% NH3 HO) to give the crude product. The crude product was purified by preparative TLC (SiO, petroleum ether / ethyl acetate = 0:1, 3% NH H O) to give the compound 1-octylnonyl 8-[2-[4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]piperazin-1-yl]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (25 mg, 16.97 μmol, 11.01% yield, 99.8% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),2.26-2.46(m,32H),1.60-1.66(m,1 2H),1.50-1.52(m,8H),1.39-1.45(m,8H),1.27-1.35(m,96H),0.89(t,J=6.8Hz,18H).LCMS:(M / 2+H + ):735.8@11.695 minutes.

[0402] 4.7: Synthesis of Compound 2252 [ka]

[0403] To a suspension of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (500 mg, 705.04 μmol, 2 equiv.), DMAP (4.31 mg, 35.25 μmol, 0.1 equiv.), TEA (71.34 mg, 705.04 μmol, 98.13 μL, 2 equiv.), and 4A molecular sieves (500 mg) in DCM (15 mL) was added (E)-but-2-enedioyl dichloride (53.92 mg, 352.52 μmol, 38.24 μL, 1 equiv.) in DCM (5 mL) dropwise under a N atmosphere at 15 °C for 0.5 h, followed by stirring at 15 °C for 8 h. The reaction mixture was filtered, and the filtrate was diluted with 20 mL of H2O and then extracted with 100 mL (50 mL x 2) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 1 / 1, 5% NH3·H2O) and preparative TLC (SiO2, petroleum ether / ethyl acetate = 0:1) to give the compound 1-octylnonyl 8-[2-[[(E)-4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-4-oxo-but-2-enoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (250 mg, 166.51 μmol, 47.23% yield, 100% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),6.87(s,2H),6.50(brs,2H),4.84-4.90(m,2H),4.07(t,J=6.8Hz,4H),3.39-3.36(m,4H),2.58(t,J=6.0Hz,4H ),2.41(m,8H),2.30(m,8H),1.60-1.67(m,12H),1.48-1.55(m,8H),1.38-1.45(m,8H),1.23-1.35(m,96H),0.88(t,J=6.8Hz,18H). LCMS: (M / 2+H + ):749.8@3.831 minutes.

[0404] 4.8: Synthesis of Compound 2253 [ka]

[0405] Step 1: A solution of 3-bromo-2-(bromomethyl)prop-1-ene (200 mg, 935.02 μmol, 1 equiv.) and KI (620.86 mg, 3.74 mmol, 4 equiv.) in acetone (10 mL) was stirred at 80° C. for 2 h. TLC showed complete consumption of 3-bromo-2-(bromomethyl)prop-1-ene and the formation of one new major spot. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give the compound 3-iodo-2-(iodomethyl)prop-1-ene (0.2 g, crude) as a black oil. 1 H NMR (400MHz, CDCl3), 5.36 (s, 2H), 4.14 (s, 4H).

[0406] Step 2: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (0.5 g, 750.63 μmol, 3 equiv.) and K2CO3 (103.74 mg, 750.63 μmol, 3 equiv.) in DMF (3 mL) was added 3-iodo-2-(iodomethyl)prop-1-ene (77.04 mg, 250.21 μmol, 1 equiv.). The resulting mixture was stirred at 15 °C for 5 h. TLC showed that 3-iodo-2-(iodomethyl)prop-1-ene was completely consumed and one new spot was formed. The combined organic phase was diluted with 20 mL of EtOAc, washed with 60 mL (20 mL × 3) of water and 40 mL (20 mL × 2) of brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to give the compound 1-octylnonyl 8-[2[[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]methyl]allyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (0.2 g, 144.48 μmol, 57.74% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),5.01(s,2H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),2.92(s,4H),2.27-2.34(m, 16H),1.61-1.64(m,16H),1.38-1.43(m,12H),1.27-1.40(m,96H),0.89(t,J=6.4Hz,18H).LCMS:(1 / 2M+H + ):692.4@3.280 minutes.

[0407] 4.9: Synthesis of Compound 2271 [ka]

[0408] Step 1: To a solution of diethyl 2-methylpropanedioate (10 g, 57.41 mmol, 9.80 mL, 1 equiv.) in THF (1000 mL) in a three-neck flask was slowly added NaH (2.30 g, 57.41 mmol, 60% purity, 1 equiv.) at 0 °C and stirred at 0 °C for 1 h. 1-Bromoheptane (10.28 g, 57.41 mmol, 9.02 mL, 1 equiv.) was added and stirred at 20 °C for 0.5 h and at 70 °C for 6.5 h. The reaction mixture was quenched by adding 2000 mL of HO at 0 °C. The mixture was extracted with 3000 mL (1000 mL × 3) of EtOAc, and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 50 / 1) to obtain the compound diethyl 2-heptyl-2-methyl-propanedioate (45 g, 165.21 mmol, yield 71.95%, 3 batches) as a colorless oil. 1 H NMR(400MHz, CDCl3),4.15-4.21(m,4H),1.85-1.87(m,2H),1.40(s,3H),1.23-1.28(m,16H),0.88(t,J=6.8Hz,3H).

[0409] Step 2: To a solution of diethyl 2-heptyl-2-methyl-propanedioate (10 g, 36.71 mmol, 1 equiv.) in EtOH (100 mL) and HO (100 mL), KOH (6.18 g, 110.14 mmol, 3 equiv.) was added. The mixture was stirred at 90 °C for 10 h. The reaction mixture was concentrated under reduced pressure to remove most of the EtOH and washed with 120 mL (40 mL × 3) of EtOAc. The aqueous phase was then adjusted to pH = 2 with 1 M aqueous HCl and extracted with 120 mL (40 mL × 3) of EtOAc. The combined organic layers were dried over Na SO , filtered, and concentrated under reduced pressure to obtain the compound 2-heptyl-2-methyl-propanedioic acid (45 g, 208.07 mmol, 94.46% yield) as a white solid without further purification.

[0410] Step 3: A solution of 2-heptyl-2-methyl-propanedioic acid (10 g, 46.24 mmol, 1 equiv.) in 1,2-dichlorobenzene (104.80 g, 712.92 mmol, 80.00 mL, 15.42 equiv.) was stirred at 180 °C for 2 h. The reaction mixture was diluted with 200 mL of HO and extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 3 / 1) to give the compound 2-methylnonanoic acid (35 g, 203.18 mmol, 87.88% yield) as a white solid.

[0411] Step 4: To a solution of LiAlH (3.08 g, 81.27 mmol, 2 equiv.) in THF (500 mL) was added 2-methylnonanoic acid (7 g, 40.64 mmol, 1 equiv.). The mixture was stirred at 0 °C for 3 h. The reaction mixture was quenched by adding 200 mL of HO at 0 °C. The mixture was extracted with 300 mL (100 mL × 3) of EtOAc, and the combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to give the compound 2-methylnonan-1-ol (15 g, 94.77 mmol, 46.64% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),3.50-3.54(m,1H),3.42-3.45(m,1H),1.61-1.64(m,1H),1.20-1.39(m,11H),1.05-1.15(m,1H),0.87-0.93(m,6H).

[0412] Step 5: To a mixture of 8-bromooctanoic acid (9.87 g, 44.23 mmol, 1 equiv.) in DCM (1000 mL) was added DMAP (1.08 g, 8.85 mmol, 0.2 equiv.), 2-methylnonan-1-ol (7 g, 44.23 mmol, 1 equiv.), and EDCI (8.48 g, 44.23 mmol, 1 equiv.) at 20 °C. The mixture was stirred at 20 °C for 12 h under a N atmosphere. The reaction mixture was diluted with 600 mL (200 mL × 3) of EtOAc and washed with 200 mL of HO and 200 mL (100 mL × 2) of 10% aqueous citric acid. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 1 / 0) to obtain the compound 2-methylnonyl 8-bromooctanoate (12 g, 33.02 mmol, yield 74.67%) as a yellow oil.

[0413] Step 6: To a solution of 1-octylnonyl 8-aminooctanoate (4.38 g, 11.01 mmol, 1 equiv.) and 2-methylnonyl 8-bromooctanoate (4 g, 11.01 mmol, 1 equiv.) in ACN (100 mL) was added K2CO3 (1.52 g, 11.01 mmol, 1 equiv.). The mixture was stirred at 80 °C for 8 h. The reaction mixture was diluted with 200 mL of HO and extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3.H2O=10 / 1 / 1 to 1 / 1 / 0.5) to obtain the compound 2-methylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3.5 g, 5.15 mmol, yield 23.37%) as a yellow oil. 1H NMR(400MHz,CDCl3),4.85-4.89(m,1H),3.93-3.98(m,1H),3.83-3.88(m,1H),2.59(t,J=7.2Hz,4H) ,2.26-2.33(m,4H),1.60-1.80(m,1H),1.40-1.60(m,10H),1.20-1.40(m,50H),0.86-0.93(m,12H).

