Triol-containing nanomaterials

Abstract

The present disclosure describes compositions, preparations, nanoparticles (such as lipid nanoparticles), and / or nanomaterials, and methods of their use.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Patent Application No. 63 / 390,882, filed July 20, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Lipid-containing particles have been used to encapsulate and as delivery vehicles for therapeutic agents such as nucleic acids, small molecule compounds, and proteins within cells and other intracellular compartments. There remains a continuing need to develop new lipids to encapsulate therapeutic agents and improve the safety, efficacy, and specificity of such nanoparticle-based delivery vehicles. Summary of the Invention

[0003] The present invention recognizes the need for compositions, preparations, nanoparticles, and / or nanomaterials, and methods of their use. Among other things, the present disclosure recognizes that the structural characteristics of compositions, preparations, nanoparticles, and / or nanomaterials strongly influence functional responses in vivo, in vitro, and ex vivo. For example, the present disclosure describes, among other things, how the selection and combination of one or more components described herein affects the functional activity of lipid nanoparticles. In some embodiments, for example, functional activity may refer to desired targeting, stabilization, bioavailability, degradation characteristics, and / or drug delivery efficacy. In some embodiments, among other things, the present disclosure describes how different ratios of one or more components affect one or more functional activities of the compositions, preparations, nanoparticles, and / or nanomaterials described herein.

[0004] Further, among other things, the present disclosure recognizes that the chemical structure of the lipid confers improved properties compared to a reference lipid structure. For example, in some embodiments, the present disclosure provides a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 1 , Y 2 , Y 3 , X 1 , X 2 and X 3 each of which is as defined herein.

[0005] Among other things, as described herein, the present disclosure may demonstrate surprising attributes of ionizable lipids comprising a triol core and / or a biodegradable tail (e.g., unexpected targeting, stabilization, bioavailability, degradation characteristics, and delivery efficacy of cargoes such as therapeutic or prophylactic agents), compositions, preparations, nanoparticles, and / or nanomaterials (e.g., LNPs and / or LNP-containing compositions, preparations, nanoparticles, nanomaterials), and methods of their use. In some embodiments, the present disclosure may demonstrate particularly surprising attributes of ionizable lipids comprising a triol core and a biodegradable tail (e.g., unexpected targeting, stabilization, bioavailability, degradation characteristics, and delivery efficacy of cargoes such as therapeutic or prophylactic agents), compositions, preparations, nanoparticles, and / or nanomaterials (e.g., LNPs and / or LNP-containing compositions, preparations, nanoparticles, nanomaterials), and methods of their use. In some embodiments, ionizable lipids, compositions, preparations, nanoparticles, and / or nanomaterials (e.g., LNPs and / or LNP-containing compositions, preparations, nanoparticles, and / or nanomaterials) comprising biodegradable tails may be characterized by improved stability relative to certain comparable reference entities. In some embodiments, the tail structures described herein degrade more readily upon administration in vivo compared to other tail structures.

[0006] In some embodiments, the present disclosure may identify the cause of a problem with a particular alternative lipid compound and / or compositions, preparations, nanoparticles, and / or nanomaterials comprising them, for example, providing insight that a particular tail structure may confer or contribute to one or more attributes for which improvement would be beneficial. In addition to such identification of the cause of the problem, the present disclosure may provide specific solutions to the problem, including lipid compounds and / or compositions, preparations, nanoparticles, and / or nanomaterials comprising them, having different tail structures. In some embodiments, the provided lipid compounds and / or compositions, preparations, nanoparticles, and / or nanomaterials comprising them may be characterized by improved stability, bioavailability, and / or degradation properties compared to suitable comparable references having different tail structures. In some particular embodiments, the provided lipid compounds and / or compositions, preparations, nanoparticles, and / or nanomaterials comprising them may be characterized by improved stability, bioavailability, and / or degradation properties compared to suitable comparable references having a linoleic acid tail characteristic (e.g., moiety). In some particular embodiments, provided lipid compounds, and / or compositions, preparations, nanoparticles, and / or nanomaterials comprising them, may be characterized by improved stability, bioavailability, and / or degradation properties compared to a suitable comparable reference having two or more linoleic acid tail features (e.g., moieties).

[0007] Among other things, the present disclosure recognizes that lipid nanoparticle (LNP) compositions include one or more ionizable lipids.

[0008] In some embodiments, provided compositions, preparations, nanoparticles, and / or nanomaterials are for use in methods of treating, delivering, producing polypeptides, or slowing / halting progression of a disease or disorder.

[0009] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials provided are for use in a method of manufacture.

[0010] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials provided are for use in methods of characterization.

[0011] Elements (eg, methods) of embodiments involving one aspect of the invention may be applied to embodiments involving other aspects of the invention, and vice versa. [Brief explanation of the drawings]

[0012] [Figure 1] 1 illustrates a bar graph showing delivery of exemplary LNP preparations (Compound 7-1, Compound 7-2) to the liver.

[0013] [Figure 2] 1 illustrates a bar graph showing delivery of exemplary LNP preparations (Compound 7-1, Compound 7-2) to the spleen.

[0014] [Figure 3] 1 depicts a bar graph showing base editing in liver cells following delivery of an exemplary LNP preparation (compound 7-7) to the liver.

[0015] [Figure 4] 1 depicts a bar graph showing base editing in liver cells following delivery of an exemplary LNP preparation (compound 7-7) to the liver.

[0016] [Figure 5] 1 illustrates a bar graph showing siRNA-mediated silencing in liver cells following delivery of an exemplary LNP preparation (compound 7-2) to the liver.

[0017] definition About: As used herein, the term "about" or "approximately," when used herein in reference to a value, refers to a value that is similar in relation to the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the reasonable degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or in either direction (greater or smaller) of the referenced value, unless otherwise stated or otherwise clear from the context (except where such number would exceed 100% of the possible values).

[0018] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system. Those of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, under appropriate circumstances. For example, in some embodiments, administration may be intraocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be intrabronchial (e.g., by intrabronchial instillation), oral, intradermal (e.g., may be or include one or more of topically on the skin, intradermal, intradermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), intramucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), intravaginal, intravitreal, etc. In some embodiments, administration can involve administration that is intermittent (e.g., multiple doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time). In some embodiments, administration can involve continuous administration (e.g., perfusion) over at least a selected period of time. In some embodiments, pharmaceutical compositions comprising lipid nanoparticles can be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration, or by using bioerodible inserts, and can be formulated in dosage forms suitable for each route of administration.

[0019] Aliphatic: The terms "aliphatic" or "aliphatic group," as used herein, refer to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic (also referred to herein as "carbocyclic," "carbocyclic," "alicyclic," or "cycloalkyl") and has a single point of attachment to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 carbon atoms. In some embodiments, an aliphatic group contains 1-4 carbon atoms. In some embodiments, an aliphatic group contains 1-3 carbon atoms, and in some embodiments, an aliphatic group contains 1-2 carbon atoms. In some embodiments, "carbocyclic" (or "alicyclic" or "carbocycle" or "cycloalkyl") refers to an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C6-C8 hydrocarbon, that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. 12 Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0020] Alkenyl: As used herein, the term "alkenyl" refers to an alkyl group, as defined herein, having one or more double bonds. In some embodiments, the term "alkenyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one double bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C2-14 , C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched hydrocarbon chain having an alkyl group. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

[0021] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0022] Alkyl: As used herein, the term "alkyl" is given its ordinary meaning in the art and can include saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, an alkyl has 1-100 carbon atoms. In certain embodiments, a straight-chain or branched-chain alkyl has about 1-20 carbon atoms in its backbone (e.g., C1-C5 for straight chain). 20 , for branched chains, C2-C 20 ), or about 1-10 carbon atoms. In some embodiments, cycloalkyl rings have from about 3-10 carbon atoms in their ring structure, and such rings are monocyclic or bicyclic, alternatively having about 5, 6, or 7 carbons in the ring structure. In some embodiments, alkyl groups can be lower alkyl groups, which lower alkyl groups have from 1-4 carbon atoms (e.g., C1-C4 for a straight chain lower alkyl).

[0023] Alkylene: The term "alkylene" or "alkylenyl" refers to a divalent alkyl group (i.e., a divalent saturated hydrocarbon chain) that is straight-chain (i.e., unbranched) or branched, substituted or unsubstituted. Any of the monovalent alkyl groups mentioned above can be alkylene by abstraction of a second hydrogen atom from the alkyl. In some embodiments, "alkylene" refers to a polymethylene group, i.e., -(CH2) n -, and n is a positive integer, preferably 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 5, or 4 to 8. A substituted alkylene is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0024] Alkynyl: As used herein, the term "alkynyl" refers to an alkyl group, as defined herein, having one or more triple bonds. In some embodiments, the term "alkynyl," used alone or as part of a larger moiety, refers to an alkyl group having at least one triple bond and (unless otherwise specified) 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ) refers to an optionally substituted straight or branched chain hydrocarbon group having an alkynyl group. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0025] Amino Acid: In its broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure HN-C(H)(R AA)-COOH, and R AA is an amino acid side chain. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid, in some embodiments, the amino acid is a D-amino acid, and in some embodiments, the amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including the carboxy- and / or amino-terminal amino acids, within a polypeptide may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or a hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the circulating half-life of a polypeptide containing a modified amino acid compared to another polypeptide containing the same unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide containing the modified amino acid compared to a polypeptide containing an otherwise identical, unmodified amino acid. As is clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid, and in some embodiments, to an amino acid residue of a polypeptide.

[0026] Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0027] Aryl: The term "aryl" refers to an alkyl group having a total of 6 to 14 ring members (e.g., C 6-14 "aryl" refers to monocyclic and bicyclic ring systems having at least one ring in the system that is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to aromatic ring systems, including but not limited to phenyl, naphthyl, anthracyl, and the like, which may bear one or more substituents. Unless otherwise specified, "aryl" groups are hydrocarbons.

[0028] Associated: As the term is used herein, two events or entities are "associated" with one another when the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they interact, directly or indirectly, such that they are in and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another but are non-covalently associated, e.g., by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetization, and combinations thereof.

[0029] Biocompatible: As used herein, the term "biocompatible" refers to a material that does not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are "biocompatible" if they are not toxic to cells. In certain embodiments, materials are "biocompatible" if their addition to cells in vitro results in 20% or less cell death and / or if their administration in vivo does not induce significant inflammation or other such adverse effects.

[0030] Biodegradable: As used herein, the term "biodegradable" refers to a material that, when introduced into cells, breaks down (e.g., by cellular mechanisms such as enzymatic degradation, hydrolysis, and / or a combination thereof) into components that the cells can reuse or discard without significant toxic effects to the cells. In certain embodiments, the components produced by the degradation of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymeric materials break down into their component monomers. In some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves cleavage of urethane bonds. Exemplary biodegradable polymers include, for example, but are not limited to, polymers of hydroxy acids such as lactic acid and glycolic acid, including poly(hydroxyl acids), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoic acids, poly(lactide-caprolactone copolymers), mixtures and copolymers thereof. Many naturally occurring Existing polymers are also biodegradable and include, for example, proteins such as albumin, collagen, gelatin, and prolamines, e.g., zein, as well as polysaccharides such as alginate, cellulose derivatives, and polyhydroxyalkanoic acids, e.g., polyhydroxybutyrate mixtures and copolymers thereof. One of ordinary skill in the art will understand or be able to determine when such polymers are their biocompatible and / or biodegradable derivatives (e.g., related to the parent polymer by substantially the same structure, differing only in the substitution or addition of certain chemical groups known in the art).

[0031] Biologically active: As used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed by detection of a direct or indirect product produced by the biological pathway or event of interest.

[0032] Divalent: As used herein, the term "divalent" refers to a chemical moiety that has two points of attachment. For example, a "divalent C 1-8 (or C 1-6 ) saturated or unsaturated, straight or branched hydrocarbon chain" refers to divalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.

[0033] Bridged Bicyclic Ring System: As used herein, the terms "bridged bicyclic ring system," "bridged bicycle," "bridged bicyclic," and "bridged bicyclic ring" refer to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, and a "bridgehead" is any skeletal atom of the ring system (except hydrogen) that is connected to three or more skeletal atoms. In some embodiments, bridged bicyclic groups have 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include the groups described below, where each group is attached to the remainder of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, bridged bicyclic groups are optionally substituted with one or more substituents described for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to: [ka]

[0034] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, tumors may be or include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies particular cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancers may be characterized by solid tumors. In some embodiments, the relevant cancers may be characterized by hematologic tumors. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphomas (Hodgkin's and non-Hodgkin's), myeloma and myeloproliferative disorders, sarcoma, melanoma, adenoma, cancer of solid tissue, squamous cell carcinoma of the mouth, throat, and lung, liver cancer, genitourinary cancers such as prostate, cervix, bladder, uterus, endometrial cancer, renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancer, breast cancer, gastrointestinal cancer, cancer of the nervous system, benign lesions such as papilloma, etc.

[0035] Carrier: As used herein, refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, a carrier may be a sterile liquid, such as water or oil, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, a carrier is or includes one or more solid components.

[0036] Carbocyclyl: As used herein, the terms "carbocyclyl," "carbocycle," and "carbocyclic" refer to a saturated or partially unsaturated cycloaliphatic monocyclic, bicyclic, or polycyclic ring system, as described herein, having 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a "carbocyclyl" (or "alicyclic") refers to an optionally substituted monocyclic C3-C8 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule, or an optionally substituted C6-C8 hydrocarbon. 12 It refers to a bicyclic hydrocarbon. The term "cycloalkyl" refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, the cycloalkyl group has 3 to 6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0037] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, circumstances, sets of conditions, etc. that are not identical to one another, but are sufficiently similar to permit their comparison so that one of skill in the art will understand that reasonable conclusions can be drawn based on the observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few altered characteristics. One of skill in the art will understand what level of identity is required in any given situation for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable in context. For example, one of skill in the art will understand that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical characteristics to justify a reasonable conclusion that differences in results obtained or observed phenomena under or with different sets of circumstances, individuals, or populations are caused by or indicate variations in those altered characteristics.

[0038] Composition: Those skilled in the art will understand that the term "composition" can be used to refer to a separate physical entity that includes one or more specified components. Generally, unless otherwise specified, a composition can be in any form, e.g., gas, gel, liquid, solid, etc.

[0039] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but that other elements or steps may be added within the composition or method. To avoid redundancy, any composition or method described as "comprising" (or "comprises") one or more named elements or steps also describes a corresponding, more limited composition or method that "consists essentially of" the same named elements or steps, meaning that the composition or method includes the essential named elements or steps, and may also include additional elements or steps that do not materially affect the basic and novel characteristics of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes a corresponding, more limited, and close-ended composition or method that "consisting of" (or "consists of") the named elements or steps, to the exclusion of any other named elements or steps. In any composition or method disclosed herein, any known or disclosed equivalent of any named essential element or step may be substituted for that element or step.

[0040] "Improve," "Increase," "Inhibit," or "Reduce": As used herein, the terms "improve," "increase," "inhibit," "reduce," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system under otherwise comparable conditions (e.g., before and / or after) in the absence of a particular agent or treatment, or in the presence of a suitable comparable reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in a comparable system known or expected to respond in a particular manner in the presence of the relevant agent or treatment.

[0041] Determining: Many methodologies described herein include a "determining" step. Those skilled in the art will understand, upon reading this specification, that such "determining" may be utilized or accomplished through the use of any of a variety of techniques available to those skilled in the art, including, for example, the specific techniques explicitly mentioned herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves reviewing and / or manipulating data or information, for example, using a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more characteristics of the sample or entity to a comparable reference.

[0042] Encapsulated: The term "encapsulated" is used herein to refer to a substance that is completely surrounded by another material.

[0043] Excipient: As used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc.

[0044] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from the DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0045] Heteroaryl: The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl," or "heteroaroalkoxy," refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5-6 membered monocyclic heteroaryl or 9-10 membered bicyclic heteroaryl), having 6, 10, or 14 pi electrons shared in the cyclic array, and having 1 to 5 heteroatoms in addition to the carbon atoms. Exemplary heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridonyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. As used herein, the terms "heteroaryl" and "heteroar-" also include groups in which a heteroaromatic ring is fused to one or more aryl, alicyclic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, and benzisoxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted.

[0046] Heteroatom: The term "heteroatom" refers to oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon, the quaternized form of any basic nitrogen, or a substitutable nitrogen of a heterocyclic ring, e.g., N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl).

[0047] Heterocycle: The terms "heterocycle," "heterocyclic," "heterocyclic radical," and "heterocyclic ring" are used interchangeably herein and refer to a stable 3- to 8-membered monocyclic, 7- to 12-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety, as defined above, that is either saturated or partially unsaturated and that, in addition to carbon atoms, has one or more, e.g., 1 to 4, heteroatoms. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, thiamorpholinyl, and [ka] Heterocyclyl groups can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions are independently and optionally substituted. Bicyclic heterocycles also include groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or alicyclic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocycles may also be spirocyclic ring systems (e.g., 7-11 membered spirocyclic fused heterocyclic rings having, in addition to carbon atoms, one or more heteroatoms (e.g., 1, 2, 3, or 4 heteroatoms) as defined above). Bicyclic heterocycles may also be bridged ring systems (e.g., 7-11 membered bridged heterocyclic rings having 1, 2, or 3 bridging atoms).

[0048] In vitro: As used herein, the term "in vitro" refers to events that take place not within a multicellular organism but within an artificial environment, e.g., in a test tube or reaction vessel, cell culture, etc.

[0049] Isolated: As used herein, refers to substances and / or entities that (1) have been separated from at least some of the components with which they are associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) have been designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they are originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a naturally occurring biopolymer, such as a polypeptide or polynucleotide, is considered to be "isolated" when: a) it is not associated, by its origin or source of derivation, with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed by or otherwise associated with components from a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cellular system that produces the polypeptide in nature is considered to be an "isolated" polypeptide.Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide in that it has been separated from other components a) with which it is associated in nature and / or b) with which it was associated when originally produced.

[0050] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0051] Linker: As used herein, this term refers to the portion of a multi-component agent that connects different components to one another. For example, one of skill in the art will understand that polypeptides whose structure comprises two or more functional or organizational domains often include a stretch of amino acids between such domains that connects them to one another. In some embodiments, a polypeptide comprising a linker element "L'" has an overall structure of the general form S1-L'-S2, where S1 and S2, which may be the same or different, represent two domains associated with one another by the linker. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more amino acids in length. In some embodiments, the linker is characterized by providing flexibility to the polypeptide rather than adopting a rigid three-dimensional structure. A variety of different linker elements that can be appropriately used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1 121-1123).

[0052] Nanoparticle: As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, a nanoparticle is a micelle in that it contains an enclosed compartment separated from the bulk solution by a micellar membrane, and is typically composed of amphiphilic entities that surround and enclose the space or compartment (e.g., define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, the lipid nanoparticles described herein can have an average hydrodynamic diameter of about 30 to about 170 nm. In some embodiments, the lipid nanoparticles described herein can have an average hydrodynamic diameter of 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, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 50 nm to 100 nm.

[0053] Nanoparticle composition: As used herein, the term "nanoparticle composition" refers to a composition containing at least one nanoparticle and at least one additional agent or component. In some embodiments, the nanoparticle composition comprises a substantially homogeneous population of the nanoparticles described herein.

[0054] Nucleic Acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to naturally occurring nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product, such as, for example, RNA or a protein. In some embodiments, a nucleic acid comprises one or more introns. In some embodiments, a nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide, hi some embodiments, the nucleic acid has enzymatic activity.

[0055] Parenteral: As used herein, the phrases "parenteral administration" and "parenterally administered" have their art-understood meaning to refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, intracapsular, intrathecal, intraspinal, and intrasternal injection and infusion.

[0056] Patient: As used herein, the term "patient" refers to any organism to which provided compositions are or can be administered, for example, experimental, diagnostic, preventative, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy to diagnose and / or treat the disease, disorder, or condition.

[0057] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue, parenteral administration, e.g., as a sterile solution or suspension, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, or sustained-release formulations, topical application, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity, intravaginally or rectally, e.g., as a pessary, cream, or foam, sublingually, ophthalmically, transdermally, or to the nose, lungs, and other mucosal surfaces.

[0058] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and / or animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0059] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as an encapsulating material that encapsulates a liquid or solid filler, diluent, excipient, or solvent, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter, and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffer solutions, polyesters, polycarbonates, and / or polyanhydrides, and other non-toxic compatible substances employed in pharmaceutical formulations.

[0060] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in pharmaceutical contexts, i.e., salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include non-toxic acid addition salts, which are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbic acid, aspartic acid, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydrogen iodide, 2-hydrogen iodide, 2-hydroxybenzoates ... Examples of suitable salts include hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc.In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates.

[0061] Prevent or prophylaxis: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of a particular disease, disorder, or condition and / or reducing their frequency and / or severity. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

[0062] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will understand that the term "polypeptide" is intended to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those of skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40%, often greater than about 50%, 60%, 70%, or 80% overall sequence identity, and more usually includes at least one region of much higher identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%, with another polypeptide of the same class in one or more highly conserved regions, usually encompassing at least 3-4, and often up to 20 or more amino acids, is encompassed within the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0063] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as individual units. The terms "polypeptide" and "protein" may be used interchangeably when a single polypeptide is an individual functional unit and does not require permanent or temporary physical association with other polypeptides to form an individual functional unit. When an individual functional unit is composed of two or more polypeptides that are physically associated with each other, the term "protein" may be used to refer to the multiple polypeptides that are physically associated and function together as an individual unit. In some embodiments, a protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. One of skill in the art will understand that in some embodiments, the term "protein" may refer to the complete polypeptide chain produced by a cell (e.g., with or without a signal sequence) and / or the form that is active within the cell (e.g., a truncated or complexed form). In some embodiments where a protein is composed of multiple polypeptide chains, such chains may be covalently associated with each other, for example, by one or more disulfide bonds, or may be associated by other means.

[0064] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing greater than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0065] Reference: As used herein, describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially contemporaneously with the testing or determination of interest. In some embodiments, the reference or control is a prior reference or control, optionally embodied in a tangible medium. Typically, as understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular potential reference or control.

[0066] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest, as described herein. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymphatic system, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (blood plasma), interstitial fluid, lymphatic fluid, and / or transcellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or irrigation (e.g., bronchoalveolar, luminal, nasal, ocular, oral, uterine, vaginal, or other lavage or irrigation). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more agents), for example, by filtration using a semi-permeable membrane. Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components, etc.

[0067] Stable Nanoparticle Compositions: As applied to compositions herein, the term "stable" means that the composition maintains one or more aspects of its physical structure (e.g., particle size range and / or distribution) over a period of time. In some embodiments, a stable nanoparticle composition is one in which the mean particle size, maximum particle size, particle size range, and / or particle size distribution (i.e., the percentage of particles above a specified size and / or outside a specified size range) are maintained under specified conditions for a period of time. In some embodiments, a stably provided composition is one in which biologically relevant activity is maintained for a period of time. In some embodiments, the period is at least about 1 hour, and in some embodiments, the period is about 5 hours, about 10 hours, about 1 day, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 8 months, about 10 months, about 12 months, about 24 months, about 36 months, or longer. In some embodiments, the period ranges from about 1 day to about 24 months, from about 2 weeks to about 12 months, or from about 2 months to about 5 months. For example, a nanoparticle composition is stable if a population of nanoparticles is subjected to long-term storage, temperature change, and / or pH change, and the majority of the nanoparticles in the composition maintain a diameter within a specified range. In some embodiments, a stable composition is stable at ambient conditions. In some embodiments, a stable composition is stable under biological conditions (i.e., in phosphate-buffered saline at 37°C).

