Nanomaterials comprising triol

Abstract

The present disclosure describes compositions, formulations, nanoparticles (e.g., lipid nanoparticles), and / or nanomaterials, and methods of use thereof.

Description

[0001] Related Applications

[0002] This application claims priority to and the benefit of U.S. Application No. 63 / 390,882, filed on July 20, 2022, the entire contents of which are hereby incorporated by reference. Background Art

[0003] Lipid-containing particles have been used to encapsulate therapeutic agents such as nucleic acids, small molecules and proteins and serve as transport vehicles for the therapeutic agents to enter cells and other intracellular compartments. There is still a need to develop new lipids to encapsulate therapeutic agents and improve the safety, efficacy and specificity of these nanoparticle-based transport vehicles. Summary of the invention

[0004] The present invention recognizes the need for compositions, preparations, nanoparticles and / or nanomaterials and methods of use thereof. In addition, the disclosure recognizes that the structural characteristics of compositions, preparations, nanoparticles and / or nanomaterials affect functional responses in vivo, in vitro and in vitro. For example, in addition, the disclosure describes that the selection and combination of one or more components described herein can affect the functional activity of lipid nanoparticles. In some embodiments, for example, functional activity can refer to desired tropism, stability, bioavailability, degradation characteristics and / or drug delivery efficacy. In some embodiments, in addition, the disclosure describes that one or more components of different ratios affect one or more functional activities of compositions, preparations, nanoparticles and / or nanomaterials described herein.

[0005] In addition, the present disclosure particularly 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 describes a compound of formula I:

[0006]

[0007] or a pharmaceutically acceptable salt thereof, wherein L 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.

[0008] In addition, as described herein, the present disclosure can exhibit unexpected properties (e.g., unexpected tropism, stability, bioavailability, degradation characteristics and delivery efficacy of cargo, such as therapeutic or prophylactic agents) of ionizable lipids and compositions, formulations, nanoparticles and / or nanomaterials (e.g., LNPs and / or compositions, formulations, nanoparticles and / or nanomaterials containing LNPs) comprising a triol core and / or a biodegradable tail, and methods of use thereof. In some embodiments, the present disclosure can exhibit particularly unexpected properties (e.g., unexpected tropism, stabilization, bioavailability, degradation characteristics and delivery efficacy of cargo, such as therapeutic or prophylactic agents) of ionizable lipids and compositions, formulations, nanoparticles and / or nanomaterials (e.g., LNPs and / or compositions, formulations, nanoparticles and / or nanomaterials containing LNPs), and methods of use thereof. In some embodiments, the ionizable lipids, compositions, formulations, nanoparticles and / or nanomaterials (e.g., LNPs and / or compositions, formulations, nanoparticles and / or nanomaterials containing LNPs) comprising a biodegradable tail may be characterized by improved stability relative to certain comparable reference entities. In some embodiments, the tail structures described herein will be more easily degraded once administered in vivo compared to other tail structures.

[0009] In some embodiments, the present disclosure can identify the source of the problem of certain alternative lipid compounds and / or compositions, preparations, nanoparticles and / or nanomaterials comprising it, for example, provide insights into certain tail structures that can confer or contribute to one or more properties, and the improvement of the property will be beneficial. In addition to this identification of the source of the problem, the present disclosure can provide certain solutions to the problem, including lipid compounds with different tail structures and / or compositions, preparations, nanoparticles and / or nanomaterials comprising it. In some embodiments, the lipid compounds provided and / or compositions, preparations, nanoparticles and / or nanomaterials comprising it are characterized by improved stability, bioavailability and / or degradation characteristics relative to appropriate comparable references with different tail structures. In some specific embodiments, the lipid compounds provided and / or compositions, preparations, nanoparticles and / or nanomaterials comprising it are characterized by improved stability, bioavailability and / or degradation characteristics relative to appropriate comparable references with linoleic acid tail features (e.g., parts). In some specific embodiments, provided lipid compounds and / or compositions, formulations, nanoparticles and / or nanomaterials comprising the same may be characterized by improved stability, bioavailability and / or degradation properties relative to an appropriate comparable reference having more than one linoleic acid tail feature (e.g., moiety).

[0010] In addition, the present disclosure recognizes that lipid nanoparticle (LNP) compositions comprise one or more ionizable lipids

[0011] In some embodiments, provided compositions, formulations, nanoparticles and / or nanomaterials can be used in methods of treatment, methods of delivery, methods of producing polypeptides, or methods of delaying / preventing progression of a disease or condition.

[0012] In some embodiments, provided compositions, formulations, nanoparticles, and / or nanomaterials can be used in manufacturing methods.

[0013] In some embodiments, provided compositions, formulations, nanoparticles, and / or nanomaterials can be used in characterization methods.

[0014] Elements of embodiments relating to one aspect of the invention (eg, a method) may be applied in embodiments relating to other aspects of the invention, and vice versa. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Depicted are bar graphs showing delivery of exemplary LNP formulations (Compound 7-1, Compound 7-2) to the liver.

[0016] Figure 2 Depicted are bar graphs showing delivery of exemplary LNP formulations (Compound 7-1, Compound 7-2) to the spleen.

[0017] Figure 3 Depicted is a bar graph showing base editing in liver cells following delivery of an exemplary LNP formulation (Compound 7-7) to the liver.

[0018] Figure 4 Depicted is a bar graph showing base editing in liver cells following delivery of an exemplary LNP formulation (Compound 7-7) to the liver.

[0019] Figure 5 Depicted is a bar graph showing siRNA mediated silencing in liver cells following delivery of an exemplary LNP formulation (Compound 7-2) to the liver.

[0020] definition

[0021] About: As used herein, the term "about" or "approximately" refers to a value similar to a reference value in the context when used herein with respect to a value. In general, those skilled in the art who are familiar with the context will appreciate that the relevant degree of deviation covered by "about" or "approximately" in the context. For example, in some embodiments, unless otherwise specified or otherwise apparent from the context, the term "about" may encompass a range of values ​​(except when the numerical value exceeds 100% of the possible value) of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% falling within any direction (greater than or less than) of the reference value.

[0022] Administration: As used herein, the term "administering" generally refers to administering a composition to a subject or system. A person of ordinary skill in the art will be aware of various routes that can be used for administration to a subject, such as a human, under appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, transdermal (which may be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration may involve intermittent administration (e.g., multiple doses spaced apart in time) and / or regular administration (e.g., each dose spaced apart by a common period of time). In some embodiments, administration may involve continuous dosing (e.g., perfusion) at least over a selected period of time. In some embodiments, pharmaceutical compositions comprising lipid nanoparticles may be formulated to be administered 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 using bioerodible inserts, and may be formulated in dosage forms suitable for each route of administration.

[0023] Aliphatic: As used herein, the term "aliphatic" or "aliphatic group" means a straight chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain, which is fully saturated or contains one or more unsaturated units; or a monocyclic hydrocarbon or a bicyclic hydrocarbon, which is fully saturated or contains one or more unsaturated units, but it is not an aromatic group with a single point of attachment to the rest of the molecule (also referred to herein as "carbocycle", "carbocyclic", "cycloaliphatic" or "cycloalkyl"). 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 "cycloaliphatic" or "carbocycle" or "cycloalkyl") refers to an optionally substituted monocyclic C3-C8 hydrocarbon or an optionally substituted C6-C8 hydrocarbon. 12 Bicyclic hydrocarbons which are fully saturated or contain one or more unsaturated units, but which are not aromatic groups having a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight or branched chain, substituted or unsubstituted alkyl, alkenyl, alkynyl, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0024] 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 having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 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 ) is an optionally substituted straight or branched hydrocarbon chain. Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.

[0025] Alkenylene: The term "alkenylene" refers to a divalent alkenyl group. Substituted alkenylene chains are polymethylene groups containing at least one double bond in which one or more hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0026] Alkyl: As used herein, the term "alkyl" is given its ordinary meaning in the art and may include saturated aliphatic groups, including straight chain alkyls, branched chain alkyls, cycloalkyls (alicyclic groups), alkyl-substituted cycloalkyls, and cycloalkyl-substituted alkyls. In some embodiments, an alkyl group has 1-100 carbon atoms. In certain embodiments, a straight chain or branched chain alkyl group has about 1-20 carbon atoms in its backbone (e.g., C1-C2 for a straight chain). 20 ; For branched chains, C2-C 20 ), and alternatively about 1-10 carbon atoms. In some embodiments, the cycloalkyl ring has about 3 to 10 carbon atoms in its ring structure, wherein these rings are monocyclic, bicyclic or polycyclic, and alternatively have about 5, 6 or 7 carbon atoms in the ring structure. In some embodiments, the alkyl group can be a lower alkyl group, wherein the lower alkyl group contains 1-4 carbon atoms (e.g., C1-C4 for straight chain lower alkyl groups).

[0027] Alkylene: The term "alkylene" or "alkylenyl" refers to a linear (i.e., unbranched) or branched, substituted or unsubstituted divalent alkyl group (i.e., a divalent saturated hydrocarbon chain). Any of the above monovalent alkyl groups can be converted to an alkylene group by abstracting the second hydrogen atom from the alkyl group. In some embodiments, "alkylene" is a polymethylene group, i.e., -(CH2) n -, wherein 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. Substituted alkylene is a polymethylene group in which one or more methylene hydrogen atoms are replaced by a substituent. Suitable substituents include the substituents described below for substituted aliphatic groups.

[0028] 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 having (unless otherwise specified) 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C 2-20 , C 2-18 , C 2-16 , C 2-14 , C 2-12 , C 2-10 , C2-8 , C 2-6 , C 2-4 or C 2-3 An optionally substituted straight or branched chain hydrocarbon group having 1,200 carbon atoms). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0029] Amino Acid: As used herein, in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain, such as by the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R AA )-COOH, where 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; in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is prepared synthetically or obtained from a natural source. In some embodiments, amino acids, including carboxyl-terminal and / or amino-terminal amino acids in polypeptides, may contain structural modifications compared to the general structure above. For example, in some embodiments, compared to the general structure, amino acids may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation and / or substitution (e.g., substitution of an amino group, a carboxylic acid group, one or more protons and / or hydroxyl groups). In some embodiments, such modifications may, for example, change the circulating half-life of a polypeptide containing a modified amino acid compared to a polypeptide containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly change the relevant activity of a polypeptide containing a modified amino acid compared to a polypeptide containing an otherwise identical unmodified amino acid. As will be apparent from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to an amino acid residue of a polypeptide.

[0030] Animal: As used herein, refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans of any sex and at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0031] Aryl: The term "aryl" refers to a radical having a total of six to fourteen ring members (e.g., C 6-14 ) wherein at least one ring in the system is aromatic, and wherein each ring in the system contains three to seven ring members. The term "aryl" can be used interchangeably with the term "aryl ring". In some embodiments, "aryl" refers to an aromatic ring system including, but not limited to, phenyl, naphthyl, anthracenyl, etc., which may carry one or more substituents. Unless otherwise indicated, "aryl" is a hydrocarbon.

[0032] Associated: Two events or entities are "associated" with each other as the term is used herein if the presence, level, extent, type, and / or form of one event or entity is related to the presence, level, extent, type, and / or form of another event or entity. For example, a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if the presence, level, and / or form of the entity is associated with the incidence and / or susceptibility to the disease, disorder, or condition (e.g., within a related population). In some embodiments, two or more entities are physically "associated" with each other if they interact, directly or indirectly, such that they are physically close to each other and / or remain in close proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other, but are non-covalently bound, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0033] Biocompatible: As used herein, the term "biocompatible" refers to materials that do 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 less than or equal to 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse reactions.

[0034] Biodegradable: As used herein, the term "biodegradable" means that when a material is introduced into a cell, the material is broken down (e.g., by cellular mechanisms, such as by enzymatic degradation, by hydrolysis, and / or by a combination thereof) into components that can be reused or processed by the cell and have no significant toxic effects on the cell. In certain embodiments, the components produced by the decomposition of the biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse reactions in vivo. In some embodiments, a biodegradable polymeric material is decomposed into its component monomers. In some embodiments, the decomposition of a biodegradable material (including, for example, a biodegradable polymeric material) involves the hydrolysis of an ester bond. Alternatively or additionally, in some embodiments, the decomposition of a biodegradable material (including, for example, a biodegradable polymeric material) involves the cleavage of a carbamate bond. Exemplary biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including, but not limited to, poly(hydroxy 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(hydroxyalkanoates, poly(lactide-co-caprolactone), mixtures and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, proteins such as albumin, collagen, gelatin, and prolamins such as zein; and polysaccharides such as alginates, cellulose derivatives, and polyhydroxyalkanoates such as polyhydroxybutyrate; mixtures and copolymers thereof. One of ordinary skill in the art will understand or be able to determine when these polymers are biocompatible and / or biodegradable derivatives thereof (e.g., related to the parent polymer by substantially the same structure, the structure differing only in the substitution or addition of specific chemical groups known in the art).

[0035] Biological activity: 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, the regulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or extent of a biological activity is assessed by detecting a direct or indirect product produced by a biological pathway or event of interest.

[0036] Divalent: As used herein, the term "divalent" refers to a chemical moiety that has two points of attachment. For example, the term "divalent C 1-8 (or C 1-6 ) saturated or unsaturated straight or branched hydrocarbon chain” refers to straight or branched divalent alkylene chains, alkenylene chains and alkynylene chains as defined herein.

[0037] Bridged bicyclic group: As used herein, the terms "bridged bicyclic group", "bridged bicyclic", "bridged bicyclic" and "bridged bicyclic ring" refer to any saturated or partially unsaturated bicyclic ring system, i.e., carbocyclic or heterocyclic, 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, wherein a "bridgehead" is any backbone atom of a ring system bonded to three or more backbone atoms (except hydrogen). In some embodiments, the bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. These bridged bicyclic groups are well known in the art and include those described below, each of which is connected to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise stated, the bridged bicyclic group is optionally substituted by one or more substituents as described for aliphatic groups. In addition or alternatively, any substitutable nitrogen of the bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include, but are not limited to:

[0038]

[0039] 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 the cells exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a related cancer may be characterized as a solid tumor. In some embodiments, a related cancer may be characterized as a hematological tumor. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers, including leukemias; lymphomas (Hodgkin's and non-Hodgkin's); myelomas and myeloproliferative disorders; sarcomas; melanomas; adenomas; solid tissue carcinomas; squamous cell carcinomas of the oral cavity, pharynx, larynx, and lung; liver cancer; genitourinary tract cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer; as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, benign lesions, such as papilloma, etc.

[0040] Carrier: As used herein, refers to a diluent, adjuvant, excipient or vehicle for administering a composition. In some exemplary embodiments, the carrier may include sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or includes one or more solid components.

[0041] Carbocyclyl: As used herein, the terms "carbocyclyl," "carbocycle," and "carbocyclic ring" refer to a saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring system having 3 to 14 members as described herein, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, "carbocyclyl" (or "cycloaliphatic") refers to an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C6-C8 hydrocarbon. 12 Bicyclic hydrocarbons, which are fully saturated or contain one or more unsaturated units, but which are not single-point connected aromatic groups to the rest of the molecule. The term "cycloalkyl" refers to an optionally substituted saturated ring system having about 3 to about 10 ring carbon atoms. In some embodiments, the cycloalkyl has 3-6 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 system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl and cycloheptenyl.

[0042] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition sets, etc., which may not be identical to each other, but are similar enough to allow comparisons to be made therebetween, so that a person skilled in the art will understand that a reasonable conclusion can be drawn based on the observed differences or similarities. In some embodiments, a comparable condition set, situation, individual or group is characterized by a plurality of substantially identical features and one or a small amount of different features. A person of ordinary skill in the art will understand that, in the context, for two or more of these agents, entities, situations, condition sets, etc., in any given case, what degree of identity is considered to be required to be comparable. For example, a person of ordinary skill in the art will understand that a set of situations, individuals or groups is comparable to each other when it is characterized by a sufficient number and type of substantially identical features to ensure a reasonable conclusion, wherein the difference in the results or observed phenomena obtained under different sets of situations, individuals or groups or using different sets of situations, individuals or groups is caused by the changes in those features that change or indicates the changes in those features that change.

[0043] Composition: Those skilled in the art will appreciate that the term "composition" may be used to refer to a discrete physical entity comprising one or more specified components. Generally, unless otherwise specified, a composition may be in any form, such as gas, gel, liquid, solid, etc.

[0044] Comprising: A composition or method described herein as "comprising" one or more specified elements or steps is open ended, meaning that the specified elements or steps are required, but other elements or steps may be added within the scope of the composition or method. To avoid redundancy, it is also understood that any composition or method described as "comprising" (or "comprising") one or more specified elements or steps also describes a corresponding, more limited composition or method "consisting essentially of the same specified elements or steps" (or "consisting essentially of the same specified elements or steps"), meaning that the composition or method includes the specified basic elements or steps, and may also include additional elements or steps that do not substantially affect the basic and novel characteristics of the composition or method. It is also understood that any composition or method described herein as "comprising" one or more specified elements or steps or "consisting essentially of" one or more specified elements or steps also describes a corresponding, more limited and closed composition or method "consisting of" (consisting of / consists of) the specified elements or steps, excluding any other unspecified elements or steps. In any composition or method disclosed herein, any designated essential element or step may be substituted with known or disclosed equivalents thereof.

[0045] "Improve," "increase," "inhibit," or "decrease": As used herein, the terms "improve," "increase," "inhibit," "decrease," or their grammatical equivalents indicate a value relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or include a measurement in a particular system (e.g., in a single individual) in the absence (e.g., before and / or after) of a particular agent or treatment under otherwise comparable conditions or in the presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or include a measurement in a comparable system that is known or expected to respond in a particular manner in the presence of the relevant agent or treatment.

[0046] Determine: The various methods described herein include a step of "determining". Those skilled in the art will appreciate upon reading this specification that such "determinations" can be implemented using any of a variety of techniques available to those skilled in the art or by using any of a variety of techniques available to those skilled in the art, including, for example, specific techniques explicitly mentioned herein. In some embodiments, determination involves manipulation of a physical sample. In some embodiments, determination involves consideration and / or manipulation of data or information, such as using a computer or other processing unit adapted to perform relevant analysis. In some embodiments, determination involves receiving relevant information and / or material from a source. In some embodiments, determination involves comparing one or more features of a sample or entity to a comparable reference.

[0047] Encapsulated: The term "encapsulated" is used herein to mean that a substance is completely surrounded by another material.

[0048] Excipient: As used herein, refers to a non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or promote a desired consistency or stabilization effect. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc.

[0049] 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 may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (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' cap formation 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.

[0050] Heteroaryl: The terms "heteroaryl" and "heteroar-", e.g., "heteroaralkyl" or "heteroaralkoxy", used alone or as part of a larger moiety, refer to monocyclic or bicyclic radicals having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); sharing 6, 10, or 14 pi electrons in the cyclic array; and having from one to five heteroatoms in addition to carbon atoms. Exemplary heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl, 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, cycloaliphatic, or heterocyclyl rings, wherein 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, quinolinyl, isoquinolinyl, 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 includes rings that are optionally substituted.

[0051] Heteroatom: The term "heteroatom" means one or more of 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, such as N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (such as in N-substituted pyrrolidyl)).

[0052] Heterocycle: The terms "heterocycle", "heterocyclyl", "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 that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, such as one to four, heteroatoms as defined above. When used with reference to the ring atoms of the heterocycle, the term "nitrogen" includes substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 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 + (such as in the pyrrolidinyl replaced by N). Heterocycle can be connected to its side group at any heteroatom or carbon atom producing a stable structure, and any ring atom can be optionally substituted. The examples of these saturated or partially unsaturated heterocyclic radicals include but are not limited to azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolyl, oxazolidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, dioxolanyl, diazepine, oxazolyl, thiazolinyl, morpholinyl, thiamorpholinyl and The heterocyclic group may be monocyclic, bicyclic, tricyclic or polycyclic, preferably monocyclic, bicyclic or tricyclic, more preferably monocyclic or bicyclic. The term "heterocyclic alkyl" refers to an alkyl group substituted by a heterocyclic group, wherein the alkyl and heterocyclic moieties are independently optionally substituted. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aryl, heteroaryl or cycloaliphatic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl and tetrahydroquinolinyl. The bicyclic heterocycle may also be a spirocyclic system (e.g., a 7 to 11-membered spirocyclic fused heterocycle having one or more heteroatoms (e.g., one, two, three or four heteroatoms) as defined above, in addition to carbon atoms). The bicyclic heterocycle may also be a bridged ring system (e.g., a 7 to 11-membered bridged heterocycle having one, two or three bridging atoms).

[0053] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

[0054] Isolated: as used herein refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was originally associated when produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity may 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 it was originally associated. In some embodiments, the 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 more 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 skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in these embodiments, the separation or purity percentage of the substance is calculated without including these carriers or excipients. By way of example only, in some embodiments, a naturally occurring biopolymer such as a polypeptide or polynucleotide is considered "isolated" when a) by virtue of its origin or source of derivation, the biopolymer is not associated in nature with some or all of the components that accompany the biopolymer in its native state; b) is substantially free of other polypeptides or nucleic acids from the same species in which the biopolymer is produced in nature; or c) is expressed by or otherwise associated with a component from a cell or other expression system that is not the species in which the biopolymer is produced in nature. Thus, for example, in some embodiments, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cellular system in which the polypeptide is produced in nature is considered 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 to the extent that the polypeptide a) has been separated from other components with which it is associated in nature; and / or b) has been separated from other components with which it was originally associated when produced.