[0414] Step 7: To a solution of 2-methylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3 g, 4.41 mmol, 1 equiv.) and tert-butyl N-(2-oxoethyl)carbamate (1.05 g, 6.62 mmol, 1.5 equiv.) in DCM (50 mL) was added NaBH(OAc) (1.87 g, 8.82 mmol, 2 equiv.). The mixture was stirred at 20 °C for 5 h. The combined organic phase was diluted with 20 mL of EtOAc, washed with 60 mL (20 mL × 3) of water and 40 mL (20 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to obtain the compound 2-methylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (2 g, 2.43 mmol, yield 55.07%) as a white solid. 1 H NMR(400MHz,CDCl3),4.85-4.89(m,1H),3.93-3.98(m,1H),3.83-3.87(m,1H),3.14(br s,2H),2.28-2.40(m,10H),1.60-1.80(m,1H),1.26-1.55(m,69H),0.86-0.93(m,12H).

[0415] Step 8: A solution of 2-methylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (2 g, 2.43 mmol, 1 equiv.) in HCl / EtOAc (4 M, 9.37 mL, 15.42 equiv.) was stirred at 20 °C for 5 h. The reaction mixture was adjusted to pH = 7 with saturated aqueous NaHCO and extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1) to obtain the compound 2-methylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.5 g, 2.07 mmol, yield 85.38%, purity 100%) as a white solid without further purification. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),3.94-3.98(m,1H),3.83-3.87(m,1H) ,2.73(t,J=6.0Hz,2H),2.46(t,J=6.0Hz,2H),2.40(t,J=7.2Hz,4H),2.27-2.3 6(m,4H),1.72-1.82(m,1H),1.60-1.65(m,4H),1.50-1.54(m,4H),1.39-1.45 (m,4H),1.23-1.34(m,46H),1.12-1.27(m,2H),0.87-0.93(m,12H).LCMS:(M+H + ):723.4@10.618 minutes.

[0416] Step 9: To a solution of 2-methylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (0.3 g, 414.82 μmol, 1 equiv.), TEA (41.98 mg, 414.82 μmol, 57.74 μL, 1 equiv.), and DMAP (10.14 mg, 82.96 μmol, 0.2 equiv.) in DCM (5 mL) was added butanedioyl dichloride (32.14 mg, 207.41 μmol, 22.80 μL, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 3 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3.H2O = 10 / 1 / 0.1 to 3 / 1 / 0.1), preparative TLC (SiO2, petroleum ether / ethyl acetate / NH3.H2O = 1:2:0.1) and column chromatography (SiO2, petroleum ether / ethyl acetate / NH3.H2O = 3 / 1 / 0.1 to 1 / 1 / 0.1). The combined organic layers were diluted with 10 mL of PE and washed with 20 mL of ACN, and the PE layer was concentrated under reduced pressure to give the compound 2-methylnonyl 8-[2-[[4-[2-[[8-(2-methylnonoxy)-8-oxo-octyl]-[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (50 mg, 32.71 μmol, 8% yield, 99% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),6.28(brs,2H),4.83-4.90(m,2H),3.93-3.98(m,2H),3.82-3.87(m,2H),3.26-3.27(brs,4H),2.26-2.51(m,24H),1.73- 1.81(m,2H),1.61-1.64(m,8H),1.48-1.51(m,8H),1.35-1.43(m,6H),1 .20-1.35(m,96H),1.10-1.18(m,2H),0.86-0.93(m,24H).LCMS:(M / 2+H +):764.6@13.275 minutes.

[0417] 4.10: Synthesis of Compound 2272 [ka]

[0418] Step 1: To a mixture of decanoic acid (17.66 g, 102.51 mmol, 19.78 mL, 1 equiv) in DCM (500 mL) was added DMAP (2.50 g, 20.50 mmol, 0.2 equiv), 7-bromoheptan-1-ol (20 g, 102.51 mmol, 1 equiv), and EDCI (19.65 g, 102.51 mmol, 1 equiv). The mixture was stirred at 20 °C under a N atmosphere for 8 h. The reaction mixture was diluted with 200 mL of HO and extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 40 / 1) to obtain the compound 7-bromoheptyl decanoate (30 g, 85.87 mmol, yield 83.77%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.07(t,J=6.8Hz,2H),3.41(t,J=6.8Hz,2H),2.31(t,J=7.2Hz,2H),1.85-1.87(m, 2H),1.62-1.63(m,4H),1.45-1.48(m,2H),1.37-1.42(m,4H),1.27-1.31(m,12H),0.89(t,J=6.8Hz,3H).

[0419] Step 2: To a solution of 1-octylnonyl 8-aminooctanoate (6 g, 15.09 mmol, 1 equiv.) and 7-bromoheptyl decanoate (5.27 g, 15.09 mmol, 1 equiv.) in ACN (50 mL) was added K2CO3 (8.34 g, 60.35 mmol, 4 equiv.). The mixture was stirred at 70 °C for 5 h. The reaction mixture was diluted with 200 mL of HO and extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, ethyl acetate:MeOH = 1 / 0 to 10 / 1) to obtain the compound 7-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (3 g, 4.50 mmol, yield 29.85%) as a yellow oil.

[0420] Step 3: To a solution of 7-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (3 g, 4.50 mmol, 1 equiv.) and tert-butyl N-(2-oxoethyl)carbamate (1.43 g, 9.01 mmol, 2 equiv.) in DCM (50 mL) was added NaBH(OAc) (1.91 g, 9.01 mmol, 2 equiv.). The mixture was stirred at 20 °C for 8 h. The mixture was diluted with 60 mL of EtOAc, washed with 180 mL (60 mL × 3) of water and 40 mL (20 mL × 2) of brine, dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to obtain the compound 7-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (2 g, 2.47 mmol, yield 54.84%) as a yellow oil. 1H NMR(400MHz,CDCl3),5.06(brs,1H),4.86-4.89(m,1H),4.06(t,J=6.8Hz,2H),3.17(brs,2H),2.2 8-2.66(m,10H),1.63-1.66(m,6H),1.48-1.62(m,15H),1.26-1.42(m,50H),0.89(t,J=6.0Hz,9H).

[0421] Step 4: A solution of 7-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (2 g, 2.47 mmol, 1 equiv) in HCl / EtOAc (4 M, 617.82 μL, 1 equiv) was stirred at 20 °C for 5 h. The crude reaction mixture was adjusted to pH = 7 with saturated NaHCO and extracted with 120 mL (40 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH.H.O=5 / 1 / 0 to 2 / 1 / 0.1) and p-TLC (petroleum ether / ethyl acetate / NH.H.O=2 / 1 / 0.1) to give the compound 7-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (1 g, 1.41 mmol, 57.06% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.06(t,J=6.8Hz,2H),2.83(t,J=6.4Hz,2H),2.59(t,J=6.4Hz,2H),2.52(t ,J=5.2Hz,4H),2.27-2.32(m,4H),1.61-1.64(m,6H),1.48-1.52(m,6H),1.27-1.32(m,50H),0.89(t,J=6.4Hz,9H). LCMS: (M+H + ):709.4@10.026 minutes.

[0422] Step 5: To a solution of 7-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.2 g, 282.02 μmol, 1 equiv.), TEA (28.54 mg, 282.02 μmol, 39.25 μL, 1 equiv.), and DMAP (6.89 mg, 56.40 μmol, 0.2 equiv.) in DCM (5 mL) was added butanedioyl dichloride (21.85 mg, 141.01 μmol, 15.50 μL, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 3 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH.H2O=5 / 1 / 0.1 to 2 / 1 / 0.1) and preparative TLC (SiO, petroleum ether / ethyl acetate / NH3.H2O=3 / 1 / 0.1) to give the compound 7-[2-[[4-[2-[7-decanoyloxyheptyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]heptyl decanoate (0.15 g, 99.97 μmol, 35.45% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),6.28(brs,1H),4.84-4.90(m,2H),4.06(t,J=6.4Hz,4H),3.28(brs,4H),2.27-2.51(m,24H),1 .62-1.65(m,10H),1.52-1.61(m,8H),1.50-1.52(m,8H),1.27-1.32(m,98H),0.89(t,J=6.4Hz,18H).LCMS:(1 / 2M+H + ):750.5@12.157 minutes.

[0423] 4.11: Synthesis of Compound 2273 [ka]

[0424] To a suspension of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (160 mg, 225.61 μmol, 1 equiv.), TEA (45.66 mg, 451.23 μmol, 62.81 μL, 2 equiv.), 4A molecular sieves (20 mg), and DMAP (5.51 mg, 45.12 μmol, 0.2 equiv.) in DCM (8 mL) was added pentanediol dichloride (19.06 mg, 112.81 μmol, 14.44 μL, 0.5 equiv.) in DCM (0.5 mL) dropwise over 0.5 h at 20 °C. The mixture was degassed and purged with N 3 times, then stirred under N 3 atmosphere at 20 °C for 4 h. The reaction mixture was filtered, diluted with 15 mL of HO, and then extracted with 80 mL (40 mL × 2) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 5 / 1 to 2 / 1, 5% NH·HO) to give the compound 1-octylnonyl 8-[2-[[5-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxyhexyl)amino]ethylamino]-5-oxo-pentanoyl]amino]ethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (54 mg, 35.19 μmol, 31.20% yield, 98.7% purity) as a yellow oil. 1 H NMR(400MHz,CDCl3),6.29(brs,2H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),3.28(brs,4H),2.47-2.57(m,10H),2.18-2.43(m ,14H),1.82-1.98(m,2H),1.59-1.69(m,12H),1.48-1.53(m,8H),1.38-1.45(m,8H),1.26-1.35(m,96H),0.88(t,J=6.8Hz,18H). LCMS: (M / 2+H + ):1514.6@12.457 minutes.