[0068] Sterolyl: As used herein, the term "sterolyl" refers to a 17-membered fused polycyclic ring moiety that is either saturated or partially unsaturated, substituted with at least one hydroxyl group, and has a single point of attachment to the remainder of the molecule at any substitutable carbon or oxygen atom. In some embodiments, the sterolyl group is a cholesterol group, or a variant or derivative thereof. In some embodiments, the cholesterol group is modified. In some embodiments, the cholesterol group is an oxidized cholesterol group (e.g., oxidized on the beta ring structure or on the hydrocarbon tail structure). In some embodiments, the cholesterol group is an esterified cholesterol group. In some embodiments, the sterolyl group is a phytosterolyl group. Exemplary sterolyl groups include, but are not limited to, 25-hydroxycosteryl (25-OH), 20α-hydroxycosteryl (20α-OH), 27-hydroxycosteryl, 6-keto-5α-hydroxycosteryl, 7-ketocosteryl, 7β-hydroxycosteryl, 7α-hydroxycosteryl, 7β-25-dihydroxycosteryl, beta-sitosterolyl, stigmasteryl, brassicasterolyl, and campesterolyl.

[0069] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including, in some embodiments, prenatal human forms). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who possesses one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or has been administered a diagnosis and / or therapy.

[0070] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the total or near total extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0071] Substituted or Optionally Substituted: As described herein, compounds of the present disclosure may contain optionally substituted and / or substituted moieties. In general, the term "substituted," whether preceded by the term "optionally," means that one or more hydrogens of the specified moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., [ka] At least [ka] refers to, [ka] At least [ka] (refers to "optionally substituted"). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and if more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. Combinations of substituents envisioned by the present disclosure are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" refers to compounds that remain substantially unchanged when subjected to conditions that permit their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Groups described as "substituted" preferably have one to four substituents, and more preferably have one or two substituents. Groups described as "optionally substituted" may be unsubstituted or "substituted," as described above.

[0072] Suitable monovalent substituents include halogen, -(CH2) 0-4 R°, -(CH2) 0-4 OR°, -O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR°, -(CH2) 0-4 CH(OR°)2, which may be substituted with R° -(CH2) 0-4 Ph, R° may be substituted -(CH2) 0-4 O(CH2) 0-1 Ph, may be substituted with R° -CH=CHPh, may be substituted with R° -(CH 0-4 O(CH2) 0-1 -Pyridyl, -NO2, -CN, -N3, -(CH2) 0-4 N(R°)2, -(CH2) 0-4 N(R°)C(O)R°, -N(R°)C(S)R°, -(CH2) 0-4 N(R°)C(O)NR°2, -N(R°)C(S)NR°2, -(CH2) 0-4 N(R°)C(O)OR°, -N(R°)N(R°)C(O)R°, -N(R°)N(R°)C(O)NR°2, -N(R°)N(R°)C(O)OR°, -(CH2)0-4 C(O)R°, -C(S)R°, -(CH2) 0-4 C(O)OR°, -(CH2) 0-4 C(O)SR°, -(CH2) 0-4 C(O)OSiR°3, -(CH2) 0-4 OC(O)R°, -OC(O)(CH2) 0-4 SR°-, -SC(S)SR°, -(CH2) 0-4 SC(O)R°, -(CH2) 0-4 C(O)NR°2, -C(S)NR°2, -C(S)SR°, -SC(S)SR°, -(CH2) 0-4 OC(O)NR°2, -C(O)N(OR°)R°, -C(O)C(O)R°, -C(O)CH2C(O)R°, -C(NOR°)R°, -(CH2) 0-4 SSR°, -(CH2) 0-4 S(O)2R°, -(CH2) 0-4 S(O)2OR°, -(CH2) 0-4 OS(O)2R°, -S(O)2NR°2, -(CH2) 0-4 S(O)R°, -N(R°)S(O)2NR°2, -N(R°)S(O)2R°, -N(OR°)R°, -C(NH)NR°2, -P(O)2R°, -P(O)R°2, -OP(O)R°2, -OP(O)(OR°)2, -SiR°3, -OSiR°3, -(C 1-4 linear or branched alkylene)ON(R°)2, or -(C 1-4 straight or branched chain alkylene)C(O)ON(R°), where each R° can be substituted as defined below and independently represents hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (a 5- to 6-membered heteroaryl ring), or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definitions, two independent occurrences of R°, taken together with their intervening atoms, form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0073] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms) are independently halogen, —(CH) 0-2 R λ ,-(Halo R λ ), -(CH2) 0-2 OH, -(CH2) 0-2 OR λ , -(CH2) 0-2 CH(OR λ )2, -O(HaloR λ ), -CN, -N3, -(CH2) 0-2 C(O)R λ , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR λ , -(CH2) 0-2 C(O)NH2, -(CH2) 0-2 C(O)NHR λ , -(CH2) 0-2 C(O)NR λ 2, -(CH2) 0-2 SR λ , -(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR λ , -(CH2) 0-2 NR λ 2, -NO2, -SiR λ 3. -OSiR λ 3. -C(O)SR λ , -(C 1-4 Straight or branched chain alkylene)C(O)OR λ , or -SSR λ and each R λ is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently represents C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include ═O and ═S.

[0074] Suitable divalent substituents include: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O- or -S(C(R * 2)) 2-3 S- is mentioned, R * Each independent occurrence of may be substituted as defined below with hydrogen, C 1-6 The "optionally substituted" group is selected from an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from aliphatic, nitrogen, oxygen, or sulfur. Preferred divalent substituents attached to adjacent substitutable carbons of the "optionally substituted" group include -O(CR * 2) 2-3 O- and R * Each independent occurrence of may be substituted as defined below with hydrogen, C 1-6 It is selected from an aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0075] R * Suitable substituents on the aliphatic group include halogen, -R λ ,-(Halo R λ ), -OH, -OR λ , -O(HaloR λ ), -CN, -C(O)OH, -C(O)OR λ , -NH2, -NHR λ , -NR λ 2 or -NO2, and each R λ is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently represents C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0076] In some embodiments, suitable substituents on a substitutable nitrogen include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CHC(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † are listed, and each R † are independently hydrogen, C which may be substituted as defined below 1-6 an aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or, regardless of the above definitions, R † which, together with their intervening atoms, form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0077] R † Suitable substituents on the aliphatic group are independently halogen, —R λ ,-(Halo R λ ), -OH, -OR λ , -O(HaloR λ ), -CN, -C(O)OH, -C(O)OR λ , -NH2, -NHR λ , -NR λ 2, or -NO2, and each R λ is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently represents C 1-4Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0078] Susceptible: An individual who is "susceptible" to a disease, disorder, or condition is at risk of developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not exhibit any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to a condition that is associated with the development of the disease, disorder, or condition. In some embodiments, the risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of an individual who suffers from the disease, disorder, or condition).

[0079] Systemic: As used herein, the phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” have their art-understood meanings referring to administration of a compound or composition such that it enters the system of the recipient.

[0080] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0081] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, suppress, alleviate, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of skill in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, and the target cell or tissue. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, ameliorates, alleviates, suppresses, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose, and in some embodiments, multiple unit doses are required to deliver the therapeutically effective amount. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (eg, age, immune system health, etc.), the disease, and the treatment being administered.

[0082] "Tissue" and / or "Organ": As used herein, unless otherwise specified, the terms "tissue" and / or "organ" refer to living cellular material in aggregate form, e.g., small portions of organs, and dispersed cells, e.g., cells dispersed, isolated, and / or grown from muscle, cardiac muscle, liver, or kidney, including bone marrow cells and progeny cells, blood-derived stem cells and progeny, and various other blood elements. In some embodiments, tissue and / or organ refers to kidney, heart, liver, stomach, spleen, pancreas, lung, brain, eye, intestine, bladder, skin or skin tissue, blood vessels, veins, arteries, heart valves, sperm, and oocytes. As used herein, the term "organ" encompasses both solid organs, e.g., kidney, heart, liver, lung, and functional parts of organs, e.g., segments of skin, arteries, venous segments, liver, kidney, transplantable lobes of the lung, etc.

[0083] Treatment: As used herein, "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, ameliorates, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of a subject who exhibits one or more established signs of the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic, and in some embodiments, treatment may be therapeutic.

[0084] For purposes of this disclosure, chemical elements are defined as follows: th In addition, general principles of organic chemistry are identified in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5 th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0085] Unless otherwise stated, structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure, such as the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E stereoisomers. Accordingly, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the present compounds are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure including the replacement of a hydrogen by deuterium or tritium, or the replacement of a carbon by a C- or C-enriched carbon are within the scope of the invention. Such compounds are useful, for example, as analytical tools, probes in biological assays, or as therapeutic agents according to the present invention.

[0086] Detailed Description of Specific Embodiments Among other things, the present invention provides compositions, preparations, nanoparticles, and / or nanomaterials for delivery of therapeutic and / or prophylactic agents. For example, the present disclosure describes lipid compounds for use in the compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, the compositions, preparations, and / or nanomaterials comprise LNPs that carry a cargo to designated target cells, tissues, and / or organs.

[0087] I. Lipid Nanoparticles (LNPs) The present invention provides compositions, preparations, and / or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise one or more components. In some embodiments, the lipid nanoparticles comprise one or more components, such as a compound, an ionizable lipid, a sterol, a conjugate-linker lipid, and a phospholipid.

[0088] In some embodiments, the lipid nanoparticles comprise one or more compounds described herein. In some embodiments, the lipid nanoparticles comprise one or more ionizable lipids described herein. In some embodiments, the lipid nanoparticles comprise one or more sterols described herein. In some embodiments, the lipid nanoparticles comprise one or more conjugate-linker lipids described herein. In some embodiments, the lipid nanoparticles comprise one or more phospholipids described herein.

[0089] A. Compound Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more compounds described herein.

[0090] In some embodiments, the present disclosure provides a compound of formula I: [ka] or a pharmaceutically acceptable salt thereof, Each L 1 and L 1’ are independently —C(O)— or —OC(O)—; Each L 2 and L 2’ are independently optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 is a hydrocarbon chain, L 3 is a covalent bond, —O—, —C(O)O—, —OC(O)—, or —OC(O)O—; R 1 optionally substituted C 1-20 aliphatic, [ka] and L CyA is a covalent bond or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 is a hydrocarbon chain, Cy Ais an optionally substituted 3- to 7-membered ring selected from saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl; L Ra optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 is a hydrocarbon chain, Each R a and R 1’ are independently optionally substituted C 1-20 is aliphatic, Y 1 is —C(O)— or —C(O)O—, Y 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 is a hydrocarbon chain, Y 3 optionally substituted C 1-20 is aliphatic, X 1 is a covalent bond, —O—, or —NR—; X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which one to three methylene units are optionally and independently replaced by -O- or -NR-; 1-12 is a hydrocarbon chain, X 3 is hydrogen or an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; Each R is independently hydrogen or optionally substituted C 1-6 The compound is aliphatic.

[0091] In some embodiments, the disclosure provides a compound of formula I: Each L 1 and L 1’ is -C(O)-, Each L2 and L 2’ are independently divalent saturated or unsaturated straight or branched chain C 1-12 is a hydrocarbon chain, L 3 is a covalent bond, —C(O)O—, or —OC(O)—; R 1 But C 1-20 Alkyl, C 2-20 alkenyl, or [ka] and Each L Ra and R 1’ But independently, C 1-20 Alkyl or C 2-20 is alkenyl, Y 1 is —C(O)— or —C(O)O—, Y 2 but a divalent saturated or unsaturated straight or branched chain C 1-6 is a hydrocarbon chain, Y 3 But C 1-20 Alkyl or C 2-20 is alkenyl, X 1 is -O- or -NR-, X 2 is a divalent saturated or unsaturated, straight or branched chain C in which 1 to 3 methylene units are optionally and independently replaced with -NR- 1-12 is a hydrocarbon chain, X 3 is hydrogen or independently selected from nitrogen, oxygen, and sulfur; C 1-6 Alkyl or C 2-6 a 3- to 7-membered heterocyclyl having 1 to 3 heteroatoms optionally substituted with alkenyl; Each R is independently hydrogen or C 1-6 Alkyl or C 2-6 It is alkenyl.

[0092] In some embodiments, the present disclosure provides a compound of formula II: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 1 , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0093] In some embodiments, the present disclosure provides a compound of formula III: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0094] In some embodiments, the present disclosure provides a compound of formula III-A: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3are as described above and in classes and subclasses herein, both alone and in combination.

[0095] In some embodiments, the present disclosure provides a compound of formula III-B: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0096] In some embodiments, the present disclosure provides a compound of formula III-Bi: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0097] In some embodiments, the present disclosure provides a compound of formula Compound IV: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0098] In some embodiments, the present disclosure provides a compound of formula IV-A: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0099] In some embodiments, the present disclosure provides a compound of formula IV-B: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0100] In some embodiments, the present disclosure provides compounds of formula IV-Bi: [ka] or a pharmaceutically acceptable salt thereof, 1’ , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0101] In some embodiments, the present disclosure provides a compound of formula V: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0102] In some embodiments, the present disclosure provides a compound of formula VA: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0103] In some embodiments, the present disclosure provides a compound of formula VB: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0104] In some embodiments, the present disclosure provides a compound of formula VBi: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0105] In some embodiments, the present disclosure provides a compound of formula VI: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3are as described above and in classes and subclasses herein, both alone and in combination.

[0106] In some embodiments, the present disclosure provides a compound of formula VI-A: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0107] In some embodiments, the present disclosure provides a compound of formula VI-B: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0108] In some embodiments, the present disclosure provides compounds of formula VI-Bi: [ka] or a pharmaceutically acceptable salt thereof, 1 , L 2 , L 2’ , L 3 , R 1 , R1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0109] In some embodiments, the present disclosure provides a compound of formula VII: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0110] In some embodiments, the present disclosure provides a compound of formula VII-A: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0111] In some embodiments, the present disclosure provides a compound of formula VII-B: [ka] or a pharmaceutically acceptable salt thereof, 2 , L2’ , L 3 , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0112] In some embodiments, the present disclosure provides a compound of formula VII-Bi: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0113] In some embodiments, the present disclosure provides a compound of formula VIII: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0114] In some embodiments, the present disclosure provides a compound of formula VIII-A: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0115] In some embodiments, the present disclosure provides a compound of formula VIII-B: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , R, X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0116] In some embodiments, the present disclosure provides a compound of formula VIII-Bi: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , L 3 , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0117] In some embodiments, the present disclosure provides a compound of formula IX: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0118] In some embodiments, the present disclosure provides a compound of formula IX-A: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0119] In some embodiments, the present disclosure provides a compound of formula IX-B: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0120] In some embodiments, the present disclosure provides a compound of formula IX-Bi: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0121] In some embodiments, the present disclosure provides a compound of formula X: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 1 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0122] In some embodiments, the present disclosure provides a compound of formula XA: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0123] In some embodiments, the present disclosure provides a compound of formula XB: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , R, Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0124] In some embodiments, the present disclosure provides a compound of formula XBi: [ka] or a pharmaceutically acceptable salt thereof, 2 , L 2’ , R 1 , R 1’ , Y 2 , Y 3 , X 2 , and X 3 are as described above and in classes and subclasses herein, both alone and in combination.

[0125] In some embodiments of any of the formulas described herein, L 1 is —C(O)— or —OC(O)—. In some embodiments, L 1 is —C(O)—. In some embodiments, L 1 is -OC(O)-.

[0126] In some embodiments of any of the formulas described herein, L 1’ is —C(O)— or —OC(O)—. In some embodiments, L 1’ is —C(O)—. In some embodiments, L 1’ is -OC(O)-.

[0127] In some embodiments, the disclosure provides compounds of formula I, wherein each L 1 and L 1’ is -C(O)-.

[0128] In some embodiments, the disclosure provides compounds of formula I, wherein each L 2 and L 2’ are independently divalent saturated or unsaturated straight or branched chain C 1-12 It is a hydrocarbon chain.

[0129] In some embodiments of any of the formulas described herein, L 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 In some embodiments, L 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L 2 optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 optionally substituted divalent saturated straight or branched chain C 4-8 In some embodiments, L 2 optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, L 2 but divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 but divalent saturated straight or branched chain C 4-8 In some embodiments, L 2 but divalent saturated straight or branched chain C 1-6 In some embodiments, L 2 is -CH-. In some embodiments, L 2is —(CH)— or —(CH)—. In some embodiments, L 2 is -(CH2)6- or -(CH2)7-.

[0130] In some embodiments of any of the formulas described herein, L 2’ optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L 2’ optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L 2’ optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, L 2’ optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, L 2’ optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, L 2’ optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, L 2’ but divalent saturated straight or branched chain C 1-12 In some embodiments, L 2’ but divalent saturated straight or branched chain C 1-6 In some embodiments, L 2’ but divalent saturated straight or branched chain C 1-3 In some embodiments, L 2’ is -(CH2)2-.

[0131] In some embodiments of any of the formulas described herein, L 3 is a covalent bond, In some embodiments, L 3is a covalent bond, —C(O)O—, or —OC(O)—. In some embodiments, L 3 is a covalent bond. In some embodiments, L 3 is —O—, —C(O)O—, —OC(O)—, or —OC(O)O—. In some embodiments, L 3 is —C(O)O— or —OC(O)—. In some embodiments, L 3 is —O—. In some embodiments, L 3 is —C(O)O—. In some embodiments, L 3 is —OC(O)—. In some embodiments, L 3 is -OC(O)O-.

[0132] In some embodiments of any of the formulas described herein, R 1 optionally substituted C 1-20 aliphatic, [ka] In some embodiments, R 1 optionally substituted C 1-20 In some embodiments, R 1 but, [ka] In some embodiments, R 1 but, [ka] In some embodiments, R 1 But C 1-20 Aliphatic or [ka] In some embodiments, R 1 optionally substituted C 6-12 In some embodiments, R 1 optionally substituted C 6-12In some embodiments, R 1 optionally substituted C 6-12 In some embodiments, R is alkenyl. 1 optionally substituted C 12-20 In some embodiments, R 1 optionally substituted C 12-20 In some embodiments, R 1 optionally substituted C 12-20 In some embodiments, R is alkenyl. 1 But C 1-20 In some embodiments, R 1 But C 6-12 In some embodiments, R 1 But C 6-12 In some embodiments, R 1 But C 6-12 In some embodiments, R is alkenyl. 1 But C 12-20 In some embodiments, R 1 But C 12-20 In some embodiments, R 1 But C 12-20 In some embodiments, R is alkenyl. 1 but, [ka] is.

[0133] In some embodiments of any of the formulas described herein, L CyA is a covalent bond or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 It is a hydrocarbon chain.

[0134] In some embodiments of any of the formulas described herein, Cy Ais a 3- to 7-membered optionally substituted ring selected from saturated or partially unsaturated carbocyclyl, 1-adamantyl, 2-adamantyl, sterolyl, and phenyl.

[0135] In some embodiments of any of the formulas described herein, L Ra optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L Ra Divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L Ra optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L Ra Divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L Ra optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, L Ra Divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, L Ra is -CH2- or -(CH2)2-.

[0136] In some embodiments of any of the formulas described herein, each R a are independently optionally substituted C 1-20 In some embodiments, each R a are independently optionally substituted C 1-12 In some embodiments, each R a are independently optionally substituted C 1-12 In some embodiments, each R a are independently optionally substituted C 1-12 In some embodiments, each R is an alkenyl. a are independently optionally substituted C4-10 In some embodiments, R a optionally substituted C 6-12 In some embodiments, R a optionally substituted C 6-12 In some embodiments, R a optionally substituted C 6-12 In some embodiments, R is alkenyl. a optionally substituted C 7-9 In some embodiments, R a optionally substituted C 7-9 In some embodiments, R a optionally substituted C 7-9 In some embodiments, each R is an alkenyl. a But independently, C 1-20 In some embodiments, R a But C 6-12 In some embodiments, R a But C 6-12 In some embodiments, R a But C 6-12 In some embodiments, R is alkenyl. a But C 7-9 In some embodiments, R a But C 7-9 In some embodiments, R a But C 7-9 In some embodiments, R is alkenyl. a but, [ka] is.

[0137] In some embodiments, -L 2 -L 3 -R 1 but, [ka] In some embodiments, -L 2 -L 3 -R 1 but, [ka] isn't it.

[0138] In some embodiments of any formula described herein, each R 1’ are independently optionally substituted C 1-20 In some embodiments, R 1’ optionally substituted C 6-12 In some embodiments, R 1’ optionally substituted C 6-12 In some embodiments, R 1’ optionally substituted C 6-12 In some embodiments, R is alkenyl. 1’ optionally substituted C 7-9 In some embodiments, R 1’ optionally substituted C 7-9 In some embodiments, R 1’ optionally substituted C 7-9 In some embodiments, each R is an alkenyl. 1’ But independently, C 1-20 In some embodiments, R 1’ But C 6-12 In some embodiments, R 1’ But C 6-12 In some embodiments, R 1’ But C 6-12 In some embodiments, R is alkenyl. 1’ But C 7-9 In some embodiments, R 1’ But C 7-9 In some embodiments, R 1’ But C 7-9 In some embodiments, R is alkenyl.1’ but, [ka] is.

[0139] In some embodiments of any of the formulas described herein, Y 1 is —C(O)— or —C(O)O—. In some embodiments, Y 1 is —C(O)—. In some embodiments, Y 1 is -C(O)O-.

[0140] In some embodiments of any of the formulas described herein, Y 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, Y is a hydrocarbon chain. 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, Y is a hydrocarbon chain. 2 optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, Y is a hydrocarbon chain. 2 optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, Y is a hydrocarbon chain. 2 but a divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, Y is a hydrocarbon chain. 2 but divalent saturated straight or branched chain C 1-6 In some embodiments, Y is a hydrocarbon chain. 2 but divalent saturated straight or branched chain C 1-3 In some embodiments, Y is a hydrocarbon chain. 2 is -CH2- or -(CH2)2-.

[0141] In some embodiments of any of the formulas described herein, Y 3 optionally substituted C 1-20 In some embodiments, Y is aliphatic.3 But C 1-20 In some embodiments, Y is aliphatic. 3 optionally substituted C 1-12 In some embodiments, Y is aliphatic. 3 optionally substituted C 1-12 In some embodiments, Y is alkyl. 3 optionally substituted C 4-8 In some embodiments, Y is aliphatic. 3 optionally substituted C 4-8 In some embodiments, Y is alkyl. 3 optionally substituted C 1-6 In some embodiments, Y is alkyl. 3 is —CH3, —CH2CH3, —(CH2)2CH3, —(CH2)3CH3, —(CH2)4CH3, —(CH2)5CH3, —(CH2)6CH3, or —(CH2)7CH3. In some embodiments, Y 3 is -CH3. In some embodiments, Y 3 is -CH2CH3. In some embodiments, Y 3 is —(CH 2 ) 2 CH 3 . In some embodiments, Y 3 is —(CH 2 ) 3 CH 3 . In some embodiments, Y 3 is —(CH 2 ) 4 CH 3 . In some embodiments, Y 3 is —(CH 2 ) 5 CH 3 . In some embodiments, Y 3 is —(CH 2 ) 6 CH 3 . In some embodiments, Y 3 is -(CH2)7CH3.

[0142] In some embodiments of any of the formulas described herein, X 1 is a covalent bond, —O—, or —NR—. In some embodiments, X 1 is a covalent bond. In some embodiments, X 1 is —O— or —NR—. In some embodiments, X 1is —O—. In some embodiments, X 1 However, it is -NR-.