[0055] In vivo: as used herein refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0056] Joint: As used herein, it refers to the part of a multi-element medicament that connects different elements to each other. For example, it will be understood by those of ordinary skill in the art that a polypeptide whose structure includes two or more functional domains or organizational domains generally includes a segment of amino acids between these domains that connect them to each other. In some embodiments, a polypeptide comprising a joint element "L'" has a general structure of the general form S1-L'-S2, wherein S1 and S2 may be the same or different and represent two domains that are bound to each other by a joint. In some embodiments, the length of a polypeptide joint 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 some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather to provide flexibility to the polypeptide. 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: 1121-1123).

[0057] Nanoparticles: As used herein, the term "nanoparticles" refers to particles with a diameter of less than 1000 nanometers (nm). In some embodiments, the diameter of the nanoparticles is less than 300nm, as defined by the National Science Foundation. In some embodiments, the diameter of the nanoparticles is less than 100nm, as defined by the National Institutes of Health. In some embodiments, the nanoparticles are micelles because they contain closed compartments separated from the bulk solution by micellar membranes, typically consisting of amphiphilic entities that surround and enclose a space or compartment (e.g., for defining an inner cavity). In some embodiments, the micellar membrane is composed of at least one polymer, such as a biocompatible and / or biodegradable polymer. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein may be about 30 to about 170nm. In some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein can be about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm or any range with endpoints defined by any two of the aforementioned values. For example, in some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein is between 50nm and 100nm.

[0058] Nanoparticle composition: As used herein, the term "nanoparticle composition" refers to a composition containing at least one nanoparticle and at least one other agent or ingredient. In some embodiments, the nanoparticle composition contains a substantially uniform collection of nanoparticles described herein.

[0059] 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 phosphodiester bonds. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, nucleic acid is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues, or consists of one or more natural nucleic acid residues. In some embodiments, nucleic acid is one or more nucleic acid analogs, comprises one or more nucleic acid analogs, or consists of one or more nucleic acid analogs. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, nucleic acid is one or more "peptide nucleic acids", comprises one or more "peptide nucleic acids", or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, which are considered to be 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-phosphoamidate bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a "nucleic acid" is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 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, embedded bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in natural nucleic acids. In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product, such as RNA or protein. In some embodiments, nucleic acid includes one or more introns. In some embodiments, nucleic acid is prepared by one or more of the following: separation from natural sources, enzymatic synthesis (in vivo or in vitro) by polymerization based on complementary templates, reproduction in recombinant cells or systems, 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; in some embodiments, the nucleic acid is partially or completely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0060] Parenteral: As used herein, the phrases "parenteral administration" and "administered parenterally" have their art-understood meanings and refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0061] Patient: As used herein, the term "patient" refers to any organism to which the provided compositions are applied or can be applied, for example, for experimental, diagnostic, preventive, 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 suffers from or is 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 the presence of cancer or one or more tumors. In some embodiments, the patient is receiving or has received a therapy to diagnose and / or treat a disease, disorder or condition.

[0062] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated 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 shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including pharmaceutical compositions suitable for: oral administration, such as drench (aqueous or non-aqueous solutions or suspensions), tablets, such as tablets targeted for buccal, sublingual and systemic absorption, boluses for application to the tongue, powders, granules, pastes; parenteral administration, such as administration by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension or a sustained release formulation; topical application, such as a cream, ointment or controlled release patch or spray applied to the skin, lungs or oral cavity; intravaginally or rectally, for example as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces.

[0063] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which 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 problems or complications commensurate with a reasonable benefit / risk ratio.

[0064] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle involved in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. 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; powdered tragacanth; malt; gelatin; talc; excipients, such as 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 glycerol, 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; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations.

[0065] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of these compounds suitable for use in a pharmaceutical environment, i.e., salts suitable for use in contact with the tissues of humans and lower animals without producing abnormal toxicity, irritation, allergic reactions, etc., and commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in the following: J. Pharmaceutical Sciences, 66: 1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, which are salts of amino groups with inorganic acids or with organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, 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 salts formed by ion exchange. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-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, and the like. Representative alkali metal 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 using counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates, as appropriate.

[0066] Prevent or prevention: As used herein, when used in conjunction with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition, and / or delaying the onset of one or more characteristics or symptoms of a specific disease, disorder, or condition and / or reducing its frequency and / or severity. In some embodiments, prevention is assessed on a population basis, such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of a disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition, then the agent is considered to "prevent" a specific disease, disorder, or condition. Prevention can be considered complete when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

[0067] Polypeptide: As used herein, the term "polypeptide" generally has the art-recognized meaning of a polymer of at least three amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently general so as to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional fragments of these complete polypeptides (i.e., fragments retaining at least one activity). In addition, it will be understood by those of ordinary skill in the art that protein sequences generally tolerate some substitutions without destroying activity. Therefore, any polypeptide is included in the related term "polypeptide" as used herein with another polypeptide of the same class, any polypeptide retaining activity and sharing at least about 30-40% overall sequence identity, generally greater than about 50%, 60%, 70% or 80%, and further generally including at least one region with higher identity (in one or more highly conserved regions, the identity is generally greater than 90% or even 95%, 96%, 97%, 98% or 99%), generally covering at least 3-4 and generally up to 20 or more amino acids. The polypeptide 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. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the protein may contain natural amino acids, non-natural 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.

[0068] Protein: As used herein, the term "protein" refers to one or more polypeptides used as discrete units. If a single polypeptide is a discrete functional unit and does not need to be physically bound to other polypeptides permanently or temporarily in order to form a discrete functional unit, the terms "polypeptide" and "protein" can be used interchangeably. If the discrete functional unit is composed of more than one polypeptide physically bound to each other, the term "protein" can be used to refer to multiple polypeptides that are physically bound and function together as discrete units. In some embodiments, the protein may include parts other than amino acids (for example, it may be a glycoprotein, proteoglycan, etc.) and / or may be processed or modified in other ways. One of ordinary skill in the art will appreciate that in some embodiments, the term "protein" may refer to a complete polypeptide chain produced by a cell (for example, with or without a signal sequence), and / or to a form that is active in a cell (for example, a truncated or complex form). In some embodiments where the protein is composed of multiple polypeptide chains, these chains may be covalently bound to each other, for example, through one or more disulfide bonds, or may be bound by other means.

[0069] Pure: As used herein, an agent or entity is "pure" if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or entity is generally considered 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.

[0070] Reference: As used herein, describes a standard or control relative to which a comparison is made. For example, in some embodiments, a pharmaceutical agent, animal, individual, colony, sample, sequence, or value of interest is compared to a reference or control pharmaceutical agent, animal, individual, colony, sample, sequence, or value. In some embodiments, a reference or control is tested and / or determined substantially while performing a test or determination of interest. In some embodiments, a reference or control is a historical reference or control optionally embodied in a tangible medium. Typically, as will be understood by those skilled in the art, a reference or control is measured or characterized under conditions or circumstances comparable to the conditions or circumstances being evaluated. Those skilled in the art will understand when there is sufficient similarity so that a specific possible reference or control can be relied upon and / or compared to a specific possible reference or control.

[0071] Sample: As used herein, the term "sample" generally refers to an aliquot of material obtained from 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 can be or comprise 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 comprises a biological tissue or fluid. In some embodiments, the biological tissue or fluid can be or comprise amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, cerumen, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, inflammatory secretions (rheum), saliva, sebum, semen, serum, smegma, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomitus, and / or combinations or components thereof. In some embodiments, the biological fluid can be or comprise intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid can be or comprise plant exudate. In some embodiments, biological tissue or sample can be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab or vaginal swab), scraping, surgery, washing or lavage (e.g., bronchoalveolar, duct, nasal, ocular, oral, uterine, vaginal or other washing or lavage). In some embodiments, biological samples are or include cells obtained from individuals. In some embodiments, the sample is a "raw sample" obtained directly from the source of interest by any appropriate means. In some embodiments, as will be apparent from the context, the term "sample" refers to a preparation obtained by processing the raw sample (e.g., by removing one or more components in the raw sample and / or by adding one or more agents to the raw sample). For example, filtering is performed using a semi-permeable membrane. Such "processed samples" may include, for example, nucleic acids or proteins extracted from the sample or obtained by subjecting the raw sample to one or more techniques such as amplification or reverse transcription of nucleic acids, separation and / or purification of certain components.

[0072] Stable nanoparticle compositions: When applied to the compositions herein, the term "stable" means that the composition maintains one or more aspects of its physical structure (e.g., size range and / or distribution of particles) over a period of time. In some embodiments, a stable nanoparticle composition is a composition in which the average particle size, maximum particle size, particle size range, and / or particle size distribution (i.e., the percentage of particles larger than a specified size and / or outside a specified size range) are maintained under specified conditions for a period of time. In some embodiments, a stable composition is provided that is a composition in which biologically relevant activity is maintained for a period of time. In some embodiments, the period of time is at least about one hour; in some embodiments, the period of time is about 5 hours, about 10 hours, about one (1) day, about one (1) week, about two (2) weeks, about one (1) month, about two (2) months, about three (3) months, about four (4) months, about five (5) months, about six (6) months, about eight (8) months, about ten (10) months, about twelve (12) months, about twenty-four (24) months, about thirty-six (36) months, or more. In some embodiments, this period of time is in the range of about one (1) day to about twenty-four (24) months, about two (2) weeks to about twelve (12) months, about two (2) months to about five (5) months, etc. For example, if a population of nanoparticles is subjected to extended storage, temperature changes, and / or pH changes, and a majority of the nanoparticles in the composition maintain a diameter within the stated range, then the nanoparticle composition is stable. In some embodiments, a stable composition is stable under ambient conditions. In some embodiments, a stable composition is stable under biological conditions (i.e., 37° C. in phosphate buffered saline).

[0073] Sterol group: As used herein, the term "sterol group" refers to a saturated or partially unsaturated 17-membered fused polycyclic moiety substituted with at least one hydroxyl group, and has a single point of connection with the rest of the molecule at any substitutable carbon atom or oxygen atom. In some embodiments, the sterol 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 a β-ring structure or a hydrocarbon tail structure). In some embodiments, the cholesterol group is an esterified cholesterol group. In some embodiments, the sterol group is a plant sterol group. Exemplary sterol groups include, but are not limited to, 25-hydroxycholesterol group (25-OH), 20α-hydroxycholesterol group (20α-OH), 27-hydroxycholesterol group, 6-keto-5α-hydroxycholesterol group, 7-ketocholesterol group, 7β-hydroxycholesterol group, 7α-hydroxycholesterol group, 7β-25-dihydroxycholesterol group, β-sitosterol group, stigmasterol group, rapeseed sterol group, and campesterol group.

[0074] 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 suffers from a relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a subject with one or more characteristics of susceptibility or risk of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and / or therapy is being and / or has been administered.

[0075] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, reach completeness and / or proceed to perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0076] Substituted or optionally substituted: As described herein, the compounds of the present disclosure may contain optionally substituted moieties and / or substituted moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. "Substituted" applies to the removal of one or more hydrogens (e.g., means at least and means at least ). Unless otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted by more than one substituent selected from a specified group, the substituent may be the same or different at each position. The combination of substituents contemplated by the present disclosure is preferably a combination of substituents that results in the formation of a stable or chemically feasible compound. As used herein, the term "stable" refers to a compound that is substantially unchanged when undergoing conditions that allow it to be produced, detected, and in certain embodiments, recovered, purified, and used for one or more purposes disclosed herein. A group described as "substituted" preferably has between 1 and 4 substituents, more preferably 1 or 2 substituents. A group described as "optionally substituted" may be unsubstituted or "substituted", as described above.

[0077] Suitable monovalent substituents include: halogen; -(CH2) 0-4 R o ; -(CH2) 0-4 OR o ; -O(CH2) 0-4 R o 、-O-(CH2) 0- 4C(O)OR o ; -(CH2) 0-4 CH(OR o )2; -(CH2) 0-4 Ph, which can be o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be o Substitution; -CH=CHPh, which can be R o Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl, which may be R o Substitution; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2; -(CH2) 0-4 N(R o )C(O)R o ;-N(R o )C(S)R o ; -(CH2) 0-4 N(R o )C(O)NR o 2;-N(R o )C(S)NR o 2; -(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2;-N(R o )N(R o )C(O)OR o ; -(CH2) 0-4 C(O)R o ;-C(S)R o ; -(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 C(O)SR o ; -(CH2)0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR o -,-SC(S)SR o ; -(CH2) 0-4 SC(O)R o ; -(CH2) 0-4 C(O)NR o 2; -C(S)NR o 2; -C(S)SR o ;-SC(S)SR o 、-(CH2) 0-4 OC(O)NR o 2; -C(O)N(OR o )R o ; -C(O)C(O)R o ; -C(O)CH2C(O)R o ;-C(NOR o )R o ; -(CH2) 0-4 SSR o ; -(CH2) 0-4 S(O)2R o ; -(CH2) 0-4 S(O)2OR o ; -(CH2) 0-4 OS(O)2R o ; -S(O)2NR o 2; -(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ; -C(NH)NR o 2;-P(O)2R o ;-P(O)R o 2;-OP(O)R o 2;-OP(O)(OR o )2;-SiR o 3;-OSiR o 3;-(C 1-4 Straight or branched alkylene)ON(R o )2; or-(C 1-4Straight or branched alkylene)C(O)ON(R o )2, where each R o may be substituted as defined below and are independently hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2-(5-6 membered heteroaromatic ring) or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definition, two independently occurring R o Together with its intervening atoms it forms a 3-12 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.

[0078] R o A suitable monovalent substituent on (or by making two independent R o and the intervening atoms to form a ring) are independently halogen, -(CH2) 0-2 R · 、-(halogen R · )、-(CH2) 0-2 OH, -(CH2) 0-2 OR · 、-(CH2) 0-2 CH(OR · )2; -O(halogen R · )、-CN、-N3、-(CH2) 0-2 C(O)R · 、-(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR · 、-(CH2) 0-2 C(O)NH2, -(CH2) 0-2 C(O)NHR · 、-(CH2) 0-2 C(O)NR · 2. -(CH2) 0-2 SR · 、-(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR · 、-(CH2) 0-2 NR · 2. -NO2, -SiR · 3. -OSiR · 3. -C(O)SR · 、-(C 1-4Straight or branched alkylene)C(O)OR · or -SSR · , where each R · unsubstituted, or, when preceded by "halo", substituted only by one or more halogens, and independently selected from C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0079] Suitable divalent substituents include the following: =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-, where each independent occurrence of R * Selected from hydrogen; C 1-6 aliphatic, which may be substituted as defined below; or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. Suitable divalent substituents of the adjacent substitutable carbon to which an "optionally substituted" group is attached include: -O(CR * 2) 2-3 O-, where each independent occurrence of R * Selected from hydrogen; C 1-6 aliphatic, which may be substituted as defined below; or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0080] R * Suitable substituents on the aliphatic group include: halogen, -R · 、-(halogen R · ), -OH, -OR · 、-O(halogen R · )、-CN、-C(O)OH、-C(O)OR · 、-NH2、-NHR · 、-NR · 2 or -NO2, where each R ·is unsubstituted, or when preceded by "halo" is present, is substituted only by one or more halogens, and is independently C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0081] In some embodiments, suitable substituents on the substitutable nitrogen include: or Each of these are independently: hydrogen; C 1-6 aliphatic, which may be substituted as defined below; unsubstituted OPh; or an unsubstituted 5-6 membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur; or notwithstanding the above definition, two independently present Together with its central atoms, it forms an unsubstituted 3-12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0082] Suitable substituents on the aliphatic group are independently: halogen, -R · 、-(halogen R · ), -OH, -OR · 、-O(halogen R · )、-CN、-C(O)OH、-C(O)OR · 、-NH2、-NHR · 、-NR · 2 or -NO2, where each R · is unsubstituted, or when preceded by "halo" is present, is substituted only by one or more halogens, and is independently C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0083] Susceptible to: An individual who is "susceptible" to a disease, disorder, or condition is at risk for 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 yet 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 conditions 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).

[0084] Systemic: As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" have their art-understood meanings and refer to administration of a compound or composition so that it enters the recipient's system.

[0085] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or causes 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, mitigate, 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.

[0086] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that causes 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 sufficient to treat, diagnose, inhibit, alleviate, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, mitigates, inhibits, prevents one or more symptoms or features of a disease, disorder, and / or condition, delays its onset, reduces its severity, and / or reduces its incidence. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. The exact dosage will vary according to factors such as subject-related variables (eg, age, immune system health, etc.), the disease, and the treatment to be achieved.

[0087] "Tissue" and / or "organ": As used herein, unless otherwise specified, the terms "tissue" and / or "organ" refer to living cell materials in aggregated form, such as small parts of organs, and dispersed cells, such as cells dispersed, separated and / or grown from muscle, myocardium, liver or kidney, including bone marrow cells and progeny cells, hematogenous stem cells and progeny, and various other blood elements. In some embodiments, tissues and / or organs refer to kidneys, hearts, livers, stomachs, spleens, pancreas, lungs, brains, eyes, intestines, bladders, skin or skin tissue, blood vessels, veins, arteries, heart valves, sperm and oocytes. As used herein, the term "organ" encompasses solid organs, such as kidneys, hearts, livers, lungs, and functional parts of organs, such as skin segments, arterial segments, venous segments, transplantable liver lobes, kidney lobes, lung lobes, etc.

[0088] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that partially or completely alleviates, ameliorates, alleviates, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment for subjects who do not show signs of the relevant disease, disorder, and / or condition and / or for subjects who show only early signs of the onset of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment for subjects who show one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment for subjects who have been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, treatment may be treatment for subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, disorder, and / or condition. Thus, in some embodiments, treatment may be prophylactic; in some embodiments, treatment may be therapeutic.

[0089] For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and March's Advanced Organic Chemistry", 5th Edition, Editors: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0090] Unless otherwise stated, the structures depicted herein are also intended to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, R and S configurations, Z and E double bond isomers, and Z and E conformational isomers about each asymmetric center. Therefore, single stereochemical isomers of the compounds of the invention as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise stated, the 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 structures of the invention including replacement of hydrogen by deuterium or tritium or replacement of carbon by 13C or 14C-enriched carbon are within the scope of the invention. These compounds can be used, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the invention. DETAILED DESCRIPTION

[0091] In addition, the present invention provides compositions, preparations, nanoparticles and / or nanomaterials for delivering therapeutic and / or prophylactic agents. For example, the disclosure describes lipid compounds for compositions, preparations, nanoparticles and / or nanomaterials. In some embodiments, compositions, preparations and / or nanomaterials include LNPs that carry cargo to specified target cells, tissues and / or organs.

[0092] I. Lipid Nanoparticles (LNP)

[0093] The present invention provides compositions, formulations 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 compounds, ionizable lipids, sterols, conjugate-linker lipids and phospholipids.

[0094] 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.

[0095] A. Compounds

[0096] Additionally, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials comprising one or more compounds described herein.

[0097] In some embodiments, the present disclosure provides a compound of Formula I:

[0098]

[0099] or a pharmaceutically acceptable salt thereof, wherein:

[0100] Each L 1 and L 1 ' is independently -C(O)- or -OC(O)-;

[0101] Each L 2 and L 2 ' is independently an optionally substituted divalent saturated or unsaturated linear or branched C 1-12 hydrocarbon chain;

[0102] L 3 is a covalent bond, -O-, -C(O)O-, -OC(O)- or -OC(O)O-;

[0103] R 1 is optionally substituted C 1-20 Fatty Acids,

[0104] L CyA is a covalent bond or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 hydrocarbon chain;

[0105] Cy A is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a 1-adamantyl group, a 2-adamantyl group, a steryl group and a phenyl group;

[0106] L Ra is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 hydrocarbon chain;

[0107] Every R a and R 1 ' is independently optionally substituted C 1-20 Fat;

[0108] Y 1 is -C(O)- or -C(O)O-;

[0109] Y 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 hydrocarbon chain;

[0110] Y 3 is optionally substituted C 1-20 Fat;

[0111] X 1 is a covalent bond, -O- or -NR-;

[0112] X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 A hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-;

[0113] X 3 is hydrogen or an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl; a phenyl group; a 3- to 7-membered heterocyclyl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and

[0114] Each R is independently hydrogen or optionally substituted C 1-6 Fatty.

[0115] In some embodiments, the present disclosure provides a compound of Formula I, wherein:

[0116] Each L 1 and L 1 ' is -C(O)-;

[0117] Each L 2 and L 2 ' is independently a divalent saturated or unsaturated straight or branched chain C 1-12 hydrocarbon chain;

[0118] L 3 is a covalent bond, -C(O)O- or -OC(O)-;

[0119] R 1 C 1-20 Alkyl, C 2-20 Alkenyl or

[0120] Each L Ra and R 1 'Independently for C 1-20 Alkyl or C 2-20 alkenyl;

[0121] Y 1 is -C(O)- or -C(O)O-;

[0122] Y 2 A divalent saturated or unsaturated straight chain or branched chain C 1-6 hydrocarbon chain;

[0123] Y 3 C 1-20 Alkyl or C 2-20 alkenyl;

[0124] X 1 is -O- or -NR-;

[0125] X 2 A divalent saturated or unsaturated straight or branched chain C 1-12 A hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -NR-;

[0126] X 3 is hydrogen or has 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur and is optionally replaced by C 1-6 Alkyl or C 2-6 an alkenyl-substituted 3- to 7-membered heterocyclic group; and

[0127] Each R is independently hydrogen or C 1-6 Alkyl or C 2-6 Alkenyl.