[0425] 4.12: Synthesis of Compound 2274 [ka]

[0426] Step 1: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (1.5 g, 2.25 mmol, 1 equiv.) in DCM (15 mL) was added tert-butyl N-(3-oxopropyl)carbamate (585.07 mg, 3.38 mmol, 1.5 equiv.) at 20 °C. The mixture was degassed and purged with N three times and then stirred under N atmosphere at 20 °C for 0.5 h. Sodium triacetoxyboranide (954.53 mg, 4.50 mmol, 2 equiv.) was added to the mixture and then stirred under N atmosphere at 20 °C for 5 h. The reaction mixture was diluted with 20 mL of HO and extracted with 300 mL (150 mL × 2) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 8 / 1 to 0 / 1) to give the compound 1-octylnonyl 8-[3-(tert-butoxycarbonylamino)propyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (1.6 g, 1.94 mmol, 86.30% yield) as a colorless oil.

[0427] Step 2: To a solution of 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (5 g, 6.18 mmol, 1 equiv.) in DCM (45 mL) was added TFA (16.50 g, 144.71 mmol, 10.71 mL, 23.42 equiv.) dropwise at 15 °C. The mixture was degassed and purged with N 3 times, then stirred under N 3 atmosphere at 15 °C for 10 h. The reaction mixture was adjusted to pH = 7.0 with 80 mL of saturated aqueous NaHCO 3 and extracted with 450 mL (150 mL × 3) of EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 3 / 1 to 0 / 1, 5% NH3·H2O) to obtain the compound 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (3.1 g, 4.37 mmol, yield 70.75%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.84-4.90(m,1H),4.06(t,J=6.8Hz,2H),2.71(t,J=6.4Hz,2H),2.48(t,J=6.8Hz,2H),2.37-2.46(m,4H) ),2.29(q,J=7.6Hz,4H),1.58-1.68(m,8H),1.44-1.48(m,4H),1.50-1.52(m,4H),1.26-1.31(m,48H),0.88(t,J=6.8Hz,9H). LCMS: (M / 2+H + ):723.4@9.826 minutes.

[0428] Step 3: To a suspension of 1-octylnonyl 8-[3-aminopropyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (250 mg, 345.68 μmol, 2 equiv.), TEA (52.47 mg, 518.53 μmol, 72.17 μL, 3 equiv.), DMAP (2.11 mg, 17.28 μmol, 0.1 equiv.), and 4A molecular sieves (40 mg) in DCM (6 mL) was added butanedioyl dichloride (26.79 mg, 172.84 μmol, 19.00 μL, 1 equiv.) in DCM (0.5 mL) dropwise over 0.5 h at 20 °C. The mixture was degassed and purged with N 3 times, then stirred under N 3 atmosphere at 20 °C for 4 h. The reaction mixture was filtered, diluted with 20 mL of HO, and then extracted with 100 mL (50 mL × 2) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1, 5% NH HO) to give the crude product. The crude product was purified by preparative TLC (SiO2, ethyl acetate:methanol=5:1, 2% NH3·H2O) to give the compound 1-octylnonyl 8-[3-[[4-[3-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]propylamino]-4-oxo-butanoyl]amino]propyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (66 mg, 43.18 μmol, 73.33% yield, 99.6% purity) as a white solid. 1 H NMR(400MHz,CDCl3),7.22(brs,2H),4.84-4.90(m,2H),4.06(t,J=6.8Hz,4H),3.29(q,J=5.6Hz,4H),2.43-2.52(m,8H),2.39(q,J=6.0Hz,8H) ),2.29(q,J=7.6Hz,8H),1.60-1.66(m,16H),1.48-1.54(m,8H),1.39-1.46(m,8H),1.27-1.35(m,96H),0.89(t,J=6.8Hz,18H).LCMS:(M / 2+H + ):764.6@12.105 minutes.

[0429] 4.13: Synthesis of Compound 2274 [ka]

[0430] Step 1: To a solution of heptadecan-9-ol (10 g, 7.80 mmol, 1 equiv.) and 8-bromooctanoic acid (9.55 g, 8.58 mmol, 1.1 equiv.) in DCM (200 mL) was added EDCI (1.79 g, 9.36 mmol, 1.2 equiv.) and DMAP (2.38 g, 3.90 mmol, 0.5 equiv.). The mixture was stirred at 15 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 15 °C and then extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were washed with 400 mL (200 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 5 / 1) to obtain the compound 1-octylnonyl 8-bromooctanoate (17.75 g, 38.46 mmol, yield 98.63%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.85-4.89(m,1H),3.40(t,J=6.8,2H),2.29(t,J=7.6Hz,2H),1.84-1 .86(m,2H),1.58-1.69(m,2H),1.39-1.57(m,6H),1.25-1.35(m,28H),0.88(t,J=6.8,6H).

[0431] Step 2: To a solution of phenylmethanamine (552.75 mg, 1.03 mmol, 562.30 μL, 1 equiv) in DMF (50 mL) was added KCO (3.56 g, 5.16 mmol, 5 equiv) and KI (2.14 g, 2.58 mmol, 2.5 equiv), followed by the addition of 1-octylnonyl 8-bromooctanoate (5 g, 2.17 mmol, 2.1 equiv) in DMF (20 mL). The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched at 15 °C by the addition of 100 mL of HO and then extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were washed with 100 mL (50 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to obtain the compound 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (3.25 g, 3.74 mmol, yield 72.55%) as a colorless oil.

[0432] Step 3: A solution of 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (2.5 g, 575.74 μmol, 1 equiv.) and Pd / C (1.25 g, 575.74 μmol, 10% purity, 1.00 equiv.) in 50 mL of EtOAc was stirred under H2 (50 Psi) at 15 °C for 4 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 0) to give the compound 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1.5 g, 1.93 mmol, 66.95% yield) as a colorless oil.

[0433] Step 4: To a solution of 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (1 g, 1.28 mmol, 1 equiv.) in DCM (10 mL) was added tert-butyl N-(2-oxoethyl)carbamate (306.78 mg, 1.93 mmol, 1.5 equiv.). The mixture was stirred at 15 °C for 30 min, and then NaBH(OAc) (544.61 mg, 2.57 mmol, 2 equiv.) was added to the mixture at 15 °C and stirred for 8 h. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain the compound 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (360 mg, 390.67 μmol, yield 30.41%) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.99(s,1H),4.84-4.90(m,2H),3.14(brs,2H),2.49-2.55(m,2H),2.39(t,J=6.8Hz,3H ),2.28(t,J=7.6Hz,4H),1.56-1.70(m,4H),1.35-1.52(m,26H),1.20-1.32(m,63H),0.89(t,J=6.4Hz,12H).

[0434] Step 5: To a solution of 1-octylnonyl 8-[2-(tert-butoxycarbonylamino)ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (360 mg, 390.67 μmol, 1 equiv.) in DCM (10 mL), TFA (3.64 g, 35.92 mmol, 5 mL, 91.95 equiv.) was added, and the mixture was stirred at 15 °C for 3 h. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 0) to give the compound 1-octylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (71 mg, 86.44 μmol, 22.13% yield) as a yellow oil.

[0435] Step 6: To a suspension of 1-octylnonyl 8-[2-aminoethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (71 mg, 86.44 μmol, 2 equiv.), TEA (13.12 mg, 129.66 μmol, 18.05 μL, 3 equiv.), DMAP (528.00 μg, 4.32 μmol, 0.1 equiv.), and 4A MS (50 mg, 1.00 equiv.) in DCM (3 mL) was added butanedioyl dichloride (6.70 mg, 43.22 μmol, 4.75 μL, 1 equiv.) in DCM (1 mL) dropwise over 30 min at 15 °C. The mixture was stirred at 15 °C for 4 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative TLC (SiO, EA:MeOH = 10:1) to obtain the compound 1-octylnonyl 8-[2-[[4-[2-[bis[8-(1-octylnonoxy)-8-oxo-octyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (20 mg, 11.36 μmol, 26.29% yield, 99% purity) as a colorless oil. 1 H NMR(400MHz,CDCl3),6.25(s,2H),4.83-4.90(m,4H),3.27(s,4H),2.26-2.52(m,24H),1.59-1.64(m,1 0H),1.48-1.52(m,12H),1.38-1.42(m,6H),1.20-1.32(m,124H),0.89(t,J=6.4Hz,24H).LCMS:(M / 2+H + ):863.0@12.517 minutes.

[0436] 4.14: Synthesis of Compound 2278 [ka] [ka]

[0437] Step 1: To a mixture of dodecanoic acid (4.93 g, 24.60 mmol, 1 equiv) in DCM (1000 mL) was added DMAP (1.50 g, 12.30 mmol, 0.5 equiv), tert-butyl N-(5-hydroxypentyl)carbamate (5 g, 24.60 mmol, 5.00 mL, 1 equiv), and EDCI (9.43 g, 49.19 mmol, 2 equiv), degassed, and purged with N three times. The mixture was stirred under N at 20 °C for 8 h. The reaction mixture was diluted with 200 mL of EtOAc and washed with 200 mL of H2O. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 1 / 0) to obtain the compound 5-(tert-butoxycarbonylamino)pentyl dodecanoate (6 g, 15.56 mmol, yield 63.26%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.52(brs,1H),4.06(t,J=6.4Hz,2H),3.10-3.13(m,2H),2.29(t,J=7.6Hz,2H),1.64- 1.66(m,4H),1.51-1.61(m,2H),1.49(s,9H),1.44-1.45(m,2H),1.26-1.38(m,16H),0.88(t,J=6.4Hz,3H).