[0143] In some embodiments of any of the formulas described herein, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which one to three methylene units are optionally and independently replaced by -O- or -NR-; 1-12 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which one to two methylene units are optionally and independently replaced by -O- or -NR-; 1-6 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, X is a hydrocarbon chain. 2 optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, X is a hydrocarbon chain. 2 but divalent saturated straight or branched chain C 1-12 In some embodiments, X is a hydrocarbon chain. 2 but divalent saturated straight or branched chain C 1-6 In some embodiments, X is a hydrocarbon chain. 2 but divalent saturated straight or branched chain C 1-3In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which one to three methylene units are independently replaced by -O- or -NR-; 1-12 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted, divalent, saturated or unsaturated, straight or branched chain C in which one to two methylene units are independently replaced by -O- or -NR-. 4-8 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which two methylene units are independently replaced by -NR- 4-8 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C in which one methylene unit is replaced by -NR- 4-8 In some embodiments, X is a hydrocarbon chain. 2 is a divalent saturated or unsaturated, straight or branched chain C in which 1 to 3 methylene units are optionally and independently replaced with -NR- 1-12 In some embodiments, X is a hydrocarbon chain. 2 is a divalent saturated or unsaturated, straight or branched chain C in which one to two methylene units are optionally and independently replaced by -NR- 1-12 In some embodiments, X is a hydrocarbon chain. 2 is a divalent saturated or unsaturated, straight or branched chain C in which one methylene unit is replaced by -NR- 4-8 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted, divalent, saturated, straight-chain or branched chain C in which one to three methylene units are independently replaced by -O- or -NR-; 1-12 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted, divalent, saturated, straight-chain or branched-chain C in which one to two methylene units are independently replaced by -O- or -NR-; 4-8 In some embodiments, X is a hydrocarbon chain.2 is an optionally substituted, divalent, saturated, straight-chain or branched-chain C in which two methylene units are independently replaced by -NR-; 4-8 In some embodiments, X is a hydrocarbon chain. 2 is an optionally substituted, divalent, saturated, straight-chain or branched chain C in which one methylene unit is replaced by -NR- 4-8 In some embodiments, X is a hydrocarbon chain. 2 is a divalent saturated straight or branched chain C in which one methylene unit is replaced by -NR- 4-8 It is a hydrocarbon chain.

[0144] In some embodiments of any of the formulas described herein, X 3 is hydrogen or an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a 3- to 7-membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5- to 6-membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is hydrogen or independently selected from nitrogen, oxygen, and sulfur; C 1-6 In some embodiments, X is an aliphatic optionally substituted 3-7 membered heterocyclyl having 1-3 heteroatoms. 3 is hydrogen. In some embodiments, X 3 is an optionally substituted ring selected from a 3-7 membered saturated or partially unsaturated carbocyclyl, a 3-7 membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or a 5-6 membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is an optionally substituted 3-7 membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3is an optionally substituted 5-6 membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-6 membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-6 membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, substituted with -R°, and R° is C 1-6 In some embodiments, X is aliphatic (e.g., methyl or ethyl). 3 is an optionally substituted 5-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 5-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur substituted with —R°, and R° is C 1-6 In some embodiments, X is aliphatic (e.g., methyl or ethyl). 3 is an optionally substituted 5-membered heterocyclyl having 1-2 nitrogen atoms. 3 is a 5-membered heterocyclyl having 1 to 2 nitrogen atoms. 3 is a 5-membered heterocyclyl having 1 to 2 nitrogen atoms substituted with -R°, and R° is C 1-6 In some embodiments, X is aliphatic (e.g., methyl or ethyl). 3 is an optionally substituted 6-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3 is a 6-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur. 3is a 6-membered heterocyclyl having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur substituted with —R°, and R° is C 1-6 In some embodiments, X is aliphatic (e.g., methyl or ethyl). 3 is optionally substituted pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. 3 is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl. 3 is pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl substituted with —R°, and R° is C 1-6 It is aliphatic (eg, methyl or ethyl).

[0145] In some embodiments, -X 2 -X 3 but, [ka] is.

[0146] In some embodiments, X 2 and X 3 At least one of the groups contains at least one ionizable nitrogen atom.

[0147] In some embodiments of any of the formulas described herein, each R is independently hydrogen or optionally substituted C 1-6 In some embodiments, each R is independently hydrogen or C 1-6 In some embodiments, R is an optionally substituted C 1-6 In some embodiments, R is C 1-6 In some embodiments, R is an optionally substituted C 1-3 In some embodiments, R is C 1-3 In some embodiments, R is —CH 3 .

[0148] In some embodiments, R° is methyl. In some embodiments, R° is ethyl.

[0149] In some embodiments of any formula described herein, the present disclosure provides compounds that include an ionizable nitrogen atom, the pKa of whose conjugate acid is from about 4 to about 12.

[0150] In some embodiments, the disclosure provides a compound selected from Table 1. [Table 1] TIFF2025528020000056.tif226165TIFF2025528020000057.tif251165TIFF2025528020000058.tif230165TIFF2025528020000059.tif243165TIFF2025528020000060.tif239165TIFF2025528020000061.tif159165 or a pharmaceutically acceptable salt thereof.

[0151] It will be understood that unless otherwise specified or prohibited by the above definitions of any of Formulas I, II, III, III-A, III-B, III-Bi, IV, IV-A, IV-B, IV-Bi, V, VA, VB, VBi, VI, VI-A, VI-B, VI-Bi, VII, VII-A, VII-B, VII-Bi, VIII, VIII-A, VIII-B, VIII-Bi, IX, IX-A, IX-B, IX-Bi, X, XA, XB, and XBi, the variables L, ... 1 , L 1’ , L 2 , L 2’ , L 3 , L CyA , Cy A , L Ra , R a , R 1 , R 1’ , R, Y 1 , Y2 , Y 3 , X 1 , X 2 , and X 3 applies to compounds of any of formulas I, II, III, III-A, III-B, III-Bi, IV, IV-A, IV-B, IV-Bi, V, VA, VB, VBi, VI, VI-A, VI-B, VI-Bi, VII, VII-A, VII-B, VII-Bi, VIII, VIII-A, VIII-B, VIII-Bi, IX, IX-A, IX-B, IX-Bi, X, XA, XB, and XBi, alone and in combination.

[0152] In some embodiments, provided compounds are provided and / or utilized in a salt form (e.g., a pharmaceutically acceptable salt form). Reference to a compound provided herein is understood to include reference to a salt thereof, unless otherwise indicated.

[0153] It will be understood that throughout this disclosure, unless otherwise indicated, a reference to a compound of Formula I is intended to also include any of Formulas II, III, III-A, III-B, III-Bi, IV, IV-A, IV-B, IV-Bi, V, VA, VB, VBi, VI, VI-A, VI-B, VI-Bi, VII, VII-A, VII-B, VII-Bi, VIII, VIII-A, VIII-B, VIII-Bi, IX, IX-A, IX-B, IX-Bi, X, XA, XB, and XBi, and species of compounds of such formulae disclosed herein.

[0154] In some embodiments, the present disclosure encompasses the recognition that provided compounds exhibit certain desirable characteristics, e.g., compared to a reference compound or other known compounds. For example, in some embodiments, provided compounds may exhibit more potent delivery to various cell types in one or more experiments described herein and / or have one or more other characteristics that make them more suitable for delivery of a cargo, such as a therapeutic or prophylactic agent, than other known compounds. While not wishing to be bound by any particular theory, the present disclosure encompasses the recognition that provided compounds characterized by comprising a triol core and / or a biodegradable tail may exhibit certain more desirable characteristics (e.g., more potent delivery to various cell types in one or more experiments described herein) than corresponding compounds lacking the same characteristics. In certain embodiments, the present disclosure encompasses the recognition that provided compounds characterized by comprising a triol core and a biodegradable tail may exhibit certain more desirable characteristics (e.g., more potent delivery to various cell types in one or more experiments described herein) than corresponding compounds lacking the same combination of characteristics.

[0155] B. Preparation of the Provided Compounds The compounds provided may generally be made by the processes illustrated in the following schemes and examples.

[0156] C. Ionizable lipids Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more ionizable lipids described herein.

[0157] For example, in some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials having about 50 mole percent or less of ionizable lipids, based on the total moles of the lipid nanoparticle's components, may be useful and / or important for the functional activity of the lipid nanoparticles, such as the desired targeting, stabilization, and drug delivery efficacy described herein.

[0158] In some embodiments, the ionizable lipid may comprise an amine-containing group on the head group. In some embodiments, the ionizable lipid is or comprises a compound described herein. In some embodiments, the ionizable lipid is present in the lipid nanoparticle (LNP) preparation at about 30 molar percent to about 70 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 33 molar percent to about 60 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 34 molar percent to about 55 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 33 molar percent to about 51 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 34.7 molar percent, based on the total moles of the lipid nanoparticle components. In some embodiments, the ionizable lipid is present at about 50 molar percent, based on the total moles of the lipid nanoparticle components.

[0159] Specifically, in some embodiments, the lipid nanoparticle composition comprises an ionizable lipid. In some embodiments, the lipid nanoparticle preparation comprises an ionizable lipid, a phospholipid, a conjugate-linker lipid, and cholesterol. In some embodiments, the ionizable lipid is or comprises a structure according to a compound described herein. In some embodiments, the ionizable lipid is present in the LNP preparation at about 30 mole percent to about 70 mole percent, based on the total moles of the lipid nanoparticle components.

[0160] D. Sterols Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more sterols described herein.

[0161] In some embodiments, the sterol is cholesterol, or a variant or derivative thereof. In some embodiments, the cholesterol is modified. In some embodiments, the cholesterol is oxidized cholesterol. In some embodiments, the cholesterol is esterified cholesterol. Unmodified cholesterol can be enzymatically acted upon to form variants with oxidized side chains or rings. In some embodiments, cholesterol can be oxidized on the beta ring structure or hydrocarbon tail structure. In some embodiments, the sterol is a phytosterol. Exemplary sterols contemplated for use in the disclosed lipid nanoparticles include, but are not limited to, 25-hydroxycholesterol (25-OH), 20α-hydroxycholesterol (20α-OH), 27-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-ketocholesterol, 7β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, side-chain oxidized cholesterol can enhance cargo delivery compared to other cholesterol variants. In some embodiments, the cholesterol is unmodified cholesterol.

[0162] In some embodiments, the LNP preparation comprises about 20 molar percent to about 50 molar percent sterols. In some embodiments, the LNP preparation comprises about 38 molar percent sterols. In some embodiments, the LNP preparation comprises about 38.5 molar percent sterols. In some embodiments, the LNP preparation comprises about 33.8 molar percent cholesterol.

[0163] E. Conjugate-Linker Lipid Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that include one or more conjugate-linker lipids described herein.

[0164] In some embodiments, the conjugate-linker lipid is or includes a polyethylene glycol (PEG) lipid or a PEG-modified lipid. In some embodiments, PEG or a PEG-modified lipid may alternatively be referred to as a PEGylated lipid or a PEG lipid. The inclusion of a PEGylated lipid may be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible, in that the PEG moiety is gradually released in the blood circulation. Exemplary PEG lipids include, but are not limited to, PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20. PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified diacylglycerol (PEG-DAG), PEG-modified dialkylglycerol, and mixtures thereof. For example, in some embodiments, the PEG lipid may be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE lipid.

[0165] In some embodiments, the conjugate-linker lipid comprises a polyethylene glycol lipid. In some embodiments, the conjugate-linker lipid comprises DiMystyrlGlycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxypolyethylene glycol (DPG-PEG), or 1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, the conjugate-linker lipid has an average molecular weight of about 500 Da to about 5000 Da. In some embodiments, the conjugate-linker lipid has an average molecular weight of about 2000 Da. In some embodiments, the LNP preparation comprises about 0 mole percent to about 5 mole percent of the conjugate-linker lipid. In some embodiments, the LNP preparation comprises about 1.5 mole percent of the conjugate-linker lipid. In some embodiments, the LNP preparation comprises about 3 mole percent of the conjugate-linker lipid.

[0166] F. Phospholipids Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials comprising one or more phospholipids described herein. In some embodiments, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials comprising one or more (poly)unsaturated lipids.

[0167] In some embodiments, one or more phospholipids may be assembled into one or more lipid bilayers. In some embodiments, one or more phospholipids may comprise a phospholipid moiety. In some embodiments, one or more phospholipids may comprise one or more fatty acid moieties. In some embodiments, one or more phospholipids may comprise a phospholipid moiety and one or more fatty acid moieties. In some embodiments, the phospholipid moiety includes, but is not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. In some embodiments, the fatty acid moiety includes, but is not limited to, 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, and docosahexaenoic acid. Non-natural species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with or crosslinked to one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced with triple bonds). Under appropriate reaction conditions, alkyne groups can undergo copper-catalyzed cycloaddition when exposed to azide. Such reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to promote membrane permeation or cell recognition, or for conjugating nanoparticle compositions to useful components (e.g., dyes), such as targeting moieties or imaging moieties.

[0168] Exemplary phospholipids include, but are not limited to, 1,2-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-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerophosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerophosphocholine (DLPC), 1,2-dilinoleoyl-sn-glycerophosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerophosphocholine (DLPC), 1,2-dilinoleo ... glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemicosinoyl-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), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl In some embodiments, the phospholipid may be DSPC, DMPC, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DMPC.

[0169] In some embodiments, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

[0170] G.Diameter Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of 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, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

[0171] In some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of 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, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, or any range having endpoints defined by any two of the foregoing values. For example, in some embodiments, the lipid nanoparticles described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

[0172] H. Polydispersity Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI of about 0.3 or any range having endpoints defined by any two of the above values: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, etc. For example, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI of about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0173] In some embodiments, the lipid nanoparticles described herein have a PDI of about 0.01 to about 0.3. In some embodiments, the lipid nanoparticles described herein have a PDI of about 0.3, or any range having endpoints defined by any two of the above values: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25. For example, in some embodiments, the lipid nanoparticles described herein have a PDI of about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0174] I. Encapsulation Efficiency Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that provide encapsulation efficiencies of about 80% to about 100%. In some embodiments, the encapsulation efficiencies of the compositions, preparations, nanoparticles, and / or nanomaterials described herein are about 100% or any range having endpoints defined by any two of the above values. For example, in some embodiments, the encapsulation efficiencies of the compositions, preparations, nanoparticles, and / or nanomaterials described herein are about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles, and / or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0175] In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 80%, or any range having endpoints defined by any two of the above values: 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%. For example, in some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, the encapsulation efficiency of the lipid nanoparticles described herein is at least about 98%, or 99%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

[0176] J.pKa Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials having a pKa of about 5 to about 9. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a pKa of about 5.0, or any range having endpoints defined by any two of the values ​​set forth above: 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein have a pKa of about 6.0, 9.0, or any range having endpoints defined by any two of the values ​​set forth above. 6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,

[0177] In some embodiments, the lipid nanoparticles described herein have a pKa of about 5 to about 9. In some embodiments, the lipid nanoparticles described herein have a pKa of about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints defined by any two of the above values. In some embodiments, the lipid nanoparticles described herein have a pKa of about 6.0, or any range having endpoints defined by any two of the above values. 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0

[0178] II. Exemplary LNP Preparations The present invention provides compositions, preparations, nanoparticles, and / or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle preparation comprises about 30 mole percent to about 70 mole percent ionizable lipid, about 5 mole percent to about 25 mole percent phospholipid, about 25 mole percent to about 45 mole percent cholesterol, and about 0 mole percent to about 5 mole percent conjugate-linker lipid.

[0179] In some embodiments, the lipid nanoparticle preparation comprises about 45 molar percent ionizable lipid, about 9 molar percent phospholipid, about 44 molar percent cholesterol, and about 2 molar percent conjugate-linker lipid, hi some embodiments, the lipid nanoparticle preparation comprises about 50 molar percent ionizable lipid, about 9 molar percent phospholipid, about 38 molar percent cholesterol, and about 3 molar percent conjugate-linker lipid.

[0180] In some embodiments, the lipid nanoparticle preparation comprises about 47.5 molar percent ionizable lipid, about 10 molar percent phospholipid, about 40 molar percent cholesterol, and about 2.5 molar percent conjugate-linker lipid.

[0181] In some embodiments, the lipid nanoparticle preparation comprises about 50 molar percent ionizable lipid, about 10 molar percent phospholipid, about 38.5 molar percent cholesterol, and about 1.5 molar percent conjugate-linker lipid.

[0182] In some embodiments, the lipid nanoparticle preparation comprises about 40 mole percent to about 60 mole percent of an ionizable lipid of any provided compound, about 5 mole percent to about 15 mole percent 1-2-distearoyl-sn-glycero-3-phosphocholine, about 1 mole percent to about 5 mole percent C14PEG2000, and about 30 mole percent to about 47 mole percent cholesterol, based on the total moles of these four components.

[0183] In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 2:1 to 50:1. In some embodiments, the LNP preparation comprises a mass ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 9:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, or about 50:1. In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):mRNA of about 11.7:1 to 19:1.

[0184] In some embodiments, the lipid nanoparticle preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 2:1 to 50:1. In some embodiments, the LNP preparation comprises a mass ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, about 50:1, about 51:1, about 52:1, about 53:1, about 54:1, about 55:1, about 56:1, about 57:1, about 58:1, about 59:1, about 60:1, about 61:1, about 62:1, about 63:1, about 64:1, about 65:1, about 66:1, about 67 In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 9:1, about 30:1, about 31:1, about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1, about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1, about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1, or about 50:1. In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid): siRNA of about 11.7:1 to 19:1.

[0185] In some embodiments, the lipid nanoparticle preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid):NA of about 2:1 to 50:1. In some embodiments, the LNP preparation comprises a mass ratio of about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about 14:1, about 15:1, about 16:1, about 17:1, about 18:1, about 19:1, about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1, about 26:1, about 27:1, about 28:1, about In some embodiments, the lipid nanoparticle (LNP) preparation comprises a mass ratio of (ionizable lipid, cholesterol, lipid-PEG, and phospholipid) to NA of about 11.7:1 to 40:1.

[0186] In some embodiments, the NA comprises a base editor described herein and a gRNA. In some embodiments, the mass ratio of the base editor:gRNA is 1:1. In some embodiments, the mass ratio of the base editor:gRNA is 2:1. In some embodiments, the mass ratio of the base editor:gRNA is 3:1. In some embodiments, the mass ratio of the base editor:gRNA is 4:1. In some embodiments, the mass ratio of the base editor:gRNA is 5:1. In some embodiments, the mass ratio of the base editor:gRNA is 6:1. In some embodiments, the mass ratio of the base editor:gRNA is 7:1. In some embodiments, the mass ratio of the base editor:gRNA is 8:1. In some embodiments, the mass ratio of the base editor:gRNA is 9:1. In some embodiments, the mass ratio of the base editor:gRNA is 10:1. In some embodiments, the mass ratio of the base editor:gRNA is 1:2. In some embodiments, the mass ratio of the base editor:gRNA is 1:3. In some embodiments, the mass ratio of base editor:gRNA is 1:4. In some embodiments, the mass ratio of base editor:gRNA is 1:5. In some embodiments, the mass ratio of base editor:gRNA is 1:6. In some embodiments, the mass ratio of base editor:gRNA is 1:7. In some embodiments, the mass ratio of base editor:gRNA is 1:8. In some embodiments, the mass ratio of base editor:gRNA is 1:9. In some embodiments, the mass ratio of base editor:gRNA is 1:10.

[0187] III. Pharmaceutical Compositions The present invention provides compositions, preparations, nanoparticles, and / or nanomaterials, including pharmaceutical compositions. In particular, in some embodiments, the pharmaceutical compositions comprise the lipid nanoparticles and lipid nanoparticle preparations described herein. For example, in some embodiments, the lipid nanoparticles and lipid nanoparticle preparations described herein can be formulated in whole or in part as pharmaceutical compositions.

[0188] In some embodiments, a pharmaceutical composition can include one or more nanoparticle compositions described herein. For example, a pharmaceutical composition can include one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents, including, but not limited to, one or more nucleic acids of different types or encoding different agents. In some embodiments, a pharmaceutical composition includes one or more pharmaceutically acceptable excipients or auxiliary ingredients, including, but not limited to, a pharmaceutically acceptable carrier.

[0189] The pharmaceutical composition can be administered to a subject. In some embodiments, the pharmaceutical composition is administered as described herein. In some in vivo approaches, the nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount as described herein.

[0190] In some embodiments, one skilled in the art will be able to devise appropriate dosage levels and administration regimens using the pharmaceutical compositions described herein for the treatment of various conditions in various patients, taking into account the recipient's treatment status, age, and general health. For example, in some embodiments, the selected dosage will depend on the desired therapeutic effect, the route of administration, and the desired duration of treatment. In some embodiments, a dosage level of about 0.001 mg to about 5 mg of nucleic acid per kg of body weight is generally administered per administration to a mammal. More specifically, in some embodiments, the preferred dose of nucleic acid in the disclosed nanoparticles is about 0.1 mg / kg to about 1.0 mg / kg. For the disclosed nanoparticles, a dosage level of about 0.2 mg to about 100 mg of the four components (ionizable lipid, cholesterol, conjugate-linker conjugate, and phospholipid) per kg of body weight is generally administered to a mammal. More specifically, in some embodiments, the preferred dose of the disclosed nanoparticles is about 0.5 mg / kg to about 5 mg / kg of the four components per kg of body weight.

[0191] In some embodiments, the pharmaceutical compositions described herein are administered locally, for example, by direct injection into the area to be treated. Typically, the injection causes a greater increase in local concentration of the composition than can be achieved by systemic administration. In some embodiments, the pharmaceutical compositions described herein can be combined with a matrix described herein to help create an increased local concentration of the polypeptide composition by reducing passive diffusion of the polypeptide from the area to be treated.

[0192] A. Preparations for Parenteral Administration In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein, including those containing lipid nanoparticles, are administered in aqueous solution via parenteral injection. In some embodiments, the preparations may also be in the form of a suspension or emulsion. Generally, pharmaceutical compositions are provided that contain an effective amount of lipid nanoparticles, and optionally contain pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions optionally contain one or more of the following additives: diluents, sterile water, buffered saline solutions of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, surfactants and solubilizers (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.The formulation can be lyophilized and redissolved / resuspended immediately before use.The formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0193] B. Controlled Delivery Polymer Matrix In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein can also be administered in controlled-release formulations. In some embodiments, controlled-release polymeric devices can be fabricated for extended systemic release following implantation of the polymeric device (e.g., rod, cylinder, film, disk, etc.) or injection (e.g., microparticles). In some embodiments, the matrix can be in the form of microparticles, such as microspheres. In some embodiments, the drug is dispersed within a solid polymer matrix or microcapsules. In some embodiments, the core is of a different material than the polymer shell of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, the peptide is dispersed or suspended in the core, which can be liquid or solid in nature, of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. In some embodiments, the polymers can be cast as thin slabs or films in the nanometer to four centimeter range, powders produced by milling or other standard techniques, or even gels, such as hydrogels.

[0194] In some embodiments, non-biodegradable matrices are used to deliver the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, biodegradable matrices are used to deliver the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, biodegradable matrices are preferred. In some embodiments, biodegradable matrices comprise natural or synthetic polymers. In some embodiments, synthetic polymers are preferred due to their better degradation and release profile characteristics. In some embodiments, the polymer is selected based on the period over which release is desired. In some embodiments, linear release may be most useful, while in other embodiments, pulsed or "bulk release" may provide more effective results. In some embodiments, the polymer may be in the form of a hydrogel (typically absorbing up to about 90% water by weight) and may optionally be crosslinked with multivalent ions or polymers.