[0128] In some embodiments, the present disclosure provides a compound of Formula II:

[0129]

[0130] or a pharmaceutically acceptable salt thereof, wherein 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 the is as described above and in the categories and subcategories herein, both individually and in combination.

[0131] In some embodiments, the present disclosure provides a compound of formula III:

[0132]

[0133] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0134] In some embodiments, the present disclosure provides a compound of formula III-A:

[0135]

[0136] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0137] In some embodiments, the present disclosure provides a compound of formula III-B:

[0138]

[0139] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0140] In some embodiments, the present disclosure provides a compound of formula III-Bi:

[0141]

[0142] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0143] In some embodiments, the present disclosure provides a compound of formula IV:

[0144]

[0145] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0146] In some embodiments, the present disclosure provides a compound of formula IV-A:

[0147]

[0148] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0149] In some embodiments, the present disclosure provides a compound of Formula IV-B:

[0150]

[0151] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0152] In some embodiments, the present disclosure provides a compound of formula IV-Bi:

[0153]

[0154] or a pharmaceutically acceptable salt thereof, wherein L 1 ', L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0155] In some embodiments, the present disclosure provides a compound of Formula V:

[0156]

[0157] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0158] In some embodiments, the present disclosure provides a compound of formula VA:

[0159]

[0160] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0161] In some embodiments, the present disclosure provides a compound of formula VB:

[0162]

[0163] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0164] In some embodiments, the present disclosure provides a compound of Formula VBi:

[0165]

[0166] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0167] In some embodiments, the present disclosure provides a compound of Formula VI:

[0168]

[0169] or a pharmaceutically acceptable salt thereof, wherein L 1 , L2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0170] In some embodiments, the present disclosure provides a compound of formula VI-A:

[0171]

[0172] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0173] In some embodiments, the present disclosure provides a compound of Formula VI-B:

[0174]

[0175] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L 3 , R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0176] In some embodiments, the present disclosure provides a compound of formula VI-Bi:

[0177]

[0178] or a pharmaceutically acceptable salt thereof, wherein L 1 , L 2 , L 2 ', L3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0179] In some embodiments, the present disclosure provides a compound of Formula VII:

[0180]

[0181] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0182] In some embodiments, the present disclosure provides a compound of formula VII-A:

[0183]

[0184] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0185] In some embodiments, the present disclosure provides a compound of Formula VII-B:

[0186]

[0187] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', R, Y 2 , Y 3 , X2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0188] In some embodiments, the present disclosure provides a compound of formula VII-Bi:

[0189]

[0190] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0191] In some embodiments, the present disclosure provides a compound of Formula VIII:

[0192]

[0193] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0194] In some embodiments, the present disclosure provides a compound of Formula VIII-A:

[0195]

[0196] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0197] In some embodiments, the present disclosure provides a compound of Formula VIII-B:

[0198]

[0199] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , R, X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0200] In some embodiments, the present disclosure provides a compound of formula VIII-Bi:

[0201]

[0202] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 ', L 3 , R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0203] In some embodiments, the present disclosure provides a compound of Formula IX:

[0204]

[0205] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 1 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0206] In some embodiments, the present disclosure provides a compound of Formula IX-A:

[0207]

[0208] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0209] In some embodiments, the present disclosure provides a compound of Formula IX-B:

[0210]

[0211] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0212] In some embodiments, the present disclosure provides a compound of formula IX-Bi:

[0213]

[0214] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0215] In some embodiments, the present disclosure provides a compound of formula X:

[0216]

[0217] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 1 , X2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0218] In some embodiments, the present disclosure provides a compound of formula XA:

[0219]

[0220] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0221] In some embodiments, the present disclosure provides a compound of formula XB:

[0222]

[0223] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', R, Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0224] In some embodiments, the present disclosure provides a compound of formula XBi:

[0225]

[0226] or a pharmaceutically acceptable salt thereof, wherein L 2 , L 2 '、R 1 , R 1 ', Y 2 , Y 3 , X 2 and X 3 Each of the is as described above and in the categories and subcategories herein, both individually and in combination.

[0227] In some embodiments of any of the formulas described herein, L 1is -C(O)- or -OC(O)-. In some embodiments, L 1 In some embodiments, L 1 It is -OC(O)-.

[0228] 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)-.

[0229] In some embodiments, the present disclosure provides a compound of Formula I, wherein: each L 1 and L 1 ' is -C(O)-.

[0230] In some embodiments, the present disclosure provides a compound of Formula I, wherein: each L 2 and L 2 ' is independently a divalent saturated or unsaturated straight or branched chain C 1-12 Hydrocarbon chain.

[0231] In some embodiments of any of the formulas described herein, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 In some embodiments, L 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L 2 is an optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 In some embodiments, L 2 is an optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, L 2 A divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 A divalent saturated straight or branched chain C 4-8 In some embodiments, L 2 A divalent saturated straight or branched chain C 1-6 In some embodiments, L2 In some embodiments, L 2 is -(CH2)4- or -(CH2)5-. In some embodiments, L 2 It is -(CH2)6- or -(CH2)7-.

[0232] In some embodiments of any of the formulas described herein, L 2 ' is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L 2 ' is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L 2 ' is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, L 2 ' is an optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 ' is an optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, L 2 ' is an optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, L 2 ' is a divalent saturated straight or branched chain C 1-12 In some embodiments, L 2 ' is a divalent saturated straight or branched chain C 1-6 In some embodiments, L 2 ' is a divalent saturated straight or branched chain C 1-3 In some embodiments, L 2 ' is -(CH2)2-.

[0233] In some embodiments of any of the formulas described herein, L 3 is a covalent bond, -O-, -C(O)O-, -OC(O)-, or -OC(O)O-. In some embodiments, L 3 is 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 In some embodiments, L 3is -C(O)O-. In some embodiments, L 3 In some embodiments, L 3 is -OC(O)O-.

[0234] In some embodiments of any of the formulas described herein, R 1 is optionally substituted C 1-20 Fatty Acids, In some embodiments, R 1 is optionally substituted C 1-20 In some embodiments, R 1 for In some embodiments, R 1 for In some embodiments, R 1 C 1-20 Aliphatic or In some embodiments, R 1 is optionally substituted C 6-12 In some embodiments, R 1 is optionally substituted C 6-12 In some embodiments, R 1 is optionally substituted C 6-12 In some embodiments, R 1 is optionally substituted C 12-20 In some embodiments, R 1 is optionally substituted C 12-20 In some embodiments, R 1 is optionally substituted C 12-20 In some embodiments, R 1 C 1-20 In some embodiments, R 1 C 6-12 In some embodiments, R 1 C 6-12 In some embodiments, R 1 C 6-12 In some embodiments, R 1 C 12-20 In some embodiments, R 1 C 12-20 In some embodiments, R 1 C 12-20 In some embodiments, R 1 for

[0235] 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 Hydrocarbon chain.

[0236] In some embodiments of any of the formulae described herein, Cy A is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a 1-adamantyl group, a 2-adamantyl group, a steryl group and a phenyl group.

[0237] In some embodiments of any of the formulas described herein, L Ra is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, L Ra A divalent saturated or unsaturated straight chain or branched chain C 1-12 In some embodiments, L Ra is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, L Ra A divalent saturated or unsaturated straight chain or branched chain C 1-6 In some embodiments, L Ra is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, L Ra A divalent saturated or unsaturated straight chain or branched chain C 1-3 In some embodiments, L Ra It is -CH2- or -(CH2)2-.

[0238] In some embodiments of any of the formulas described herein, each R a is independently optionally substituted C 1-20 In some embodiments, each R a is independently optionally substituted C 1-12 In some embodiments, each R a is independently optionally substituted C 1-12 In some embodiments, each R a is independently optionally substituted C 1-12 In some embodiments, each R a is independently optionally substituted C 4-10 In some embodiments, R a is optionally substituted C 6-12 In some embodiments, R ais optionally substituted C 6-12 In some embodiments, R a is optionally substituted C 6-12 In some embodiments, R a is optionally substituted C 7-9 In some embodiments, R a is optionally substituted C 7-9 In some embodiments, R a is optionally substituted C 7-9 In some embodiments, each R a Independently for C 1-20 In some embodiments, R a C 6-12 In some embodiments, R a C 6-12 In some embodiments, R a C 6-12 In some embodiments, R a C 7-9 In some embodiments, R a C 7-9 In some embodiments, R a C 7-9 In some embodiments, R a for

[0239] In some embodiments, -L 2 -L 3 -R 1 for In some embodiments, -L 2 -L 3 -R 1 Not for

[0240] In some embodiments of any of the formulas described herein, each R 1' is independently optionally substituted C 1-20 In some embodiments, R 1' is optionally substituted C 6-12 In some embodiments, R 1' is optionally substituted C 6-12 In some embodiments, R 1' is optionally substituted C 6-12 In some embodiments, R 1'is optionally substituted C 7-9 In some embodiments, R 1' is optionally substituted C 7-9 In some embodiments, R 1' is optionally substituted C 7-9 In some embodiments, each R 1' Independently for C 1-20 In some embodiments, R 1' C 6-12 In some embodiments, R 1 ' is C 6-12 In some embodiments, R 1 ' is C 6-12 In some embodiments, R 1 ' is C 7-9 In some embodiments, R 1 ' is C 7-9 In some embodiments, R 1 ' is C 7-9 In some embodiments, R 1 'for

[0241] In some embodiments of any of the formulas described herein, Y 1 is -C(O)- or -C(O)O-. In some embodiments, Y 1 In some embodiments, Y 1 It is -C(O)O-.

[0242] In some embodiments of any of the formulas described herein, Y 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, Y 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, Y 2 is an optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, Y 2 is an optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, Y 2 A divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, Y 2 A divalent saturated straight or branched chain C 1-6In some embodiments, Y 2 A divalent saturated straight or branched chain C 1-3 In some embodiments, Y 2 It is -CH2- or -(CH2)2-.

[0243] In some embodiments of any of the formulas described herein, Y 3 is optionally substituted C 1-20 In some embodiments, Y 3 C 1-20 In some embodiments, Y 3 is optionally substituted C 1-12 In some embodiments, Y 3 is optionally substituted C 1-12 In some embodiments, Y 3 is optionally substituted C 4-8 In some embodiments, Y 3 is optionally substituted C 4-8 In some embodiments, Y 3 is optionally substituted C 1-6 In some embodiments, Y 3 is -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, -(CH2)4CH3, -(CH2)5CH3, -(CH2)6CH3 or -(CH2)7CH3. In some embodiments, Y 3 In some embodiments, Y 3 is -CH2CH3. In some embodiments, Y 3 is -(CH2)2CH3. In some embodiments, Y 3 is -(CH2)3CH3. In some embodiments, Y 3 is -(CH2)4CH3. In some embodiments, Y 3 is -(CH2)5CH3. In some embodiments, Y 3 is -(CH2)6CH3. In some embodiments, Y 3 It is -(CH2)7CH3.

[0244] In some embodiments of any of the formulas described herein, X 1 is a covalent bond, -O- or -NR-. In some embodiments, X 1 In some embodiments, X 1 In some embodiments, X 1In some embodiments, X 1 It is -NR-.

[0245] 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 1-12 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 1-6 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 1-3 In some embodiments, X 2 A divalent saturated straight or branched chain C 1-12 In some embodiments, X 2 A divalent saturated straight or branched chain C 1-6 In some embodiments, X 2 A divalent saturated straight or branched chain C 1-3 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C4-8 In some embodiments, X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 In some embodiments, X 2 A divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, X 2 A divalent saturated or unsaturated straight or branched chain C 1-12 In some embodiments, X 2 A divalent saturated or unsaturated straight or branched chain C 4-8 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 1-12 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 In some embodiments, X 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 In some embodiments, X 2 A divalent saturated straight or branched chain C 4-8 A hydrocarbon chain in which one methylene unit is replaced by -NR-.

[0246] In some embodiments of any of the formulas described herein, X 3 X is hydrogen or an optionally substituted ring selected from 3 to 7 membered saturated or partially unsaturated carbocyclyl, phenyl, 3 to 7 membered heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, or 5 to 6 membered heteroaryl having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is hydrogen or has 1-3 independently selected from nitrogen, oxygen and sulfur and is optionally replaced by C 1-6 In some embodiments, X 3In some embodiments, X 3 is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclyl, a phenyl, 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. In some embodiments, X 3 is an optionally substituted 3 to 7 membered heterocyclyl having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is an optionally substituted 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a 5- to 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a group having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, o A substituted 5- to 6-membered heterocyclic group, wherein R o C 1-6 In some embodiments, X 3 is an optionally substituted 5-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a 5-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a group having 1 to 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, o A substituted 5-membered heterocyclic group, wherein R o C 1-6 In some embodiments, X 3 is an optionally substituted 5-membered heterocyclic group having 1-2 nitrogen atoms. 3 is a 5-membered heterocyclic group having 1-2 nitrogen atoms. 3 Has 1-2 nitrogen atoms, o A substituted 5-membered heterocyclic group, wherein R o C 1-6 In some embodiments, X 3 is an optionally substituted 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a 6-membered heterocyclyl having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur. 3 is a group having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, oA substituted 6-membered heterocyclic group, wherein R o C 1-6 In some embodiments, X 3 X is optionally substituted pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl. 3 X is pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl. 3 For being-R o substituted pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl, wherein R o C 1-6 Aliphatic (eg, methyl or ethyl).

[0247] In some embodiments, -X 2 -X 3 for

[0248] In some embodiments, X 2 and X 3 At least one of the comprises at least one ionizable nitrogen atom.

[0249] In some embodiments of any of the formulae 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 hydrogen. In some embodiments, R is optionally substituted C 1-6 In some embodiments, R is C 1-6 In some embodiments, R is optionally substituted C 1-3 In some embodiments, R is C 1-3 Aliphatic. In some embodiments, R is -CH3.

[0250] In some embodiments, R o In some embodiments, R o It is ethyl.

[0251] In some embodiments of any of the formulae described herein, the present disclosure provides compounds comprising an ionizable nitrogen atom wherein the pKa of its conjugate acid is between about 4 and about 12.

[0252] In some embodiments, the present disclosure provides a compound selected from Table 1.

[0253] Table 1.

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263] or a pharmaceutically acceptable salt thereof.

[0264] It should be understood that unless otherwise indicated or prohibited by the definition of any of the foregoing 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 variable L as defined above and described in the classes and subclasses herein 1 , L 1 ', L 2 , L 2 ', L 3 , L CyA , Cy A , L Ra , R a , R 1 , R 1' , R, Y 1 , Y 2 , Y 3 , X 1 , X 2 and X 3The embodiments apply 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, both individually and in combination.

[0265] In some embodiments, provided compounds are provided and / or utilized in salt form (e.g., pharmaceutically acceptable salt form). Unless otherwise indicated, reference to a compound provided herein should be understood to include reference to a salt thereof.

[0266] It should be understood that throughout this disclosure, unless otherwise indicated, reference to a compound of Formula I is also intended to 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, as well as species of compounds of such formulae disclosed herein.

[0267] In some embodiments, the disclosure encompasses the following recognition: for example, compared to a reference compound or other known compound, the provided compound displays certain desired features. For example, in some embodiments, the provided compound is shown to be more effectively delivered to various cell types in one or more experiments described herein, and / or has one or more other features that make it more suitable for delivery of a carrier such as a therapeutic agent or a preventive agent than other known compounds. Without wishing to be bound by any particular theory, the disclosure encompasses the following recognition: the provided compound characterized as including a triol core and / or a biodegradable tail may display certain more desired features (e.g., more effectively delivered to various cell types in one or more experiments described herein) than the corresponding compound lacking the same features. In specific embodiments, the disclosure encompasses the following recognition: the provided compound characterized as including a triol core and a biodegradable tail may display certain more desired features (e.g., more effectively delivered to various cell types in one or more experiments described herein) than the corresponding compound lacking the same feature combination.

[0268] B. Preparation of provided compounds

[0269] The provided compounds can generally be prepared by the methods described in the schemes and examples that follow.

[0270] C. Ionizable lipids

[0271] Additionally, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials comprising one or more ionizable lipids described herein.

[0272] For example, in some embodiments, compositions, formulations, nanoparticles and / or nanomaterials having about 50 mol% or less ionizable lipids based on the total moles of components of the lipid nanoparticle may be useful and / or critical for the functional activity of the lipid nanoparticle, such as the desired tropism, stability and drug delivery efficacy described herein.

[0273] In some embodiments, ionizable lipids may include amine-containing groups on the head group. In some embodiments, ionizable lipids are or include compounds described herein. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present in lipid nanoparticle (LNP) preparations with about 30 mol % to about 70 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 33 mol % to about 60 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 34 mol % to about 55 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 33 mol % to about 51 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 34.7 mol %. In some embodiments, based on the total moles of lipid nanoparticle components, ionizable lipids are present with about 50 mol %.

[0274] In addition, in some embodiments, the lipid nanoparticle composition comprises an ionizable lipid. In some embodiments, the lipid nanoparticle formulation 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 formulation at about 30 mol % to about 70 mol % based on the total moles of the lipid nanoparticle components.

[0275] D. Sterol

[0276] Additionally, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials comprising one or more sterols described herein.

[0277] In some embodiments, sterol is cholesterol or its variant or derivative. In some embodiments, cholesterol is modified. In some embodiments, cholesterol is oxidized cholesterol. In some embodiments, cholesterol is esterified cholesterol. Unmodified cholesterol can form side chain or epoxidized variants by enzyme action. In some embodiments, cholesterol can be oxidized on the β ring structure or on the hydrocarbon tail structure. In some embodiments, sterol is plant sterol. Exemplary sterols that are considered to be useful for 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 base, 7β-25-dihydroxycholesterol, β-sitosterol, stigmasterol, rapeseed sterol, campesterol or a combination thereof. In some embodiments, cholesterol with side chain oxidation can enhance carrier delivery relative to other cholesterol variants. In some embodiments, cholesterol is unmodified cholesterol.

[0278] In some embodiments, the LNP formulation comprises about 20 mol % to about 50 mol % of sterols. In some embodiments, the LNP formulation comprises about 38 mol % of sterols. In some embodiments, the LNP formulation comprises about 38.5 mol % of sterols. In some embodiments, the LNP formulation comprises about 33.8 mol % of cholesterol.

[0279] E. Conjugate-Linker Lipid

[0280] Additionally, the disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials comprising one or more of the conjugate-linker lipids described herein.

[0281] In some embodiments, conjugate-linker lipid is or comprises polyethylene glycol (PEG)-lipid or PEG-modified lipid.In some embodiments, PEG or PEG-modified lipid may alternatively be referred to as PEGylated lipid or PEG-lipid.The inclusion of PEGylated lipid can be used to enhance the stability of lipid nanoparticle colloid in vitro and the circulation time in vivo.In some embodiments, because the PEG part is gradually released in the blood circulation, PEGization is reversible.Exemplary PEG-lipid includes but is not limited to PEG conjugated with saturated or unsaturated alkyl chains of length 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 its mixture.For example, in some embodiments, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or PEG-DSPE lipid.

[0282] In some embodiments, conjugate-joint lipids include polyethylene glycol lipids. In some embodiments, conjugate-joint lipids include dimyristyl glycerol (DMG), 1,2-dipalmitoyl-rac-glycerol, methoxy polyethylene glycol (DPG-PEG) or 1,2-distearoyl-rac-glycerol-3-methylpolyoxyethylene (DSG-PEG). In some embodiments, the average molecular weight of conjugate-joint lipids is about 500Da to about 5000Da. In some embodiments, the average molecular weight of conjugate-joint lipids is about 2000Da. In some embodiments, LNP preparations include about 0 mol% to about 5 mol% of conjugate-joint lipids. In some embodiments, LNP preparations include about 1.5 mol% of conjugate-joint lipids. In some embodiments, LNP preparations include about 3 mol% of conjugate-joint lipids.

[0283] F. Phospholipids

[0284] In addition, the present disclosure describes compositions, formulations, nanoparticles and / or nanomaterials comprising one or more phospholipids described herein. In some embodiments, the present disclosure describes compositions, formulations, nanoparticles and / or nanomaterials comprising one or more (poly)unsaturated lipids.

[0285] In some embodiments, one or more phospholipids can be assembled into one or more lipid bilayers. In some embodiments, one or more phospholipids may include a phospholipid moiety. In some embodiments, one or more phospholipids may include one or more fatty acid moieties. In some embodiments, one or more phospholipids may include 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, linolenic acid, α-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 or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkynes can undergo copper-catalyzed cycloadditions when exposed to azides. These reactions can be useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition, or for conjugating nanoparticle compositions with useful components, such as targeting or imaging moieties (e.g., dyes).