[0438] Step 2: 5-(tert-Butoxycarbonylamino)pentyl dodecanoate (6 g, 15.56 mmol, 1 equiv) in HCl / EtOAc (2 M, 60.00 mL, 15.42 equiv) was stirred for 5 hours at 20° C. The mixture was filtered, and the filter cake was concentrated under reduced pressure to give the compound 5-aminopentyl dodecanoate (4 g, 12.43 mmol, 79.85% yield, HCl) as a white solid without further purification. LCMS: (M+H + ):386.3@0.887 minutes.

[0439] Step 3: To a mixture of 7-bromoheptan-1-ol (3.43 g, 17.58 mmol, 1 equiv.) in DCM (1000 mL) was added DMAP (429.46 mg, 3.52 mmol, 0.2 equiv.), 2-octyldecanoic acid (5 g, 17.58 mmol, 1 equiv.), and EDCI (3.37 g, 17.58 mmol, 1 equiv.), followed by degassing and purging with N2 three times. The mixture was stirred under N2 atmosphere at 20 °C for 8 h. The reaction mixture was diluted with 600 mL (200 mL × 3) of EtOAc and washed with 200 mL of HO. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 1 / 0) to obtain the compound 7-bromoheptyl 2-octyldecanoate (5 g, 10.83 mmol, yield 61.63%) as a yellow oil.

[0440] Step 4: To a solution of 5-aminopentyl dodecanoate (2 g, 6.21 mmol, 1 equiv., HCl), 7-bromoheptyl 2-octyldecanoate (2.87 g, 6.21 mmol, 1 equiv.) in ACN (100 mL) was added K2CO3 (2.58 g, 18.64 mmol, 3 equiv.). The mixture was stirred at 80 °C for 5 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate / NH3.H2O=10 / 1 / 1 to 1 / 1 / 0.5) to obtain the compound 7-(5-dodecanoyloxypentylamino)heptyl 2-octyldecanoate (1.5 g, 2.25 mmol, yield 36.25%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.05-4.09(m,4H),2.59-2.63(m,4H),2.27-2.31(m,3H),1.60-1.70 (m,8H),1.45-1.60(m,4H),1.35-1.45(m,8H),1.20-1.35(m,44H),0.88(t,J=6.8Hz,9H).

[0441] Step 5: To a solution of 7-(5-dodecanoyloxypentylamino)heptyl 2-octyldecanoate (1.5 g, 2.25 mmol, 1 equiv.), tert-butyl N-(2-bromoethyl)carbamate (2.52 g, 11.26 mmol, 30.22 μL, 5 equiv.) in DMF (10 mL), KCO (1.56 g, 11.26 mmol, 5 equiv.), and KI (373.81 mg, 2.25 mmol, 1 equiv.) were added and stirred at 80 °C for 5 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain the compound 7-[2-(tert-butoxycarbonylamino)ethyl-(5-dodecanoyloxypentyl)amino]heptyl 2-octyldecanoate (1 g, 1.24 mmol, yield 54.87%) as a yellow oil.

[0442] Step 6: A mixture of 7-[2-(tert-butoxycarbonylamino)ethyl-(5-dodecanoyloxypentyl)amino]heptyl 2-octyldecanoate (1 g, 1.24 mmol, 1 equiv.) in HCl / EtOAc (2 M, 4.76 mL, 15.42 equiv.) was stirred at 20 °C for 5 h. The crude reaction mixture was adjusted to pH = 7 with saturated aqueous NaHCO3 and extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1) to give the compound 7-[2-aminoethyl(5-dodecanoyloxypentyl)amino]heptyl 2-octyldecanoate (0.7 g, 977.19 μmol, 79.08% yield, 99% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),4.07(t,J=6.8Hz,4H),2.73(t,J=6.0Hz,2H),2.47(t,J=6.0Hz,2H),2.39-2.44(m,4H),2. 30(t,J=7.2Hz,3H),1.61-1.66(m,6H),1.44-1.47(m,6H),1.20-1.36(m,50H),0.89(t,J=6.8Hz,9H).LCMS:(M+H + ):709.3@10.360 minutes.

[0443] Step 7: To a solution of 7-[2-aminoethyl(5-dodecanoyloxypentyl)amino]heptyl 2-octyldecanoate (0.3 g, 423.03 μmol, 1 equiv.), TEA (42.81 mg, 423.03 μmol, 58.88 μL, 1 equiv.), and DMAP (10.34 mg, 84.61 μmol, 0.2 equiv.) in DCM (5 mL) was added butanedioyl dichloride (32.78 mg, 211.51 μmol, 23.25 μL, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 3 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate / NH.H2O = 1 / 0 / 0.1 to 3 / 1 / 0.1) and preparative TLC (SiO, petroleum ether / ethyl acetate / NH3.H2O = 1:2:0.1) to give the compound 7-[5-dodecanoyloxypentyl-[2-[[4-[2-[5-dodecanoyloxypentyl-[7-(2-octyldecanoyloxy)heptyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl]amino]heptyl 2-octyldecanoate (0.1 g, 65.98 μmol, yield 15.60%, purity 99%) as a yellow oil. 1H NMR(400MHz,CDCl3),6.26(brs,2H),4.04-4.09(m,8H),3.20-3.30(m,4H),2.35-2.60(m,16H),2.30( t,J=7.2Hz,6H),1.62-1.64(m,18H),1.40-1.45(m,10H),1.20-1.34(m,96H),0.88(t,J=6.8Hz,18H).

[0444] 4.15: Synthesis of Compound 2279 [ka]

[0445] Step 1: To a solution of 2-hexyldecanoic acid (2.5 g, 9.75 mmol, 1 equiv.) and 6-bromohexan-1-ol (1.77 g, 9.75 mmol, 1.28 mL, 1 equiv.) in DCM (50 mL) was added EDCI (2.24 g, 11.70 mmol, 1.2 equiv.) and DMAP (595.55 mg, 4.87 mmol, 0.5 equiv.). The mixture was stirred at 15 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 15 °C and then extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were washed with 400 mL (200 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 20 / 1) to obtain the compound 6-bromohexyl 2-hexyldecanoate (14 g, 33.37 mmol, yield 85.58%, 4 batches) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.08(t,J=6.8Hz,2H),3.42(t,J=6.8Hz,2H),2.28-2.35(m,1H),1.84-1 .91(m,2H),1.57-1.69(m,4H),1.38-1.48(m,6H),1.26-1.29(m,20H),0.88(t,J=6.8Hz,6H).

[0446] Step 2: To a solution of phenylmethanamine (851.48 mg, 7.95 mmol, 866.20 μL, 1 equiv.) in DMF (75 mL) was added K2CO3 (5.49 g, 39.73 mmol, 5 equiv.) and KI (3.30 g, 19.87 mmol, 2.5 equiv.), followed by the addition of a solution of 6-bromohexyl 2-hexyldecanoate (7 g, 16.69 mmol, 2.1 equiv.) in DMF (25 mL). The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched at 15 °C by the addition of 200 mL of HO and extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were washed with 200 mL (100 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to obtain the compound 6-[benzyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (9 g, 11.48 mmol, yield 72.21%) as a colorless oil. 1 H NMR(400MHz,CDCl3),7.27-7.33(m,4H),7.20-7.25(m,1H),4.05(t,J=6.8Hz,4H),2.27-2.4 1(m,4H),1.56-1.62(m,10H),1.40-1.48(m,8H),1.26-1.32(m,50H),0.88(t,J=7.2Hz,12H).

[0447] Step 3: A solution of Pd / C (1 g, 10% purity) and 6-[benzyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (4.5 g, 5.74 mmol, 1 equiv.) in EtOAc (500 mL) was stirred under H2 at 50 psi at 15 °C for 8 h. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 1 / 0) to give the compound 6-[6-(2-hexyldecanoyloxy)hexylamino]hexyl 2-hexyldecanoate (1.8 g, 2.59 mmol, 45.19% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.07(t,J=6.4Hz,4H),2.63(t,J=7.6Hz,4H),2.28-2.35(m,2H),1.52-1.66(m,12H),1.26-1.45(m,52H),0.88(t,J=7.2Hz,12H).

[0448] Step 4: To a solution of 6-[6-(2-hexyldecanoyloxy)hexylamino]hexyl 2-hexyldecanoate (800 mg, 1.15 mmol, 1 equiv.) in DMF (10 mL) was added KCO (796.39 mg, 5.76 mmol, 5 equiv.) and KI (191.31 mg, 1.15 mmol, 1 equiv.), followed by a solution of tert-butyl N-(2-bromoethyl)carbamate (1.16 g, 5.19 mmol, 4.5 equiv.) in DMF (5 mL). The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched at 15 °C by the addition of 20 mL of HO and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 3 / 1) to give the compound 6-[2-(tert-butoxycarbonylamino)ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (560 mg, 668.78 μmol) as a colorless oil.