[0195] The matrix can be formed by solvent evaporation, spray drying, solvent extraction, and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed to make microspheres for drug delivery, as described, for example, by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988), the disclosures of which are incorporated herein by reference in their entireties.

[0196] In some embodiments, the described compositions, preparations, nanoparticles, and / or nanomaterials can be formulated for local release to treat the area of ​​implantation or injection, or for systemic delivery, which typically delivers a much smaller dose than for systemic treatment. They can be implanted or injected subcutaneously into muscle, fat, or swallowed.

[0197] C. Cargo Among other things, the present invention provides compositions, preparations, nanoparticles, and / or nanomaterials comprising the cargo described herein. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials comprise a therapeutic or prophylactic agent for delivery to a subject. In some embodiments, the therapeutic or prophylactic agent is encapsulated by the lipid nanoparticles. In some embodiments, the lipid nanoparticles are loaded with one or more nucleic acids.

[0198] D. Therapeutic and / or Prophylactic Agents The cargo delivered via the LNP preparation can be a biologically active agent. In some embodiments, the cargo can be an mRNA, a guide RNA (gRNA), a nucleic acid, an RNA-guided DNA-binding agent, an expression vector, a template nucleic acid, an antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof), cholesterol, a hormone, a peptide, a protein, a chemotherapeutic agent and other types of anti-tumor agent, a low molecular weight drug, a vitamin, a cofactor, a nucleoside, a nucleotide, an oligonucleotide, an enzymatic nucleic acid, an antisense nucleic acid, a triplex-forming oligonucleotide, an antisense DNA or RNA composition, a chimeric DNA:RNA composition, an allozyme, an aptamer, a ribozyme, a decoy and its analogs, a plasmid, and other types of vectors. The compound may be or include one or more biologically active agents, such as targets, small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNAs), short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and "self-replicating RNAs" (which encode replicase enzyme activity and can direct their own replication or amplification in vivo), peptide nucleic acids (PNAs), locked nucleic acid ribonucleotides (LNAs), morpholino nucleotides, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), sisiRNAs (small internal segmented interfering RNAs), and iRNAs (asymmetric interfering RNAs). The above list of biologically active agents is illustrative only and is not intended to be limiting. Such compounds may be purified or partially purified, naturally occurring or synthetic, and may be chemically modified.

[0199] The cargo delivered via the LNP preparation may be RNA, such as an mRNA molecule encoding a protein of interest. For example, in some embodiments, mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA binder, or a Cas nuclease is described herein. Provided are LNP preparations containing a Cas nuclease mRNA, e.g., a Class 2 Cas nuclease mRNA that enables the expression in cells of a Class 2 Cas nuclease, such as Cas9 or a Cpfl protein. Additionally, the cargo may contain one or more guide RNAs or nucleic acids encoding the guide RNAs. A template nucleic acid, e.g., for repair or recombination, may also be included in the composition, or the template nucleic acid may be used in the methods described herein. In some embodiments, the cargo comprises an mRNA encoding Streptococcus pyogenes Cas9, optionally with an S. pyogenes gRNA. In some embodiments, the cargo comprises an mRNA encoding Neisseria meningitidis Cas9, optionally with an nme gRNA.

[0200] "mRNA" refers to a polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). mRNA can contain a phosphate-sugar backbone that includes ribose residues or analogs thereof, such as 2'-methoxyribose residues. In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. Generally, mRNA does not contain substantial amounts of thymidine residues (e.g., 0 residues, or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 0.2%, or 0.1%). 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, mRNA can contain modified uridines at some or all of its uridine positions.

[0201] E. CRISPR / Cas cargo In some embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise mRNA encoding an RNA-guided DNA binder, such as a Cas nuclease. In certain embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9.

[0202] As used herein, "RNA-guided DNA binding agent" refers to a polypeptide or polypeptide complex having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavase / nickases and their inactivated forms ("dCas DNA binders"). As used herein, "Cas nuclease" encompasses Cas cleavase, Cas nickase, and dCas DNA binders. Cas cleavase / nickases and dCas DNA binders include the Csm or Cmr complex of a type III CRISPR system, its CaslO, Csml, or Cmr2 subunit, the Cascade complex of a type I CRISPR system, its Cas3 subunit, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavase / nickases that also have RNA-guided DNA cleavase or nickase activity (e.g., H840A, D10A, or N863 A variants), and Class 2 dCas DNA binders, in which the cleavase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins, and modifications thereof. The Cpfl protein, Zetsche et al., Cell, 163:1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpfl sequence of Zetsche is incorporated herein by reference in its entirety, see, e.g., Zetsche, Tables S1 and S3.See, for example, Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015), the contents of which are incorporated herein in their entireties.

[0203] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA together with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., a Cas cleavase, a Cas nickase, or a dCas DNA-binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA-binding agent, such as Cas9, to a target sequence, the guide RNA hybridizes to the target sequence, an agent binds to the target sequence, and if the agent is a cleavase or nickase, binding can be followed by cleavage or nicking.

[0204] In some embodiments, the cargo for LNP preparation comprises at least one guide RNA comprising a guide sequence that directs an RNA-guided DNA-binding agent, which may be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. The gRNA may guide the Cas nuclease or a Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a Class 2 Cas nuclease and provides specificity for cleavage by the Class 2 Cas nuclease. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type V CRISPR / Cas complex, such as a Cpfl / guide RNA complex. The Cas nuclease and cognate gRNA may be paired. The gRNA scaffold structure paired with each Class 2 Cas nuclease varies with the particular CRISPR / Cas system.

[0205] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or the combination of crRNA and trRNA (also known as tracrRNA). Guide RNA may include modified RNAs described herein. The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence that has a modification or variation compared to the naturally occurring sequence.

[0206] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence may be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, may also be used as a guide. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain one, two, three, or four mismatches, and the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain one to four mismatches, and the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, and the guide sequence comprises 20 nucleotides.

[0207] The target sequence of an RNA-guided DNA-binding protein, such as a Cas protein, includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of the sequence) because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments in which the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence without the PAM), except for the substitution of U for T in the guide sequence.

[0208] The length of the targeting sequence can depend on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have different optimal targeting sequence lengths. Thus, the targeting sequence can comprise 50 or more nucleotides in length. In some embodiments, the length of the targeting sequence is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the naturally occurring nucleotide sequence guide sequence.

[0209] CRISPR / Cas systems. In certain embodiments, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0210] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a "Cpfl sgRNA" capable of mediating RNA-guided DNA cleavage by a Cpfl protein. In some embodiments, the gRNA comprises sufficient crRNA and tracrRNA to form an active complex with a Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA comprises sufficient crRNA to form an active complex with a Cpfl protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0211] Certain embodiments of the present invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes, which are nucleic acids that encode one or more guide RNAs.

[0212] Certain embodiments of the present disclosure also provide for the delivery of adenine base editors ("ABEs") using the LNP compositions, preparations, nanoparticles, and / or nanomaterials described herein. ABEs and their methods of use are described, for example, in U.S. Pat. No. 10,113,163 and U.S. Patent Publication No. 2021 / 0130805, the contents of each of which are incorporated herein by reference in their entirety.

[0213] Certain embodiments of the present disclosure also provide for the delivery of cytosine base editors (CBEs) using the LNP compositions, preparations, nanoparticles, and / or nanomaterials described herein. CBEs and methods for their use are described, for example, in U.S. Patent Nos. 10,167,457 and 9,840,699, the contents of each of which are incorporated herein by reference in their entirety.

[0214] The term "base editor (BE)" or "nucleic acid base editor (NBE)" refers to an agent comprising a polypeptide that can make modifications to bases (e.g., A, T, C, G, or U) in a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor can deaminate a base in a nucleic acid. In some embodiments, the base editor can deaminate a base in a DNA molecule. In some embodiments, the base editor can deaminate adenine (A) in DNA. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the base editor is a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 protein fused to an adenosine deaminase. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to an adenosine deaminase. In some embodiments, the base editor is a nuclease-inactivated Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor is fused to an inhibitor of base excision repair, such as a UGI domain or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, to guide the napDNAbp to a specific nucleic acid sequence. For example, a Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that has complementarity to the guide RNA. In some embodiments, the napDNAbp is a class 2 microbial CRISPR-Cas effector. In some embodiments, the napDNAbp is a Cas9 domain, e.g., a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated Cas9 (dCas9).Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. However, nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind to RNA. For example, napDNAbp can associate with a nucleic acid that guides the napDNAbp to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure.

[0215] F. Modified RNA In certain embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise modified nucleic acids, including modified RNA.

[0216] Modified nucleosides or nucleotides can be present in an RNA, e.g., a gRNA or mRNA. A gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA to account for the presence of one or more non-natural and / or naturally occurring components or arrangements, e.g., used in place of or in addition to the standard A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with non-standard nucleosides or nucleotides and is referred to herein as "modified."

[0217] Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the unlinked and / or linked phosphate oxygens in the phosphodiester backbone linkages (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of a component of the ribose sugar, e.g., the 2' hydroxyl on the ribose sugar (exemplary sugar modifications); (iii) complete replacement of the phosphate moiety with a "dephosphorylation" linker (exemplary backbone modifications); (iv) a modification or substitution of a naturally occurring nucleobase, including substitution with a non-standard nucleobase (exemplary base modifications); (v) a substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., removal, modification, or substitution of a terminal phosphate group, or conjugation of a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) a modification or substitution of a sugar (exemplary sugar modifications). Certain embodiments include 5'-end modifications to the mRNA, gRNA, or nucleic acid. Certain embodiments include 3'-end modifications to the mRNA, gRNA, or nucleic acid. The modified RNA may include modifications at the 5' and 3' ends. The modified RNA may contain one or more modified residues at non-terminal locations. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

[0218] Unmodified nucleic acids may be susceptible to degradation, for example, by intracellular nucleases or nucleases found in serum. For example, nucleases may hydrolyze nucleic acid phosphodiester bonds. Thus, in one aspect, RNAs (e.g., mRNAs, gRNAs) described herein may contain one or more modified nucleosides or nucleotides, for example, to introduce stability against intracellular or serum-based nucleases. In some embodiments, modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a population of cells both in vivo and ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, which involve the induction of cytokine expression and release, particularly interferon, and cell death.

[0219] Thus, in some embodiments, the RNA or nucleic acid in the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprises at least one modification that confers increased or enhanced stability to the nucleic acid, including, for example, improved resistance to nuclease digestion in vivo. As used herein, the terms "modified" and "modified" refer to at least one alteration that, in relation to the nucleic acids provided herein, preferably enhances stability and renders the RNA or nucleic acid more stable (e.g., resistant to nuclease digestion) than wild-type or naturally occurring versions of the RNA or nucleic acid. As used herein, the terms "stable" and "stability" as such refer to the nucleic acids of the invention, particularly with respect to RNA, for example, to increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that can normally degrade such RNA. Increased stability can include, for example, reduced susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such RNA in the target cell, tissue, subject, and / or cytoplasm. The stabilized RNA molecules provided herein demonstrate a longer half-life compared to their naturally occurring, unmodified counterparts (e.g., wild-type versions of mRNA). Also contemplated by the terms "modification" and "modified" as they relate to the mRNA of the LNP preparations disclosed herein are modifications that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of a sequence that functions in the initiation of protein translation (e.g., a Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15(20):8125-48 (1987), the contents of which are incorporated herein by reference in their entirety.)

[0220] In some embodiments, the RNA or nucleic acid of the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein has been chemically or biologically modified to make it more stable. Exemplary modifications to RNA include base depletion (e.g., by deletion or by substituting one nucleotide for another) or base modification, e.g., chemical modification of the base. As used herein, the phrase "chemical modification" includes modifications that introduce chemicals different from those found in naturally occurring RNA, e.g., covalent modifications such as the introduction of modified nucleotides (e.g., nucleotide analogs or the inclusion of pendant groups that do not naturally occur in such RNA molecules).

[0221] In some embodiments of backbone modifications, the phosphate group of the modified residue may be modified by replacing one or more of its oxygens with a different substituent. Furthermore, modified residues, e.g., modified residues present in modified nucleic acids, may include complete replacement of the unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, backbone modifications of the phosphate backbone may include modifications resulting in either an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms may chiralize the phosphorus atom. The phosphorus atom of a stereoisomer may possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing bridging oxygens (i.e., the oxygens linking the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Substitution can occur at either or both linking oxygens. The phosphate group can be replaced by a non-phosphorus-containing linking group in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties that can replace the phosphate group can include, without limitation, methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0222] G. mRNA In some embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. The mRNA may comprise one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may comprise a modified open reading frame, for example, to encode a nuclear localization sequence or to use alternative codons to encode a protein.

[0223] The mRNA in the disclosed compositions, preparations, nanoparticles, and / or nanomaterials can encode, for example, a secreted hormone, enzyme, receptor, polypeptide, peptide, or other normally secreted protein of interest. In one embodiment of the present invention, the mRNA can optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA, or improve or otherwise facilitate protein production.

[0224] Additionally, suitable modifications include altering one or more nucleotides of a codon so that the codon encodes the same amino acid but is more stable than the codon found in the wild-type version of the mRNA. For example, an inverse correlation between RNA stability and a higher number of cytidine (C) and / or uridine (U) residues has been demonstrated, and RNA lacking C and U residues has been found to be stable against most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994), the disclosure of which is incorporated herein by reference in its entirety). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substituting one codon encoding a particular amino acid with another codon encoding the same or a related amino acid. Contemplated modifications of the mRNA nucleic acids of the present invention also include the incorporation of pseudouridine. The incorporation of pseudouridine into the mRNA nucleic acids of the present invention can enhance stability and translation ability, as well as reduce immunogenicity in vivo. See, for example, Kariko, K., et al., Molecular Therapy 16(11):1833-1840 (2008), the contents of which are incorporated herein by reference in their entirety. Substitutions and modifications to the mRNA of the present invention can be carried out by methods readily known to those skilled in the art.

[0225] Constraints on reducing the number of C and U residues in a sequence are likely to be greater in the coding regions of an mRNA compared to untranslated regions (i.e., it is likely not possible to eliminate all of the C and U residues present in the message while retaining the message's ability to encode the desired amino acid sequence). However, the degeneracy of the genetic code presents an opportunity to allow the number of C and / or U residues present in a sequence to be reduced, while maintaining the same coding capacity (i.e., several different possibilities for modification of an RNA sequence may be possible, depending on which amino acids are encoded by the codons).

[0226] The term modified also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the invention (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a longer polyA tail), alteration of the 3' or 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and the inclusion of elements that alter the structure of the mRNA molecule (e.g., to form secondary structures).

[0227] Poly-A tails are believed to stabilize natural messengers. Thus, in one embodiment, long poly-A tails can be added to mRNA molecules, thus making the mRNA more stable. Poly-A tails can be added using a variety of techniques recognized in the art. For example, long poly-A tails can be added to synthetic or in vitro transcribed mRNA using poly-A polymerase (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256, the contents of which are incorporated herein by reference in their entirety). Transcription vectors can also encode long poly-A tails. In addition, poly-A tails can be added by direct transcription from PCR products. In one embodiment, the length of the poly-A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In one embodiment, the length of the poly-A tail is adjusted to control the stability of the modified mRNA molecules of the present invention and, therefore, protein transcription. For example, because the length of the poly-A tail can affect the half-life of an mRNA molecule, the length of the poly-A tail can be adjusted to modify the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in cells. In one embodiment, stabilized mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases) so that they can be delivered to target cells without a transport vehicle.

[0228] In some embodiments, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences that are not naturally present in wild-type mRNA. In one embodiment, 3' and / or 5' flanking sequences naturally adjacent to the mRNA and encoding a second, unrelated protein can be incorporated into the nucleotide sequence of an mRNA molecule encoding a therapeutic or functional protein to modify it. For example, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzyme) can be incorporated into the 3' and / or 5' region of a sense mRNA nucleic acid molecule to increase the stability of the sense mRNA molecule. See, e.g., US2003 / 0083272, the contents of which are incorporated herein by reference in their entirety. A more detailed description of mRNA modifications can be found on pages 57-68 of US2017 / 0210698(A1), the contents of which are incorporated herein by reference in their entirety.

[0229] H. Template nucleic acid The compositions, preparations, nanoparticles, and / or nanomaterials, and methods disclosed herein may include a template nucleic acid. The template may be used to modify or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein, such as a Cas nuclease, e.g., a class 2 Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In some embodiments, two or more templates may be provided so that editing can occur at two or more target sites. For example, different templates may be provided to edit a single gene in a cell or two different genes in a cell.

[0230] In some embodiments, the template can be used in homologous recombination. In some embodiments, homologous recombination can result in the incorporation of the template sequence or a portion of the template sequence into a target nucleic acid molecule. In some embodiments, the template can be used for homology-directed repair, which involves invasion of a DNA strand at a break site within the nucleic acid. In some embodiments, homology-directed repair can result in the inclusion of the template sequence in an edited target nucleic acid molecule. In some embodiments, the template can be used for gene editing mediated by non-homologous end joining. In some embodiments, the template sequence does not share similarity with the nucleic acid sequence near the break site. In some embodiments, the template or a portion of the template sequence is integrated. In some embodiments, the template includes flanking inverted terminal repeat (ITR) sequences.

[0231] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. It may also, or alternatively, correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or a sequence that is in a location different from its natural location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell.

[0232] In some embodiments, the endogenous sequence can be a chromosomal sequence or an extrachromosomal sequence, hi some embodiments, the endogenous sequence can be a plasmid sequence of the cell.

[0233] In some embodiments, the template contains ssDNA or dsDNA containing flanking inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0234] In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., a method described herein). In some embodiments, nucleic acids are purified using a chromatography-based method, such as an HPLC-based method or equivalent method (e.g., a method described herein). In some embodiments, nucleic acids are purified using both a precipitation method (e.g., LiCl precipitation) and an HPLC-based method. In some embodiments, nucleic acids are purified by tangential flow filtration (TFF).

[0235] IV. Methods of Producing LNPs Methods for producing lipid nanoparticles are known in the art. In some embodiments, the described compositions, preparations, nanoparticles, and / or nanomaterials are produced using microfluidics. For example, exemplary methods for using microfluidics to form lipid nanoparticles are described in Leung, AKK, et al., J Phys Chem, 116:18440-18450 (2012); Chen, D., et al., J Am Chem Soc, 134:6947-6951 (2012); and Belliveau, NM, et al., Molecular Therapy-Nucleic Acids, 1:e37 (2012), the disclosures of which are incorporated herein by reference in their entireties.

[0236] Briefly, a cargo, such as those described herein, is prepared in a first buffer solution. Other lipid nanoparticle components (such as ionizable lipids, conjugate-linker lipids, cholesterol, and phospholipids) are prepared in a second buffer solution. In some embodiments, a syringe pump introduces the two solutions into a microfluidic device. The two solutions contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.

[0237] Methods for screening the disclosed lipid nanoparticles are described in International Patent Application No. PCT / US2018 / 058171, which is incorporated herein by reference in its entirety. In some embodiments, the screening method characterizes vehicle delivery preparations to identify preparations that have the desired targeting and deliver a functional cargo to the cytoplasm of specific cells. In some embodiments, the screening method uses a reporter that has a function that can be detected when delivered to a cell. For example, detecting a functional reporter within a cell indicates that the LNP preparation delivers a functional cargo to the cell. Notably, in some embodiments, a chemical composition identifier is included in each different delivery vehicle formulation to keep track of the chemical composition specific to each different delivery vehicle formulation. In some embodiments, the chemical composition identifier is a nucleic acid barcode. In some embodiments, the sequence of the nucleic acid barcode is paired with the chemical components used to formulate the LNP preparation into which it is loaded, such that when the nucleic acid barcode is sequenced, the chemical composition of the delivery vehicle that delivered the barcode is identified. Representative barcodes include, but are not limited to, those described by Sago, 2018 PNAS, Sago, JACS 2018, the disclosures of which are incorporated herein by reference in their entireties. Representative reporters include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotype modifiers. DNA (genomic and DNA barcode) can be isolated using QuickExtract (Lucigen) and sequenced using Illumina MiniSeq as described by Sago et al. PNAS 2018, Sago et al. JACS 2018, Sago, Lokugamage et al. Nano Letters 2018, the disclosures of which are incorporated herein by reference in their entireties.

[0238] V. How to use Among other things, the present disclosure describes methods of using the compositions, preparations, nanoparticles, and / or nanomaterials described herein. For example, in some embodiments, the present disclosure describes methods of using the compositions, preparations, nanoparticles, and / or nanomaterials as described herein to deliver cargo to specific cells, tissues, or organs. As another example, in some embodiments, the present disclosure describes methods of treating a disease or disorder and / or methods of slowing and / or halting the progression of a disease or disorder using the compositions, preparations, nanoparticles, and / or nanomaterials described herein. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein are for use in medicine.

[0239] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials described herein deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof in the absence of a targeting ligand. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials are useful for treating or preventing disease in a subject in need thereof.

[0240] A. Methods for Delivering Cargo to Cells, Tissues, or Organs In particular, in some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein target specific cell types or classes of cells (e.g., cells of a particular organ or system), tissues, and organs. In some embodiments, the present disclosure provides methods of delivering one or more cargoes described herein to a subject in need thereof. In some embodiments, such methods include in vivo and / or in vitro delivery. In some embodiments, such methods include in vivo delivery. In some embodiments, such methods include in vitro delivery. In some embodiments, the present disclosure provides methods of delivering one or more therapeutic and / or prophylactic nucleic acids to a subject in need thereof, as described herein.

[0241] In some embodiments, the compositions, preparations, nanoparticles, and / or nanoparticles comprise a therapeutic and / or prophylactic of interest that can be delivered specifically to liver cells of a subject. Exemplary liver cells include, but are not limited to, hepatocytes.

[0242] In some embodiments, the compositions, preparations, nanoparticles, and / or nanoparticles comprise a treatment and / or prophylaxis of interest that can be delivered specifically to a subject's spleen cells. Exemplary spleen cells include, but are not limited to, spleen monocytes, spleen T cells, spleen memory B cells, or spleen B cells.

[0243] In some embodiments, the compositions, preparations, nanoparticles, and / or nanoparticles comprise a therapeutic and / or prophylactic of interest that can be delivered specifically to a subject's bone marrow cells. Exemplary bone marrow cells include, but are not limited to, myelomonocytic, myeloblastic B cells, myeloblastic memory B cells, or myeloblastic T cells.

[0244] In some embodiments, the compositions, preparations, nanoparticles, and / or nanoparticles comprise a therapeutic and / or prophylactic of interest that can be delivered specifically to a subject's immune cells. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells.

[0245] In some embodiments, the compositions, preparations, nanoparticles, and / or nanoparticles comprise a treatment and / or prophylaxis of interest that can be delivered specifically to the hematopoietic stem cells of a subject. Unless otherwise specified, it is understood that the terms "hematopoietic stem cells (HSCs)" and "hematopoietic stem and progenitor cells (HSPCs)" are used interchangeably in this disclosure.

[0246] In some embodiments, lipid nanoparticles can be formulated to be delivered to mammalian liver hepatocytes, liver immune cells, splenic T cells, or lung endothelial cells in the absence of a targeting ligand. Specific delivery to a particular class or type of cell indicates that a higher percentage of the lipid nanoparticles are delivered to the target type or class of cells. In some embodiments, specific delivery can be 2-fold, 5-fold, 10-fold, 15-fold, or even 20-fold greater than delivery using conventional nanoparticle systems (e.g., MC3-containing LNPs).