[0286] 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-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 (18:0Diether PC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleyl-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, , 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (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.

[0287] 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.

[0288] G. Diameter

[0289] Additionally, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials having an average hydrodynamic diameter of about 30 to about 220 nm. In some embodiments, the average hydrodynamic diameter of the compositions, formulations, nanoparticles, and / or nanomaterials described herein is 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 with endpoints bounded by any two of the foregoing values. For example, in some embodiments, the compositions, formulations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter of between 50 nm and 200 nm.

[0290] In some embodiments, the average hydrodynamic diameter of lipid nanoparticles described herein is about 30 to about 220nm. In some embodiments, the average hydrodynamic diameter of lipid nanoparticles described herein is about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm, 200nm, 205nm, 210nm, 215nm, 220nm or any range with endpoints defined by any two of the aforementioned values. For example, in some embodiments, the average hydrodynamic diameter of the lipid nanoparticles described herein is between 50 nm and 200 nm.

[0291] H. Polydispersity

[0292] In addition, the present disclosure describes a polydispersity index (PDI) of about 0.01 to about 0.3 compositions, formulations, nanoparticles and / or nanomaterials. In some embodiments, the PDI of compositions, formulations, nanoparticles and / or nanomaterials described herein is about 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, 0.3, or any range of endpoints defined by any two of the aforementioned values. For example, in some embodiments, the PDI of compositions, formulations, nanoparticles and / or nanomaterials described herein is about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0293] In some embodiments, the PDI of the lipid nanoparticles described herein is about 0.01 to about 0.3. In some embodiments, the PDI of the lipid nanoparticles described herein is about 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, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, the PDI of the lipid nanoparticles described herein is about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09.

[0294] I. Encapsulation Efficiency

[0295] In addition, the present disclosure describes compositions, preparations, nanoparticles and / or nanomaterials, wherein the encapsulation efficiency of the compositions, preparations, nanoparticles and / or nanomaterials provided is about 80% to about 100%. In some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles and / or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range of endpoints defined by any two of the aforementioned values. For example, in some embodiments, the encapsulation efficiency of the compositions, preparations, nanoparticles and / or nanomaterials 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 compositions, formulations, nanoparticles and / or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0296] In some embodiments, the encapsulation efficiency of lipid nanoparticles described herein is about 80% to about 100%. In some embodiments, the encapsulation efficiency of lipid nanoparticles described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 100%, or any range of endpoints defined by any two of the aforementioned values. For example, in some embodiments, the encapsulation efficiency of 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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0297] J.pKa

[0298] Additionally, the present disclosure describes compositions, formulations, nanoparticles, and / or nanomaterials having a pKa of about 5 to about 9. In some embodiments, the pKa of the compositions, formulations, nanoparticles, and / or nanomaterials described herein is about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or any range having endpoints bounded by any two of the foregoing values. In some embodiments, the pKa of the compositions, formulations, nanoparticles, and / or nanomaterials described herein is about 6.0, 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, or any range with endpoints bounded by any two of the foregoing values.

[0299] In some embodiments, the pKa of the lipid nanoparticles described herein is about 5 to about 9. In some embodiments, the pKa of the lipid nanoparticles described herein is 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 foregoing values. In some embodiments, the pKa of the lipid nanoparticles described herein is about 6.0, 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, or any range having endpoints defined by any two of the foregoing values.

[0300] II. Exemplary LNP Formulations

[0301] The present invention provides compositions, preparations, nanoparticles and / or nanomaterials comprising lipid nanoparticles. In some embodiments, the lipid nanoparticle preparations include about 30 mol % to about 70 mol % of ionizable lipids, about 5 mol % to about 25 mol % of phospholipids, about 25 mol % to about 45 mol % of cholesterol and about 0 mol % to about 5 mol % of conjugate-linker lipids.

[0302] In some embodiments, the lipid nanoparticle formulation comprises about 45 mol % ionizable lipids, about 9 mol % phospholipids, about 44 mol % cholesterol, and about 2 mol % conjugate-linker lipids. In some embodiments, the lipid nanoparticle formulation comprises about 50 mol % ionizable lipids, about 9 mol % phospholipids, about 38 mol % cholesterol, and about 3 mol % conjugate-linker lipids.

[0303] In some embodiments, the lipid nanoparticle formulation comprises about 47.5 mol% ionizable lipid, about 10 mol% phospholipid, about 40 mol% cholesterol, and about 2.5 mol% conjugate-linker lipid.

[0304] In some embodiments, the lipid nanoparticle formulation comprises about 50 mol% ionizable lipid, about 10 mol% phospholipid, about 38.5 mol% cholesterol, and about 1.5 mol% conjugate-linker lipid.

[0305] In some embodiments, the lipid nanoparticle formulation comprises about 40 mol % to about 60 mol % of an ionizable lipid of any provided compound, about 5 mol % to about 15 mol % of 1-2-distearoyl-sn-glycero-3-phosphocholine, about 1 mol % to about 5 mol % of C14PEG2000, and about 30 mol % to about 47 mol % of cholesterol, based on the total moles of these four components.

[0306] In some embodiments, the lipid nanoparticle (LNP) formulation comprises (ionizable lipid, cholesterol, lipid-PEG and phospholipid) at a mass ratio of about 2: 1 and 50: 1: mRNA. In some embodiments, the LNP formulation 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 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 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA. In some embodiments, the lipid nanoparticle (LNP) formulation comprises a mass ratio of about 11.7:1 and 19:1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): mRNA.

[0307] In some embodiments, the lipid nanoparticle formulation comprises (ionizable lipid, cholesterol, lipid-PEG and phospholipid) at a mass ratio of about 2: 1 and 50: 1: siRNA. In some embodiments, the LNP formulation 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 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): siRNA. In some embodiments, the lipid nanoparticle (LNP) formulation comprises a mass ratio of about 11.7: 1 and 19: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): siRNA.

[0308] In some embodiments, the lipid nanoparticle formulation comprises (ionizable lipid, cholesterol, lipid-PEG and phospholipid):NA in a mass ratio of about 2:1 and 50:1. In some embodiments, the LNP formulation comprises (ionizable lipid, cholesterol, lipid-PEG and phospholipid):NA in 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 : 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 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): NA. In some embodiments, the lipid nanoparticle (LNP) formulation comprises a mass ratio of about 11.7: 1 and 40: 1 (ionizable lipid, cholesterol, lipid-PEG and phospholipid): NA.

[0309] In some embodiments, NA comprises a base editor and a gRNA as described herein. In some embodiments, the mass ratio of base editor: gRNA is 1: 1. In some embodiments, the mass ratio of base editor: gRNA is 2: 1. In some embodiments, the mass ratio of base editor: gRNA is 3: 1. In some embodiments, the mass ratio of base editor: gRNA is 4: 1. In some embodiments, the mass ratio of base editor: gRNA is 5: 1. In some embodiments, the mass ratio of base editor: gRNA is 6: 1. In some embodiments, the mass ratio of base editor: gRNA is 7: 1. In some embodiments, the mass ratio of base editor: gRNA is 8: 1. In some embodiments, the mass ratio of base editor: gRNA is 9: 1. In some embodiments, the mass ratio of base editor: gRNA is 10: 1. In some embodiments, the mass ratio of base editor: gRNA is 1: 2. In some embodiments, the mass ratio of 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.

[0310] III. Pharmaceutical Compositions

[0311] The present invention provides compositions, preparations, nanoparticles and / or nanomaterials comprising pharmaceutical compositions. In addition, in some embodiments, the pharmaceutical compositions comprise 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 into pharmaceutical compositions.

[0312] In some embodiments, a pharmaceutical composition may include one or more nanoparticle compositions described herein. For example, a pharmaceutical composition may include one or more nanoparticle compositions (including one or more different therapeutic and / or prophylactic agents, including but not limited to one or more different types of nucleic acids) or encode different agents. In some embodiments, a pharmaceutical composition includes one or more pharmaceutically acceptable excipients or auxiliary ingredients, including but not limited to pharmaceutically acceptable carriers.

[0313] The pharmaceutical composition can be administered to a subject. In some embodiments, the pharmaceutical composition is administered as described herein. In some in vivo methods, the nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount as described herein.

[0314] In some embodiments, taking into account the treatment table environment, age and general health of the recipient, ordinary technicians will be able to use the pharmaceutical composition described herein to design appropriate dosage levels and dosage regimens to treat various conditions of various patients. For example, in some embodiments, the selected dosage depends on the desired therapeutic effect, route of administration and the duration of the desired treatment. In some embodiments, the dosage level of about 0.001mg to about 5mg nucleic acid per kg body weight per dose is usually applied to mammals. More specifically, in some embodiments, the preferred dosage of nucleic acid in the disclosed nanoparticle is about 0.1mg / kg to about 1.0mg / kg. For the disclosed nanoparticle, the dosage level of about 0.2mg to about 100mg four components (ionizable lipids, cholesterol, conjugate-linker conjugate and phospholipids) per kg body weight is usually applied to mammals. More specifically, in some embodiments, the preferred dosage of the disclosed nanoparticle is about 0.5mg / kg to about 5mg / kg four components per kg body weight.

[0315] In some embodiments, the pharmaceutical compositions described herein are administered topically, for example, by direct injection into the site to be treated. Typically, injection results in an increase in local concentration of the composition that is greater than that which can be achieved by systemic administration. In some embodiments, the pharmaceutical compositions described herein can be combined with a matrix as described herein to help produce an increased local concentration of a polypeptide composition by reducing passive diffusion of the polypeptide out of the site to be treated.

[0316] A. Formulations for Parenteral Administration

[0317] In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials disclosed herein (including compositions, formulations, nanoparticles and / or nanomaterials containing lipid nanoparticles) are administered by parenteral injection in the form of aqueous solutions. In some embodiments, the formulations can also be in the form of suspensions or emulsions. In general, a pharmaceutical composition is provided, which includes an effective amount of lipid nanoparticles, and optionally includes a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. These compositions optionally include one or more of the following: diluent, sterile water, buffered saline with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives, such as detergents and solubilizers (e.g., TWEEN20 (polysorbate-20), TWEEN80 (polysorbate-80)), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g., thimerosal, 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 prior to 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.

[0318] B. Controlled Delivery Polymer Matrices

[0319] In some embodiments, the compositions, preparations, nanoparticles and / or nanomaterials disclosed herein can also be administered in the form of controlled release formulations. In some embodiments, controlled release polymeric devices can be prepared for long-term systemic release after implantation of polymeric devices (such as rods, cylinders, membranes, disks) or injections (such as micron particles). In some embodiments, the matrix can be in the form of micron particles (such as microspheres). In some embodiments, the agent is dispersed in a solid polymer matrix or microcapsule. In some embodiments, the core is a material different from the polymer shell of any one of the described compositions, preparations, nanoparticles and / or nanomaterials. In some embodiments, the peptide is dispersed or suspended in the core of any one of the described compositions, preparations, nanoparticles and / or nanomaterials, and the core can be liquid or solid in nature. Unless specifically defined herein, micron particles, microspheres and microcapsules can be used interchangeably. In some embodiments, polymers can be cast into thin plates or films in the range of several nanometers to four centimeters, powders produced by grinding or other standard techniques, or even gels (such as hydrogels).

[0320] In some embodiments, non-biodegradable matrix is ​​used to deliver the described composition, preparation, nanoparticle and / or nanomaterial. In some embodiments, biodegradable matrix is ​​used to deliver the described composition, preparation, nanoparticle and / or nanomaterial. In some embodiments, biodegradable matrix is ​​preferred. In some embodiments, biodegradable matrix comprises natural or synthetic polymer. In some embodiments, due to the better characterization of degradation and release curve, synthetic polymer is preferred. In some embodiments, polymer is selected based on the time period of expected release. In some embodiments, linear release may be the most useful, but in other embodiments, pulse release or "bulk release (bulk release)" can provide more effective results. In some embodiments, polymer can be in the form of hydrogel (usually absorbing up to about 90% by weight of water), and can be optionally cross-linked with multivalent ions or polymers.

[0321] 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 method developed for preparing microspheres for drug delivery, for example, as described in 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 hereby incorporated herein by reference in their entirety.

[0322] In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials described can be formulated for local release to treat the implanted or injected area, which will generally deliver a much smaller dose than that used to treat the entire body or systemic delivery. These devices can be implanted or injected subcutaneously into muscle, fat or swallowed.

[0323] C.Cargo

[0324] In addition, the present invention provides compositions, preparations, nanoparticles and / or nanomaterials comprising the carriers described herein. In some embodiments, compositions, preparations, nanoparticles and / or nanomaterials include therapeutic agents or prophylactic agents for delivery to a subject. In some embodiments, the therapeutic agent or prophylactic agent is encapsulated by lipid nanoparticles. In some embodiments, the lipid nanoparticles are loaded with one or more nucleic acids.

[0325] D. Therapeutic and / or preventive agents

[0326] The carrier delivered via the LNP formulation may be a bioactive agent. In some embodiments, the carrier is or comprises one or more bioactive agents, such as mRNA, guide RNA (gRNA), nucleic acid, RNA-guided DNA binder, expression vector (vector), template nucleic acid, antibody (e.g., monoclonal antibody, chimeric antibody, humanized antibody, nanobody and fragments thereof, etc.), cholesterol, hormone, peptide, protein, chemotherapeutic agent and other types of anti-tumor agent, low molecular weight drug, vitamin, cofactor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA: RNA composition, allozyme, aptamer, ribozyme, decoy (decoy) and its analogs, plasmids and other types of vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acids (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and "self-replicating RNA" (encoding replicase activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acids (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acids (TNA), glycol nucleic acids (GNA), sisiRNA (small internal segmented interfering RNA), and RNA (asymmetric interfering RNA). The above list of biologically active agents is exemplary only and is not intended to be limiting. These compounds can be purified or partially purified, and can be naturally occurring or synthetic, and can be chemically modified.

[0327] The carrier delivered via the LNP formulation can be RNA, such as an mRNA molecule encoding a protein of interest. For example, in some embodiments, mRNA for expressing proteins such as green fluorescent protein (GFP), RNA-guided DNA binders or Cas nucleases is described herein. LNP formulations are provided, which include Cas nuclease mRNAs, such as 2 types of Cas nuclease mRNAs that are allowed to be expressed in cells of 2 types of Cas nucleases (such as Cas9 or Cpfl proteins). In addition, the carrier may contain one or more guide RNAs or nucleic acids encoding guide RNAs. Template nucleic acids (such as for repair or reorganization) may also be included in the composition, or template nucleic acids may be used in the methods described herein. In some embodiments, the carrier includes mRNA encoding Streptococcus pyogenes Cas9, optionally with S.pyogenes gRNA. In some embodiments, the carrier includes mRNA encoding Neisseria meningitidis Cas9, optionally with Neisseria meningitidis (nme) gRNA.

[0328] "mRNA" refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNA). mRNA may comprise a phosphate-sugar backbone containing ribose residues or their analogs (e.g., 2'-methoxyribose residues). In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. In general, mRNA does not contain a large amount of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3 or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1% thymidine content). mRNA may contain modified uridine at some or all of its uridine positions.

[0329] E. CRISPR / Cas delivery vehicle

[0330] In some embodiments, the disclosed compositions, formulations, nanoparticles and / or nanomaterials include mRNA encoding RNA-guided DNA binders (such as Cas nucleases). In specific embodiments, the disclosed compositions, formulations, nanoparticles and / or nanomaterials include mRNA encoding Class 2 Cas nucleases (such as Streptococcus pyogenes Cas9).

[0331] As used herein, "RNA-guided DNA binders" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders include Cas cleavage enzymes / nickases and inactivated forms thereof ("dCas DNA binders"). As used herein, "Cas nucleases" encompass Cas cleavage enzymes, Cas nickases, and dCas DNA binders. Cas cleavage enzymes / nickases and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, Cas10, Csml or Cmr2 subunits thereof, Cascade complexes of type I CRISPR systems, Cas3 subunits thereof, and class 2 Cas nucleases. As used herein, "class 2 Cas nucleases" are single-chain polypeptides having RNA-guided DNA binding activity. Class 2 Cas nucleases include: Class 2 Cas cleavage enzymes / nickases (e.g., H840A, D10A or N863 A variants), the cleavage enzymes / nickases further having RNA-guided DNA cleavage enzymes / nickase activity; and Class 2 dCas DNA binders, wherein the cleavage enzyme / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, 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. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated herein by reference in its entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., 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 hereby incorporated herein in their entirety.

[0332] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas nuclease, for example, a Cas cleavage enzyme, a Cas nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder, such as Cas9, to a target sequence, and the guide RNA hybridizes to the target sequence and the agent binds to the target sequence; in the case where the agent is a cleavage enzyme or a nickase, the binding may be followed by cleavage or nicking.

[0333] In some embodiments, the carrier for LNP formulation includes at least one guide RNA, the guide RNA comprising a guide sequence that guides the RNA-guided DNA binder (which may be a nuclease (e.g., Cas nuclease, such as Cas9)) to the target DNA. The gRNA can guide the Cas nuclease or the 2nd class Cas nuclease to the target sequence on the target nucleic acid molecule. In some embodiments, the gRNA binds to the 2nd class Cas nuclease and provides the specificity of cutting. In some embodiments, the gRNA and the Cas nuclease can form a ribonucleoprotein (RNP), for example, 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 the homologous gRNA can be paired. The gRNA scaffold structure paired with each 2nd class Cas nuclease varies with the specific CRISPR / Cas system.

[0334] "Guide RNA", "gRNA" and simply "guide" are used interchangeably herein to refer to crRNA (also known as CRISPR RNA), or a combination of crRNA and trRNA (also known as tracrRNA). Guide RNA may include modified RNA as described herein. crRNA and trRNA can be combined 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. trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to a naturally occurring sequence.

[0335] As used herein, "guide sequence" refers to a sequence that is complementary to a target sequence within a guide RNA and is used to guide the guide RNA to the target sequence to bind or modify (e.g., cut) by a DNA binder guided by RNA. "Guide sequence" may also be referred to as a "targeting sequence" or "spacer sequence". The length of the guide sequence may be 20 base pairs, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences may also be used as guides, for example, with a length of 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the target sequence is, for example, in a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the complementarity or degree of identity between the guide sequence and its corresponding target sequence may be about at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 consecutive nucleotides. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein 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 the target region may contain 1-4 mismatches, wherein the guide sequence contains 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, wherein the guide sequence contains 20 nucleotides.

[0336] The target sequence of the RNA-guided DNA binding protein such as the Cas protein includes both the positive and negative strands of the genomic DNA (i.e., the given sequence and the reverse complementary sequence of the sequence), because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Therefore, in the case where the guide sequence is referred to as "complementary to the target sequence", it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complementary sequence of the target sequence. Therefore, in some embodiments, in the case where the guide sequence binds to the reverse complementary sequence of the target sequence, except that U replaces T in the guide sequence, the guide sequence is identical to some nucleotides of the target sequence (e.g., a target sequence that does not include PAM).

[0337] The length of the targeting sequence may depend on the CRISPR / Cas system and the components used. For example, different 2 types of Cas nucleases from different bacterial species have different optimal targeting sequence lengths. Therefore, the targeting sequence may comprise a length of 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 or more than 50 nucleotides. In some embodiments, the targeting sequence length is 0, 1, 2, 3, 4 or 5 nucleotides longer or shorter than the guide sequence of the naturally occurring nucleotide sequence.

[0338] CRISPR / Cas system. In certain embodiments, the Cas nuclease and the 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.

[0339] In some embodiments, the sgRNA is a "Cas9sgRNA" capable of mediating RNA-guided DNA cleavage by a Cas9 protein. In some embodiments, the sgRNA is a "CpflsgRNA" capable of mediating RNA-guided DNA cleavage by a Cpf1 protein. In some embodiments, the gRNA comprises a crRNA and a tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In some embodiments, the gRNA comprises a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0340] Certain embodiments of the present invention also provide nucleic acids encoding gRNAs described herein, e.g., expression cassettes. "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 encoding one or more guide RNAs.

[0341] Certain embodiments of the present disclosure also provide for the delivery of adenine base editors ("ABEs") using LNA compositions, formulations, nanoparticles and / or nanomaterials described herein. ABEs and methods of using them 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 hereby incorporated by reference in their entirety.

[0342] Certain embodiments of the present disclosure also provide for the delivery of cytosine base editors ("CBEs") using LNA compositions, formulations, nanoparticles and / or nanomaterials described herein. ABEs and methods of using them are described, for example, in U.S. Pat. Nos. 10,167,457 and 9,840,699, the contents of each of which are hereby incorporated by reference in their entirety.

[0343] The term "base editor (BE)" or "nucleobase editor (NBE)" refers to an agent comprising a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the editor is capable of deaminating a base within a nucleic acid. In some embodiments, the editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating 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-inactive Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, the base editor is fused to a base excision repair inhibitor (e.g., a UGI domain or a dISN domain). In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and a base excision repair inhibitor, such as a UGI or dISN domain. The term "nucleic acid programmable DNA binding protein" or "napDNAbp" refers to a protein that binds to a nucleic acid (e.g., DNA or RNA) (such as a guide nucleic acid that guides napDNAbp to a specific nucleic acid sequence). For example, the Cas9 protein can be bound to a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, napDNAbp is a Class 2 microbial CRISPR-Cas effector. In some embodiments, napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, C2c1, C2c2, C2C3, and Argonaute. However, it should be understood that nucleic acid programmable DNA binding proteins also include nucleic acid programmable proteins that bind RNA. For example, napDNAbp can bind to a nucleic acid that guides napDNAbp to RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, but they may not be specifically listed in the present disclosure.