[0449] Step 5: A solution of 6-[2-(tert-butoxycarbonylamino)ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (560 mg, 668.78 μmol, 1 equiv.) in DCM (4 mL) and TFA (3.59 g, 31.51 mmol, 2.33 mL, 47.12 equiv.) was stirred at 15° C. for 3 hours. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, EA:MeOH=10:1) to give the compound 6-[2-aminoethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (420 mg, 552.61 μmol, 82.63% yield, 97% purity) as a yellow oil. 1H NMR(400MHz,CDCl3),4.07(t,J=6.8Hz,4H),2.79(t,J=6.0Hz,2H),2.53(t,J=6.0Hz,2H),2.45(t,J=7.2Hz,4H),2. 29-2.34(m,2H),2.21(brs,2H),1.59-1.65(m,8H),1.43-1.45(m,8H),1.26-1.42(m,48H),0.89(t,J=7.2Hz,12H). LCMS: (M+H + ):737.5@11.219 minutes.

[0450] Step 6: To a suspension of 6-[2-aminoethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (100 mg, 135.64 μmol, 2 equiv.), TEA (20.59 mg, 203.46 μmol, 28.32 μL, 3 equiv.), and DMAP (828.56 μg, 6.78 μmol, 0.1 equiv.) in DCM (3 mL) was added butanedioyl dichloride (10.51 mg, 67.82 μmol, 7.45 μL, 1 equiv.) in DCM (1 mL) dropwise at 15 °C. The mixture was stirred at 15 °C for 2 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, EA:MeOH = 10:1) to give the compound 6-[2-[[4-[2-[bis[6-(2-hexyldecanoyloxy)hexyl]amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-[6-(2-hexyldecanoyloxy)hexyl]amino]hexyl 2-hexyldecanoate (32 mg, 20.15 μmol, yield 29.71%, purity 98%) as a colorless oil. 1H NMR(400MHz,CDCl3),6.27(brs,2H),4.07(t,J=6.8Hz,8H),3.29(brs,4H),2.29-2.52(m,20H),1 .60-1.65(m,14H),1.40-1.46(m,18H),1.26-1.36(m,96H),0.88(t,J=7.2Hz,24H).LCMS:(M / 2+H + ):778.9@16.635 minutes.

[0451] 4.16: Synthesis of Compound 2280 [ka]

[0452] Step 1: To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxyhexyl)amino]octanoate (2.10 g, 3.15 mmol, 1.5 equiv.) and tert-butyl N-(2-bromoethyl)-N-methyl-carbamate (500 mg, 2.10 mmol, 1 equiv.) in DCE (20 mL) was added AcOH (12.61 mg, 209.98 μmol, 12.01 μL, 0.1 equiv.) at 0°C and stirred for 30 min. NaBH(OAc)3 (667.54 mg, 3.15 mmol, 1.5 equiv.) was then added to the mixture. The mixture was stirred at 20°C for 8 h. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 20 / 1 to 1 / 1) to obtain the compound 1-octylnonyl 8-[2-[tert-butoxycarbonyl(methyl)amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (1.5 g, 1.82 mmol, yield 86.77%) as a yellow oil.

[0453] Step 2: To a solution of 1-octylnonyl 8-[2-[tert-butoxycarbonyl(methyl)amino]ethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (1.5 g, 1.82 mmol, 1 equiv.) in DCM (10 mL) was added TFA (5 mL). The mixture was stirred at 20 °C for 3 hours. The mixture was concentrated under reduced pressure, the pH was adjusted to 8 with saturated NaHCO₃, and then extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were washed with 60 mL (20 mL × 3) of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give the compound 1-octylnonyl 8-[2-(methylamino)ethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (1.3 g, crude) as a brown oil.

[0454] Step 3: To a solution of 1-octylnonyl 8-[2-(methylamino)ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (186.66 mg, 258.10 μmol, 2 equiv.) in DCM (10 mL) was added TEA (65.29 mg, 645.25 μmol, 89.81 μL, 5 equiv.), DMAP (7.88 mg, 64.52 μmol, 0.5 equiv.), and butanedioyl dichloride (20 mg, 129.05 μmol, 14.18 μL, 1 equiv.) at 0° C. The mixture was stirred at 20° C. for 2 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1, 0.1% NH.HO) to give the compound 1-octylnonyl 8-[2-[methyl-[4-[methyl-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]amino]-4-oxo-butanoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (43 mg, 27.57 μmol, yield 21.36%, purity 98%) as a colorless oil. 1H NMR(400MHz,CDCl3),4.85-4.88(m,2H),4.06(t,J=6.8Hz,4H),3.37-3.43(m,3H),3.06-3.08(m,4H),2.94(s,2H),2.53-2.68(m, 7H),2.41-2.42(m,6H),2.26-2.30(m,8H),1.61-1.63(m,14H),1.51-1.60(m,10H),1.26-1.42(m,104H),0.89(t,J=6.8Hz,18H). LCMS: (M / 2+H + ):764.9@16.206 minutes.

[0455] 4.17: Synthesis of Compound 2281 [ka]

[0456] Step 1: To a solution of 1-octylnonyl 8-[2-(methylamino)ethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (300 mg, 414.82 μmol, 1 equiv.) in THF (5 mL) was added TEA (83.95 mg, 829.64 μmol, 115.48 μL, 2 equiv.) and tetrahydrofuran-2,5-dione (62.27 mg, 622.23 μmol, 1.5 equiv.). The mixture was stirred at 20°C for 8 hours. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, EtOAc / MeOH = 1 / 0~3 / 1, 0.1% NH3·H2O) to give the compound 4-[methyl-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]amino]-4-oxo-butanoic acid (200 mg, 242.93 μmol, 58.56% yield) as a colorless oil.

[0457] Step 2: To a solution of 4-[methyl-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]amino]-4-oxo-butanoic acid (200 mg, 242.93 μmol, 1 equiv.) and 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (206.74 mg, 291.52 μmol, 1.2 equiv.) in DCM (5 mL) was added EDCI (55.88 mg, 291.52 μmol, 1.2 equiv.) and DMAP (14.84 mg, 121.47 μmol, 0.5 equiv.) at 0° C. The mixture was stirred at 20° C. for 8 h. The reaction mixture was quenched by adding 20 mL of H2O at 0°C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 30 mL (10 mL × 3) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Welch Xltimate C4 100 × 30 × 10 μm; mobile phase: [water (HCl)-ACN]; B%: 70% to 100%, 15 min). The pH was then adjusted to 8 with saturated NaHCO3 and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were concentrated under reduced pressure. This was then purified by p-TLC (EtOAc / MeOH=3 / 1, with 0.1% NH3.HO) to give the compound 1-octylnonyl 8-[2-[[4-[methyl-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]amino]-4-oxobutanoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (30 mg, 63.39 μmol, yield 7.83%, purity 98%) as a colorless oil. 1H NMR(400MHz,CDCl3),6.27-6.35(m,1H),4.87(t,J= 6.4Hz,2H),4.06(t,J=6.8Hz,3H),3.98-4.00(m,1H),3.33-3.41(m,1H),3.26-3.28(m,1 H),2.93-3.04(m,3H),2.26-2.53(m,20H),1.23-1.67(m,130H),0.88(t,J=5.2Hz,18H). LCMS: (M+H + ):1514.2@11.778 minutes.

[0458] 4.18: Synthesis of Compound 2282 [ka] [ka]

[0459] Step 1: To a solution of 8-(tert-butoxycarbonylamino)octanoic acid (10 g, 38.56 mmol, 1 equiv.) and heptadecan-9-amine (11.82 g, 46.27 mmol, 1.2 equiv.) in DCM (100 mL) was added EDCI (8.87 g, 46.27 mmol, 1.2 equiv.) and DMAP (2.36 g, 19.28 mmol, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 8 h. The reaction mixture was quenched by adding 100 mL of HO at 0 °C and then extracted with 300 mL (100 mL × 3) of EtOAc. The combined organic layers were washed with 300 mL (100 mL × 3) of saturated brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30 / 1 to 2 / 1) to obtain the compound N-[8-(1-octylnonylamino)-8-oxo-octyl]carbamate tert-butyl ester (12 g, 24.15 mmol, yield 62.64%) as a white solid.

[0460] Step 2: To a solution of tert-butyl N-[8-(1-octylnonylamino)-8-oxo-octyl]carbamate (8 g, 16.12 mmol, 1 equiv.) in DCM (60 mL) was added TFA (30 mL). The mixture was stirred at 20 °C for 3 hours. The mixture was concentrated under reduced pressure, the pH was adjusted to 8 with saturated NaHCO₃, and then extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were washed with 150 mL (50 mL × 3) of brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure to give compound 8-amino-N-(1-octylnonyl)octanamide (6 g, crude) as a yellow oil.

[0461] Step 3: To a solution of 6-bromohexanoic acid (10 g, 51.27 mmol, 1 equiv.) and undecan-1-amine (10.54 g, 61.52 mmol, 1.2 equiv.) in DCM (100 mL) was added EDCI (11.79 g, 61.52 mmol, 1.2 equiv.) and DMAP (3.13 g, 25.63 mmol, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 8 h. The reaction mixture was quenched by adding 100 mL of HO at 0 °C and then extracted with EtOAc (100 mL × 3). The combined organic layers were washed with 300 mL (100 mL × 3) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=30 / 1 to 3 / 1) to obtain the compound 6-bromo-N-undecyl-hexanamide (12 g, 34.45 mmol, yield 67.19%) as a white solid.