[0247] B. Methods for Producing Polypeptides In particular, in some embodiments, methods using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used in methods of producing polypeptides. In particular, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells in a subject in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells in a subject in need thereof. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein comprise one or more nucleic acid sequences to be delivered to cells.

[0248] In some embodiments, one or more nucleic acids are expressed in a cell. In some embodiments, expression of a nucleic acid sequence involves one or more of: (1) production of an RNA template from a DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation), (3) translation of the RNA into a polypeptide or protein, and / or (4) post-translational modification of the polypeptide or protein.

[0249] C. Methods of Gene Regulation In particular, in some embodiments, methods using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used for gene regulation. In particular, in some embodiments, the lipid nanoparticles described herein can be used to reduce and / or increase gene expression in target cells in a subject in need thereof. For example, in some embodiments, the lipid nanoparticles described herein can deliver one or more nucleic acids to target cells of a subject without a targeting ligand. In some embodiments, the nucleic acid is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is an siRNA. In some embodiments, the nucleic acid is a nucleic acid described herein. As another example, in some embodiments, the lipid nanoparticles described herein can deliver a cargo to target cells of a subject without a targeting ligand. In some embodiments, the cargo is any cargo described herein.

[0250] In particular, in some embodiments, methods of editing genes in cells in a subject in need thereof using the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein.

[0251] In some embodiments, the cells targeted for gene regulation are immune cells. The immune cells can be T cells, such as CD8+ T cells, CD4+ T cells, or T regulatory cells. Other exemplary immune cells for gene editing include, but are not limited to, macrophages, dendritic cells, B cells, or natural killer cells. In some embodiments, the cells targeted for gene regulation are hepatocytes.

[0252] Exemplary genes that may be targeted include, but are not limited to, T cell receptor, B cell receptor, CTLA4, PD1, FOXO1, FOXO3, AKT, CCR5, CXCR4, LAG3, TIM3, killer immunoglobulin-like receptor, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNFR-II, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1, IL-2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1, IL-2, IL-1, IL-2, IL-1, IL-1, IL-2, IL-1, IL-2, IL-3, IL-1, IL-2, IL-1 ... -7R, OX-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK-R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NFAT, IL2R, and IL7. Other exemplary genes that can be targeted include, but are not limited to, OCT, G6Pase, Mut, PCCA, PCCB, PCSK9, ALAS1, and PAH. Exemplary tumor-associated antigens that can be recognized by T cells and that are contemplated for targeting include, but are not limited to, MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1 / Melan A, MC1R, GP100, tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2 / Neu, hTERT, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN, and β-catenin.

[0253] D. Subjects to be treated In some embodiments, the subject being treated is a mammal experiencing cancer, an autoimmune disease, an infectious disease, an organ transplant, organ failure, a protein deficiency, or a combination thereof. In some embodiments, the subject is human. In some embodiments, the methods described herein can cause hepatocytes to translate specific proteins. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to hepatocytes. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic T cells. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic B cells. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to splenic monocytes. In some embodiments, the methods described herein can be used to deliver one or more DNAs, mRNAs, sgRNAs, or siRNAs to bone marrow cells.

[0254] The order or sequence of steps for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0255] While the present invention has been particularly shown and described with reference to certain preferred embodiments, it should be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention as defined by the appended claims. [Example]

[0256] Example The present disclosure exemplifies the compositions, preparations, formulations, nanoparticles, and / or nanomaterials described herein. The present disclosure also exemplifies methods of preparing, characterizing, and validating the compositions, preparations, formulations, nanoparticles, and / or nanomaterials described herein.

[0257] Example 1: Materials and Methods This example provides exemplary materials and methods for preparing, characterizing, and validating certain compositions, formulations, nanoparticles, and / or nanomaterials described herein. A detailed description of this example can be found in Example 1 of WO2022 / 140252, which is incorporated herein by reference.

[0258] Example 2: Potency per Screen This example provides exemplary compositions, preparations, nanoparticles, and / or nanomaterials, as well as materials and methods for screening the efficacy of such compositions, preparations, nanoparticles, and / or nanomaterials described herein. The exemplary LNP screen described in Example 1 of WO2022 / 140252 can be used to demonstrate that each pool of LNPs is highly potent across many tissues.

[0259] Example 3: Exemplary LNP preparation and delivery to various cell types This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials for delivery to various cell types described herein. In some embodiments, this example can be used to demonstrate that the provided lipids exhibit delivery to various cell types.

[0260] Based on the results derived from the screening in Example 1, exemplary LNP preparations are identified for use in spleen delivery, particularly for use in B cell delivery. These identified lipids are formulated into LNP preparations and screened using the Cre reporter system described herein. Three Ai14 mice are used per group. The payload contains 0.3 mg / kg Cre mRNA. Data are collected 168 hours after injection. Results are compared with the MC3-LNP preparation as a control.

[0261] Example 4: Exemplary LNP preparations are delivered to various cell types This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials with delivery to various cell types described herein. This example can be used to demonstrate that the provided lipids exhibit delivery across various cell types.

[0262] LNP preparations are selected to confirm efficacy results using the Cre reporter system and Ai14 mouse model described herein. Three Ai14 mice are used per group in each experiment. Data are collected 72 hours after injection. The screening platform described herein can identify LNP preparations to determine which type of LNP preparation is most potent for a particular cell type.

[0263] Example 5: Exemplary LNP preparations deliver functional mRNA This example provides exemplary LNP compositions, preparations, nanoparticles, and / or nanomaterials that deliver functional mRNA to various cell types. This example can be used to demonstrate that the provided lipids can deliver functional mRNA in mice.

[0264] Exemplary LNP preparations were selected to determine the ability of each preparation to deliver functional mRNA in mice. LNP preparations, each carrying 0.15 mg / kg of hEPO mRNA, were administered at a mass ratio of 11.7 and 19 in two to three C57BL6 mice. hEPO expression in plasma was measured 6 hours after injection of the LNP preparations.

[0265] Example 6: Tolerability and Efficacy Studies This example provides exemplary materials and methods for preparing, characterizing, and validating the compositions, preparations, nanoparticles, and / or nanomaterials described herein. This example can be used to demonstrate that the provided lipids can deliver functional mRNA in mice.

[0266] Exemplary LNP preparations are selected to determine the tolerability and efficacy of hEPO mRNA delivery in rats as described herein. Each LNP preparation containing 1.0 mg / kg of hEPO mRNA is injected into Sprague-Dawley rats (N=2). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are collected from rat plasma (U / L) 24 hours after injection for each exemplary LNP preparation. Monocyte chemoattractant protein-1 (MCP-1) is collected from rat plasma (ng / mL) 6 hours after injection for each LNP preparation. Saline is used as a control. After vehicle (control) and exemplary LNP preparations are administered at various time points (0, 2, 4, 6, 24, 48, 96 hours) after injection, hEPO is collected from rat plasma (ng / mL).

[0267] Example 7: Synthesis of ionizable lipids This example provides exemplary materials and methods for preparing, characterizing, and validating the ionizable lipids described herein. As described in the following examples, in certain exemplary embodiments, the compounds are prepared according to the following general procedures. Although the general methods illustrate the synthesis of specific compounds of the present disclosure, it will be understood that the following general methods and other methods known to those skilled in the art can be applied to all compounds and subclasses and species of each of these compounds, as described herein.

[0268] General notes: Unless otherwise stated, all reactions were carried out using anhydrous-grade solvents under a nitrogen atmosphere in magnetically stirred flasks or vials. Anhydrous solvents were purchased from Sigma-Aldrich and used as received. Flash column chromatography was performed using a Biotage Selekt or Teledyne-Isco Combiflash Nextgen300+ equipped with prepacked silica gel cartridges. Thin-layer chromatography was performed using Merck silica gel 60 plates, and compounds were visualized using iodine. Nuclear magnetic resonance (NMR) spectroscopy was performed using either a Varian INOVA 500 MHz or a Bruker AVANCE 400 MHz spectrometer; chemical shifts are reported in δ parts per million (ppm) referenced to tetramethylsilane at δ = 0.00 ppm for CDCl3 samples and the residual solvent peak (δ = 2.50 ppm) for DMSO samples. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was performed using a QDa detector (ESI + ) was performed using one of the following general methods.

[0269] Method A (5-minute run): Column—XTERRA RP 18 (4.6 × 50 mm), 5 μm. Mobile phase: Initially 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], then 2% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] over 2.65 minutes. This mobile phase composition was held for up to 3.75 minutes. Finally, at 4.90 minutes, the initial conditions were returned to, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF]. This mobile phase composition was held for up to 5.10 minutes. Flow rate = 1.2 mL / min.

[0270] Method B1 (12 min run): Column - XTERRA RP 18 (4.6 x 50 mm), 5 μm, (Mobile phase: initially 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH (70:30) ACN:THF solution], held at this initial condition for 0.75 min, then 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH (70:30) ACN:THF solution] for 3.0 min, then 2% [0.1% The mobile phase composition was held for a maximum of 9.0 minutes, and finally returned to the initial conditions at 11.00 minutes, i.e., 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in ACN:THF], and held for a maximum of 12.10 minutes. Flow rate = 1.2 mL / min.

[0271] Method B2 (5 min run): Column - XTERRA RP 18 (4.6 x 50 mm), 5 μm, (Mobile phase: initially 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH (70:30) ACN:THF solution], held at this initial condition for 0.75 min, then 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH (70:30) ACN:THF solution] for 3.0 min, then 2% [0.1 The mobile phase composition was maintained for a maximum of 9.0 min, and then returned to the initial conditions, i.e., 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in ACN:THF (70:30)], over a period of 4.90 min. The mobile phase composition was maintained for a maximum of 5.10 min. Flow rate = 1.2 mL / min.

[0272] Method C (12 min run): Column - XTERRA RP 18 (4.6 x 50 mm), 5 μm, (Mobile phase: initially 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH (70:30) ACN:THF solution], held at this initial condition for 0.75 min, then 65% [0.1% HCOOH in water] and 35% [0.1% HCOOH (70:30) ACN:THF solution] for 3.0 min, then 2% [0.1% The mobile phase composition was held for a maximum of 9.0 minutes, and finally returned to the initial conditions at 11.00 minutes, i.e., 80% [0.1% HCOOH in water] and 20% [0.1% HCOOH in ACN:THF], and held for a maximum of 12.10 minutes. Flow rate = 1.2 mL / min. List of abbreviations Ac: Acetyl ACN: acetonitrile d: doublet DCC: N,N'-dicyclohexylcarbodiimide DCM: dichloromethane DIPEA: N,N-diisopropylethylamine DMAP: 4-(dimethylamino)pyridine DMSO: dimethyl sulfoxide EDC: N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride Eq: equivalent Et: Ethyl i-Pr: Isopropyl m: multiplet Me: Methyl p:Pentet PPTS: Pyridinium p-toluenesulfonate q:Quartet Rt: retention time s:singlet t: triplet TBAF: Tetrabutylammonium fluoride TBS: tert-butyldimethylsilyl TEA: Triethylamine THF: tetrahydrofuran

[0273] general synthesis Exemplary lipids were prepared according to the following general synthetic scheme, using Example 7-1 for illustration. [ka] TIFF2025528020000063.tif34165

[0274] Example 7-1: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoic acid salt [ka] Step 1: 4,4-bis(octyloxy)butanenitrile General Procedure A: To a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 equiv.) was added 4,4-diethoxybutanenitrile (1.5 g, 9.5 mmol, 1 equiv.) and 1-octanol (3.7 g, 29 mmol, 3 equiv.). The vial was tightly capped, and the resulting mixture was heated at 105 °C for 72 h. The mixture was then allowed to cool to room temperature. The crude material was purified by silica gel column chromatography using a gradient of 0 to 100% dichloromethane in hexanes to give 4,4-bis(octyloxy)butanenitrile (1.08 g, 35%) as a colorless oil. 1 H NMR (400MHz, chloroform-d)δ 0.87(t,J=6.7Hz,6H),1.17-1.41(m,20H),1.54-1.62(m,4H),1.88-1.98(m,2H),2 .41(t,J=7.4Hz,2H),3.37-3.47(m,2H),3.54-3.64(m,2H),4.54(t,J=5.3Hz,1H).

[0275] [ka] Step 2: 4,4-bis(octyloxy)butanoic acid General Procedure B: To a vial containing 4,4-bis(octyloxy)butanenitrile (2.3 g, 7.1 mmol, 1 equiv.) was added potassium hydroxide (1.2 g, 21 mmol, 3 equiv.), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was tightly capped, and the resulting mixture was heated to 110° C. for 18 hours. The mixture was then allowed to cool to room temperature. The mixture was diluted with ethyl acetate (20 mL), and the pH was adjusted to approximately 5 by the addition of 1 M HCl. The resulting biphasic mixture was separated, and the aqueous phase was extracted twice more with ethyl acetate (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated to give 4,4-bis(octyloxy)butanoic acid (1.1 g, 45% yield) as a pale yellow oil. 1HNMR(400MHz,DMSO-d6)δ 0.85(t,J=6.5Hz,6H),1.15-1.36(m,21H),1.46(q,J=6.7Hz,4H),1.72(q,J=7.0Hz,2H),2.21 (t,J=7.5Hz,2H),3.32-3.39(m,1H),3.43-3.52(m,2H),4.45(t,J=5.5Hz,1H),12.05(s,1H).

[0276] [ka] Step 3: 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate General procedure C: To a stirred solution of (9Z,12Z)-octadeca-9,12-dienoic acid (5.5 g, 19.62 mmol) in DCM (50 mL) was added EDC (5.64 g, 29.43 mmol), DMAP (48 mg, 3.92 mmol), and DIPEA (10.27 mL, 58.87 mmol). The resulting mixture was stirred at 25 °C for 30 min. 2-(hydroxymethyl)propane-1,3-diol (2.1 g, 19.62 mmol) was then added thereto and further stirred at 25 °C for 16 h. Water (20 mL) was added, and the mixture was extracted with DCM (2 × 100 mL). The organic layer was dried over anhydrous NaSO and evaporated under reduced pressure. The crude compound thus obtained was purified by flash chromatography eluting with 70% EtOAc-hexane to give 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (3.2 g, 44%) as a colorless liquid. 1 H NMR (400MHz, chloroform-d)δ 0.88(t,J=6.8Hz,3H),1.16-1.43(m,15H),1.51-1.76(m,2H),1.92-2.11(m,5H),2.32(t,J=7.6Hz,2 H),2.39(s,1H),2.76(t,J=6.4Hz,2H),3.68-3.82(m,4H),4.24(d,J=6.3Hz,2H),5.26-5.43(m,4H).

[0277] [ka] Step 4: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate General Procedure D: To a stirred solution of 4,4-bis(octyloxy)butanoic acid (0.4 g, 1.16 mmol) in DCM (30 mL) was added EDC (334 mg, 1.74 mmol), DMAP (28 mg, 0.233 mmol), and DIPEA (0.5 mL, 2.90 mmol). The resulting mixture was stirred at 25 °C for 30 min. 3-Hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (0.4 g, 1.16 mmol) was then added thereto and further stirred at 25 °C for 16 h. Water (10 mL) was added, and the mixture was extracted with DCM (2 × 50 mL). The organic layer was dried over anhydrous NaSO and evaporated under reduced pressure. The crude compound thus obtained was purified by flash chromatography eluting with 30% EtOAc-hexane to give 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (231 mg, 30%) as a colorless liquid. 1 H NMR (400MHz, chloroform-d)δ 0.83-0.92(m,9H),1.15-1.43(m,38H),1.55-1.69(m,2H),1.93(q,J=7.0H z,2H),2.04(q,J=7.0Hz,4H),2.19(t,J=6.8Hz,2H),2.31(t,J=7.6Hz,2H) ,2.40(t,J=7.4Hz,2H),2.76(t,J=6.3Hz,2H),3.34-3.44(m,2H),3.50-3. 65(m,4H),4.17(t,J=5.7Hz,4H),4.48(t,J=5.5Hz,1H),5.14-5.54(m,4H).

[0278] [ka] Step 5: Decane-1,4,-diol General Procedure E: To a solution of 5-hexyldihydrofuran-2(3H)-one (2.0 g, 11.75 mmol, 1 equiv.) in THF (15 mL) at 0 °C was added lithium aluminum hydride (35 mL, 1 M in THF, 35 mmol, 3 equiv.) dropwise. After the addition, the reaction mixture was allowed to warm to 25 °C and stirred for 12 h. Water and 15% NaOH (aq.) were then added to the reaction mixture at 0 °C. After further stirring for 15 min, the mixture was filtered through a Celite pad. The Celite pad was washed with ether (100 mL), and the combined filtrates were concentrated. The crude material was purified by silica gel column chromatography using a gradient of 0 to 50% ethyl acetate in hexane to give decane-1,4-diol (1.6 g, 78%) as a colorless liquid. 1 H NMR (400 MHz, chloroform-d) δ 0.90 (t, J = 6.3 Hz, 3H), 1.23-1.39 (m, 6H), 1.42-1.53 ​​(m, 4H), 1.67-1.75 (m, 4H), 3.65-3.74 (m, 3H).

[0279] [ka] Step 6: 1-((tert-butyldimethylsilyl)oxy)decan-4-ol General Procedure F: To a solution of decane-1,4-diol (500 mg, 2.87 mmol) in DCM (10 mL) at 0 °C, imidazole (293 mg, 4.30 mmol) and tert-butyldimethylsilyl chloride (520 mg, 3.45 mmol) were added. After the addition, the reaction mixture was warmed to 25 °C and stirred for 2 h. The reaction mixture was then diluted with water (3 mL) and extracted with ethyl acetate (2 × 40 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0 to 10% ethyl acetate in hexane to give 1-((tert-butyldimethylsilyl)oxy)decan-4-ol (660 mg, 80%) as a pale yellow liquid. 1 H NMR (400 MHz, chloroform-d) δ 0.06 (s, 6H), 0.85-0.90 (m, 11H), 1.27 (bs, 8H), 1.41-1.45 (m, 4H), 1.59-1.67 (m, 4H), 3.59 (d, J = 5.1 Hz, 1H), 3.65 (t, J = 5.3 Hz, 2H).

[0280] [ka] Step 7: 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate General Procedure G: To a solution of 1-((tert-butyldimethylsilyl)oxy)decan-4-ol (100 mg, 0.35 mmol) in DCM (3 mL) were added pyridine (0.06 mL, 0.69 mmol), DMAP (13 mg, 0.10 mmol), and 4-nitrophenyl carbonochloridate (175 mg, 0.86 mmol) and stirred at 25 °C for 1 h. Then, 2-(pyrrolidin-1-yl)ethan-1-amine (99 mg, 0.86 mmol) and DIPEA (0.18 mL, 1.04 mmol) were added and stirred at 25 °C for 12 h. The reaction mixture was then diluted with dichloromethane (40 mL), washed with 1 M sodium carbonate (2 × 5 mL), water (5 mL), brine, and finally dried over anhydrous NaSO. The resulting dichloromethane layer was concentrated and purified by silica gel column chromatography using a gradient of 0 to 2% methanol in DCM to give 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate (123 mg, 73%). 1 H NMR (400 MHz, chloroform-d) δ 0.03 (s, 6H), 0.84-0.87 (m, 12H), 1.22-1.26 (m, 10H), 1.49-1.58 (m, 6H), 1.94-2.03 (m, 2H), 2.66 (bs, 6H), 3.33-3.35 (m, 2H), 3.59-3.60 (m, 2H), 4.73 (s, 1H), 5.30 (s, 1H).

[0281] [ka] Step 8: 1-Hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate General Procedure H: To a solution of 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate (1.5 g, 3.5 mmol) in THF (10 mL) was added TBAF 1 (M) in THF (8.7 mL, 8.75 mmol) at 0° C. under a nitrogen atmosphere. The reaction was stirred at 25° C. for 8 hours. The reaction was quenched with water (10 mL) and extracted with 10% MeOH-DCM (2×30 mL). The organic layer was washed with brine (10 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash chromatography eluting with 10% MeOH-DCM to give 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate (800 mg, 80%) as a pale yellow viscous liquid. 1 H NMR (400MHz, chloroform-d)δ 0.86(t,J=6.6Hz,3H),1.17-1.39(m,8H),1.46-1.64(m,4H),1.65-1.82(m,4H),2.51(s,3H),2 .47-2.62(m,3H),3.19-3.36(m,2H),3.47(s,3H),3.60-3.68(m,2H),4.75(s,1H),5.22(s,1H).

[0282] [ka] Step 9: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid General procedure J: To a stirred solution of 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate (1.01 g, 3.21 mmol) in acetone (30 mL) was added Jones reagent (2 M CrO in HSO, 1.93 mL, 3.85 mmol) dropwise at 0 °C. The reaction mixture was stirred at 25 °C for 16 h. Upon completion, isopropanol (2 mL) was added and the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure. The crude mass was diluted with water, the pH of the aqueous layer was adjusted to 6, and the mixture was extracted with 5% MeOH-DCM (3 × 40 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure to give 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid (872 mg, 82%) as a light greenish viscous liquid, which was used without further purification. 1 H NMR (400 MHz, chloroform-d) δ 0.86 (s, 3H), 1.11-1.36 (m, 15H), 1.38-1.75 (m, 3H), 1.74-2.12 (m, 4H), 2.13-2.19 (m, 1H), 2.26-2.44 (m, 1H), 2.65-2.88 (m, 1H), 3.59-3.72 (m, 1H), 3.86-4.38 (m, 1H), 4.76 (s, 1H), 5.29 (s, 1H).

[0283] Step 10: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoic acid salt (Example 7-1) General Procedure K: To a stirred solution of 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid (200 mg, 0.609 mmol) in DCM (10 mL) were added DMAP (15 mg, 0.122 mmol) and DCC (188 mg, 0.91 mmol). The resulting mixture was stirred at 25° C. for 30 minutes. To it was then added 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (423 mg, 0.60 mmol), and the mixture was then further stirred at 25° C. for 16 hours. Water (20 mL) was added, and the reaction mixture was extracted with DCM (2×50 mL). The organic layer was dried over anhydrous NaSO and evaporated under reduced pressure. The crude compound thus obtained was purified by flash chromatography eluting with 2% MeOH-DCM to give 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (120 mg, 24%) as a colorless liquid. UPLC-MS (Method B1): Rt 5.72 min, m / z calculated [M+H]: 1005.8, found: 1006.2. 1 H NMR (400 MHz, chloroform-d) δ 0.79-0.92 (m, 9H), 1.17-1.41 (m, 49H), 1.45-1.64 (m, 9H), 1.64-1.85 (m, 3H), 1.91 (q, J = 7.7 Hz, 6H), 2.04 (q, J = 6.9 Hz, 4H), 2.29 (t, J = 7.6 Hz, 2H), 2.31-2.44 (m ,4H),2.76(t,J=6.6Hz,3H),2.81-2.99(m,3H),3.34-3.47(m,4H),3.50-3.60(m ,2H),3.99-4.24(m,4H),4.47(t,J=5.5Hz,1H),4.74(s,1H),5.26-5.42(m,4H). [ka]

[0284] Example 7-2: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoic acid salt [ka] Step 1: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile To a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 equiv.) was added 4,4-diethoxybutanenitrile (1.5 g, 9.5 mmol, 1 equiv.) and cis-5-octen-1-ol (3.7 g, 29 mmol, 3 equiv.). The vial was tightly capped, and the resulting mixture was heated at 105 °C for 72 h. The mixture was then allowed to cool to room temperature. The crude material was purified by flash column chromatography (0 to 100% dichloromethane in hexanes). 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (1.14 g, 37%) was obtained as a colorless oil. 1 H NMR (500MHz, chloroform-d)δ 5.43-5.27(m,4H),4.56(t,J=5.3Hz,1H),3.61(dt,J=9.3,6.6Hz,2H),3.44(dt,J=9.3,6.6Hz,2H),2 .42(t,J=7.4Hz,2H),2.12-1.91(m,9H),1.66-1.54(m,5H),1.49-1.36(m,4H),0.96(t,J=7.6Hz,6H).