[0344] F. Modified RNA

[0345] In certain embodiments, the disclosed compositions, formulations, nanoparticles, and / or nanomaterials comprise modified nucleic acids, including modified RNA.

[0346] Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides is, for example, referred to as "modified" RNA to describe the presence of one or more non-natural and / or naturally occurring components or configurations used to replace typical A, G, C, and U residues or in addition to typical A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with typical nucleosides or nucleotides, referred to herein as "modified".

[0347] Modified nucleosides and nucleotides may include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of the composition of a ribose sugar, e.g., a 2' hydroxyl group on a ribose sugar (exemplary sugar modifications); (iii) large-scale replacement of phosphate moieties with "dephospho" linkers (exemplary backbone modifications); (iv) modification or replacement of naturally occurring nucleobases (including with atypical nucleobases) (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of an oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group, or conjugation of a moiety, cap or linker (these 3' or 5' cap modifications may comprise sugar and / or backbone modifications); and (vii) modification or replacement of a sugar (exemplary sugar modifications). Certain embodiments include 5' end modifications to mRNA, gRNA or nucleic acid. Certain embodiments include 3' end modifications to mRNA, gRNA or nucleic acid. The modified RNA may contain 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

[0348] Unmodified nucleic acids may be susceptible to degradation by, for example, intracellular nucleases or nucleases found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Therefore, in one aspect, the gRNA described herein (e.g., mRNA, gRNA) may contain one or more modified nucleosides or nucleotides, for example to introduce stability to intracellular or serum-based nucleases. In some embodiments, when introduced into in vivo and ex vivo cell populations, the modified gRNA molecules described herein may exhibit a reduced innate immune response. The term "innate immune response" includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids, and the cellular responses involve induction of cytokine (particularly interferon) expression and release and cell death.

[0349] Therefore, in some embodiments, the RNA or nucleic acid in the disclosed compositions, formulations, nanoparticles and / or nanomaterials disclosed herein comprises at least one modification that imparts increased or enhanced stability to the nucleic acid, including, for example, improved resistance to in vivo digestion by nucleases. As used herein, the terms "modification" and "modified" when associated with the nucleic acids provided herein include at least one change that preferably enhances stability and makes the RNA or nucleic acid more stable (e.g., resistant to nuclease digestion) than wild-type or naturally occurring RNA or nucleic acid patterns. As used herein, the terms "stable" and "stability" when associated with the nucleic acids of the present invention and particularly with respect to RNA, refer to, for example, increased or enhanced resistance to degradation by nucleases (i.e., endonucleases or exonucleases) that are generally capable of degrading such RNA. Increased stability may include, for example, reduced sensitivity to hydrolysis or other destruction caused by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions in target cells or tissues, thereby increasing or enhancing the retention of such RNA in target cells, tissues, subjects and / or cytoplasm. The stable RNA molecules provided herein exhibit a longer half-life relative to their naturally occurring unmodified counterparts (e.g., wild-type forms of mRNA). The terms "modification" and "modified" when related to the mRNA of the LNP formulations disclosed herein, these terms also encompass changes that improve or enhance mRNA nucleic acid translation, including, for example: sequences that play a role in protein translation initiation (e.g., Kozak consensus sequences). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987), the contents of which are hereby incorporated herein by reference in their entirety).

[0350] In some embodiments, the RNA or nucleic acid of the compositions, formulations, nanoparticles and / or nanomaterials disclosed herein has undergone chemical or biological modification to make it more stable. Exemplary modifications to RNA include the consumption of bases (e.g., by deletion or by substitution of one nucleotide for another) or base modifications, such as chemical modifications of bases. As used herein, the phrase "chemical modification" includes the introduction of chemical substances visible in naturally occurring RNA, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or comprising side groups that are not naturally present in these RNA molecules).

[0351] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygens with different substituents. In addition, the modified residue (for example, the modified residue present in the modified nucleic acid) may include large-scale replacement of unmodified phosphate moieties with the modified phosphate group described herein. In some embodiments, the backbone modification of the phosphate backbone may include changes that result in uncharged joints or charged joints with asymmetric charge distribution. The example of modified phosphate groups includes thiophosphate, selenophosphate, boranophosphate, boranophosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester. The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or atomic groups can make the phosphorus atom chiral. Stereo phosphorus atoms can have "R" configuration (herein Rp) or "S" configuration (herein Sp). The main chain can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting phosphate to nucleoside) with nitrogen (phosphoramidate of bridge), sulfur (phosphorothioate of bridge) and carbon (methylene phosphonate of bridge). Replacement can occur at any one connecting oxygen or two connecting oxygens. In some main chain modifications, the phosphate group can be replaced by a phosphorus-free connector. In some embodiments, the charged phosphate group can be replaced by a neutral part. The example of the part of the replaceable phosphate group can include but is not limited to, such as methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide joint, sulfonate, sulfonamide, thioformal, methylal, oxime, methylene imino, methylene methyl imino, methylene hydrazo (methylenehydrazo), methylene dimethyl hydrazo (methylenedimethylhydrazo) and methylene oxygen methyl imino.

[0352] G.mRNA

[0353] In some embodiments, the disclosed compositions, formulations, nanoparticles and / or nanomaterials comprise mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binder, such as a Cas nuclease or a Class 2 Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binder, such as a Cas nuclease or a 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 encode a protein using alternative codons.

[0354] The mRNA in the disclosed compositions, formulations, nanoparticles and / or nanomaterials can encode, for example, secreted hormones, enzymes, receptors, polypeptides, peptides, or other proteins of interest that are normally secreted. In one embodiment of the invention, the mRNA can optionally have a chemical or biological modification that, for example, improves the stability and / or half-life of such mRNA, or improves or otherwise promotes protein production.

[0355] In addition, suitable modifications include changes in one or more nucleotides of a codon, such that the codon encodes the same amino acid, but is more stable than the codon found in the wild-type form of mRNA. For example, it has been shown that there is an inverse relationship between the stability of RNA and a higher number of cytidine (C) and / or uridine (U) residues, and it has been found that RNA without C and U residues is stable to most RNases (Heidenreich et al., J Biol Chem 269, 2131-8 (1994), the disclosure of which is hereby 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 replacing another codon encoding the same or related amino acid with a codon encoding a specific amino acid. The expected modification of the mRNA nucleic acid of the present invention also includes the incorporation of pseudouridine. Pseudouridine is incorporated into the mRNA nucleic acid of the present invention to enhance stability and translation ability, and reduce immunogenicity in vivo. See, e.g., Kariko, K. et al., Molecular Therapy 16(11):1833-1840 (2008), the contents of which are hereby incorporated by reference in their entirety. The mRNA of the present invention can be substituted and modified by methods readily understood by those of ordinary skill in the art.

[0356] The constraints on reducing the number of C and U residues in the sequence may be greater in the coding region of the mRNA than in the untranslated regions (i.e., it may not be possible to eliminate all C and U residues present in the message while still retaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code provides the opportunity to reduce the number of C and / or U residues present in the sequence while maintaining the same coding capacity (i.e., depending on which amino acids are encoded by the codons, there may be several different possibilities for RNA sequence modifications).

[0357] The term modification also includes, for example, the incorporation of non-nucleotide bonds or modified nucleotides into the mRNA sequence of the invention (e.g., modification of one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). These modifications include adding bases to the mRNA sequence (e.g., including a poly A tail or a longer poly A tail), altering the 3'UTR or 5'UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and including elements that alter the structure of the mRNA molecule (e.g., which form a secondary structure).

[0358] The poly (A) tail is believed to stabilize the natural messenger. Therefore, in one embodiment, a long poly (A) tail can be added to the mRNA molecule, thereby making the mRNA more stable. The poly (A) tail can be added using a variety of techniques recognized in the art. For example, a long poly (A) tail can be added to a synthetic or in vitro transcribed mRNA using poly (A) polymerase (Yokoe et al. Nature Biotechnology. 1996; 14: 1252-1256, the contents of which are hereby incorporated herein by reference in their entirety). The transcription vector can also encode a long poly (A) tail. In addition, the poly (A) tail can be added by directly transcribing from a PCR product. In one embodiment, the length of the poly (A) tail is at least about 90, 200, 300, 400, at least 500 nucleotides. In one embodiment, the length of the poly (A) tail is adjusted to control the stability of the modified mRNA molecule of the present invention, and thus control the transcription of the protein. For example, since 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 change the level of resistance of the mRNA to nucleases and thereby control the time course of protein expression in the cell. In one embodiment, the stabilized mRNA molecules are sufficiently resistant to degradation in vivo (e.g., by nucleases) that they can be delivered to target cells without a transfer vehicle.

[0359] 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, the 3' and / or 5' flanking sequences of natural flanking mRNA and encoding the second unrelated protein can be incorporated into the nucleotide sequence of the mRNA molecule encoding therapeutic or functional protein to modify it. For example, 3' or 5' sequences from stable mRNA molecules (e.g., globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) can be incorporated into 3' and / or 5' regions of the mRNA nucleic acid molecules to increase the stability of the mRNA molecules. See, for example, US2003 / 0083272, the contents of which are hereby incorporated herein by reference in their entirety. A more detailed description of mRNA modifications can be found in pages 57-68 of US2017 / 0210698A1, the contents of which are incorporated herein by reference in their entirety.

[0360] H. Template nucleic acid

[0361] The compositions, formulations, nanoparticles and / or nanomaterials and methods disclosed herein may include template nucleic acids. The template can be used to change or insert a nucleic acid sequence at or near the target site of an RNA-guided DNA binding protein (such as a Cas nuclease, e.g., a Class 2 Cas nuclease). In some embodiments, the method comprises 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 be performed 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.

[0362] In some embodiments, the template can be used for homologous recombination. In some embodiments, homologous recombination can integrate the template sequence or a portion of the template sequence into the target nucleic acid molecule. In some embodiments, the template can be used for homology directed repair, which involves the invasion of the DNA chain at the cleavage site in the nucleic acid. In some embodiments, homology directed repair can include the template sequence in the edited target nucleic acid molecule. In some embodiments, the template can be used for gene editing mediated by non-homologous end connection. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, a portion of the template or template sequence is incorporated. In some embodiments, the template includes a flanking reverse terminal repeat (ITR) sequence.

[0363] 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 at a different position from the native position in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell.

[0364] In some embodiments, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell.

[0365] 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, plasmid, minicircle, nanocircle, or PCR product.

[0366] In some embodiments, nucleic acid is purified. In some embodiments, nucleic acid is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation or equivalent methods, for example, as described herein). In some embodiments, a chromatography-based method is used, such as a HPLC-based method or an equivalent method (e.g., as described herein) nucleic acid is purified. In some embodiments, nucleic acid is purified using a precipitation method (e.g., LiCl precipitation) and a HPLC-based method. In some embodiments, nucleic acid is purified by tangential flow filtration (TFF).

[0367] IV. Methods of Making LNPs

[0368] The method of manufacturing lipid nanoparticles is known in the art. In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials described are manufactured using microfluidics. For example, exemplary methods of using microfluidics to form lipid nanoparticles are described by 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 hereby incorporated by reference in their entirety.

[0369] In brief, a carrier, such as the carrier 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 carrier.

[0370] The method for screening the disclosed lipid nanoparticles is 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 the delivery formulation of the vehicle to identify the formulation with the desired tropism and delivering the functional carrier to the cytoplasm of a specific cell. In some embodiments, the screening method uses a reporter with a functionality that can be detected when delivered to the cell. For example, the detection of the functional reporter in the cell indicates that the LNP formulation delivers the functional carrier to the cell. In addition, in some embodiments, the chemical composition identifier is included in each different delivery vehicle formulation to track 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 component for preparing the LNP formulation of the chemical component loaded therein, so as to identify the chemical composition of the delivery vehicle of the delivery barcode when sequencing the nucleic acid barcode. Representative barcodes include, but are not limited to, barcodes described by Sago, 2018 PNAS, Sago, JACS 2018, the disclosures of which are hereby incorporated by reference in their entirety. Representative reporter molecules include, but are not limited to, siRNA, mRNA, nuclease proteins, nuclease mRNA, small molecules, epigenetic modifiers, and phenotypic modifiers. DNA (genomic and DNA barcodes) can be separated 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 hereby incorporated by reference in their entirety).

[0371] V. How to use

[0372] In addition, the present disclosure describes methods using compositions, preparations, nanoparticles and / or nanomaterials described herein. For example, in some embodiments, the present disclosure describes methods for delivering a vehicle to specific cells, tissues or organs described herein using compositions, preparations, nanoparticles and / or nanomaterials. As another example, in some embodiments, the present disclosure describes methods for treating a disease or disorder and / or delaying and / or preventing a disease or disorder from progressing using compositions, preparations, nanoparticles and / or nanomaterials described herein. In some embodiments, compositions, preparations, nanoparticles and / or nanomaterials described herein are used in medicine.

[0373] In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials described herein deliver therapeutic or prophylactic agents to specific cells or organs of subjects in need. In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials deliver therapeutic or prophylactic agents to specific cells or organs of subjects in need in the absence of targeting ligands. In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials can be used to treat or prevent diseases in subjects in need.

[0374] A. Methods of delivering cargo to cells, tissues or organs

[0375] In addition, in some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials disclosed herein target specific types or categories of cells (e.g., cells of a specific organ or its system), tissues and / or organs. In some embodiments, the disclosure provides methods of delivering one or more carriers described herein to a subject in need. In some embodiments, these methods include in vivo and / or in vitro delivery. In some embodiments, these methods include in vivo delivery. In some embodiments, these methods include in vitro delivery. In some embodiments, the disclosure provides methods of delivering one or more therapeutic and / or preventive nucleic acids to a subject in need as described herein.

[0376] In some embodiments, the composition, formulation, nanoparticle and / or nanomaterial comprises a therapeutic and / or prophylactic agent of interest that can be delivered specifically to liver cells of a subject.Exemplary liver cells include, but are not limited to, hepatocytes.

[0377] In some embodiments, the composition, formulation, nanoparticle and / or nanomaterial comprises a therapeutic and / or prophylactic agent of interest that can be specifically delivered 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.

[0378] In some embodiments, the composition, formulation, nanoparticle and / or nanomaterial comprises a therapeutic agent and / or prophylactic agent of interest that can be specifically delivered to the subject's bone marrow cells. Exemplary bone marrow cells include, but are not limited to, bone marrow mononuclear cells, bone marrow B cells, bone marrow memory B cells, or bone marrow T cells.

[0379] In some embodiments, the composition, formulation, nanoparticle and / or nanomaterial comprises a therapeutic and / or prophylactic agent of interest that can be specifically delivered to immune cells of a subject. Exemplary immune cells include, but are not limited to, CD8+, CD4+, or CD8+CD4+ cells.

[0380] In some embodiments, the composition, formulation, nanoparticle and / or nanomaterial comprises a therapeutic agent and / or prophylactic agent of interest that can be specifically delivered to a subject's hematopoietic stem cell. Unless otherwise indicated, it should be understood that the terms "hematopoietic stem cell (HSC)" and "hematopoietic stem and progenitor cells (HSPC)" are used interchangeably in the present disclosure.

[0381] In some embodiments, lipid nanoparticle can be formulated to be delivered to mammal liver hepatocyte, liver immune cell, spleen T cell or lung endothelial cell in the absence of targeting ligand.Specific delivery to the cell of particular class or type shows that the lipid nanoparticle of higher ratio is delivered to the cell of target type or class.In some embodiments, specific delivery can cause than using conventional nanoparticle system (for example, the LNP containing MC3) delivery 2 times, 5 times, 10 times, 15 times or 20 times.

[0382] B. Methods for Producing Polypeptides

[0383] In addition, in some embodiments, the methods of using the compositions, formulations, nanoparticles and / or nanomaterials disclosed herein are used for methods of producing polypeptides. In addition, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells of a subject in need. For example, in some embodiments, the lipid nanoparticles described herein can be used to produce polypeptides in target cells of a subject in need. In some embodiments, the compositions, formulations, nanoparticles and / or nanomaterials disclosed herein comprise one or more nuclear sequences to be delivered to a cell.

[0384] 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 the following: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, 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.

[0385] C. Methods of Gene Regulation

[0386] In addition, in some embodiments, the use methods of compositions, preparations, nanoparticles and / or nanomaterials disclosed herein are used for gene regulation. In addition, in some embodiments, lipid nanoparticles described herein can be used to reduce and / or increase gene expression in target cells of a subject in need. For example, in some embodiments, lipid nanoparticles described herein can deliver one or more nucleic acids to a subject's target cell without a targeting ligand. In some embodiments, nucleic acid is an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid is siRNA. In some embodiments, nucleic acid is a nucleic acid described herein. As another example, in some embodiments, lipid nanoparticles described herein can deliver a carrier to a subject's target cell without a targeting ligand. In some embodiments, the carrier is any carrier described herein.

[0387] Furthermore, in some embodiments, methods of using the compositions, formulations, nanoparticles, and / or nanomaterials disclosed herein to edit genes in cells of a subject in need thereof.

[0388] In some embodiments, the cell targeted for gene regulation is an immune cell. The immune cell may be a T cell, such as a CD8+T cell, a CD4+T cell, or a T regulatory cell. 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, targeting is used for cells for gene regulation in hepatocytes.

[0389] Exemplary genes that can be targeted include, but are not limited to, T cell receptor, B cell receptor, CTLA4, PD1, FOXO1, FOXO3, AKTs, 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-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 may be recognized by T cells and are contemplated for targeting include, but are not limited to, MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1 / MelanA, 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.

[0390] D. Subjects to be treated

[0391] In some embodiments, the subject to be treated is a mammal suffering from cancer, autoimmune disease, infectious disease, organ transplantation, organ failure, protein deficiency or a combination thereof. In some embodiments, the subject is human. In some embodiments, the methods described herein can cause liver cells to translate certain proteins. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA or siRNA to liver cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA or siRNA to spleen T cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA or siRNA to spleen B cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA or siRNA to spleen mononuclear cells. In some embodiments, the methods described herein can be used to deliver one or more DNA, mRNA, sgRNA or siRNA to bone marrow cells.

[0392] It should be understood that the order of steps or the order in which certain actions are performed is not important, as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0393] While the present invention has been particularly shown and described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the following claims.

[0394] example

[0395] The present disclosure exemplifies the compositions, formulations, formulations, nanoparticles and / or nanomaterials described herein. The present disclosure also exemplifies methods of making, characterizing and validating the compositions, formulations, formulations, nanoparticles and / or nanomaterials described herein.

[0396] Example 1: Materials and Methods

[0397] 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 WO 2022 / 140252, which is incorporated herein by reference.

[0398] Example 2: Efficacy of each screening

[0399] This example provides exemplary compositions, formulations, nanoparticles and / or nanomaterials, and materials and methods for screening the efficacy of these compositions, formulations, nanoparticles and / or nanomaterials described herein. The exemplary LNP screening described in Example 1 of WO 2022 / 140252 can be used to demonstrate that each LNP pool is highly effective in many tissues.

[0400] Example 3: Exemplary LNP Formulations and Delivery to Various Cell Types

[0401] This example provides exemplary LNP compositions, formulations, nanoparticles and / or nanomaterials for delivery to various cell types described herein. This example can be used to demonstrate that in some embodiments, provided lipids show delivery to various cell types.

[0402] Based on the results derived from the screening in Embodiment 1, exemplary LNP formulations were identified for spleen delivery, and specifically for B cell delivery. These identified lipids were formulated into LNP formulations and screened using the Cre reporter system described herein. Three Ai14 mice were used per group. The payload contained 0.3 mg / kg Cre mRNA. Data were collected 168 hours after injection. The results were compared with the MC3-LNP formulation as a control.

[0403] Example 4: Delivery of Exemplary LNP Formulations to Various Cell Types

[0404] This example provides exemplary LNP compositions, formulations, nanoparticles and / or nanomaterials delivered to various cell types described herein. This example can be used to demonstrate that the provided lipids show delivery in various cell types.

[0405] LNP formulations were selected to confirm efficacy results using the Cre reporter system and Ai14 mouse model described herein. Three Ai14 mice per group were used in each experiment. Data were collected 72 hours after injection. The screening platform described herein can identify LNP formulations to determine which type of LNP formulation will be most effective for a specific cell type.

[0406] Example 5: Demonstration of LNP formulations delivering functional mRNA

[0407] This example provides exemplary LNP compositions, formulations, nanoparticles and / or nanomaterials that deliver functional mRNA to different cell types. This example can be used to demonstrate that the provided lipids are capable of delivering functional mRNA in mice.

[0408] The exemplary LNP formulations were selected to determine the ability of each formulation to deliver functional mRNA in mice. Each LNP formulation carried 0.15 mg / kg hEPO mRNA and was administered in 2-3 C57BL6 mice at a mass ratio of 11.7 and 19. hEPO expression in plasma was measured six hours after LNP formulation injection.