[0462] Step 4: To a solution of 8-amino-N-(1-octylnonyl)octanamide (3.42 g, 8.62 mmol, 1.5 equiv) in DMF (30 mL) was added DIEA (1.48 g, 11.48 mmol, 2.00 mL, 2 equiv), KI (476.52 mg, 2.88 mmol, 0.5 equiv), and 6-bromo-N-undecyl-hexanamide (2 g, 5.74 mmol, 1 equiv). The mixture was stirred at 35°C for 8 h. The reaction mixture was quenched by adding 60 mL of HO at 0°C and then extracted with EtOAc (30 mL × 3). The combined organic layers were washed with 90 mL (30 mL × 3) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, EtOAc / MeOH = 1 / 0 to 4 / 1 + 0.1% NH.H.sub.2O) to obtain the compound N-(1-octylnonyl)-8-[[6-oxo-6-(undecylamino)hexyl]amino]octanamide (1 g, 1.51 mmol, 26.23% yield) as a yellow solid.

[0463] Step 5: To a solution of N-(1-octylnonyl)-8-[[6-oxo-6-(undecylamino)hexyl]amino]octanamide (1 g, 1.51 mmol, 1 equiv.) in DMF (10 mL) was added K2CO3 (1.04 g, 7.52 mmol, 5 equiv.), KI (249.96 mg, 1.51 mmol, 1 equiv.), and tert-butyl N-(2-bromoethyl)carbamate (506.14 mg, 2.26 mmol, 1.5 equiv.). The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched by adding 30 mL of HO at 0 °C and then extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, PE: EtOAc = 0:1 + 0.1% NH.H0) to give the compound N-[2-[[8-(1-octylnonylamino)-8-oxo-octyl]-[6-oxo-6-(undecylamino)hexyl]amino]ethyl] tert-butylcarbamate (530 mg, 656.49 μmol, 43.60% yield) as a yellow oil.

[0464] Step 6: To a solution of tert-butyl N-[2-[[8-(1-octylnonylamino)-8-oxo-octyl]-[6-oxo-6-(undecylamino)hexyl]amino]ethyl]carbamate (530 mg, 656.49 μmol, 1 equiv.) in DCM (6 mL) was added TFA (3 mL). The mixture was stirred at 20 °C for 3 h. The mixture was concentrated under reduced pressure, then the pH was adjusted to 8 with saturated NaHCO and extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 30 mL (10 mL × 3) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, EtOAc / MeOH = 1 / 0 to 1 / 1, 0.1% NH3·H2O) to give the compound 8-[2-aminoethyl-(6-oxo-6-(undecylamino)hexyl]amino]-N-(1-octylnonyl)octanamide (320 mg, 452.48 μmol, yield 68.92%) as a colorless oil. 1 H NMR(400MHz,CDCl3),6.12-6.15(m,1H),5.29(d,J=8.8Hz,1H),3.85-3.91(m,1H),3.18-3.22(m,2H),3.12-3.15(m,2 H),2.87-2.90(m,2H),2.62(s,5H),2.05-2.21(m,9H),1.56-1.64(m,7H),1.26-1.33(m,52H),0.89(t,J=6.4Hz,9H).

[0465] Step 7: To a solution of 8-[2-aminoethyl-[6-oxo-6-(undecylamino)hexyl]amino]-N-(1-octylnonyl)octanamide (319.43 mg, 451.67 μmol, 2 equiv.) in DCM (5 mL) was added TEA (114.26 mg, 1.13 mmol, 157.17 μL, 5 equiv.), DMAP (13.80 mg, 112.92 μmol, 0.5 equiv.), and butanedioyl dichloride (35 mg, 225.84 μmol, 24.82 μL, 1 equiv.). The mixture was stirred at 0 °C for 2 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, EtOAc / MeOH = 1 / 0 to 3 / 1) to obtain the compound N,N'-bis[2-[[8-(1-octylnonylamino)-8-oxo-octyl]-[6-oxo-6-(undecylamino)hexyl]amino]ethyl]butanediamide (86 mg, 57.47 μmol, yield 25.45%, purity 100%) as a colorless oil. 1 H NMR(400MHz,CDCl3),6.18(t,J=5.2Hz,2H),5.44(d,J=9.2Hz,2H),3.87-3.99(m,2H),3.19-3.32(m,8H),2.14-2.75(m,1 4H),2.10-2.20(m,8H),1.99(s,2H),1.64-1.69(m,8H),1.45-1.49(m,16H),1.26-1.31(m,102H),0.88(t,J=6.4Hz,18H). LCMS: (M+H + ):1496.3@8.078 minutes.

[0466] 4.19: Synthesis of Compound 2284 [ka]

[0467] To a solution of 2-methylbutanedioic acid (7.45 mg, 56.40 μmol, 1 equiv.) in DMF (3 mL) was added HBTU (85.56 mg, 225.61 μmol, 4 equiv.) and HOBt (30.48 mg, 225.61 μmol, 4 equiv.). Next, a solution of 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (100 mg, 141.01 μmol, 2.5 equiv.) and DIEA (29.16 mg, 225.61 μmol, 39.30 μL, 4 equiv.) in DMSO (1.5 mL) was added. The mixture was stirred at 15 °C for 8 h. The reaction mixture was quenched by adding 10 mL of HO at 15° C. and then extracted with 30 mL (10 mL×3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL×2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, EA:MeOH=10:1) to give compound 1-octylnonyl 8-[2-[[3-methyl-4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-4-oxo-butanoyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (31 mg, 20.43 μmol, yield 36.22%, purity 99.8%) as a colorless oil. 1H NMR(400MHz, CDCl3),6.08-6.36(m,2H),4.73-4.86(m,2H),3.98(t,J=7.2Hz,4H),2.91-3.39(m,4H),1.97-2.88(m,23H), 1.53-1.58(m,12H),1.41-1.47(m,8H),1.29-1.38(m,8H),1.16-1.26(m,96H),1.10(d,J=7.2Hz,3H),0.79-0.83(m,18H). LCMS: (M / 2+1): 757.9@13.893 min.

[0468] 4.20: Synthesis of compound 2288

change

[0469] ステップ1: To a solution of 2-[tert-butoxycarbonyl(carboxymethyl)amino]acetic acid (109.62 mg, 470.03 μmol, 1 equiv.) in DMF (10 mL) were added EDCI (270.31 mg, 1.41 mmol, 3 equiv.) and HOBt (31.76 mg, 235.01 μmol, 0.5 equiv.). The mixture was stirred at 15 °C for 8 hours. Then, 1-octylnonyl 8-[2-aminoethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (1 g, 1.41 mmol, 3 equiv.) was added to the mixture. The mixture was stirred at 15 °C for 3 hours. The reaction mixture was quenched by adding 10 mL of HO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, EtOAc:MeOH = 1:0) to give the compound 1-octylnonyl 8-[2-[[2-[tert-butoxycarbonyl-[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-2-oxo-ethyl]amino]acetyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (438 mg, 271.12 μmol, 57.68% yield) as a colorless oil. 1 H NMR(400MHz,CDCl3),4.80-4.93(m,2H),4.06(t,J=6.8Hz,4H),3.79-3.94(m,3H),3.27-3.42(m,4H),2.51-2.66(m,4H),2.35- 2.49(m,7H),2.22-2.35(m,8H),1.56-1.67(m,19H),1.47-1.56(m,10H),1.44(s,10H),1.21-1.36(m,98H),0.84-0.93(m,18H).

[0470] Step 2: To a solution of 1-octylnonyl 8-[2-[[2-[tert-butoxycarbonyl-[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-2-oxo-ethyl]amino]acetyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (400 mg, 247.60 μmol, 1 equiv) in DCM (4 mL) was added TFA (3.08 g, 27.01 mmol, 2 mL, 109.10 equiv) and stirred for 3 h at 15° C. The reaction mixture was quenched by the addition of 10 mL of saturated aqueous NaHCO3 at 15° C. and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, EA:MeOH = 10:1) to give the compound 1-octylnonyl 8-[2-[[2-[[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-2-oxo-ethyl]amino]acetyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (230 mg, 151.77 μmol, 61.30% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.79-4.95(m,2H),4.06(t,J=6.8Hz,4H),3.28-3.37(m,4H),2.23-2.62(m,17H),1.22-1.75(m,134H),0.86-0.92(m,18H).

[0471] Step 3: To a solution of 3-pyrrolidin-1-ylpropanoic acid (100 mg, 698.41 μmol, 1 equiv.) in DCM (5 mL) was added (COCl) (443.23 mg, 3.49 mmol, 305.68 μL, 5 equiv.) and DMF (5.10 mg, 69.84 μmol, 5.37 μL, 0.1 equiv.). The mixture was stirred at 15 °C for 2 h. The reaction mixture was concentrated under reduced pressure to give compound 3-pyrrolidin-1-ylpropanoyl chloride (138.3 mg, 698.17 μmol, 99.97% yield, purity -, HCl) as a yellow solid.