[0285] [ka] Step 2: 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid To a vial containing 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanenitrile (1.14 g, 3.54 mmol, 1 equiv.) was added potassium hydroxide (0.60 g, 10.6 mmol, 3 equiv.), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was tightly capped, and the resulting mixture was heated to 110° C. for 18 h. The mixture was then allowed to cool to room temperature. The mixture was diluted with ethyl acetate (20 mL), and the pH was adjusted to approximately 5 by the addition of 1 M HCl. The resulting biphasic mixture was separated, and the aqueous phase was extracted twice more with ethyl acetate (2×20 mL). The organic extracts were combined, dried over sodium sulfate, filtered, and concentrated to give 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (1.16 g, 96% yield) as a sticky white solid. 1 H NMR (400MHz, chloroform-d)δ 5.41-5.24(m,4H),4.45(t,J=5.6Hz,1H),3.51(dt,J=9.0,6.7Hz,2H),3.39(dt,J=9.0,6.7Hz,2H),2.17(t,J=7. 6Hz,2H),2.08-1.98(m,8H),1.81(q,J=7.3Hz,2H),1.59-1.52(m,4H),1.44-1.32(m,4H),0.94(t,J=7.5Hz,6H).

[0286] [ka] Step 3: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate To a mixture of 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid (591 mg, 1 equiv., 1.74 mmol) in dichloromethane (10 mL), 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (640 mg, 1 equiv., 1.74 mmol), DIPEA (673 mg, 904 μL, 3 equiv., 5.21 mmol), and DMAP (42.4 mg, 0.2 equiv., 347 μmol) were added. EDC (666 mg, 2 equiv., 3.47 mmol) was added last and stirred at 23 °C for 18 h. The reaction mixture was then concentrated and purified by flash column chromatography (0–40% ethyl acetate in hexanes). 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (450 mg, 38%) was obtained as a colorless oil. 1 H NMR (500MHz, chloroform-d)δ 5.43-5.27(m,8H),4.49(t,J=5.5Hz,1H),4.23-4.12(m,4H),3.65-3.60(m,2H),3.58(dt,J =9.3,6.6Hz,2H),3.41(dt,J=9.3,6.6Hz,2H),2.81-2.73(m,2H),2.41(t,J=7.5Hz,2H),2. 32(dd,J=7.9,7.2Hz,2H),2.25-2.15(m,2H),2.12-1.97(m,12H),1.94(ddd,J=8.0,7.2,5. 5Hz,2H),1.67-1.53(m,8H),1.45-1.26(m,17H),0.96(t,J=7.6Hz,5H),0.92-0.87(m,3H).

[0287] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoic acid salt (Example 7-2) General procedure L: To a stirred solution of 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid (50 mg, 0.15 mmol) in DCM (2 mL) was added EDC (37.95 mg, 0.20 mmol), DMAP (3.93 mg, 0.03 mmol), 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (115.71 mg, 0.16 mmol), and DIPEA (0.10 mL, 0.60 mmol). The reaction mixture was stirred at 25° C. for 16 h. Upon completion, the reaction mixture was concentrated, diluted with DCM (50 mL), and washed with a saturated solution of NaHCO (2 × 25 mL), water (2 × 25 mL), and brine (2 × 15 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure. The crude material was purified by CombiFlash column chromatography, eluting with 0–5% methanol in DCM to afford 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (46 mg, 30%) as a colorless liquid. UPLC-MS (Method A): Rt 2.39 min, m / z calculated [M+H]: 1001.8, found: 1001.8. 1 H NMR (400 MHz, chloroform-d) δ 0.87 (q, J = 6.7 Hz, 6H), 0.94 (t, J = 7.5 Hz, 6H), 1.19-1.46 (m, 28H), 1.46-1.69 (m, 14H), 1.82-1.96 (m, 7H), 1.96-2.09 (m, 12H), 2.29 (t, J = 7.6H) z,2H),2.33-2.44(m,5H),2.76(t,J=6.4Hz,2H),3.32-3.46(m,4H),3.51-3.61(m, 2H),4.05-4.20(m,6H),4.47(t,J=5.4Hz,1H),4.67-4.81(m,1H),5.26-5.42(m,8H) [ka]

[0288] Example 7-3: 15-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-9-hexyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azahexadecan-16-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt [ka] Step 1: 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(dimethylamino)propyl)carbonate To a stirred solution of 1-((tert-butyldimethylsilyl)oxy)decan-4-ol (1 g, 3.46 mmol) in DCM (20 mL) was added pyridine (0.56 mL, 6.93 mmol), DMAP (212 mg, 1.73 mmol), and 4-nitrophenyl carbonochloridate (1.75 g, 8.66 mmol). The reaction mixture was stirred at 25 °C for 2 h. Then, 3-(dimethylamino)propan-1-ol (894 mg, 8.66 mmol) and DIPEA (1.85 mL, 10.39 mmol) were added and further stirred at 25 °C for 12 h. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1 M sodium carbonate (2 × 75 mL), water (75 mL), brine (50 mL), and finally dried over anhydrous NaSO and concentrated under reduced pressure. The crude material thus obtained was purified by flash column chromatography eluting with 3-4% MeOH-DCM to give 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(dimethylamino)propyl)carbonate (480 mg, 33%) as a colorless liquid. 1 H NMR (400MHz, chloroform-d)δ -0.11-0.20(m,6H),0.78-0.97(m,11H),1.14-1.41(m,14H),1.58-1.74(m,2H),1.79-1.87(m,2H),2 .16-2.28(m,5H),2.35(t,J=7.3Hz,2H),3.49-3.74(m,2H),4.16(t,J=6.3Hz,2H),4.59-4.83(m,1H).

[0289] [ka] Step 2: 3-(Dimethylamino)propyl(1-hydroxydecan-4-yl)carbonate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate with 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(dimethylamino)propyl)carbonate. Isolated 110 mg, 54%. 1 H NMR(400MHz,DMSO-d6)δ 0.85(t,J=7.0Hz,3H),1.16-1.32(m,9H),1.86-2.05(m,2H),2.64(s,6H),2.87-3.04(m,2H),3.16(d,J= 5.2Hz,3H),3.28-3.42(m,2H),4.03-4.21(m,3H),4.44(t,J=4.7Hz,1H),4.57-4.68(m,1H),9.96(s,1H).

[0290] [ka] Step 3: 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)decanoic acid Prepared according to general procedure J, substituting 3-(dimethylamino)propyl(1-hydroxydecan-4-yl)carbonate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. UPLC-MS (Method A): Rt 0.35 min, m / z calculated [M+H]: 318.2, found: 318.3.

[0291] Step 4: 15-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-9-hexyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azahexadecan-16-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt (Example 7-3) Prepared according to general procedure L, substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)decanoic acid. Isolated 32 mg, 12%. UPLC-MS (Method A): Rt 2.23 min, m / z calculated [M+H]: 990.8, found: 990.7. [ka]

[0292] Example 7-4: 12-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecan-13-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt [ka] Step 1: 1-((tert-butyldimethylsilyl)oxy)octan-2-ol To a stirred solution of octane-1,2-diol (500 mg, 2.87 mmol) in DCM (10 mL) was added imidazole (293 mg, 4.30 mmol) and tert-butyldimethylsilyl chloride (520 mg, 3.45 mmol) at 0 °C. The reaction mixture was stirred at 25 °C for 2 h. Upon completion, the reaction mixture was diluted with water (3 mL) and extracted with ethyl acetate (2 × 40 mL). The combined organic layers were dried over anhydrous NaSO and concentrated under reduced pressure. The crude material thus obtained was purified by Combiflash chromatography eluting with 10% EtOAc-hexane to give 1-((tert-butyldimethylsilyl)oxy)octan-2-ol (660 mg, 80%) as a pale yellow liquid. 1 H NMR (400MHz, chloroform-d)δ 0.06(s,6H),0.78-1.00(m,12H),1.14-1.52(m,10H),2.41(d,J=3.3Hz,1H),3.37(dd,J=8.3,10.5Hz,1H),3.57-3.64(m,2H).

[0293] [ka] Step 2: 1-((tert-butyldimethylsilyl)oxy)octan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 1-((tert-butyldimethylsilyl)oxy)octan-2-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated 522 mg, 48%. 1 H NMR (400MHz, chloroform-d)δ 0.04(s,6H),0.87(s,11H),1.25(m,6H),1.57-1.62(m,4H),1.75(s,4H),2.56( t,J=6.3Hz,2H),3.27(s,2H),3.62(d,J=5.0Hz,2H),4.72(s,1H),5.14(s,1H).

[0294] [ka] Step 3: 1-hydroxyoctan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)octan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 130 mg, 65%. 1 H NMR (400 MHz, chloroform-d): δ 0.86 (t, J = 6.6 Hz, 3H), 1.00 (t, J = 7.4 Hz, 1H), 1.16-1.41 (m, 8H), 1.41-1.61 (m, 3H), 1.61-1.73 (m, 1H), 1.83 (s, 4H), 2.41-2.84 (m, 4H), 3.23-3.34 (m, 1H), 3.38 (t, J = 8.5 Hz, 1H), 3.47 (s, 1H), 3.58 (dd, J = 6.8, 12.1 Hz, 1H), 3.66-3.74 (m, 1H), 4.77 (s, 1H), 5.58 (s, 1H).

[0295] Step 4: 12-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecan-13-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt (Example 7-4) General procedure M: To a stirred solution of 1-hydroxyoctan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate (50 mg, 0.17 mmol) in DCM (5 mL) was added pyridine (0.03 mL, 0.34 mmol), DMAP (4.2 mg, 0.03 mmol), and 4-nitrophenyl carbonochloridate (70.41 mg, 0.34 mmol). The reaction mixture was stirred at 25° C. for 2 h. Then, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate (145.7 mg, 0.21 mmol) and DIPEA (0.09 mL, 0.52 mmol) were added. The reaction mixture was further stirred at 25° C. for 12 h. Upon completion, the reaction mixture was diluted with water (15 mL), extracted with DCM (2 × 25 mL), washed with 1 M NaCO solution (10 mL) and brine (5 mL). The organic layer was dried over anhydrous NaSO and concentrated under reduced pressure. The crude compound thus obtained was subjected to CombiFlash column chromatography eluting with 5% MeOH-DCM to give 12-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecan-13-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt (50.32 mg, 29%) as a pale yellow liquid. UPLC-MS (Method B1): Rt 5.53 min, m / z calculated [M+H]: 1007.8, found: 1008.2. [ka]

[0296] Example 7-5: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecan-13-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt Prepared according to general procedure M, substituting 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-heptadeca-9,12-dienoate. Isolated 123 mg, 50%. UPLC-MS (Method A): Rt 2.20 min, m / z calculated [M+H]: 1003.8, found: 1004.0. [ka]

[0297] Example 7-6: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((8-((2-butyloctanoyl)oxy)octanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate [ka] Step 1: 8-Hydroxyoctyl 2-butyloctanoate General Procedure N: To a stirred solution of 2-butyloctanoic acid (1 equiv.) in DCM (5 mL / 0.5 mmol) was added DIPEA (3 equiv.), EDC (1.5 equiv.), and DMAP (0.5 equiv.) at 25 °C. The reaction mixture was stirred for 15 min, then 1,8-octanediol (3.0 equiv.) was added and stirred for an additional 16 h at 25 °C. Upon completion, the reaction mixture was diluted with DCM (2 × 50 mL) and washed with saturated NaHCO solution (2 × 25 mL), followed by water and brine (25 mL). The organic layer was separated, passed through anhydrous NaSO, and dried on a rotary evaporator. The crude material thus obtained was purified by Combiflash chromatography, eluting with 15–20% EtOAc–hexane to give 8-hydroxyoctyl 2-butyloctanoate (1.6 g, 49%) as a colorless oil. 1 H NMR (400 MHz, chloroform-d) δ 0.82-0.91 (m, 6H), 1.13-1.47 (m, 25H), 1.49-1.69 (m, 4H), 2.24-2.36 (m, 1H), 3.63 (q, J = 5.8 Hz, 2H), 4.05 (t, J = 6.6 Hz, 2H).

[0298] [ka] Step 2: 8-((2-butyloctanoyl)oxy)octanoic acid Prepared according to general procedure J, substituting 8-hydroxyoctyl 2-butyloctanoate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 1.4 g (crude). 1 H NMR (400MHz, DMSO-d6) δ0.80~0.88 (m, 6H), 1.03~1.64 (m, 31H), 2.09~2.37 (m, 2H), 4.01 (t, J=6.5Hz, 2H), 11.95 (s, 1H).

[0299] [ka] Step 3: 3-Hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate Prepared according to general procedure C, substituting 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for (9Z,12Z)-octadeca-9,12-dienoic acid. Isolated 2.5g, 47%. 1 H NMR(400MHz,chloroform-d)δ 0.97(t,J=7.6Hz,6H),1.36-1.47(m,4H),1.50-1.67(m,6H),1.89-2.13(m,10H),2.15-2.33(m,1H),2.43(t,J=7.2Hz,2H),3 .43(q,J=6.2Hz,2H),3.54-3.64(m,2H),3.69-3.92(m,4H),4.28(d,J=6.2Hz,2H),4.50(t,J=5.6Hz,1H),5.24-5.51(m,4H).

[0300] [ka] Step 4: 8-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-8-oxooctyl 2-butyloctanoate Prepared according to general procedure D, substituting 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 8-((2-butyloctanoyl)oxy)octanoic acid for 4,4-bis(octyloxy)butanoic acid. Isolated 150 mg, 46%. 1 H NMR(400MHz,CDCl3)δ 0.85-0.96(m,14H),1.24-1.62(m,32H),1.91-2.41(m,16H),3.38-3.62(m,6H),4.03-4.17(m,6H),4.40-4.50(m,1H),5.31-5.35(m,4H)

[0301] Step 5: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((8-((2-butyloctanoyl)oxy)octanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate (Example 7-6) Prepared according to general procedure L, substituting 8-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 8-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-8-oxooctyl 2-butyloctanoate. Isolated 72 mg, 35%. UPLC-MS (Method A): Rt 2.19 min, m / z calculated [M+H]: 1063.8, found: 1064.0. [ka]

[0302] Example 7-7: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate [ka] Step 1: 7-Hydroxyheptyl 2-butyloctanoate Prepared according to general procedure N, substituting 1,7-heptanediol for 1,8-octanediol. Isolated 1.5g, 49%. 1 H NMR (400 MHz, chloroform-d) δ 0.82-0.91 (m, 6H), 1.18-1.33 (m, 12H), 1.36-1.74 (m, 15H), 2.24-2.36 (m, 1H), 3.64 (t, J = 6.4 Hz, 2H), 4.08 (t, J = 6.5 Hz, 2H).

[0303] [ka] Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid Prepared according to general procedure J, substituting 7-hydroxyheptyl 2-butyloctanoate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 1.5 g (crude). 1 H NMR(400MHz,DMSO-d6)δ 0.79-0.88(m,6H),1.10-1.36(m,12H),1.32-1.61(m,8H),2.18(t,J=7.3Hz,2H),2.20-2.35(m,1H),4.00(t,J=6.3Hz,2H),11.97(s,1H).

[0304] [ka] Step 3: 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate Prepared according to general procedure D, substituting 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and 7-((2-butyloctanoyl)oxy)heptanoic acid for 4,4-bis(octyloxy)butanoic acid. Isolated 120 mg, 45%. 1H NMR (400MHz, chloroform-d)δ 0.87(t,J=7.1Hz,6H),0.94(t,J=7.5Hz,6H),1.15-1.49(m,23H),1.56- 1.72(m,9H),1.89-1.98(m,2H),1.97-2.09(m,8H),2.13-2.26(m,2H),2. 27-2.45(m,5H),3.40(t,J=8.8Hz,2H),3.53-3.66(m,4H),4.05(t,J=6.8 Hz,2H),4.17(d,J=5.2Hz,4H),4.48(t,J=6.0Hz,1H),5.12-5.67(m,4H).

[0305] Step 4: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate (Example 7-7) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate. Isolated 105 mg, 47%. UPLC-MS (Method A): Rt 2.04 min, m / z calculated [M+H]: 1049.8, found: 1049.8. [ka]

[0306] Examples 7-8: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-hexyl-4,9,15-trioxo-1-(pyrrolidin-1-yl)-5,8,10,14-tetraoxa-3-azadocosan-22-yl 2-butyloctanoate Prepared according to general procedure M, substituting 8-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-8-oxooctyl 2-butyloctanoate for 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated 92 mg, 33%. UPLC-MS (Method A): Rt 2.17 min, m / z calculated [M+H]: 1065.8, found: 1066.2. H NMR (400 MHz, chloroform-d) δ 0.82-0.91(m,12H),0.94(t,J=7.5Hz,4H),1.13-1.49(m,31H),1.49-1.67(m,10H),1.72-1.8 7(m,4H),1.86-1.96(m,3H),1.96-2.10(m,9H),2.23-2.47(m,8H),2.48-2.79(m,5H),3.23-3 .45(m,4H),3.52-3.59(m,2H),4.04(t,J=6.6Hz,2H),4.07-4.18(m,5H),4.19(t,J=5.5Hz,2H ),4.27(dd,J=3.2,11.6Hz,1H),4.47(t,J=5.5Hz,1H),4.84-5.08(m,1H),5.24-5.42(m,4H). [ka]

[0307] Example 7-9: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-hexyl-4,9,15-trioxo-1-(pyrrolidin-1-yl)-5,8,10,14-tetraoxa-3-azahenicosan-21-yl 2-butyloctanoate Prepared according to general procedure M, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated 92 mg, 33%. UPLC-MS (Method A): Rt 2.18 min, m / z calculated [M+H]: 1051.8, found: 1052.1.

[0308] Examples 7-10: 16-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-3-ethyl-10-hexyl-8,13-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt Step 1: 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbonate Prepared according to general procedure G, substituting 3-(diethylamino)propan-1-ol for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 3.5 g, yield 56%. 1 H NMR (400MHz, chloroform-d)δ 0.02(s,6H),0.87(s,9H),1.04(t,6H),1.20-1.37(m,6H),1.45-1.74(m,2H),1.85(t,2H),2.35- 2.80(m,12H),3.51-3.69(m,2H),4.13-4.18(m,2H),4.60-4.82(m,1H),6.79(d,2H),8.13(d,1H).

[0309] Step 2: 3-(Diethylamino)propyl(1-hydroxydecan-4-yl)carbonate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbonate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.6 g, yield 49%. 1 H NMR (400 MHz, chloroform-d) δ 0.86 (t, 3H), 1.00 (t, 6H), 1.15–1.42 (m, 9H), 1.56–1.73 (m, 6H), 1.76–1.88 (m, 2H), 2.46–2.57 (m, 6H), 3.64 (t, 2H), 4.16 (t, 2H), 4.59–4.88 (m, 1H).

[0310] Step 3: 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid Prepared according to general procedure J, substituting 3-(diethylamino)propyl(1-hydroxydecan-4-yl)carbonate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 400 mg crude. 1 H NMR(400MHz,DMSO-d6)δ 0.86(t,3H),1.07-1.35(m,17H),1.44-1.62(m,2H),1.63-2.06(m,4H) ,2.15-2.30(m,1H),2.92-3.19(m,5H),4.09-4.18(m,2H),4.65(s,1H).

[0311] Step 4: 16-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-3-ethyl-10-hexyl-8,13-dioxo-7,9,14-trioxa-3-azaheptadecan-17-yl(9Z,12Z)-octadeca-9,12-dienoic acid salt (Examples 7-10) Prepared according to general procedure L, substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid. Isolated mass 56.94 mg, 36% yield. UPLC-MS: Performed method B2 on UPLC. Rt 5.36 min, MS calculated: 1018.79 [M+H], MS found 1019.01 [M+H].

[0312] Examples 7-11: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate Step 1: 7-Hydroxyheptyl 2-butyloctanoate To a stirred solution of 2-butyloctanoic acid (2 g, 9.98 mmol) in DCM (20 mL) was added EDC·HCl (2.67 g, 12.97 mmol), DMAP (244 mg, 1.99 mmol), and DIPEA (5.2 mL, 29.95 mmol). The reaction mixture was stirred at 25 °C for 30 min. Heptane-1,7-diol (1.9 g, 14.97 mmol) was then added to the reaction mixture and stirred for 16 h. Upon completion, the reaction mixture was diluted with water (150 mL) and extracted with DCM (2 × 100 mL). The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure to give the crude material. The crude compound was purified by CombiFlash column chromatography, eluting with 10% ethyl acetate in hexanes, to give 17 (1.8 g, 57%) as a colorless liquid. 1 H NMR (400 MHz, chloroform-d) δ 0.84-0.86 (m, 6H), 1.23 (m, 13H), 1.42-1.46 (m, 4H), 1.55-1.61 (m, 6H), 2.26-2.29 (m, 1H), 3.62 (t, 2H), 4.05 (t, 2H).

[0313] Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid To a stirred solution of 7-hydroxyheptyl 2-butyloctanoate (1 g, 3.18 mmol) in acetone (20 mL) was added Jones reagent (2.4 mL, 4.77 mmol) dropwise under ice-cooled conditions. The cooling bath was removed and stirring was continued for 2 h. Upon completion, iPrOH (20 mL) was added and the mixture was filtered through a bed of Celite®. The filtrate was concentrated under reduced pressure, the crude mass was diluted with water (30 mL), the pH of the aqueous layer was adjusted to 6-7, and the mixture was extracted with 10% MeOH-DCM (3 × 60 mL). The combined organic layers were dried over NaSO and filtered. The filtrate was concentrated under reduced pressure to give 7-((2-butyloctanoyl)oxy)heptanoic acid (900 mg, crude) as a pale green liquid, which was used in the next step without further purification. 1 H NMR(400MHz,DMSO-d6)δ 0.82-0.84(m,6H),1.20-1.28(m,16H),1.40-1.54(m,8H),2.18(t,2H),2.26-2.27(m,1H),4.00(t,2H),11.96(s,1H).

[0314] Step 3: 7-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate Prepared according to general procedure D, substituting 7-(3-hydroxy-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated mass 580 mg, 42.73%. 1 H NMR(400MHz,chloroform-d)δ 0.84-0.87(m,12H),1.25-1.34(m,38H),1.52-1.61(m,5H),1.83-1.94(m,2H),2.16-2.19(m,1H),2.29 -2.41(m,5H),3.35-3.41(m,2H),3.52-3.64(m,4H),4.04(t,2H),4.11-4.17(m,4H),4.47(t,1H).

[0315] Step 4: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate (Examples 7-11) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate. Isolated mass 93 mg, 30%. UPLC-MS: Method A performed for UPLC. Rt 2.30 min, MS calculated: 1053.82 [M+H], MS found 1054.22 [M+H].

[0316] Example 7-12: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(3-butylheptyl)heptanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-butylheptyl)heptanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 90 mg, yield 28%. UPLC-MS: Method A was performed for UPLC. Rt 2.19 min, MS calculated: 1035.77 [M+H], MS found 1036.20 [M+H].