[0409] Example 6: Tolerance and efficacy experiments

[0410] This example provides exemplary materials and methods for preparing, characterizing and validating the compositions, formulations, nanoparticles and / or nanomaterials described herein. This example can be used to demonstrate that the provided lipids are capable of delivering functional mRNA in mice.

[0411] Select exemplary LNP preparations to determine the tolerance and efficacy of hEPO mRNA delivery in rats, as described herein. Each LNP preparation containing 1.0 mg / kg hEPO mRNA is injected into Sprague-Dawley rats (N=2). For each exemplary LNP preparation, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (U / L) are collected from rat plasma 24 hours after injection. For each LNP preparation, monocyte chemoattractant protein-1 (MCP-1) (ng / mL) is collected from rat plasma 6 hours after injection. Saline is used as a control. After the vehicle (control) and exemplary LNP preparations are applied at each time point (0, 2, 4, 6, 24, 48, 96 hours) after injection, hEPO (ng / mL) is collected from rat plasma.

[0412] Example 7: Synthesis of ionizable lipids

[0413] The present embodiment provides the exemplary materials and methods for preparing, characterizing and verifying the ionizable lipids described herein. As described in the following examples, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It should be understood that although the general method describes the synthesis of certain compounds of the present disclosure, the following general method and other methods known to those of ordinary skill in the art can be applied to all compounds described herein and subclasses and species of each compound in these compounds.

[0414] General Notes: Unless otherwise noted, all reactions were performed using anhydrous grade solvents in flasks or vials under a nitrogen atmosphere with magnetic stirring. 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+ with pre-packed 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 a Varian INOVA 500 MHz or a Bruker AVANCE 400 MHz spectrometer; chemical shifts were reported in δ parts per million (ppm) with reference to tetramethylsilane at δ = 0.00 ppm for CDCl3 samples and the residual solvent peak (δ = 2.50 ppm) for DMSO samples. Analytical results were obtained using an HPLC-MS / MS with a QDa detector (ESI + Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analyses were performed on a Waters Acquity UPLC H-class Plus equipped with HPLC-MS / MS equipment using one of the following general methods.

[0415] Method A (5 min run): Column: XTERRA RP 18 (4.6×50 mm), 5 μm, Mobile phase: Initially 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF]; then to 2% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70:30) ACN:THF], 2.65 min, keep this mobile phase composition for up to 3.75 min, and finally return to the initial conditions, i.e., 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF], 4.90 min, keep this mobile phase composition for up to 5.10 min. Flow rate = 1.2 mL / min.

[0416] Method B1 (12 min run): Column: XTERRA RP 18 (4.6×50 mm), 5 μm, (mobile phase: initially 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70:30) ACN:THF]; maintain this initial condition for 0.75 min; then to 65% [0.1% HCOOH / water] and 35% [0.1% HCOOH / (70:30) ACN:THF], 3.0 min, then to 2% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70: 30) ACN: THF], 6.0 min, keep this mobile phase composition for 9.0 min, and finally return to the initial conditions, i.e. 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70: 30) ACN: THF], 11.00 min, keep this mobile phase composition for 12.10 min. Flow rate = 1.2 mL / min.

[0417] Method B2 (5 min run): Column: XTERRA RP 18 (4.6×50 mm), 5 μm, (mobile phase: initially 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70:30) ACN:THF]; maintain this initial condition for 0.75 min; then to 65% [0.1% HCOOH / water] and 35% [0.1% HCOOH / (70:30) ACN:THF], 3.0 min, then to 2% [0.1% HCOO H / water] and 98% [0.1% HCOOH / (70:30)ACN:THF] for 6.0 min, keeping this mobile phase composition for 9.0 min, and finally returning to the initial conditions, i.e. 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70:30)ACN:THF] for 4.90 min, keeping this mobile phase composition for 5.10 min. Flow rate = 1.2 mL / min.

[0418] Method C (12 min run): Column: XTERRARP 18 (4.6×50 mm), 5 μm, (Mobile phase: initially 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70:30) ACN:THF]; maintain this initial condition for 0.75 min; then to 65% [0.1% HCOOH / water] and 35% [0.1% HCOOH / (70:30) ACN:THF], 3.0 min, then to 2% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70: 30) ACN: THF], 6.0 min, keep this mobile phase composition for 9.0 min, and finally return to the initial conditions, i.e. 80% [0.1% HCOOH / water] and 20% [0.1% HCOOH / (70: 30) ACN: THF], 11.00 min, keep this mobile phase composition for 12.10 min. Flow rate = 1.2 mL / min.

[0419] List of abbreviations

[0420] Ac:Acetyl

[0421] ACN: Acetonitrile

[0422] d: Twin Peaks

[0423] DCC: N,N′-dicyclohexylcarbodiimide

[0424] DCM: dichloromethane

[0425] DIPEA: N,N-diisopropylethylamine

[0426] DMAP: 4-(dimethylamino)pyridine

[0427] DMSO: dimethyl sulfoxide

[0428] EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

[0429] Eq: equivalent

[0430] Et: Ethyl

[0431] i-Pr: isopropyl

[0432] m: multiple peaks

[0433] Me:Methyl

[0434] p: Quintet

[0435] PPTS: Pyridinium p-toluenesulfonate

[0436] q: quartet

[0437] Rt: retention time

[0438] s: Single peak

[0439] t: triplet

[0440] TBAF: Tetrabutylammonium fluoride

[0441] TBS: tert-butyldimethylsilyl

[0442] TEA: triethylamine

[0443] THF: Tetrahydrofuran

[0444] Universal synthesis

[0445] Exemplary lipids were prepared according to the following general synthetic scheme, which is illustrated using Example 7-1.

[0446]

[0447]

[0448] Example 7-1: (9Z,12Z)-octadecane-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl ester

[0449]

[0450] Step 1: 4,4-Bis(octyloxy)butyronitrile

[0451] General Procedure A:

[0452] To a vial containing pyridinium p-toluenesulfonate (0.12 g, 0.48 mmol, 0.05 Eq) was added 4,4-diethoxybutyronitrile (1.5 g, 9.5 mmol, 1 Eq) and 1-octanol ((3.7 g, 29 mmol, 3 Eq). The vial was capped tightly and the resulting mixture was heated at 105 °C for 72 h. Afterwards, the mixture was cooled to room temperature. The crude material was purified by silica gel column chromatography using a gradient of 0 to 100% dichloromethane / hexanes to give 4,4-bis(octyloxy)butyronitrile (1.08 g, 35%) as a colorless oil. 1H NMR (400 MHz, CHLOROFORM-d) δ 0.87 (t, J = 6.7 Hz, 6H), 1.17–1.41 (m, 20H), 1.54–1.62 (m, 4H), 1.88–1.98 (m, 2H), 2.41 (t, J = 7.4 Hz, 2H), 3.37–3.47 (m, 2H), 3.54–3.64 (m, 2H), 4.54 (t, J = 5.3 Hz, 1H).

[0453]

[0454] Step 2: 4,4-Bis(octyloxy)butyric acid

[0455] General Procedure B:

[0456] Potassium hydroxide (1.2 g, 21 mmol, 3 Eq) was added to a vial containing 4,4-bis(octyloxy)butyronitrile (2.3 g, 7.1 mmol, 1 Eq), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was capped tightly and the reaction mixture was heated at 110 ° C for 18 h. Thereafter, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL) and the pH was adjusted to about 5 by adding 1M 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)butyric acid (1.1 g, 45% yield) as a light yellow oil. 1 H NMR(400MHz, DMSO-d6)δ0.85(t,J=6.5Hz,6H),1.15–1.36(m,21H),1.46(q,J=6.7Hz,4H),1.72(q,J=7.0H z, 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).

[0457]

[0458] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester

[0459] General Procedure C:

[0460] To a stirred solution of (9Z,12Z)-octadec-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. Subsequently, 2-(hydroxymethyl)propane-1,3-diol (2. The mixture was stirred for 16 h at 25 °C. Water (20 mL) was added and the mixture was extracted with DCM (2 × 100 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude compound thus obtained was purified by flash chromatography eluted with 70% EtOAc-hexane to give (9Z, 12Z)-octadecane-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (3.2 g, 44%) as a colorless liquid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 0.88 (t, J = 6.8 Hz, 3H), 1.16–1.43 (m, 15H), 1.51–1.76 (m, 2H), 1.92–2.11 (m, 5H), 2.32 (t, J = 7.6 Hz, 2H), 2.39 (s, 1H), 2.76 (t, J = 6.4 Hz, 2H), 3.68–3.82 (m, 4H), 4.24 (d, J = 6.3 Hz, 2H), 5.26–5.43 (m, 4H).

[0461]

[0462] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl ester

[0463] General Procedure D:

[0464] To a stirred solution of 4,4-bis(octyloxy)butyric acid (0.4g, 1.16mmol) in DCM (30mL), EDC (334mg, 1.74mmol), DMAP (28mg, 0.233mmol) and DIPEA (0.5mL, 2.90mmol) were added. The resulting mixture was stirred at 25 °C for 30min. Subsequently, (9Z, 12Z)-octadecane-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (0.4g, 1.16mmol) was added thereto and further stirred at 25 °C for 16h. Water (10mL) was added, and the mixture was extracted with DCM (2×50mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude compound thus obtained was purified by flash chromatography eluting with 30% EtOAc-hexanes to afford (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester (231 mg, 30%) as a colorless liquid. 1 H NMR(400MHz, chloroform-d)δ0.83–0.92(m,9H),1.15–1.43(m,38H),1.55–1.69(m,2H), 1.93(q,J=7.0Hz,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),3.5 0–3.65(m,4H),4.17(t,J=5.7Hz,4H),4.48(t,J=5.5Hz,1H),5.14–5.54(m,4H).

[0465]

[0466] Step 5: Decane-1,4-diol

[0467] General Procedure E:

[0468] Lithium aluminum hydride (35mL, 1M in THF, 35mmol, 3Eq) was added dropwise to a solution of 5-hexyldihydrofuran-2 (3H) -one (2.0g, 11.75mmol, 1Eq) in THF (15mL) at 0°C. After addition, the reaction mixture was warmed to 25°C and stirred for 12h. Subsequently, water and 15% NaOH (aqueous solution) were added to the reaction mixture at 0°C. After further stirring for 15 minutes, the mixture was filtered through a diatomaceous earth pad. The diatomaceous earth pad was washed with ether (100mL), and the combined filtrate was concentrated. The crude material was purified by silica gel column chromatography using a gradient of 0 to 50% ethyl acetate / hexane to obtain decane-1,4,-diol (1.6g, 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).

[0469]

[0470] Step 6: 1-((tert-Butyldimethylsilyl)oxy)decan-4-ol

[0471] General Procedure F:

[0472] Imidazole (293mg, 4.30mmol) and tert-butyldimethylsilyl chloride (520mg, 3.45mmol) were added to a solution of decane-1,4,-diol (500mg, 2.87mmol) in DCM (10mL) at 0°C. After addition, the reaction mixture was warmed to 25°C and stirred for 2 hours. Subsequently, the reaction mixture was diluted with water (3mL) and extracted with ethyl acetate (2×40mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude material was purified by silica gel column chromatography using a gradient of 0 to 10% ethyl acetate / hexane to obtain 1-((tert-butyldimethylsilyl)oxy)decane-4-ol (660mg, 80%) as a light 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).

[0473]

[0474] Step 7: 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0475] General Procedure G:

[0476] To a solution of 1-((tert-butyldimethylsilyl)oxy)decan-4-ol (100 mg, 0.35 mmol) in DCM (3 mL) was added pyridine (0.06 mL, 0.69 mmol), DMAP (13 mg, 0.10 mmol) and 4-nitrophenyl chloroformate (175 mg, 0.86 mmol) and stirred at 25° C. for 1 h. Subsequently, 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. Thereafter, the reaction mixture was diluted with dichloromethane (40 mL), washed with 1 M sodium carbonate (2×5 mL), water (5 mL), brine, and finally dried over anhydrous Na2SO4. The resulting dichloromethane layer was concentrated and purified by silica gel column chromatography using a gradient of 0 to 2% methanol / DCM to give 1-((tert-butyldimethylsilyl)oxy)dec-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).

[0477]

[0478] Step 8: 1-Hydroxydec-4-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0479] General Procedure H:

[0480] To a solution of 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate (1.5 g, 3.5 mmol) in THF (10 mL) was added 1(M)TBAF in THF (8.7 mL, 8.75 mmol) at 0°C under nitrogen atmosphere. The reaction mass was stirred at 25°C for 8 h. 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 Na2SO4, and concentrated under reduced pressure. The crude material thus obtained was purified by combiflash chromatography, eluting with 10% MeOH-DCM to give 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate (800 mg, 80%) as a light yellow viscous liquid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 0.86 (t, J = 6.6 Hz, 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).

[0481]

[0482] Step 9: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid

[0483] General Procedure J:

[0484] At 0 ° C, to a stirred solution of (2-(pyrrolidin-1-yl)ethyl)carbamic acid 1-hydroxydecyl-4-yl ester (1.01 g, 3.21 mmol) in acetone (30 mL) was added Jones reagent (H2SO4 containing 2M CrO3, 1.93 mL, 3.85 mmol) dropwise. The reaction mixture was stirred at 25 ° C for 16 h. After completion, isopropanol (2 mL) was added, and the mixture was filtered through diatomaceous earth. The filtrate was concentrated under reduced pressure. The crude material was diluted with water, and 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 Na2SO4 and concentrated under reduced pressure to give 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid (872 mg, 82%) as a light green viscous liquid, which was used without further purification. 1H NMR(400MHz, chloroform-d)δ0.86(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).

[0485] Step 10: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl ester (Example 7-1)

[0486] General Procedure K:

[0487] DMAP (15 mg, 0.122 mmol) and DCC (188 mg, 00.91 mmol) were added to a stirred solution of 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid (200 mg, 0.609 mmol) in DCM (10 mL). The resulting mixture was stirred at 25 ° C for 30 min. Subsequently, (9Z, 12Z)-octadecane-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester (423 mg, 0.60 mmol) was added thereto, and the mixture was further stirred at 25 ° C for 16 h. Water (20 mL) was added, and the reaction mixture was extracted with DCM (2×50 mL). The organic layer was dried over anhydrous Na2SO4 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)-octadec-9,12-dienoate (120 mg, 24%) as a colorless liquid. UPLC-MS (Method B1): Rt 5.72 min, m / z calcd [M+H]: 1005.8, found: 1006.2. 1H NMR (400MHz, chloroform-d) δ0.79–0.92(m,9H),1.17–1.41(m,49H),1.45–1.64(m,9H),1.64– 1.85(m,3H),1.91(q,J=7.7Hz,6H),2.04(q,J=6.9Hz,4H),2.29(t,J=7.6Hz,2H),2.3 1-2.44(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).

[0488]

[0489] Example 7-2: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester

[0490]

[0491] Step 1: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butyronitrile

[0492] 4,4-diethoxybutyronitrile (1.5g, 9.5mmol, 1Eq) and cis-5-octen-1-ol (3.7g, 29mmol, 3Eq) were added to a vial containing pyridinium p-toluenesulfonate (0.12g, 0.48mmol, 0.05Eq). The vial was tightly capped and the resulting mixture was heated at 105 ° C for 72h. Thereafter, the mixture was cooled to room temperature. The crude material was purified by flash column chromatography (0 to 100% dichloromethane / hexane). 4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butyronitrile (1.14g, 37%) was obtained as a colorless oil. 1 HNMR (500MHz, chloroform-d) δ5.43–5.27(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).

[0493]

[0494] Step 2: 4,4-Bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid

[0495] To a vial containing 4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butyronitrile (1.14 g, 3.54 mmol, 1 Eq) was added potassium hydroxide (0.60 g, 10.6 mmol, 3 Eq), followed by ethanol (3.5 mL) and water (3.5 mL). The vial was capped tightly and the reaction mixture was heated at 110 °C for 18 h. Thereafter, the mixture was cooled to room temperature. The mixture was diluted with ethyl acetate (20 mL) and the pH was adjusted to about 5 by adding 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-ene-1-yl)oxy)butyric acid (1.16 g, 96% yield) as a white sticky solid. 1 H NMR (400MHz, chloroform-d) δ5.41–5.24(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).

[0496]

[0497] Step 3: (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester

[0498] To a mixture of 4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butanoic acid (591 mg, 1 Eq, 1.74 mmol) in dichloromethane (10 mL) was added (9Z, 12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester (640 mg, 1 Eq, 1.74 mmol), DIPEA (673 mg, 904 μL, 3 Eq, 5.21 mmol) and DMAP (42.4 mg, 0.2 Eq, 347 μmol). Finally, EDC (666 mg, 2 Eq, 3.47 mmol) was added and stirred at 23 ° C for 18 h. Thereafter, the reaction mixture was concentrated and purified by flash column chromatography (0 to 40% ethyl acetate / hexane). 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate (450 mg, 38%) was obtained as a colorless oil. 1 HNMR (500 MHz, chloroform-d) δ5.43–5.27 (m, 8H), 4.49 (t, J = 5.5 Hz, 1H), 4.23–4.12 (m, 4H), 3.65–3.60 (m, 2H), 3.58 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.6 Hz, 2H), 2.81–2.73 (m, 2H), 2.41 (t, J = 7.5 Hz,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).

[0499] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (Example 7-2)

[0500] General Procedure L:

[0501] 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), (9Z, 12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-ene-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester (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. After completion, the reaction mixture was concentrated and diluted with DCM (50 mL), washed with NaHCO saturated solution (2×25 mL), water (2×25 mL) and brine (2×15 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude material was purified by combiflash column chromatography eluting with 0-5% methanol / DCM to give (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (46 mg, 30%) as a colorless liquid. UPLC-MS (Method A): Rt 2.39 min, m / z calculated value [M+H]: 1001.8, found value: 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 = 7.6 Hz, 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,2 H),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)

[0502]

[0503] Example 7-3: (9Z,12Z)-octadec-9,12-dienoic acid 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-azahexadecane-16-yl ester

[0504]

[0505] Step 1: 1-((tert-Butyldimethylsilyl)oxy)dec-4-yl (3-(dimethylamino)propyl) carbonate

[0506] Pyridine (0.56 mL, 6.93 mmol), DMAP (212 mg, 1.73 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.66 mmol) were added to a stirred solution of 1-((tert-butyldimethylsilyl)oxy)decane-4-ol (1 g, 3.46 mmol) in DCM (20 mL). The reaction mixture was stirred at 25 ° C for 2 h. Subsequently, 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 Na2SO4 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)dec-4-yl (3-(dimethylamino)propyl) carbonate (480 mg, 33%) as a colorless liquid. 1 H NMR (400 MHz, 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 = 7.3 Hz, 2H), 3.49–3.74 (m, 2H), 4.16 (t, J = 6.3 Hz, 2H), 4.59–4.83 (m, 1H).

[0507]

[0508] Step 2: 3-(Dimethylamino)propyl (1-hydroxydec-4-yl) carbonate

[0509] Prepared according to General Procedure H substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (3-(dimethylamino)propyl) carbonate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 110 mg, 54%. 1 H NMR(400MHz, DMSO-d6)δ0.85(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).

[0510]

[0511] Step 3: 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)decanoic acid

[0512] Prepared according to General Procedure J, substituting 3-(dimethylamino)propyl (1-hydroxydec-4-yl) carbonate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. UPLC-MS (Method A): Rt 0.35 min, m / z calcd [M+H]: 318.2, found: 318.3.

[0513] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 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-azahexadecane-16-yl ester (Example 7-3)

[0514] Prepared according to General Procedure L, substituting 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated 32 mg, 12%. UPLC-MS (Method A): Rt 2.23 min, m / z calcd [M+H]: 990.8, found: 990.7.

[0515]

[0516] Example 7-4: (9Z,12Z)-octadec-9,12-dienoic acid 12-(((4,4-bis(octyloxy)butyryl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecane-13-yl ester

[0517]

[0518] Step 1: 1-((tert-Butyldimethylsilyl)oxy)octan-2-ol

[0519] At 0 ° C, imidazole (293 mg, 4.30 mmol) and tert-butyldimethylsilyl chloride (520 mg, 3.45 mmol) were added to a stirred solution of octane-1,2-diol (500 mg, 2.87 mmol) in DCM (10 mL). The reaction mixture was stirred at 25 ° C for 2 h. After completion, the reaction mixture was diluted with water (3 mL) and extracted with ethyl acetate (2×40 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude material thus obtained was purified by combiflash chromatography, eluted with 10% EtOAc-hexane to obtain 1-((tert-butyldimethylsilyl)oxy)octan-2-ol (660 mg, 80%) as a light yellow liquid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 0.06 (s, 6H), 0.78–1.00 (m, 12H), 1.14–1.52 (m, 10H), 2.41 (d, J=3.3 Hz, 1H), 3.37 (dd, J=8.3, 10.5 Hz, 1H), 3.57–3.64 (m, 2H).

[0520]

[0521] Step 2: 1-((tert-butyldimethylsilyl)oxy)octan-2-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0522] 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)δ0.04(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).

[0523]

[0524] Step 3: 1-Hydroxyoctan-2-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0525] 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)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 130 mg, 65%. 1 H NMR(400MHz, chloroform-d)δ0.86(t,J=6.6Hz,3H),1.00(t,J=7.4Hz,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=8.5Hz,1H),3.47(s,1H),3.5 8(dd,J=6.8,12.1Hz,1H),3.66–3.74(m,1H),4.77(s,1H),5.58(s,1H).