[0472] Step 4: 1-Octylnonyl 8-[2-[[2-[[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-2-oxo-ethyl]amino]acetyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (100 mg, 65.99 μmol, 1 equiv.), T To a suspension of EA (100.16 mg, 989.82 μmol, 137.77 μL, 15 equiv) and DMAP (4.03 mg, 32.99 μmol, 0.5 equiv) in DCM (3 mL) was added 3-pyrrolidin-1-ylpropanoyl chloride (130.72 mg, 659.88 μmol, 10 equiv, HCl) in DCM (1 mL) dropwise at 15 °C. The mixture was stirred at 15 °C for 3 h under a N atmosphere. The reaction mixture was quenched by adding 10 mL of saturated aqueous NaHCO at 15 °C and then extracted with 30 mL (10 mL × 3) of EtOAc. The combined organic layers were washed with 20 mL (10 mL × 2) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO, EA:MeOH=10:1) to give compound 1-octylnonyl 8-[2-[[2-[[2-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethylamino]-2-oxo-ethyl]-(3-pyrrolidin-1-ylpropanoyl)amino]acetyl]amino]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (17 mg, 10.36 μmol, yield 15.70%, purity 100%) as a colorless oil. 1 H NMR(400MHz,CDCl3),8.73(brs,1H),6.64(brs,1H),4.83-4.91(m,2H),4.06(t,J=6.8Hz,6H),3.93(s,2H),3.25-3.40(m,4H),2 .22-2.88(m,28H),1.78(s,4H),1.63(s,12H),1.48-1.54(m,8H),1.38-1.47(m,8H),1.24-1.33(m,96H),0.89(t,J=7.2Hz,18H). LCMS: (M+H + ):1640.4@5.693 minutes.

[0473] 4.21: Synthesis of Compound 2299 [ka]

[0474] To a solution of 1-octylnonyl 8-[(6-oxo-6-undecoxy-hexyl)-(2-piperazin-1-ylethyl)amino]octanoate (300 mg, 385.46 μmol, 1 equiv.) and TEA (78.01 mg, 770.93 μmol, 107.30 μL, 2 equiv.) in DCM (10 mL) was added bis(trichloromethyl)carbonate (18.30 mg, 61.67 μmol, 0.16 equiv.) in DCM (5 mL) dropwise at 0 °C for 0.5 h. The mixture was degassed and purged with N three times, then stirred under N at 25 °C for 8 h. The reaction mixture was quenched by adding 30 mL of HO under N at 0 °C and then extracted with 150 mL (50 mL × 3) of EtOAc. The combined organic layers were washed with 50 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 5 / 1 to 0 / 1), preparative TLC (SiO2, ethyl acetate / methanol = 5:1, 2% NH3·H2O), and preparative TLC (SiO2, ethyl acetate / methanol = 40:1, 2% NH3·H2O) to give the compound 1-octylnonyl 8-[2-[4-[4-[2-[[8-(1-octylnonoxy)-8-oxo-octyl]-(6-oxo-6-undecoxy-hexyl)amino]ethyl]piperazine-1-carbonyl]piperazin-1-yl]ethyl-(6-oxo-6-undecoxy-hexyl)amino]octanoate (60 mg, 36.57 μmol, 18.98% yield, 99.65% purity) as a colorless oil. 1H NMR(400MHz,CDCl3),4.83-4.91(m,2H),4.06(t,J=6.8Hz,4H),3.27(t,J=4.0Hz,8H),2.51-2.60(m,4H),2.35-2.47(m,18H) ,2.29(q,J=7.6Hz,8H),1.60-1.67(m,16H),1.5-1.52(m,8H),1.33-1.40(m,6H),1.26-1.32(m,96H),0.87(t,J=7.2Hz,18H). LCMS: (M / 2+H + ):791.9@10.691 minutes.

[0475] 4.22: Synthesis of Compound 2302 [ka]

[0476] Step 1: To a mixture of 6-bromohexanoic acid (22.64 g, 116.07 mmol, 1 equiv.) in DCM (1 mL) was added DMAP (2.84 g, 23.21 mmol, 0.2 equiv.), undecan-1-ol (20 g, 116.07 mmol, 1 equiv.), and EDCI (22.25 g, 116.07 mmol, 1 equiv.). The mixture was stirred at 25 °C under a N atmosphere for 12 h. The reaction mixture was diluted with 200 mL of HO and extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=1 / 0 to 40 / 1) to obtain the compound undecyl 6-bromohexanoate (36 g, 103.05 mmol, yield 88.78%) as a yellow oil.

[0477] Step 2: To a solution of 1,3-diaminopropan-2-ol (15 mg, 166.44 μmol, 1 equiv.), undecyl 6-bromohexanoate (290.72 mg, 832.19 μmol, 5 equiv.) in DMF (10 mL) was added K2CO3 (115.01 mg, 832.19 μmol, 5 equiv.), and KI (55.26 mg, 332.88 μmol, 2 equiv.). The mixture was stirred at 40 °C for 5 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: Phenomenex Luna C18 100 × 30 mm × 5 μm; mobile phase: [water (HCl)-ACN]; B%: 60% to 90%, 10 min) to give the crude product. The crude product was dissolved in HO (10 mL), adjusted to pH = 7 with saturated aqueous NaHCO3, and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by preparative TLC (SiO2, PE:EtOAc = 3:1) and column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give the compound 6-[[3-[bis(6-oxo-6-undecoxy-hexyl)amino]-2-hydroxy-propyl]-(6-oxo-6-undecoxy-hexyl)amino]undecyl hexanoate (50 mg, 42.96 μmol, yield 25.81%) as a yellow oil. 1 H NMR(400MHz,CDCl3),4.06(t,J=6.8Hz,8H),3.65(brs,1H),2.28-2.47(m,20H),1 .58-1.68(m,18H),1.43-1.47(m,8H),1.25-1.35(m,70H),0.89(t,J=6.8Hz,12H). LCMS: (M+H + ):1163.5@14.292 minutes.

[0478] 4.23: Synthesis of Compound 2303 [ka]

[0479] Step 1: To a solution of 8-bromooctanoic acid (20 g, 89.64 mmol, 1 equiv.) and heptadecan-9-ol (27.60 g, 107.56 mmol, 1.2 equiv.) in DCM (400 mL) was added EDCI (20.64 g, 107.56 mmol, 1.2 equiv.) and DMAP (5.48 g, 44.84 mmol, 0.5 equiv.) at 0 °C. The mixture was stirred at 20 °C for 8 h. The reaction mixture was quenched by adding 200 mL of HO at 0 °C and then extracted with 600 mL (200 mL × 3) of EtOAc. The combined organic layers were washed with 600 mL (200 mL × 3) of brine, dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 50 / 1) to obtain the compound 1-octylnonyl 8-bromooctanoate (33 g, 71.50 mmol, yield 79.76%, purity 100%) as a colorless oil.

[0480] Step 2: To a solution of phenylmethanamine (1.1 g, 10.27 mmol, 1.12 mL, 1 equiv.) in DMF (50 mL) was added K2CO3 (7.09 g, 51.33 mmol, 5 equiv.) and KI (5.11 g, 30.80 mmol, 3 equiv.). Then, a solution of 1-octylnonyl 8-bromooctanoate (9.62 g, 20.84 mmol, 2.03 equiv.) in DMF (50 mL) was added to the mixture. The mixture was stirred at 80 °C for 8 h. The reaction mixture was quenched by adding 500 mL of HO at 0 °C and then extracted with 900 mL (300 mL × 3) of EtOAc. The combined organic layers were washed with 900 mL (300 mL × 3) of saturated brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 1 / 0 to 10 / 1) to obtain the compound 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (23 g, 26.48 mmol, yield 86.00%) as a yellow oil. 1 H NMR(400MHz,CDCl3),7.24-7.31(m,4H),7.22-7.24(m,1H),4.87(t,J=6.4Hz,2H),3.53(s,2H),2.38(t,J=7.2H z,3H),2.27(t,J=7.2Hz,4H),1.58-1.62(m,4H),1.43-1.52(m,12H),1.25-1.28(m,64H),0.89(t,J=6.4Hz,9H).

[0481] Step 3: To a solution of Pd / C (4 g, 10% purity) and Pd(OH) / C (4 g, 5.70 mmol, 20% purity, 3.81 e-1 equiv.) in EtOAc (100 mL) was added 1-octylnonyl 8-[benzyl-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (13 g, 14.96 mmol, 1 equiv.). The mixture was stirred under an atmosphere of H (50 psi) at 20 °C for 8 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO, EtOAc / MeOH = 1 / 0 to 3 / 1, 0.1% NH.H.sub.2O) to give the compound 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (8 g, 10.18 mmol, yield 67.98%, purity 99%) as a colorless oil.

[0482] Step 4: To a solution of 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (0.5 g, 642.41 μmol, 1 equiv.), 2-(bromomethyl)oxirane (439.97 mg, 3.21 mmol, 265.04 μL, 5 equiv.) in ACN (5 mL), KCO (266.36 mg, 1.93 mmol, 3 equiv.), and KI (106.64 mg, 642.41 μmol, 1 equiv.) were added and stirred at 80 °C for 8 h. The reaction mixture was diluted with 20 mL of HO and extracted with 60 mL (20 mL × 3) of EtOAc. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1) to obtain the compound 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]-(oxiran-2-ylmethyl)amino]octanoate (0.4 g, 479.40 μmol, yield 74.62%) as a yellow oil.