[0317] Example 7-13: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 85.88 mg, yield 27%. UPLC-MS: Method A was performed for UPLC. Rt 2.20 min, MS calculated: 1063.81 [M+H], MS found 1065.29 [M+H].

[0318] Example 7-14: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(3-hexylnonyl)heptanedioic acid salt Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-hexylnonyl)heptanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 78 mg, yield 23%. UPLC-MS: Method A was performed for UPLC. Rt 2.26 min, MS calculated: 1091.84 [M+H], MS found 1092.34 [M+H].

[0319] Example 7-15: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Step 1: 3-Hydroxy-2-(hydroxymethyl)propyl 4,4-bis(octyloxy)butanoate Heptanedioic acid salt Prepared according to general procedure C, substituting 4,4-bis(octyloxy)butanoic acid for (9Z,12Z)-octadeca-9,12-dienoic acid. Isolated mass 1.8 g, 43% yield. 1 H NMR (400MHz, chloroform-d)δ 0.85-0.88(m,6H),1.25(m,20H),1.52-1.54(m,4H),1.65(m,4H),1.90-1.95(m,2H),2.00-2.03(m,1H),2. 40(t,2H),3.36-3.41(m,2H),3.52-3.64(m,3H),3.71-3.80(m,4H),4.24-4.29(m,2H),4.46-4.47(m,1H).

[0320] Step 2: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 7-(3-pentyloctyl)heptanedioate Prepared according to general procedure D, substituting 7-oxo-7-((3-pentyloctyl)oxy)heptanoic acid (as described in WO 2022 / 159472 (A1)) for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(octyloxy)butanoate. Isolated mass 327 mg, yield 37%. 1H NMR (400MHz, chloroform-d)δ 0.85-0.88(m,12H),1.24-1.26(m,40H),1.52-1.63(m,13H),1.89-1.94(m,2H),2.16-2.19(m,1H),2.26-2.33(m, 4H),2.39(t,2H),3.36-3.41(m,2H),3.52-3.61(m,4H),4.04-4.08(m,2H),4.11-4.20(m,4H),4.46-4.48(m,1H).

[0321] Step 3: 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt (Examples 7-15) Prepared according to general procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate. Isolated mass 76 mg as a pale yellow liquid, 22% yield. UPLC-MS: Performed as method A for UPLC. Rt 2.32 min, MS calculated: 1067.84 [M+H], MS found 1068.34 [M+H].

[0322] Example 7-16: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(3-hexylnonyl)adipate Prepared according to general procedure L, substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(3-hexylnonyl)adipate (described in WO 2022 / 159472 (A1)). Isolated mass 82 mg as a deep yellow liquid, 25% yield. UPLC-MS: Method A was performed for UPLC. Rt 2.24 min, MS calculated: 1077.82 [M+H], MS found 1078.36 [M+H].

[0323] Example 7-17: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(3-hexylnonyl)adipate Step 1: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(3-hexylnonyl) adipate Prepared according to general procedure D, substituting 6-((3-hexylnonyl)oxy)-6-oxohexanoic acid (as described in WO 2022 / 159472 (A1)) for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(octyloxy)butanoate. Isolated mass 580 mg as a yellow liquid, 42.73% yield. 1 H NMR (400MHz, chloroform-d)δ 0.84-0.87(m,12H),1.25-1.34(m,38H),1.52-1.61(m,5H),1.83-1.94(m,2H),2.16-2.19(m,1H),2.29 -2.41(m,5H),3.35-3.41(m,2H),3.52-3.64(m,4H),4.04(t,2H),4.11-4.17(m,4H),4.47(t,1H).

[0324] Step 2: 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(3-hexylnonyl)adipate (Examples 7-17) Prepared according to general procedure L, substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(3-hexylnonyl)adipate. Mass isolated as a colorless gum 78 mg, yield 24%. UPLC-MS: Performed as method A for UPLC. Rt 2.30 min, MS calculated: 1081.85 [M+H], MS found 1082.42 [M+H].

[0325] Example 7-18: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)8-(3-butylheptyl)octanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)8-(3-butylheptyl)octanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 76 mg, yield 25%. UPLC-MS: Method A was performed for UPLC. Rt 2.18 min, MS calculated: 1049.79 [M+H], MS found 1050.30 [M+H].

[0326] Example 7-19: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)8-(3-pentyloctyl)octanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)8-(3-pentyloctyl)octanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 92 mg, yield 29%. UPLC-MS: Method A was performed for UPLC. Rt 2.21 min, MS calculated: 1077.82 [M+H], MS found 1078.36 [M+H].

[0327] Example 7-20: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-ethyl-4,9,15-trioxo-1-(pyrrolidin-1-yl)-5,10,14-trioxa-3-azahenicosan-21-yl 2-butyloctanoate Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid. Isolated mass 117 mg, 44% as a yellow gel. UPLC-MS: Method A was performed for UPLC. Rt 2.06 min, MS calculated: 993.72, MS observed: 994.03 [M+H].

[0328] Example 7-21: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-4,9,15-trioxo-6-propyl-1-(pyrrolidin-1-yl)-5,10,14-trioxa-3-azahenicosan-21-yl 2-butyloctanoate The compound can be prepared according to general procedure L, substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid and 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate. Yield: 119 mg, 44%, yellow thick liquid. UPLC-MS: Method A was performed for UPLC. Rt 2.08 min, MS calculated: 1007.75, MS observed: 1008.24 [M+H]. 1H NMR(400MHz,CDCl3)δ 0.83-0.91(m,9H),0.91-0.97(m,6H),1.25-1.45(m,21H),1.46-1.65( m,15H),1.84-1.96(m,7H),1.95-2.09(m,9H),2.24-2.32(m,3H),2.33 -2.45(m,5H),2.47-3.07(m,7H),3.34-3.44(m,4H),3.51-3.61(m,2H) ,4.01-4.14(m,8H),4.47(t,1H),4.71-4.80(m,1H),5.24-5.41(m,4H).

[0329] Example 7-22: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoate Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid (as described in WO 2022 / 159463(A1)). Isolated mass 87 mg, 33%, light brown gum. UPLC-MS: Method A was performed for UPLC. Rt 2.11 min, MS calculated: 1021.77, MS observed: 1022.27 [M+H].

[0330] Example 7-23: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoate Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (as described in WO 2022 / 159463(A1)). Isolated mass 81 mg, 29%, light brown gum. UPLC-MS: Method A was performed for UPLC. Rt 2.15 min, MS calculated: 1035.78, MS observed: 1036.34 [M+H].

[0331] Example 7-24: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Step 1: 6-((tert-butyldimethylsilyl)oxy)hexan-3-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 6-((tert-butyldimethylsilyl)oxy)hexan-3-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated mass 1.6 g, 45% yield. 1H NMR (400 MHz, CDCl3) δ 0.03 (s, 6H), 0.86-0.93 (m, 13H), 1.53-1.62 (m, 8H), 2.62-2.93 (m, 7H), 3.27-3.43 (m, 2H), 3.57-3.62 (m, 2H), 4.67-4.73 (m, 1H), 5.38-5.62 (m, 1H).

[0332] Step 2: 6-Hydroxyhexan-3-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 6-((tert-butyldimethylsilyl)oxy)hexan-3-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 900 mg, yield 93%. 1 H NMR(400MHz,CDCl3)δ 0.90(t,3H),1.54-1.62(m,5H),1.73-1.81(m,4H),1.88-1.99(m,2H),2.42-2.57(m,4H), 2.60(s,2H),3.19-3.37(m,2H),3.62-3.67(m,2H),4.64-4.74(m,1H),5.21-5.28(m,1H).

[0333] Step 3: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid Prepared according to general procedure J, substituting 6-hydroxyhexan-3-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 700 mg, crude. UPLC-MS: Column - XTERRA RP 18 (4.6 × 50 mm), 5μ, (Mobile phase: initial 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], then 2.0% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] in 1.2 min. This mobile phase composition was held for a maximum of 2.50 min, then returned to the initial composition, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] in 0.47 min. This mobile phase composition was held for a maximum of 12.10 min. Flow rate = 1.2 mL / min. Rt 0.47 min, MS calculated: 273.18, MS observed: 273.54 [M+H].

[0334] Step 4: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt (Example 7-24). Prepared according to general procedure L, substituting 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)), and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid. Isolated mass 110 mg, yield 41%. UPLC-MS: Method A was performed for UPLC. Rt 2.11 min, MS calculated: 1007.75, MS observed: 1008.13 [M+H].

[0335] Example 7-25: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Step 1: 1-((tert-butyldimethylsilyl)oxy)heptan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 1-((tert-butyldimethylsilyl)oxy)heptan-4-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated mass 4 g, 64% yield. 1 H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.85-0.93(m,12H),1.24-1.32(m,2H),1.46-1.62(m,6H),1.98-2.05(m,4 H),3.04(s,4H),3.48(s,2H),3.52-3.62(m,4H),4.65-4.79(m,1H),5.66-5.99(m,1H).

[0336] Step 2: 1-Hydroxyheptan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)heptan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.8 g, 85% yield. 1 H NMR(400MHz,CDCl3)δ 0.88(t,3H),1.23-1.51(m,4H),1.56-1.64(m,4H),1.86-2.04(m,4H),2.67-3.05( m,6H),3.36-3.53(m,2H),3.59-3.64(m,2H),4.73-4.78(m,1H),5.93-5.97(m,1H).

[0337] Step 3: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid Prepared according to general procedure J, substituting 1-hydroxyheptan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.3 g, yield 73%. 1 H NMR(400MHz,DMSO-d6)δ 0.87(t,3H),1.24-1.31(m,2H),1.41-1.50(m,2H),1.64-1.70(m,1H),1.73-1.89(m,3H),1.93-2.05(m,2H),2.19-2. 29(m,2H),2.87-3.01(m,2H),3.17(s,2H),3.48-3.61(m,2H),4.57-4.75(m,1H),10.32-10.60(m,1H),12.08(s,1H).

[0338] Step 4: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt (Example 7-25). Prepared according to general procedure L, substituting 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid. Isolated mass 150 mg, yield 55%. UPLC-MS: Method A was performed for UPLC. Rt 2.13 min, MS calculated: 1021.77, MS observed: 1022.15 [M+H]

[0339] Example 7-26: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Step 1: 1-((tert-butyldimethylsilyl)oxy)octan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 1-((tert-butyldimethylsilyl)oxy)octan-4-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated mass 1.3 g, 47% yield. 1H NMR(400MHz,CDCl3)δ 0.04(s,6H),0.85-0.91(m,12H),1.22-1.30(m,4H),1.48-1.60(m,6H),1.77-1.81(m,4H),2. 49-2.66(m,6H),3.28-3.34(m,2H),3.57-3.61(m,2H),4.60-4.80(m,1H),5.12-5.30(m,1H).

[0340] Step 2: 1-hydroxyoctan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)octan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 750 mg, yield 94%. 1 H NMR(400MHz,DMSO-d6)δ 0.85(t,3H),1.18-1.30(m,5H),1.33-1.52(m,6H),1.64-1.76(m,4H),2.53-2.65(m,4H) ,3.06-3.20(m,3H),3.32-3.35(m,2H),4.27-4.48(m,1H),4.56-4.81(m,1H),6.94(s,1H)

[0341] Step 3: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid Prepared according to general procedure J, substituting 1-hydroxyoctan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 620 mg, crude. UPLC-MS: Column - XTERRA RP 18 (4.6 × 50 mm), 5μ, (Mobile phase: initial 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], then 2.0% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] in 1.2 min. This mobile phase composition was held for a maximum of 2.50 min, then returned to the initial composition, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] in 2.85 min. This mobile phase composition was held for a maximum of 3.00 min. Flow rate = 1.5 mL / min. Rt 0.34 min, MS calculated: 301.21, MS found: 301.48 [M+H].

[0342] Step 4: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt (Example 7-26). Prepared according to general procedure L, substituting 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)), and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid. Isolated mass 118 mg, yield 43%. UPLC-MS: Method A was performed for UPLC. Rt 2.18 min, MS calculated: 1035.78, MS observed: 1036.18 [M+H].

[0343] Example 7-27: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Step 1: 1-((tert-butyldimethylsilyl)oxy)nonan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 1-((tert-butyldimethylsilyl)oxy)nonan-4-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated mass 4 g, 75% yield. 1 H NMR(400MHz,CDCl3)δ 0.05(s,6H),0.85-0.92(m,12H),1.25-1.35(m,6H),1.45-1.63(m,6H),1.78-1.87(m,4H),2. 51-2.68(m,6H),3.25-3.37(m,2H),3.59-3.64(m,2H),4.73-4.78(m,1H),5.15-5.27(m,1H).

[0344] Step 2: 1-Hydroxynonan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)nonan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.7 g, yield 78%. 1 H NMR(400MHz,CDCl3)δ 0.84(t,3H),0.98(t,2H),1.22-1.29(m,5H),1.35-1.50(m,3H),1.61-1.69(m,2H),1.79-1.86(m,4H),2.68- 2.73(m,3H),2.94-3.13(m,2H),3.22-3.37(m,4H),3.57-3.61(m,2H),4.69-4.73(m,1H),5.62-5.66(m,1H).

[0345] Step 3: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid Prepared according to general procedure J, substituting 1-hydroxynonan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.4 g, crude yield. UPLC-MS: Column - XTERRA RP 18 (4.6 × 50 mm), 5μ, (Mobile phase: initial 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF], then 2.0% [0.1% HCOOH in water] and 98% [0.1% HCOOH in (70:30) ACN:THF] in 1.2 min. This mobile phase composition was held for a maximum of 2.50 min, then returned to the initial composition, i.e., 50% [0.1% HCOOH in water] and 50% [0.1% HCOOH in (70:30) ACN:THF] in 2.85 min. This mobile phase composition was held for a maximum of 3.10 min. Flow rate = 1.5 mL / min. Rt 0.34 min, MS calculated: 315.23, MS observed: 315.51 [M+H].

[0346] Step 4: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt (Example 7-27). Prepared according to general procedure L, substituting 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472 (A1)), and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid. Isolated mass 139 mg, yield 49%. UPLC-MS: Method A was performed for UPLC. Rt 2.19 min, MS calculated: 1049.80, MS observed: 1050.24 [M+H].

[0347] Example 7-28: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-methyl-4,9,15-trioxo-1-(pyrrolidin-1-yl)-5,10,14-trioxa-3-azahenicosan-21-yl 2-butyloctanoate Step 1: 5-((tert-butyldimethylsilyl)oxy)pentan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 5-((tert-butyldimethylsilyl)oxy)pentan-2-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated as a yellow liquid, mass 1.6 g, 32% yield. 1H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.85-0.93(m,9H),1.21(d,3H),1.53-1.59(m,4H),1.72-1.83(m,4H),2.45- 2.71(m,6H),3.23-3.36(m,2H),3.57-3.62(m,2H),4.69-4.84(m,1H),5.14-5.37(m,1H).

[0348] Step 2: 5-Hydroxypentan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 5-((tert-butyldimethylsilyl)oxy)pentan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 900 mg, 83% yield as a pale yellow liquid. 1 H NMR(400MHz,CDCl3)δ 1.22(d,3H),1.53-1.67(m,4H),1.70-1.87(m,4H),2.24-2.36(m,1H),2.49-2.7 1(m,6H),3.20-3.39(m,2H),3.65(d,2H),4.81-4.85(m,1H),5.30-5.39(m,1H).

[0349] Step 3: 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid Prepared according to general procedure J, substituting 5-hydroxypentan-2-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 120 mg, which was used in the next step without further purification. UPLC-MS: Method C performed on UPLC. Rt 0.32 min, MS calculated: 259.16 [M+H], MS found: 259.47 [M+H].

[0350] Step 4: 12-(((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)methyl)-6-methyl-4,9,15-trioxo-1-(pyrrolidin-1-yl)-5,10,14-trioxa-3-azahenicosan-21-yl 2-butyloctanoate (Examples 7-28). Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid. Isolated mass 102.41 mg, 31%, yellow gel. UPLC-MS: Method A was performed for UPLC. Rt 2.02 min, MS calculated: 979.7 [M+H], MS observed: 980.24 [M+H]. 1 H NMR(400MHz,CDCl3)δ 0.85-0.93(m,6H),0.97(t,6H),1.22-1.29(m,15H),1.37-1.45(m,9H) ,1.60-1.65(m,18H),1.93-2.07(m,12H),2.29-2.34(m,3H),2.37-2.43 (m,5H),2.79-3.00(m,4H),3.38-3.48(m,4H),3.55-3.59(m,2H),4.08( t,2H),4.14(d,6H),4.50(t,1H),4.79-4.85(m,1H),5.27-5.41(m,4H).

[0351] Example 7-30: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoyl)oxy)methyl)propyl)7-(3-pentyloctyl)heptanedioic acid salt Prepared according to general procedure L, substituting 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472(A1)) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(3-pentyloctyl)heptanedioate (as described in WO 2022 / 159472(A1)), and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid. Isolated mass 78.36 mg, yield 30%. UPLC-MS: Method A was performed for UPLC. Rt 2.09 min, MS calculated: 993.7 [M+H], MS observed: 994.13 [M+H].

[0352] Example 7-32: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(2-butyloctyl)heptanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(2-butyloctyl)heptanedioate (described in WO 2022 / 159472 (A1)). Isolated mass 167 mg, yield 59%. UPLC-MS: Method B2 was performed for UPLC. Rt 2.19 min, MS calculated: 1049.80, MS found: 1050.28 [M+H].

[0353] Example 7-33: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioic acid salt Step 1: 7-oxo-7-(pentadeca-1,14-dien-8-yloxy)heptanoic acid The compound can be prepared according to general procedure C, substituting heptanedioic acid for (9Z,12Z)-octadeca-9,12-dienoic acid and pentadeca-1,14-dien-8-ol for 2-(hydroxymethyl)propane-1,3-diol.

[0354] Step 2: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioic acid salt Prepared according to general procedure D, substituting 7-oxo-7-(pentadeca-1,14-dien-8-yloxy)heptanoic acid for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate. Isolated mass 750 mg, 59% yield as a colorless liquid. 1 H NMR (400 MHz, CDCl3) δ 0.94(t,6H),1.27(s,6H),1.35(d,6H),1.45-1.68(m,16H),1.87-1.95(m,2H) ,1.97-2.09(m,12H),2.16-2.22(m,1H),2.26-2.34(m,4H),2.40(t,1H),2.63 (t,1H),2.81(t,1H),3.32-3.44(m,2H),3.51-3.68(m,5H),4.09-4.18(m,4H) ,4.41-4.52(m,1H),4.81-5.02(m,5H),5.24-5.40(m,4H),5.70-5.87(m,2H).

[0355] Step 3: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioic acid salt (Example 7-33). Prepared according to general procedure L, substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (as described in WO2022 / 159463(A1)). Isolated mass 108 mg, 31.25% yield as a light brown sticky gum. UPLC-MS: Performed method B2 on UPLC. Rt 1.10 min, MS calculated: 1073.79 [M+H], MS found 1074.37 [M+H]. 1 H NMR (400 MHz, DMSO-d6) δ 0.83-0.95(m,9H),1.10-1.36(m,28H),1.42-1.58(m,14H),1.66-1.81(m,8H ),1.95-2.02(m,10H),2.25-2.34(m,8H),3.03-3.17(m,4H),3.34-3.37(m,2H ),3.45-3.50(m,2H),4.01-4.10(m,6H),4.40-4.48(m,2H),4.57-4.65(m,2H) ,4.72-4.81(m,2H),4.91-5.00(m,4H),5.27-5.37(m,4H),5.74-5.81(m,2H).

[0356] Example 7-34: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate for 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadeca-1,14-dien-8-yl)heptanedioate. Mass isolated as a pale yellow gum: 155 mg, 43.35% yield. UPLC-MS: Method B2 performed for UPLC. Rt 6.90 min, MS calculated: 1087.81 [M+H], MS found 1088.36 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ 0.78-0.86(m,3H),0.90(t,6H),1.09-1.27(m,32H),1.30-1.40(m,8H),1.44-1.56(m ,14H),1.68-1.81(m,6H),1.96-2.02(m,9H),2.23-2.32(m,7H),3.11-3.19(m,2H),3. 34-3.38(m,2H),3.44-3.52(m,2H),4.05(d,5H),4.44(t,1H),4.58-4.65(m,1H),4.70 -4.80(m,1H),4.89-5.02(m,4H),5.24-5.39(m,4H),5.69-5.85(m,2H),7.02(bs,1H).

[0357] Example 7-35: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(pentadecan-8-yl)heptanedioic acid salt Step 1: 7-oxo-7-(pentadecan-8-yloxy)heptanoic acid Prepared according to general procedure C, substituting heptanedioic acid for (9Z,12Z)-octadeca-9,12-dienoic acid and pentadecan-8-ol for 2-(hydroxymethyl)propane-1,3-diol. Isolated as a colorless liquid, mass 1.9 g, 58% yield. 1 H NMR(400MHz,DMSO-d6)δ 0.85(t,6H),1.16-1.29(m,22H),1.37-1.53(m,8H),2.17(t,2H),2.25(t,2H),4.72-4.90(m,1H),11.99(s,1H).

[0358] Step 2: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 7-(pentadecan-8-yl)heptanedioic acid salt Prepared according to general procedure D, substituting 7-oxo-7-(pentadecan-8-yloxy)heptanoic acid for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate. Isolated as a colorless liquid, mass 1.3 g, 41% yield. 1 H NMR(400MHz,CDCl3)δ 0.86(t,6H),0.94(t,6H),1.16-1.28(m,20H),1.35-1.43(m,6H),1.45-1.52(m ,4H),1.53-1.58(m,6H),1.60-1.65(m,4H),1.92(q,2H),2.00-2.05(m,6H),2. 15-2.21(m,1H),2.26-2.33(m,4H),2.40(t,2H),3.34-3.42(m,2H),3.51-3.64 (m,4H),4.13-4.19(m,4H),4.48(t,1H),4.78-4.88(m,1H),5.25-5.42(m,4H).

[0359] Step 3: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propyl)7-(pentadecan-8-yl)heptanedioic acid salt (Example 7-35) Prepared according to general procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadecan-8-yl)heptanedioate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadecan-8-yl)heptanedioate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (described in WO 2022 / 159463 (A1)). Isolated mass 110 mg as a pale yellow liquid, 32% yield. UPLC-MS: Method B2 was performed for UPLC. Rt 1.16 min, MS calculated: 1077.82 [M+H], MS observed 1078.38 [M+H]. 1 H NMR (400 MHz, DMSO-d6) δ 0.84(t,9H),0.90(t,6H),1.19-1.26(m,32H),1.31-1.38(m,4H),1.40-1. 54(m,14H),1.66-1.81(m,8H),1.89-2.06(m,9H),2.20-2.35(m,10H),3.07 -3.15(m,2H),3.34-3.37(m,2H),3.46-3.51(m,2H),4.05(d,6H),4.44(t,1 H),4.59-4.65(m,1H),4.74-4.80(m,1H),5.26-5.37(m,4H),6.97(bs,1H).

[0360] Example 7-36: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl)7-(pentadecan-8-yl)heptanedioic acid salt Prepared according to general procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)7-(pentadecan-8-yl)heptanedioate for 3-((4,4-bis((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Isolated mass 142 mg, 41%) as a pale yellow liquid. UPLC-MS: Method B2 performed for UPLC. Rt 7.12 min, MS calculated: 1091.84 [M+H], MS found 1092.32 [M+H]. HPLC: 1 H NMR (400 MHz, DMSO-d6) δ 0.84(t,9H),0.90(t,6H),1.12-1.28(m,33H),1.31-1.37(m,4H),1.45-1.53( m,10H),1.60-1.66(m,5H),1.73-1.79(m,3H),1.95-2.03(m,8H),2.22-2.34( m,10H),2.37-2.44(m,8H),3.05(d,2H),3.45-3.52(m,2H),4.05(d,6H),4.44 (t,1H),4.59-4.66(m,1H),4.75-4.80(m,1H),5.26-5.35(m,4H),6.89(s,1H).