[0526] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 12-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridec-13-yl ester (Example 7-4)

[0527] General Program M:

[0528] Pyridine (0.03 mL, 0.34 mmol), DMAP (4.2 mg, 0.03 mmol) and 4-nitrophenyl chloroformate (70.41 mg, 0.34 mmol) were added to a stirred solution of 1-hydroxyoctane-2-yl (2-(pyrrolidin-1-yl)ethyl)carbamate (50 mg, 0.17 mmol) in DCM (5 mL). The reaction mixture was stirred at 25 ° C for 2 h. Subsequently, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z, 12Z)-octadec-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. After completion, the reaction mixture was diluted with water (15 mL) and extracted with DCM (2×25 mL), washed with 1 M Na2CO3 solution (10 mL) and brine (5 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude compound thus obtained was subjected to combiflash column chromatography, eluted with 5% MeOH-DCM to give (9Z, 12Z)-octadec-9,12-dienoic acid 12-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-6-hexyl-4,9-dioxo-1-(pyrrolidin-1-yl)-5,8,10-trioxa-3-azatridecane-13-yl ester (50.32 mg, 29%) as a light yellow liquid. UPLC-MS (Method B1): Rt 5.53 min, m / z calculated value [M+H]: 1007.8, found value: 1008.2.

[0529]

[0530] Example 7-5: (9Z,12Z)-octadec-9,12-dienoic acid 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-azatridecane-13-yl ester

[0531] Prepared according to General Procedure M, substituting (9Z,12Z)-heptadeca-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester for (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester. Isolated 123 mg, 50%. UPLC-MS (Method A): Rt 2.20 min, m / z calcd [M+H]: 1003.8, found: 1004.0.

[0532]

[0533] 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

[0534]

[0535] Step 1: 2-Butyloctanoate 8-Hydroxyoctyl ester

[0536] General Procedure N:

[0537] At 25 ° C, DIPEA (3 eq), EDC (1.5 eq) and DMAP (0.5 eq) were added to a stirred solution of 2-butyloctanoic acid (1 eq) in DCM (5 mL / 0.5 mmol). The reaction mixture was stirred for 15 min and then 1,8-octanediol (3.0 eq) was added and further stirred for 16 h at 25 ° C. After completion, the reaction mixture was diluted with DCM (2 × 50 mL) and washed with saturated NaHCO3 solution (2 × 25 mL), followed by water and brine (25 mL). The organic layer was separated and passed through anhydrous Na2SO4 and dried under a rotary evaporator. The crude material thus obtained was purified by combiflash chromatography, eluted with 15-20% EtOAc-hexane to obtain 2-butyloctanoic acid 8-hydroxyoctyl ester (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).

[0538]

[0539] Step 2: 8-((2-butyloctanoyl)oxy)octanoic acid

[0540] Prepared according to general procedure J, substituting 8-hydroxyoctyl 2-butyloctanoate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 1.4 g (crude material). 1H 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).

[0541]

[0542] Step 3: 3-Hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate

[0543] Prepared according to General Procedure C, substituting 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoic acid for (9Z,12Z)-octadec-9,12-dienoic acid. Isolated 2.5 g, 47%. 1 H NMR (400MHz, chloroform-d) δ0.97(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).

[0544]

[0545] Step 4: 2-Butyloctanoate 8-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propoxy)-8-oxooctyl ester

[0546] Prepared according to General Procedure D, substituting 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 8-((2-butyloctanoyl)oxy)octanoic acid for 4,4-bis(octyloxy)butyric acid. Isolated 150 mg, 46%. 1 H NMR(400MHz, CDCl3)δ0.85–0.96(m,14H),1.24–1.62(m,32H),1.91–2.41(m,16 H),3.38–3.62(m,6H),4.03–4.17(m,6H),4.40–4.50(m,1H),5.31–5.35(m,4H)

[0547] 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)

[0548] Prepared according to General Procedure L, 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(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadec-9,12-dienoate. Isolated 72 mg, 35%. UPLC-MS (Method A): Rt 2.19 min, m / z calcd [M+H]: 1063.8, found: 1064.0.

[0549]

[0550] 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

[0551]

[0552] Step 1: 2-Butyloctanoate 7-Hydroxyheptyl ester

[0553] Prepared according to general procedure N, substituting 1,7-heptanediol for 1,8-octanediol. Isolated 1.5 g, 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).

[0554]

[0555] Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid

[0556] Prepared according to general procedure J, substituting 7-hydroxyheptyl 2-butyloctanoate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated 1.5 g (crude material). 1H NMR(400MHz,DMSO-d6)δ0.79–0.88(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).

[0557]

[0558] Step 3: 2-Butyloctanoate 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl ester

[0559] Prepared according to General Procedure D, substituting 3-hydroxy-2-(hydroxymethyl)propyl 4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-hydroxy-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 7-((2-butyloctanoyl)oxy)heptanoic acid for 4,4-bis(octyloxy)butanoic acid. Isolated 120 mg, 45%. 1 H NMR(400MHz, chloroform-d)δ0.87(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 =6.8Hz, 2H), 4.17 (d, J = 5.2Hz, 4H), 4.48 (t, J = 6.0Hz, 1H), 5.12–5.67 (m, 4H).

[0560] 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)

[0561] 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 for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated 105 mg, 47%. UPLC-MS (Method A): Rt 2.04 min, m / z calcd [M+H]: 1049.8, found: 1049.8.

[0562]

[0563] Example 7-8: 2-Butyloctanoate 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 ester

[0564] 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)-octadec-9,12-dienoate. Isolated 92 mg, 33%. UPLC-MS (Method A): Rt 2.17 min, m / z calcd [M+H]: 1065.8, found: 1066.2. 1H NMR (400MHz, chloroform-d) δ0.82–0.91(m,12H),0.94(t,J=7.5Hz,4H),1.13–1.49(m,31H),1.49–1.67(m, 10H),1.72–1.87(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,5 H),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=5.5 Hz, 2H), 4.27 (dd, J = 3.2, 11.6 Hz, 1H), 4.47 (t, J = 5.5 Hz, 1H), 4.84–5.08 (m, 1H), 5.24–5.42 (m, 4H).

[0565]

[0566] Example 7-9: 2-Butyloctanoic acid 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-azaheneicosane-21-yl ester

[0567] 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)-octadec-9,12-dienoate. Isolated 92 mg, 33%. UPLC-MS (Method A): Rt 2.18 min, m / z calcd [M+H]: 1051.8, found: 1052.1.

[0568] Example 7-10: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester

[0569] Step 1: 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (3-(diethylamino)propyl) carbonate

[0570] 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, 56% yield. 1 H NMR(400MHz, chloroform-d)δ0.02(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,2 H),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).

[0571] Step 2: 3-(Diethylamino)propyl (1-hydroxydec-4-yl) carbonate

[0572] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (3-(diethylamino)propyl) carbonate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.6 g, 49% yield. 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).

[0573] Step 3: 4-(((3-(Diethylamino)propoxy)carbonyl)oxy)decanoic acid

[0574] Prepared according to General Procedure J, substituting 3-(diethylamino)propyl (1-hydroxydec-4-yl) carbonate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 400 mg, crude material. 1 H NMR(400MHz,DMSO-d6)δ0.86(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).

[0575] Step 4: (9Z,12Z)-octadec-9,12-dienoic acid 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 ester (Example 7-10)

[0576] Prepared according to General Procedure L substituting 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 56.94 mg, 36% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 5.36 min, MS calcd: 1018.79 [M+H], MS found 1019.01 [M+H].

[0577] Example 7-11: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate

[0578] Step 1: 2-Butyloctanoate 7-Hydroxyheptyl ester

[0579] To a stirred solution of 2-butyloctanoic acid (2g, 9.98mmol) in DCM (20mL) was added EDC HCl (2.67g, 12.97mmol), DMAP (244mg, 1.99mmol), DIPEA (5.2mL, 29.95mmol). The reaction mixture was stirred at 25 ° C for 30min. Subsequently, heptane-1,7-diol (1.9g, 14.97mmol) was added to the reaction mixture and stirred for 16h. After completion, the reaction mixture was diluted with water (150mL) and extracted with DCM (2×100mL). The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure to obtain a crude material. The crude compound was purified by CombiFlash column chromatography, eluted with 10% ethyl acetate / hexane to obtain 17 (1.8g, 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).

[0580] Step 2: 7-((2-butyloctanoyl)oxy)heptanoic acid

[0581] Jones reagent (2.4 mL, 4.77 mmol) was added dropwise to a stirred solution of 7-hydroxyheptyl 2-butyloctanoate (1 g, 3.18 mmol) in acetone (20 mL) under ice cooling. The cooling bath was removed and stirring was continued for 2 h. Upon completion, iPrOH (20 mL) was added and the mixture was stirred by The mixture was filtered off with 4% paraformaldehyde (2- butyl octanoyl) oxy) heptanoic acid (900 mg, crude material) and the mixture was concentrated under reduced pressure. The filtrate was concentrated under reduced pressure, and the crude material was diluted with water (30 mL), and 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 Na2SO4 and filtered. The filtrate was concentrated under reduced pressure to give 7-((2-butyloctanoyl)oxy)heptanoic acid (900 mg, crude material) as a light green liquid, which was used in the next step without further purification. 1H NMR (400MHz, DMSO-d6) δ0.82–0.84(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).

[0582] Step 3: 2-Butyloctanoate 7-(3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl ester

[0583] 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)-octadec-9,12-dienoate. Isolated mass 580 mg, 42.73%. 1 H NMR (400 MHz, 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).

[0584] Step 4: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((7-((2-butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoate (Example 7-11)

[0585] Prepared according to General Procedure L substituting 2-butyloctanoic acid 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl ester for 2-butyloctanoic acid 7-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl ester. Isolated mass 93 mg, 30%. UPLC-MS: For UPLC, Method A was performed. Rt 2.30 min, MS Calcd: 1053.82 [M+H], MS Found 1054.22 [M+H]

[0586] 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) heptanoate

[0587] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-butylheptyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 90 mg, 28% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.19 min, MS calcd: 1035.77 [M+H], MS found 1036.20 [M+H].

[0588] 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) heptanoate

[0589] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl 2-butyloctanoate. Isolated mass 85.88 mg, 27% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.20 min, MS calcd: 1063.81 [M+H], MS found 1065.29 [M+H].

[0590] 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) heptanoate

[0591] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-hexylnonyl)ester (as described in WO 2022 / 159472 A1) for 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl 2-butyloctanoate. Isolated mass 78 mg, 23% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.26 min, MS calcd: 1091.84 [M+H], MS found 1092.34 [M+H].

[0592] Example 7-15: 1-(3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl) 7-(3-pentyloctyl) pimelate

[0593] Step 1: 3-Hydroxy-2-(Hydroxymethyl)propyl 4,4-bis(octyloxy)butyrate

[0594] Pimelate

[0595] Prepared according to general procedure C, substituting 4,4-bis(octyloxy)butanoic acid for (9Z,12Z)-octadec-9,12-dienoic acid. Isolated mass 1.8 g, 43% yield. 1 H NMR (400 MHz, 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).

[0596] Step 2: 1-(3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)

[0597] Prepared according to General Procedure D, replacing 4,4-bis(octyloxy)butyric acid with 7-oxo-7-((3-pentyloctyl)oxy)heptanoic acid (as described in WO 2022 / 159472A1) and replacing (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester with 4,4-bis(octyloxy)butyric acid 3-hydroxy-2-(hydroxymethyl)propyl ester. Isolated mass 327 mg, 37% yield. 1 H NMR (400 MHz, 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).

[0598] 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) pimelate (Example 7-15)

[0599] Prepared according to General Procedure L substituting 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl) ester for 2-butyloctanoic acid 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl ester. Isolated mass 76 mg, 22% yield as a light yellow liquid. UPLC-MS: For UPLC, Method A was performed. Rt 2.32 min, MS calcd: 1067.84 [M+H], MS found 1068.34 [M+H].

[0600] 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

[0601] Prepared according to General Procedure L, substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl adipate (3-hexylnonyl) as described in WO 2022 / 159472 A1 for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 82 mg, 25% yield as a yellow viscous liquid. UPLC-MS: For UPLC, Method A was performed. Rt 2.24 min, MS calcd: 1077.82 [M+H], MS found 1078.36 [M+H].

[0602] Example 7-17: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(((4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoyl)oxy)methyl)propyl (3-hexylnonyl) adipate

[0603] Step 1: 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl adipate (3-hexylnonyl)

[0604] Prepared according to General Procedure D, substituting 6-((3-hexylnonyl)oxy)-6-oxohexanoic acid (as described in WO 2022 / 159472A1) for 4,4-bis(octyloxy)butyric acid, and substituting 3-hydroxy-2-(hydroxymethyl)propyl-4,4-bis(octyloxy)butyrate for 3-hydroxy-2-(hydroxymethyl)propyl-(9Z,12Z)-octadec-9,12-dienoate. Isolated mass 580 mg, 42.73% yield, as a yellow liquid. 1 H NMR (400 MHz, 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).

[0605] 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 (Example 7-17).

[0606] Prepared according to General Procedure L substituting 3-((4,4-bis(octyloxy)butyryl)oxy)-2-(hydroxymethyl)propyl (3-hexylnonyl) adipate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 78 mg, 24% yield as a colorless gum. UPLC-MS: For UPLC, Method A was performed. Rt 2.30 min, MS Calcd: 1081.85 [M+H], MS Found 1082.42 [M+H].

[0607] 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) suberate

[0608] Prepared according to General Procedure L, suberic acid 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) ester 8-(3-butylheptyl) ester (as described in WO 2022 / 159472 A1) replacing (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester. Isolated mass 76 mg, 25% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.18 min, MS calcd: 1049.79 [M+H], MS found 1050.30 [M+H].

[0609] 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) suberate

[0610] Prepared according to General Procedure L suberic acid 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) ester 8-(3-pentyloctyl) ester (as described in WO 2022 / 159472 A1) instead of (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester. Isolated mass 92 mg, 29% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.21 min, MS calcd: 1077.82 [M+H], MS found 1078.36 [M+H].

[0611] Example 7-20: 2-Butyloctanoic acid 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-azaheneicosane-21-yl ester

[0612] Prepared according to General Procedure L substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 117 mg, 44% as yellow gel. UPLC-MS: For UPLC, Method A was performed. Rt 2.06 min, MS calcd: 993.72, MS found: 994.03 [M+H].

[0613] Example 7-21: 2-Butyloctanoic acid 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-azaheneicosane-21-yl ester

[0614] The compound can be prepared according to General Procedure L, replacing 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid and replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadec-9,12-dienoate with 2-butyloctanoate. Yield: 119 mg, 44%, yellow viscous liquid. UPLC-MS: For UPLC, Method A was performed. Rt 2.08 min, MS calculated value: 1007.75, MS experimental value: 1008.24 [M+H]. 1 H NMR(400MHz, CDCl3)δ0.83–0.91(m,9H),0.91–0.97(m,6H),1.25–1.45(m,2 1H),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).

[0615] 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

[0616] Prepared according to General Procedure L, replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl 2-butyloctanoate and replacing 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid (as described in WO 2022 / 159463 A1) with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 87 mg, 33%, light brown gum. UPLC-MS: For UPLC, Method A was performed. Rt 2.11 min, MS calcd: 1021.77, MS found: 1022.27 [M+H].

[0617] 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

[0618] Prepared according to General Procedure L replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl 2-butyloctanoate and replacing 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: For UPLC, Method A was performed. Rt 2.15 min, MS calcd: 1035.78, MS found: 1036.34 [M+H].

[0619] 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) heptanoate

[0620] Step 1: 6-((tert-butyldimethylsilyl)oxy)hex-3-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0621] 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).

[0622] Step 2: 6-Hydroxyhexan-3-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0623] 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)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 900 mg, 93% yield. 1 H NMR(400MHz, CDCl3)δ0.90(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).

[0624] Step 3: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid

[0625] Prepared according to General Procedure J, substituting 6-hydroxyhexan-3-yl (2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 700 mg, crude material. UPLC-MS: Column: XTERRA RP 18 (4.6×50 mm), 5μ, (mobile phase: initially 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF]; then to 2.0% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70:30) ACN:THF], 1.2 min, keep this mobile phase composition for up to 2.50 min; then return to the initial composition, i.e., 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF], 0.47 min, keep this mobile phase composition for up to 12.10 min. Flow rate = 1.2 mL / min. Rt 0.47 min, MS calcd: 273.18, MS found: 273.54 [M+H].

[0626] 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) pimelate (Example 7-24).

[0627] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)hexanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 110 mg, 41% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.11 min, MS calcd: 1007.75, MS found: 1008.13 [M+H].

[0628] 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) heptanoate

[0629] Step 1: 1-((tert-butyldimethylsilyl)oxy)hept-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0630] 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)δ0.03(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,4H),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).

[0631] Step 2: 1-Hydroxyhept-4-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0632] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)hept-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.8 g, 85% yield. 1 H NMR(400MHz, CDCl3)δ0.88(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).

[0633] Step 3: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid

[0634] Prepared according to General Procedure J substituting 1-hydroxyhept-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.3 g, 73% yield.1 H NMR(400MHz, DMSO-d6)δ0.87(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,2 H),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).

[0635] 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) pimelate (Example 7-25).

[0636] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)heptanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 150 mg, 55% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.13 min, MS calculated value: 1021.77, MS observed value: 1022.15 [M+H]

[0637] 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) heptanoate

[0638] Step 1: 1-((tert-butyldimethylsilyl)oxy)octan-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0639] 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. 1 H NMR(400MHz, CDCl3)δ0.04(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).

[0640] Step 2: 1-Hydroxyoctan-4-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0641] 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)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 750 mg, 94% yield. 1 H NMR(400MHz,DMSO-d6)δ0.85(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)

[0642] Step 3: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid

[0643] 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 material. UPLC-MS: Column: XTERRA RP 18 (4.6×50 mm), 5μ, (mobile phase: initially 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF]; then to 2.0% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70:30) ACN:THF], 1.2 min, and keep this mobile phase composition for up to 2.50 min; then return to the initial composition, i.e., 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF], 2.85 min, and keep this mobile phase composition for up to 3.00 min. Flow rate = 1.5 mL / min. Rt 0.34 min, MS calculated value: 301.21, MS experimental value: 301.48 [M+H].

[0644] 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) pimelate (Example 7-26).

[0645] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)octanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 118 mg, 43% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.18 min, MS calcd: 1035.78, MS found: 1036.18 [M+H].

[0646] 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) heptanoate

[0647] Step 1: 1-((tert-butyldimethylsilyl)oxy)nonan-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0648] 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)δ0.05(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).

[0649] Step 2: 1-Hydroxynonan-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0650] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)nonan-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 1.7 g, 78% yield. 1 H NMR(400MHz, CDCl3)δ0.84(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).

[0651] Step 3: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid

[0652] 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: initially 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF]; then to 2.0% [0.1% HCOOH / water] and 98% [0.1% HCOOH / (70:30) ACN:THF], 1.2 min, and keep this mobile phase composition for 2.50 min; then return to the initial composition, i.e., 50% [0.1% HCOOH / water] and 50% [0.1% HCOOH / (70:30) ACN:THF], 2.85 min, and keep this mobile phase composition for 3.10 min. Flow rate = 1.5 mL / min. Rt 0.34 min, MS calculated value: 315.23, MS experimental value: 315.51 [M+H].

[0653] 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) pimelate (Example 7-27).

[0654] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 139 mg, 49% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.19 min, MS calcd: 1049.80, MS found: 1050.24 [M+H].

[0655] Example 7-28: 2-Butyloctanoic acid 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-azaheneicosane-21-yl ester

[0656] Step 1: 5-((tert-butyldimethylsilyl)oxy)pentan-2-yl (2-(pyrrolidin-1-yl)ethyl)carbamate

[0657] Prepared according to General Procedure G, substituting 5-((tert-butyldimethylsilyl)oxy)pentan-2-ol for 1-((tert-butyldimethylsilyl)oxy)decan-4-ol. Isolated mass 1.6 g, 32% yield as a yellow liquid. 1 H NMR(400MHz, CDCl3)δ0.03(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).

[0658] Step 2: 5-Hydroxypentan-2-yl (2-(Pyrrolidin-1-yl)ethyl)carbamate

[0659] 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)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 900 mg, 83% yield as a light yellow liquid. 1 H NMR(400MHz, CDCl3)δ1.22(d,3H),1.53–1.67(m,4H),1.70–1.87(m,4H),2.24–2.36(m,1H) ,2.49–2.71(m,6H),3.20–3.39(m,2H),3.65(d,2H),4.81–4.85(m,1H),5.30–5.39(m,1H).

[0660] Step 3: 4-(((2-(Pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid

[0661] Prepared according to General Procedure J, substituting 5-hydroxypentan-2-yl (2-(pyrrolidin-1-yl)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 120 mg, which was used in the next step without further purification. UPLC-MS: For UPLC, Method C was performed. Rt 0.32 min, MS Calcd: 259.16 [M+H], MS Found: 259.47 [M+H].