[0483] Step 5: A mixture of 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (100 mg, 128.48 μmol, 1 equivalent) and 1-octylnonyl 8-[[8-(1-octylnonoxy)-8-oxo-octyl]-(oxiran-2-ylmethyl)amino]octanoate (214.40 mg, 256.96 μmol, 2 equivalents) was heated at 120° C. under a N atmosphere for 5 hours. The reaction mixture was purified by preparative TLC (SiO, EtOAc:MeOH=6:1) to give the compound 1-octylnonyl 8-[[3-[bis[8-(1-octylnonoxy)-8-oxo-octyl]amino]-2-hydroxy-propyl]-[8-(1-octylnonoxy)-8-oxo-octyl]amino]octanoate (70 mg, 43.44 μmol, 33.78% yield) as a color...

Claims

1. Compounds of formula (I): 、 a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing During the ceremony, each A is independently optionally interrupted or substituted with one or more heteroatoms, or substituted with OH, SH, or halogen; 1 -C 16 Branched or unbranched alkyl or C 1 -C 16 is branched or unbranched alkenyl; each B is independently optionally substituted with a heteroatom or substituted with OH, SH, or halogen; 1 -C20 branched or unbranched alkyl or C 1 -C20 branched or unbranched alkenyl; each X is independently a biodegradable moiety; and W is, or During the ceremony, R 5 (CH2)sOH, OH, SH, NR 10 R 11 and Each R 6 are independent, H, C 1 -C 3 Branched or unbranched alkyl, C 2 -C 3 branched or unbranched alkenyl, or cycloalkyl; Each R 7 and each R 8 are independent, H, C 1 -C 3 Branched or unbranched alkyl, C 2 -C 3 Branched or unbranched alkenyl, halogen, OH, SH, (CH 2 ) s R 17 , NR 10 R 11 and R 10 and R 11 are each independently H, C 1 -C 3 alkyl or R 10 and R 11 are taken together to form a heterocyclic ring; or R 7 and R 8 are taken together to form a ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; each v is independently 0, 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 or a divalent heterocyclic group, wherein R 12 But H, C 1 -C 7 branched or unbranched alkyl, or C 2 -C 7 branched or unbranched alkenyl, provided that when Z is present, the adjacent R 1 and R 2 OH, NR 10 R 11 or without being SH; V is a branched or unbranched C 2 -C 10 alkylene, C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, or C 2 -C 10 heteroalkylene optionally substituted with one or more OH, SH, and / or halogen groups; T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic group optionally substituted with one or more -(CH 2 ) v OH, -(CH 2 ) v SH, and / or -(CH 2 ) v -halogen groups; R 14 is a heterocyclic group, NR 10 R 11 , C(O)NR 10 R 11 , or C(S)NR 10 R 11 ; R 16 is H, ═O, ═S, or CN; R 17 is OH, SH, or N(CH 3 ) 2 ; and Compounds wherein Q is O, S, or NR 13 , where each R 13 is H, C 1 -C 5 alkyl.

2. B is C 3 -C 20 The compound of claim 1 , wherein the aryl group is alkyl.

3. Compound of formula (II): 、 a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing During the ceremony, Each R 1 and each R 2 are independent, H, C 1 -C 3 Branched or unbranched alkyl, OH, halogen, SH, or NR 10 R 11 or Each R 1 and each R 2 are independently taken together with the carbon atoms to which they are attached to form a cyclic ring; R 10 and R 11 are each independently H, C 1 -C 3 branched or unbranched alkyl, or R 10 and R 11 are taken together to form a heterocyclic ring; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each X is independently a biodegradable moiety; Each R 3 and each R 4 are independently H, C 3 -C 14 branched or unbranched alkyl (C 3 -C 10 may be branched or unbranched alkyl), or C 3 -C 10 branched or unbranched alkenyl, provided that R 3 and R 4 at least one of is not H; W is, or During the ceremony, R 5 (CH2)sOH, OH, SH, NR 10 R 11 and Each R 6 are independent, H, C 1 -C 3 Branched or unbranched alkyl, C 2 -C 3 branched or unbranched alkenyl, or cycloalkyl; Each R 7 and each R 8 are independent, H, C 1 -C 3 Branched or unbranched alkyl, C 2 -C 3 Branched or unbranched alkenyl, halogen, OH, SH, (CH 2 ) s R 17 , NR 10 R 11 wherein R 10 and R 11 are each independently H, C 1 -C 3 alkyl, or each R 10 and each R 11 taken together with the carbon atoms to which they are attached form a heterocyclic ring; or R 7 and R 8 are grouped together to form a ring; each s is independently 1, 2, 3, 4, or 5; each u is independently 1, 2, 3, 4, or 5; each v is independently 0, 1, 2, 3, 4, or 5; t is 1, 2, 3, 4, or 5; Each Z is independently absent, O, S, or NR 12 or a divalent heterocyclic group, wherein R 12 is H, C 1 -C 7 branched or unbranched alkyl, or C 2 -C 7 is branched or unbranched alkenyl; V is a branched or unbranched C 2 -C 10 alkylene, C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, or C 2 -C 10 heteroalkylene optionally substituted with one or more OH, SH, and / or halogen groups; T is -NHC(O)O-, -OC(O)NH-, or a divalent heterocyclic group optionally substituted with one or more -(CH 2 ) v OH, -(CH 2 ) v SH, and / or -(CH 2 ) v -halogen groups; R 14 is a heterocyclic group, NR 10 R 11 , C(O)NR 10 R 11 , or C(S)NR 10 R 11 ; R 16 is H, ═O, ═S, or CN; R 17 is OH, SH, or N(CH 3 ) 2 ; and Q is O, S, CH2, or NR 13 wherein each R 13 is H, C 1 -C 5 A compound that is alkyl.

4. Each X is independently -OC(O)-, -C(O)O-, -SS-, -N(R 18 )C(O)-, -C(O)N(R 18 )-, -C(O-R 13 )-O-, -C(O)O(CH 2 ) a -, -OC(O)(CH 2 ) a -, -C(O)N(R 18 )(CH 2 ) a -, -N(R 18 )C(O)(CH 2 ) a -, -C(O-R 13 )-O-(CH 2 ) a -, wherein each R 18 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, and each R 13 is independently C 3 -C 10 The compound of claim 1 or 3, wherein each a is independently 0 to 16.

5. X is -OCO-, -COO-, -NHCO-, -CONH-, -C(O-R 13 ) -O-, -COO(CH 2 ) r- , -CONH(CH 2 ) r -, or -C(O-R 13 )—O—(CH 2 ) r -, -O(CO)O-, wherein R 13 But C 3 -C 10 5. The compound of claim 4, wherein r is branched or unbranched alkyl and r is 1, 2, 3, 4, or 5.

6. The compound according to claim 1 or 3, wherein X is —OCO— or —COO—.

7. W is 4. The compound of claim 1 or 3, wherein Z is absent, O, S, or NH.

8. W is 4. The compound of claim 1 or 3, wherein: and V is C 2 -C 10 alkylene, C 2 -C 10 alkenylene, or C 2 -C 10 heteroalkylene.

9. The compound according to claim 8, wherein V is branched or unbranched C 2 -C 3 alkylene optionally substituted with OH, or branched or unbranched C 2 -C 3 alkenylene.

10. The compound of claim 1 or 3, wherein each R 6 is independently H or methyl.

11. R 7 and R 8 4. The compound of claim 1 or 3, wherein at least one of is H.

12. The compound of claim 11, wherein R 7 and R 8 are each H.

13. 4. The compound of claim 3, wherein m is 5, 6, 7, 8, or 9.

14. The compound of claim 3, wherein s is 1 or 2.

15. The compound of claim 3, wherein u is 1 or 2.

16. 4. The compound of claim 1 or 3, wherein the pKa of the protonated form of the compound is from about 5.1 to about 8.

0.

17. The compound of claim 16, wherein the pKa of the protonated form of the compound is from about 5.7 to about 6.

5.

18. The compound of claim 1, wherein the compound is one of the following formulas:

19. A combination of a compound according to claim 1 or 3 with a lipid component.

20. The combination of claim 19, comprising the compound and lipid component in a ratio of approximately 1:

1.

21. 20. The combination of claim 19, which is an LNP composition.

22. 20. The combination of claim 19, wherein the lipid component comprises a helper lipid and a PEG lipid, and may include a neutral lipid.

23. 20. The combination of claim 19, further comprising a nucleic acid component.

24. 24. The combination of claim 23, wherein the nucleic acid component is an RNA or DNA component.

25. 24. The combination of claim 23 having an N / P ratio of about 3 to 10.

26. The combination described in claim 24, wherein the nucleic acid component is an RNA component, and the RNA component comprises mRNA.

27. 10. Use of a composition comprising one or more compounds of claim 1 or 3 in the manufacture of a medicament for delivering a therapeutic cargo to a target organ in a subject in need thereof.

28. The target organ is the pancreas or the lung, and less than 50%, 30%, or 10% of the therapeutic cargo is delivered to the liver of the subject; and / or greater than 50%, 70%, or 90% of the therapeutic cargo is delivered to the pancreas and / or lungs of the subject; 28. The use according to claim 27.

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