[0361] Example 7-37: 2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Step 1: 2-(hydroxymethyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure D, substituting 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadeca-9,12-dienoate for 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate. Isolated as a colorless gum, mass 2.05 g, 58% yield. 1 H NMR(400MHz,CDCl3)δ 0.94(t,12H),1.36-1.45(m,9H),1.54-1.59(m,9H),1.88-1.96(m,4H),2.01-2.06(m,12H),2.40(t,3H),2.63 (t,1H),2.81(t,1H),3.34-3.44(m,3H),3.51-3.68(m,8H),4.02-4.22(m,5H),4.47(t,2H),5.26-5.43(m,8H).

[0362] Step 2: 2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) (Examples 7-37) Prepared according to general procedure L, substituting 2-(hydroxymethyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 2-(hydroxymethyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (described in WO 2022 / 159463 (A1)). Isolated mass 128 mg, yield 25.47%. UPLC-MS: Method A was performed for UPLC. Rt 5.46 min, MS calculated: 1047.77 [M+H], MS observed: 1049.29 [M+H]. 1H NMR(400MHz,DMSO-d6)δ 0.81-0.85(m,3H),0.90(t,12H),1.18-1.26(m,12H),1.29-1.39(m,10H),1.41-1.51(m,11H),1.69-1.79(m,8H),1.95-2.03 (m,16H),2.31(t,9H),3.35-3.39(m,3H),3.47-3.51(m,4H),4.06(d,6H),4.44(t,2H),4.59-4.64(m,1H),5.26-5.37(m,8H).

[0363] Example 7-38: 2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate) Prepared according to general procedure L, substituting 2-(hydroxymethyl)propane-1,3-diylbis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate). Isolated mass 157 mg, yield 44.4%. UPLC-MS: Method A performed on UPLC. Rt 2.28 min, MS calculated: 1061.79 [M+H], MS found 1062.29 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ 0.82-0.86(m,3H),0.90(t,12H),1.18-1.26(m,14H),1.29-1.38(m,12H),1.41-1.52(m,12H),1.72-1.79(m,7H),1.93-2. 04(m,18H),2.31(t,7H),3.44-3.54(m,5H),4.05(d,6H),4.44(t,2H),4.58-4.66(m,1H),5.25-5.35(m,8H),7.00(bs,1H).

[0364] Example 7-39: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(2-(hept-6-en-1-yl)non-8-en-1-yl)adipate Step 1. 2,2-Di(hept-6-en-1-yl)malonate dimethyl To a stirred solution of dimethyl malonate (3 g, 22.72 mmol) in THF (90 mL) was added NaH (1.64 g, 68.16 mmol) under ice-cooled conditions under a nitrogen atmosphere. After 30 min, 7-bromohept-1-ene (12 g, 68.16 mmol) was added, and the mixture was refluxed for 16 h. The reaction mixture was cooled to 25 °C, quenched with 1 N HCl, and extracted with EtO (2 × 250 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous NaSO, concentrated under reduced pressure, and then purified by CombiFlash (2–10% EtOAc in hexanes) to provide the product, 2,2-di(hept-6-en-1-yl)dimethyl malonate (5.5 g, 75% yield), as a colorless liquid. 1 H NMR(400MHz,CDCl3)δ 1.08-1.18(m,4H),1.25-1.40(m,8H),1.81-1.89(m,4H),2.01(q,4H),3.69(s,6H),4.88-5.01(m,4H),5.70-5.90(m,2H).

[0365] Step 2. 2,2-Di(hept-6-en-1-yl)malonic acid To a stirred solution of dimethyl 2,2-di(hept-6-en-1-yl)malonate (5.3 g, 16.35 mmol) in EtOH:HO (50 mL) was added KOH (8.63 g, 49.04 mmol) under ice-cooled conditions under a nitrogen atmosphere. The reaction mixture was then refluxed for 16 h. The reaction mixture was cooled to 25 °C and quenched with 1 N HCl. The product was extracted with EtOAc (2 × 250 mL), dried over anhydrous NaSO, and concentrated under reduced pressure to give 2,2-di(hept-6-en-1-yl)malonic acid (4.2 g, 87% yield) as a pale yellow oil, which was used directly in the next step. 1 H NMR(400MHz,CDCl3)δ 1.23-1.41(m,12H),1.90-2.06(m,8H),4.89-5.02(m,4H),5.69-5.84(m,2H).

[0366] Step 3. 2-(hept-6-en-1-yl)non-8-enoic acid 2,2-Di(hept-6-en-1-yl)malonic acid (4 g, 13.5 mmol) was heated to 190 °C for 2 h. The reaction mass was cooled to 25 °C and then purified by CombiFlash (20-40% EtOAc in hexanes) to provide 2-(hept-6-en-1-yl)non-8-enoic acid (3.2 g, 94% yield). 1 H NMR(400MHz,CDCl3)δ 1.24-1.38(m,12H),1.41-1.50(m,2H),1.57-1.65(m,2H),2.00-2.04(m,4H),2.31-2.38(m,1H),4.89-5.02(m,4H),5.71-5.86(m,2H).

[0367] Step 4. 2-(hept-6-en-1-yl)non-8-en-1-ol To an ice-cold solution of 2-(hept-6-en-1-yl)non-8-enoic acid (3, 11.89 mmol) in THF (50 mL) was added LAH (2.4 M solution in THF) (17.33 mL, 41.6 mmol). The reaction mixture was stirred at 70 °C for 3 h, then cooled to 0 °C and quenched by the addition of NaSO-5H0. The reaction mixture was filtered with EtOAc (200 mL). The filtrate was concentrated under reduced pressure and then purified by CombiFlash (20-40% EtOAc in hexanes) to provide 2-(hept-6-en-1-yl)non-8-en-1-ol (2.4 g, 84.7% yield) as a colorless liquid. 1 H NMR(400MHz,CDCl3)δ 1.23-1.49(m,17H),2.01-2.07(m,4H),3.54(d,2H),4.94(d,2H),4.99(d,2H),5.73-5.87(m,2H).

[0368] Step 5. 6-((2-(hept-6-en-1-yl)non-8-en-1-yl)oxy)-6-oxohexanoic acid To a stirred solution of adipic acid (2.94 g, 20.15 mmol) in DCM (30 mL) was added DIPEA (2.6 g, 20.15 mmol), EDC·HCl (1.93 g, 10.01 mmol), and DMAP (0.25 g, 2.015 mmol) sequentially at 25 °C. The reaction mixture was stirred for 10 minutes, and then 2-(hept-6-en-1-yl)non-8-en-1-ol (1.6, 6.72 mmol) was added thereto. The reaction mixture was stirred at 25 °C for 16 hours while being monitored by TLC (30% EtOAc in hexanes). After completion, the reaction mixture was diluted with water (30 mL), and the product was extracted with DCM (2 × 50 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, and then purified by CombiFlash (30-50% EtOAc in hexanes) to give (1.6 g, 65% yield) as a colorless liquid. 1H NMR(400MHz,DMSO-d6)δ 1.15-1.40(m,17H),1.40-1.71(m,4H),2.00(q,4H),2.20(t,2H),2.29( t,2H),3.91(d,2H),4.89-5.04(m,4H),5.71-5.86(m,2H),12.01(s,1H).

[0369] Step 6. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(2-(hept-6-en-1-yl)non-8-en-1-yl) adipate Prepared according to general procedure D, substituting 6-((2-(hept-6-en-1-yl)non-8-en-1-yl)oxy)-6-oxohexanoic acid for 4,4-bis(octyloxy)butanoic acid and 3-hydroxy-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate for 3-hydroxy-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Isolated mass 1.1 g, yield 55%. 1 H NMR (400MHz, chloroform-d)δ 0.94(t,12H),1.33-1.46(m,6H),1.55-1.71(m,8H),1.85-2.11(m,8H),2.13-2.26(m,2H),2.26-2.47(m,12H),3.34-3.45( m,4H),3.63(d,4H),3.96(d,4H),4.07-4.25(m,6H),4.48(t,2H),4.86-5.05(m,4H),5.21-5.49(m,4H),5.72-5.87(m,4H).

[0370] Step 7. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl(2-(hept-6-en-1-yl)non-8-en-1-yl)adipate (Example 7-39) Prepared according to general procedure L, substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(2-(hept-6-en-1-yl)non-8-en-1-yl)adipate. Isolated mass 160 mg as a pale brown liquid, 46% yield. UPLC-MS: Method A was performed for UPLC. Rt 6.83 min, MS calculated: 1087.81 [M+H], MS found 1088.45 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ 0.82-0.94(m,3H),0.95-1.29(m,52H),1.43-1.51(m,6H),1.67-1.80(m,5H),1.96-2.02(m,8H),2.21-2.39(m,11H),3. 92(d,4H),4.06(d,6H),4.42-4.47(m,1H),4.59-4.64(m,1H),4.91-5.07(m,8H),5.24-5.37(m,4H),5.78-5.83(m,1H).

[0371] Example 7-40: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(dimethylamino)ethyl)carbamate 2-(pyrrolidin-1-yl)ethan-1-amine N 1 ,N 1 Prepared according to general procedure G, substituting -dimethylethane-1,2-diamine. Isolated mass 750 mg, yield 84%. 1H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.86(d,12H),1.18-1.32(m,8H),1.45-1.60(m,6H),2.24(s,6H),2.38 -2.44(m,2H),3.25(d,2H),3.57-3.61(m,2H),4.67-4.77(m,1H),5.12-5.19(m,1H).

[0372] Step 2. 1-Hydroxydecan-4-yl(2-(dimethylamino)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(dimethylamino)ethyl)carbamate. Isolated mass 420 mg, yield 78%. 1 H NMR(400MHz,CDCl3)δ 0.86(t,3H),1.20-1.31(m,7H),1.62-1.68(m,8H),2.86(s,6H),3.16-3.27(m,2H),3.51-3.77(m,4H),4.72-4.82(m,1H),6.38(s,1H).

[0373] Step 3. 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoic acid Prepared according to general procedure J, substituting 1-hydroxydecan-4-yl(2-(dimethylamino)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 410 mg, yield 93%. 1 H NMR(400MHz,DMSO-d6)δ 0.86(t,3H),1.19-1.30(m,8H),1.47(s,2H),1.64-1.82(m,2H),2.15-2.28(m,2H),2.78(s,6H),3.05-3 .14(m,2H),3.73-3.83(m,1H),4.30-4.40(m,1H),4.60-4.66(m,1H),7.31(s,1H),11.87-12.17(m,1H).

[0374] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoate (Example 7-40) Prepared according to general procedure L, substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoic acid and 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate. Isolated mass 122 mg, yield 30%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.88 min, MS calculated: 1023.77 [M+H], MS observed: 1024.32 [M+H]. 1 H NMR (400 MHz, DMSO-d6) δ 0.80-0.87(m,9H),0.90(t,6H),1.17-1.29(m,24H),1.32-1.37(m,5H),1.41- 1.57(m,14H),1.64-1.68(m,1H),1.72-1.80(m,3H),1.95-2.03(m,8H),2.13( s,6H),2.25-2.34(m,10H),2.98-3.08(m,2H),3.35-3.51(m,3H),4.00(t,2H) ,4.06(d,6H),4.44(t,1H),4.58-4.65(m,1H),5.23-5.39(m,4H),6.86(s,1H).

[0375] Example 7-41: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(diethylamino)ethyl)carbamate 2-(pyrrolidin-1-yl)ethan-1-amine N 1 ,N 1 Prepared according to general procedure G, substituting -diethylethane-1,2-diamine. Isolated mass 2.1 g, 70% yield. UPLC-MS: Performed on UPLC, method B2. Rt 0.58 min, MS calculated: 431.36 [M+H], MS found 431.56 [M+H].

[0376] Step 2. 1-Hydroxydecan-4-yl(2-(diethylamino)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(diethylamino)ethyl)carbamate. Isolated mass 950 mg, yield 66%. UPLC-MS: Performed method B2 for UPLC. Rt 0.34 min, MS calculated: 317.27 [M+H], MS found 317.33 [M+H].

[0377] Step 3. 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoic acid Prepared according to general procedure J, substituting 1-hydroxydecan-4-yl(2-(diethylamino)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 700 mg, yield 83%. UPLC-MS: Performed method B2 on UPLC. Rt 0.36 min, MS calculated: 331.25 [M+H], MS found 331.47 [M+H].

[0378] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoate (Example 7-41) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 178 mg, yield 56%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.86 min, MS calculated: 1051.81 [M+H], MS observed 1052.32 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ 0.80-0.87(m,6H),0.91(t,6H),0.97-1.28(m,28H),1.32-1.64(m,18H) ,1.72-1.81(m,2H),1.83-2.10(m,11H),2.14-2.35(m,12H),2.88-3.05 (m,4H),3.27-3.39(m,8H),3.44-3.54(m,2H),4.00(t,2H),4.06(d,4H) ,4.44(t,1H),4.57-4.66(m,1H),5.09-5.53(m,4H),8.98-9.37(m,1H).

[0379] Example 7-42: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((3-(diethylamino)propyl)carbamoyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbamate 2-(pyrrolidin-1-yl)ethan-1-amine N 1 ,N 1 Prepared according to general procedure G, substituting -diethylpropane-1,3-diamine. Isolated mass 850 mg, yield 87%. 1 H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.86(d,12H),1.18-1.32(m,8H),1.45-1.60(m,6H),2.24(s,6H),2.38 -2.44(m,2H),3.25(d,2H),3.57-3.61(m,2H),4.67-4.77(m,1H),5.12-5.19(m,1H).

[0380] Step 2. 1-Hydroxydecan-4-yl(3-(diethylamino)propyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbamate. Isolated mass 520 mg, yield 82%. UPLC-MS: Performed method B2 for UPLC. Rt 0.35 min, MS calculated: 331.29 [M+H], MS found 331.42 [M+H].

[0381] Step 3. 4-(((3-(diethylamino)propyl)carbamoyl)oxy)decanoic acid Prepared according to general procedure J, substituting 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(3-(diethylamino)propyl)carbamate. Isolated mass 489 mg, yield 90%. UPLC-MS: Performed method B2 on UPLC. Rt 0.34 min, MS calculated: 345.27 [M+H], MS found 345.56 [M+H].

[0382] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((3-(diethylamino)propyl)carbamoyl)oxy)decanoate (Example 7-42) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((3-(diethylamino)propyl)carbamoyl)oxy)decanoic acid. Isolated mass 125 mg, yield 34%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.88 min, MS calculated: 1065.82 [M+H], MS observed 1066.31 [M+H]. 1H NMR(400MHz,DMSO-d6)δ 0.73-1.03(m,21H),1.11-1.29(m,23H),1.31-1.59(m,21H),1.63-1.68 (m,1H),1.75-1.81(m,3H),1.93-2.04(m,8H),2.24-2.35(m,10H),2.37 -2.43(m,4H),2.93-3.00(m,2H),3.33-3.52(m,4H),4.00(t,2H),4.05( d,6H),4.44(t,1H),4.56-4.65(m,1H),5.23-5.39(m,4H),6.99(s,1H).

[0383] Example 7-43: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(ethyl(methyl)amino)ethyl)carbamate 2-(pyrrolidin-1-yl)ethan-1-amine N 1 -ethyl-N 1 Prepared according to general procedure G, substituting -methylethane-1,2-diamine. Isolated mass 580 mg, yield 63%. UPLC-MS: Performed method B2 on UPLC. Rt 0.6 min, MS calculated: 417.34 [M+H], MS found 417.58 [M+H].

[0384] Step 2. 1-Hydroxydecan-4-yl(2-(ethyl(methyl)amino)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(ethyl(methyl)amino)ethyl)carbamate. Isolated mass 320 mg, yield 76%. 1H NMR(400MHz,CDCl3)δ 0.84-0.89(m,3H),1.22-1.29(m,8H),1.49-1.65(m,13H),2.77-2.85(m,2H),3.48(s,3H),3.65-3.72(m,2H),4.73-4.79(m,1H).

[0385] Step 3. 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoic acid Prepared according to general procedure J, substituting 1-hydroxydecan-4-yl(2-(ethyl(methyl)amino)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 290 mg, yield 87%. 1 H NMR(400MHz,DMSO-d6)δ 0.83-0.88(m,3H),1.02-1.06(m,1H),1.16-1.29(m,13H),1.46-1.50(m,2H),1.62-1.68(m,1H),1.75-1.81(m,1H),2.21-2 .25(m,2H),2.74-2.76(m,2H),3.05-3.11(m,2H),3.14-3.18(m,2H),4.60-4.66(m,1H),7.30(s,1H),11.95-12.11(m,1H).

[0386] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoate (Example 7-43) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 160 mg, yield 41%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.92 min, MS calculated: 1037.79 [M+H], MS observed: 1038.28 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ 0.79-0.85(m,9H),0.88-0.96(m,9H),1.11-1.29(m,27H),1.32-1.52 (m,18H),1.73-1.79(m,3H),1.95-2.02(m,8H),2.12(s,3H),2.27-2.3 3(m,10H),2.99-3.03(m,2H),3.44-3.54(m,3H),4.00(t,2H),4.06(d ,6H),4.44(t,1H),4.56-4.66(m,2H),5.20-5.34(m,4H),6.83(s,1H).

[0387] Example 7-44: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(piperidin-1-yl)ethyl)carbamate Prepared according to general procedure G, substituting 2-(piperidin-1-yl)ethan-1-amine for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 620 mg, yield 62%. 1H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.88(s,12H),1.19-1.34(m,10H),1.43-1.57(m,10H),2.35-2.52(m ,6H),3.21-3.33(m,2H),3.57-3.62(m,2H),4.70-4.81(m,1H),5.20-5.34(m,1H).

[0388] Step 2. 1-Hydroxydecan-4-yl(2-(piperidin-1-yl)ethyl)carbamate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(piperidin-1-yl)ethyl)carbamate. Isolated mass 330 mg, yield 71%. 1 H NMR(400MHz,DMSO-d6)δ 0.83-0.88(m,3H),1.22-1.28(m,8H),1.38-1.52(m,7H),1.66-1.78(m,4H),2.08(s,3H),2. 82-2.92(m,2H),3.03-3.12(m,2H),3.35-3.46(m,5H),4.34-4.42(m,1H),4.57-4.64(m,1H).

[0389] Step 3. 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid Prepared according to general procedure J, substituting 1-hydroxydecan-4-yl(2-(piperidin-1-yl)ethyl)carbamate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 290 mg, 88%. 1H NMR(400MHz,DMSO-d6)δ 0.83-0.88(m,3H),1.03(d,4H),1.24-1.26(m,4H),1.44-1.53(m,2H),1.67-1.79(m,6H),2.19-2.26(m,2H),2.83-2.92(m ,2H),3.02-3.10(m,2H),3.32-3.41(m,4H),3.77(t,1H),4.29-4.40(m,1H),4.58-4.67(m,1H),7.34(s,1H),12.08(s,1H).

[0390] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoate (Example 7-44) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 165 mg, yield 44%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.89 min, MS calculated: 1063.81 [M+H], MS observed: 1064.32 [M+H]. 1H NMR (400MHz, DMSO-d6) δ0.79~0.94 (m, 15H), 1.04~1.29 (m, 26H), 1.31~1. 38(m, 7H), 1.40~1.58(m, 18H), 1.73~1.79(m, 3H), 1.95~2.04(m, 8H), 2.2 3~2.38(m, 14H), 2.97~3.08(m, 2H), 3.42~3.53(m, 2H), 4.00(t, 2H), 4.05 (d, 6H), 4.44(t, 1H), 4.57~4.64(m, 1H), 5.23~5.39(m, 4H), 6.82(s, 1H).

[0391] Example 7-45: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbonate To a solution of 3-(diethylamino)propan-1-ol (2 equiv.) in THF (20 mL) was added NaH (2 equiv.) at 0° C. and then stirred for 30 min. To this suspension was added 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(4-nitrophenyl)carbonate (1 equiv.). The reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was diluted with DCM (250 mL), water (50 mL), and brine (50 mL). The resulting organic layer was dried over anhydrous NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography using 2–5% MeOH-DCM to give 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbonate (820 mg, 83% yield). 1H NMR(400MHz,CDCl3)δ 0.03(s,6H),0.87(s,12H),1.00(t,6H),1.22-1.30(m,8H),1.57-1.65(m,6H) ,1.80(t,2H),2.50(d,6H),3.57-3.61(m,2H),4.16(t,2H),4.63-4.75(m,1H).

[0392] Step 2. 3-(Diethylamino)propyl(1-hydroxydecan-4-yl)carbonate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate with 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(3-(diethylamino)propyl)carbonate. Isolated mass 510 mg, yield 84%. UPLC-MS: Performed method B2 for UPLC. Rt 0.4 min, MS calculated: 332.27 [M+H], MS found 332.55 [M+H].

[0393] Step 3. 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid Prepared according to general procedure J, substituting 3-(diethylamino)propyl(1-hydroxydecan-4-yl)carbonate for 1-hydroxydecan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 480 mg. UPLC-MS: Performed method B2 on UPLC. Rt 0.42 min, MS calculated: 346.25 [M+H], MS found 346.52 [M+H].

[0394] Step 4. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoate 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(4-nitrophenyl)carbonate (Examples 7-45) Prepared according to general procedure L, substituting 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadeca-9,12-dienoate with 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl 2-butyloctanoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid with 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid. Isolated mass 125 mg, yield 40%. UPLC-MS: Method B2 was performed for UPLC. Rt 6.85 min, MS calculated: 1066.81 [M+H], MS observed 1067.25 [M+H]. 1 H NMR(400MHz,CDCl3)δ 0.84-0.89(m,6H),0.94(t,6H),1.12-1.29(m,15H),1.31-1.45(m,12H),1.44- 1.76(m,32H),1.84-1.95(m,4H),1.94-2.09(m,8H),2.23-2.34(m,4H),2.33-2 .44(m,4H),3.34-3.43(m,2H),3.52-3.59(m,2H),4.02-4.07(m,2H),4.11(d,6) H),4.18-4.22(m,2H),4.45-4.50(m,1H),4.68-4.74(m,1H),5.22-5.39(m,4H).

[0395] Example 7-46: 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)decanoate Step 1. 1-((tert-butyldimethylsilyl)oxy)decan-4-yl((1-ethylpiperidin-3-yl)methyl)carbonate To a solution of (1-ethylpiperidin-3-yl)methanol (2 equiv.) in THF (20 mL) was added NaH (2 equiv.) at 0° C. and then stirred for 30 min. To this suspension was added 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(4-nitrophenyl)carbonate (1 equiv.). The reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was diluted with DCM (250 mL), water (50 mL), and brine (50 mL). The resulting organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude residue was purified by flash column chromatography using 2-5% MeOH-DCM. Isolated mass 880 mg, yield 87%. UPLC-MS: Method B2 was performed for UPLC. Rt 0.64 min, MS calculated: 458.36 [M+H], MS found: 458.36 [M+H].

[0396] Step 2. (1-Ethylpiperidin-3-yl)methyl(1-hydroxydecan-4-yl)carbonate Prepared according to general procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)decan-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate with 1-((tert-butyldimethylsilyl)oxy)decan...

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