[0662] Step 4: 2-Butyloctanoic acid 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-azaheneicosane-21-yl ester (Example 7-28).

[0663] 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 for 2-butyloctanoate, and substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 102.41 mg, 31%, yellow gel. UPLC-MS: For UPLC, Method A was performed. Rt 2.02 min, MS calcd: 979.7 [M+H], MS found: 980.24 [M+H]. 1 H NMR(400MHz, CDCl3)δ0.85–0.93(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).

[0664] 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) heptanoate

[0665] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(3-pentyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and substituting 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)pentanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 78.36 mg, 30% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.09 min, MS calcd: 993.7 [M+H], MS found: 994.13 [M+H].

[0666] 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) heptanoate

[0667] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(2-butyloctyl)ester (as described in WO 2022 / 159472 A1) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 167 mg, 59% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 2.19 min, MS calcd: 1049.80, MS found: 1050.28 [M+H].

[0668] 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)heptanedioate 7-(pentadeca-1,14-dien-8-yl)

[0669] Step 1: 7-oxo-7-(pentadeca-1,14-dien-8-yloxy)heptanoic acid

[0670] The compound can be prepared according to General Procedure C, substituting heptanediol for (9Z,12Z)-octadeca-9,12-dienoic acid and pentadeca-1,14-dien-8-ol for 2-(hydroxymethyl)propane-1,3-diol).

[0671] Step 2: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)heptanedioate 7-(pentadeca-1,14-dien-8-yl)

[0672] 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-4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-hydroxy-2-(hydroxymethyl)propyl-(9Z,12Z)-octadec-9,12-dienoate. Isolated mass 750 mg, 59% yield as a colorless liquid. 1 H NMR(400MHz, CDCl3)δ0.94(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).

[0673] 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) heptanoate (Example 7-33).

[0674] Prepared according to General Procedure L replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate with 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(pentadeca-1,14-dien-8-yl)ester and replacing 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (as described in WO 2022 / 159463A1) with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 108 mg, 31.25% yield as a light brown viscous gum. UPLC-MS: For UPLC, method B2 was performed. Rt 1.10 min, MS calcd: 1073.79 [M+H], MS found 1074.37 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.83–0.95(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).

[0675] 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) heptanoate

[0676] Prepared according to General Procedure L substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) heptane 7-(pentadeca-1,14-dien-8-yl) ester for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 155 mg, 43.35% yield as a light yellow gum. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.90 min, MS calcd: 1087.81 [M+H], MS found 1088.36 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.78–0.86(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),3.11–3.1 9(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).

[0677] 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) heptanoate

[0678] Step 1: 7-Oxo-7-(pentadecan-8-yloxy)heptanoic acid

[0679] Prepared according to general procedure C using heptanediol for (9Z,12Z)-octadeca-9,12-dienoic acid and replacing 2-(hydroxymethyl)propane-1,3-diol with pentadecane-8-ol. Isolated mass 1.9 g, 58% yield as a colorless liquid. 1 H NMR (400MHz, DMSO-d6) δ0.85(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).

[0680] Step 2: 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl) 7-(pentadecan-8-yl) pimelate

[0681] 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-4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-hydroxy-2-(hydroxymethyl)propyl-(9Z,12Z)-octadec-9,12-dienoate. Isolated mass 1.3 g, 41% yield as a colorless liquid. 1 H NMR(400MHz, CDCl3)δ0.86(t,6H),0.94(t,6H),1.16–1.28(m,20H),1.35–1.43(m,6 H),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).

[0682] 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) heptanoate (Example 7-35)

[0683] Prepared according to General Procedure L, substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(pentadecan-8-yl)ester for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (as described in WO 2022 / 159463 A1) for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 110 mg, 32% yield as a light yellow liquid. UPLC-MS: For UPLC, Method B2 was performed. Rt 1.16 min, MS calcd: 1077.82 [M+H], MS observed 1078.38 [M+H]. 1 HNMR(400MHz,DMSO-d6)δ0.84(t,9H),0.90(t,6H),1.19–1.26(m,32H),1.31–1.3 8(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),4.4 4(t,1H),4.59–4.65(m,1H),4.74–4.80(m,1H),5.26–5.37(m,4H),6.97(bs,1H).

[0684] 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) heptanoate

[0685] Prepared according to General Procedure L substituting 1-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl)pimelate 7-(pentadecan-8-yl) for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 142 mg, 41%) as a light yellow liquid. UPLC-MS: For UPLC, Method B2 was performed. Rt 7.12 min, MS Calcd: 1091.84 [M+H], MS Found 1092.32 [M+H]. HPLC: 1 H NMR(400MHz,DMSO-d6)δ0.84(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).

[0686] 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

[0687] Step 1: 2-(Hydroxymethyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate

[0688] 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-4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate for 3-hydroxy-2-(hydroxymethyl)propyl-(9Z,12Z)-octadec-9,12-dienoate. Isolated mass 2.05 g, 58% yield as a colorless gum. 1HNMR(400MHz, CDCl3)δ0.94(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).

[0689] 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 (Example 7-37)

[0690] Prepared according to General Procedure L, replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate with 2-(hydroxymethyl)propane-1,3-diyl bis(4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoate and replacing 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)nonanoic acid (as described in WO 2022 / 159463 A1) with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 128 mg, 25.47% yield. UPLC-MS: For UPLC, Method A was performed. Rt 5.46 min, MS calcd: 1047.77 [M+H], MS observed 1049.29 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.81–0.85(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).

[0691] 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

[0692] Prepared according to General Procedure L substituting 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)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadec-9,12-dienoate. Isolated mass 157 mg, 44.4% yield. UPLC-MS: For UPLC, Method A was performed. Rt 2.28 min, MS calcd: 1061.79 [M+H], MS found 1062.29 [M+H]. 1 H NMR(400MHz, DMSO-d6)δ0.82–0.86(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,7 H),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).

[0693] 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 adipate (2-(hept-6-en-1-yl)non-8-en-1-yl)

[0694] Step 1. Dimethyl 2,2-di(hept-6-en-1-yl)malonate

[0695] Under nitrogen atmosphere, under ice cooling conditions, NaH (1.64 g, 68.16 mmol) was added to a stirred solution of dimethyl malonate (3 g, 22.72 mmol) in THF (90 mL). After 30 minutes, 7-bromohept-1-ene (12 g, 68.16 mmol) was added, and the mixture was refluxed for 16 hours. The reaction mixture was cooled to 25 ° C, quenched with 1N HCl and extracted with Et2O (2 × 250 mL). The combined organic layer was washed with water (100 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure, then purified by CombiFlash (2-10% EtOAc / hexane) to obtain the product 2,2-di (hept-6-ene-1-yl) dimethyl malonate (5.5 g, 75% yield) as a colorless liquid. 1 H NMR (400MHz, CDCl3) δ1.08–1.18(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).

[0696] Step 2. 2,2-Di(hept-6-en-1-yl)malonic acid

[0697] Under nitrogen atmosphere, under ice cooling conditions, KOH (8.63 g, 49.04 mmol) was added to a stirred solution of 2,2-di(hept-6-ene-1-yl)malonate (5.3 g, 16.35 mmol) in EtOH:HO (50 mL). Subsequently, the reaction mixture was refluxed for 16 hours. The reaction mixture was cooled to 25 ° C, quenched with 1N HCl, and the product was extracted with EtOAc (2 × 250 mL), dried over anhydrous NaSO4 and concentrated under reduced pressure to give 2,2-di(hept-6-ene-1-yl)malonic acid (4.2 g, 87% yield) as a light yellow oil, which was used directly in the next step. 1 H NMR (400MHz, CDCl3) δ1.23–1.41(m,12H), 1.90–2.06(m,8H), 4.89–5.02(m,4H), 5.69–5.84(m,2H).

[0698] Step 3. 2-(Hept-6-en-1-yl)non-8-enoic acid

[0699] 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 / hexanes) to give 2-(hept-6-en-1-yl)non-8-enoic acid (3.2 g, 94% yield). 1 H NMR (400MHz, CDCl3) δ1.24–1.38(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).

[0700] Step 4. 2-(Hept-6-en-1-yl)non-8-en-1-ol

[0701] LAH (2.4M solution in THF) (17.33 mL, 41.6 mmol) was added to an ice-cooled solution of 2-(hept-6-ene-1-yl)non-8-enoic acid (3, 11.89 mmol) in THF (50 mL). The reaction mixture was stirred at 70 ° C for 3 hours, then cooled to 0 ° C and quenched by adding Na2SO4-5H2O. The reaction mixture was filtered with EtOAc (200 mL). The filtrate was concentrated under reduced pressure and then purified by CombiFlash (20-40% EtOAc / hexanes) to give 2-(hept-6-ene-1-yl)non-8-ene-1-ol (2.4 g, 84.7% yield) as a colorless liquid. 1 H NMR (400MHz, CDCl3) δ1.23–1.49(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).

[0702] Step 5. 6-((2-(hept-6-en-1-yl)non-8-en-1-yl)oxy)-6-oxohexanoic acid

[0703] 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), DMAP (0.25 g, 2.015 mmol) in sequence at 25°C. The reaction mixture was stirred for 10 minutes, followed by the addition of 2-(hept-6-ene-1-yl)non-8-ene-1-ol (1.6, 6.72 mmol). The reaction mixture was stirred at 25°C for 16 hours, monitored by TLC (30% EtOAc / hexanes). Upon 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 and dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure, then purified by CombiFlash (30-50% EtOAc / hexanes) to give (1.6 g, 65% yield) as a colorless liquid. 1 H NMR(400MHz,DMSO-d6)δ1.15–1.40(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).

[0704] Step 6. 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl adipate (2-(hept-6-en-1-yl)non-8-en-1-yl)

[0705] 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 substituting (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester for (9Z,12Z)-octadec-9,12-dienoic acid 3-hydroxy-2-(hydroxymethyl)propyl ester. Isolated mass 1.1 g, 55% yield. 1HNMR(400MHz, chloroform-d)δ0.94(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).

[0706] 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 adipate (2-(hept-6-en-1-yl)non-8-en-1-yl)ester (Example 7-39)

[0707] Prepared according to General Procedure L substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl adipate (2-(hept-6-en-1-yl)non-8-en-1-yl)ester for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butyryl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate. Isolated mass 160 mg, 46% yield as a light brown liquid. UPLC-MS: For UPLC, Method A was performed. Rt 6.83 min, MS calcd: 1087.81 [M+H], MS found 1088.45 [M+H]. 1 H NMR(400MHz, DMSO-d6)δ0.82–0.94(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).

[0708] 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

[0709] Step 1. 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(dimethylamino)ethyl)carbamate

[0710] Prepared according to general procedure G with N 1 ,N 1 -Dimethylethane-1,2-diamine for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 750 mg, 84% yield. 1 H NMR(400MHz, CDCl3)δ0.03(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).

[0711] Step 2. 1-Hydroxydec-4-yl (2-(Dimethylamino)ethyl)carbamate

[0712] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl(2-(dimethylamino)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 420 mg, 78% yield. 1 H NMR(400MHz, CDCl3)δ0.86(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).

[0713] Step 3. 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoic acid

[0714] Prepared according to General Procedure J substituting 1-hydroxydec-4-yl (2-(dimethylamino)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 410 mg, 93% yield. 1H NMR(400MHz,DMSO-d6)δ0.86(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).

[0715] 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)

[0716] Prepared according to General Procedure L replacing 4-(((2-(dimethylamino)ethyl)carbamoyl)oxy)decanoic acid with 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid and replacing 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl(9Z,12Z)-octadec-9,12-dienoate with 2-butyloctanoate. Isolated mass 122 mg, 30% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.88 min, MS calcd: 1023.77 [M+H], MS found 1024.32 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.80–0.87(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).

[0717] 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

[0718] Step 1. 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(diethylamino)ethyl)carbamate

[0719] Prepared according to general procedure G with N 1 ,N 1 -Diethylethane-1,2-diamine for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 2.1 g, 70% yield. UPLC-MS: For UPLC, method B2 was performed. Rt 0.58 min, MS calculated: 431.36 [M+H], MS found 431.56 [M+H].

[0720] Step 2. 1-Hydroxydec-4-yl (2-(diethylamino)ethyl)carbamate

[0721] Prepared according to General Procedure H substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(diethylamino)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 950 mg, 66% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.34 min, MS calcd: 317.27 [M+H], MS found 317.33 [M+H].

[0722] Step 3. 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoic acid

[0723] Prepared according to General Procedure J, substituting 1-hydroxydec-4-yl (2-(diethylamino)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 700 mg, 83% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.36 min, MS calcd: 331.25 [M+H], MS found 331.47 [M+H].

[0724] 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)

[0725] Prepared according to General Procedure L substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-(((2-(diethylamino)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 178 mg, 56% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.86 min, MS calcd: 1051.81 [M+H], MS found 1052.32 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.80–0.87(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).

[0726] 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

[0727] Step 1. 1-((tert-butyldimethylsilyl)oxy)dec-4-yl(3-(diethylamino)propyl)carbamate

[0728] Prepared according to general procedure G with N 1 ,N 1 -Diethylpropane-1,3-diamine for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 850 mg, 87% yield. 1H NMR(400MHz, CDCl3)δ0.03(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).

[0729] Step 2. 1-Hydroxydec-4-yl (3-(Diethylamino)propyl)carbamate

[0730] Prepared according to General Procedure H substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (3-(diethylamino)propyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 520 mg, 82% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.35 min, MS calcd: 331.29 [M+H], MS found 331.42 [M+H].

[0731] Step 3. 4-(((3-(Diethylamino)propyl)carbamoyl)oxy)decanoic acid

[0732] Prepared according to General Procedure J, substituting 1-hydroxydec-4-yl (3-(diethylamino)propyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 489 mg, 90% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.34 min, MS calcd: 345.27 [M+H], MS found 345.56 [M+H].

[0733] 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)

[0734] Prepared according to General Procedure L, substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-(((3-(diethylamino)propyl)carbamoyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 125 mg, 34% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.88 min, MS calcd: 1065.82 [M+H], MS found 1066.31 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.73–1.03(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).

[0735] 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

[0736] Step 1. 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(ethyl(methyl)amino)ethyl)carbamate

[0737] Prepared according to general procedure G with N 1 -ethyl-N 1 -Methylethane-1,2-diamine for 2-(pyrrolidin-1-yl)ethan-1-amine. Isolated mass 580 mg, 63% yield. UPLC-MS: For UPLC, method B2 was performed. Rt 0.6 min, MS calculated: 417.34 [M+H], MS found 417.58 [M+H].

[0738] Step 2. 1-Hydroxydec-4-yl (2-(ethyl(methyl)amino)ethyl)carbamate

[0739] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(ethyl(methyl)amino)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 320 mg, 76% yield. 1 H NMR (400MHz, CDCl3) δ0.84–0.89(m,3H),1.22–1.29(m,8H),1.49–1.65(m,1 3H),2.77–2.85(m,2H),3.48(s,3H),3.65–3.72(m,2H),4.73–4.79(m,1H).

[0740] Step 3. 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoic acid

[0741] Prepared according to General Procedure J substituting 1-hydroxydec-4-yl (2-(ethyl(methyl)amino)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 290 mg, 87% yield. 1 H NMR(400MHz, DMSO-d6)δ0.83–0.88(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).

[0742] 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)

[0743] Prepared according to General Procedure L, substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-(((2-(ethyl(methyl)amino)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 160 mg, 41% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.92 min, MS calcd: 1037.79 [M+H], MS found 1038.28 [M+H]. 1 H NMR(400MHz,DMSO-d6)δ0.79–0.85(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 ),2.27–2.33(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).

[0744] 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

[0745] Step 1. 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(piperidin-1-yl)ethyl)carbamate

[0746] 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, 62% yield. 1H NMR(400MHz, CDCl3)δ0.03(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).

[0747] Step 2. 1-Hydroxydec-4-yl (2-(piperidin-1-yl)ethyl)carbamate

[0748] Prepared according to General Procedure H, substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl(2-(piperidin-1-yl)ethyl)carbamate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 330 mg, 71% yield. 1 H NMR(400MHz,DMSO-d6)δ0.83–0.88(m,3H),1.22–1.28(m,8H),1.38–1.52(m,7H),1.66–1.78(m,4H),2.0 8(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).

[0749] Step 3. 4-(((2-(Piperidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid

[0750] Prepared according to General Procedure J substituting 1-hydroxydec-4-yl (2-(piperidin-1-yl)ethyl)carbamate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 290 mg, 88%. 1 H NMR(400MHz, DMSO-d6)δ0.83–0.88(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).

[0751] 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)

[0752] Prepared according to General Procedure L substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propoxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-(((2-(piperidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 165 mg, 44% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.89 min, MS calcd: 1063.81 [M+H], MS found 1064.32 [M+H]. 1 H 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).

[0753] 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

[0754] Step 1. 1-((tert-Butyldimethylsilyl)oxy)dec-4-yl (3-(diethylamino)propyl) carbonate

[0755] At 0 ° C, NaH (2eq) was added to a solution of 3- (diethylamino) propan-1-ol (2eq) in THF (20mL), followed by stirring for 30 minutes. Carbonic acid 1- ((tert-butyldimethylsilyl) oxy) decane-4-yl ester (4-nitrophenyl) ester (1eq) was added to this suspension. The reaction mixture was stirred at 25 ° C for 16 hours. The reaction mixture was diluted with DCM (250mL) and water (50mL) and saline (50mL). 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 to obtain carbonate 1- ((tert-butyldimethylsilyl) oxy) decane-4-yl ester (3- (diethylamino) propyl) ester (820mg, 83% yield). 1 H NMR(400MHz, CDCl3)δ0.03(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).

[0756] Step 2. 3-(Diethylamino)propyl (1-hydroxydec-4-yl) carbonate

[0757] Prepared according to General Procedure H substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (3-(diethylamino)propyl) carbonate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 510 mg, 84% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.4 min, MS calcd: 332.27 [M+H], MS found 332.55 [M+H].

[0758] Step 3. 4-(((3-(Diethylamino)propoxy)carbonyl)oxy)decanoic acid

[0759] Prepared according to General Procedure J substituting 3-(diethylamino)propyl(1-hydroxydec-4-yl)carbonate for 1-hydroxydec-4-yl(2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 480 mg. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.42 min, MS Calcd: 346.25 [M+H], MS Found 346.52 [M+H].

[0760] 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

[0761] 1-((tert-Butyldimethylsilyl)oxy)dec-4-yl (4-nitrophenyl) carbonate (Example 7-45)

[0762] 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 for 2-butyloctanoate, and substituting 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate for 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl (9Z,12Z)-octadec-9,12-dienoate and 4-(((3-(diethylamino)propoxy)carbonyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 125 mg, 40% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.85 min, MS calcd: 1066.81 [M+H], MS found 1067.25 [M+H]. 1 H NMR(400MHz, CDCl3)δ0.84–0.89(m,6H),0.94(t,6H),1.12–1.29(m,15H),1.31–1.4 5(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,4 H),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,6H),4.18–4.22(m,2H),4.45–4.50(m,1H),4.68–4.74(m,1H),5.22–5.39(m,4H).

[0763] 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

[0764] Step 1. 1-((tert-Butyldimethylsilyl)oxy)dec-4-yl ((1-ethylpiperidin-3-yl)methyl) carbonate

[0765] At 0 ° C, NaH (2 eq) was added to a solution of (1-ethylpiperidin-3-yl)methanol (2 eq) in THF (20 mL), followed by stirring for 30 minutes. Carbonic acid 1-((tert-butyldimethylsilyl)oxy)dec-4-yl ester (4-nitrophenyl) ester (1 eq) was added to this suspension. The reaction mixture was stirred at 25 ° C for 16 hours. The reaction mixture was diluted with DCM (250 mL) and 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. Separation mass 880 mg, 87% yield. UPLC-MS: For UPLC, method B2 was performed. Rt 0.64 min, MS calculated value: 458.36 [M+H], MS experimental value 458.36 [M+H].

[0766] Step 2. (1-Ethylpiperidin-3-yl)methyl (1-hydroxydec-4-yl) carbonate

[0767] Prepared according to General Procedure H substituting 1-((tert-butyldimethylsilyl)oxy)dec-4-yl ((1-ethylpiperidin-3-yl)methyl)carbonate for 1-((tert-butyldimethylsilyl)oxy)dec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 450 mg, 68% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.36 min, MS calcd: 344.27 [M+H], MS found 344.51 [M+H].

[0768] Step 3. 4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)decanoic acid

[0769] Prepared according to General Procedure J, substituting (1-ethylpiperidin-3-yl)methyl (1-hydroxydec-4-yl) carbonate for 1-hydroxydec-4-yl (2-(pyrrolidin-1-yl)ethyl)carbamate. Isolated mass 410 mg, crude material, used directly in the next reaction. UPLC-MS: For UPLC, Method B2 was performed. Rt 0.33 min, MS calcd: 358.25 [M+H], MS found 358.44 [M+H].

[0770] 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-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)decanoate (Example 7-46)

[0771] Prepared according to General Procedure L, substituting 2-butyloctanoate, 7-(3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyloxy)-7-oxoheptyl for (9Z,12Z)-octadec-9,12-dienoate, 3-((4,4-bis(((Z)-oct-5-en-1-yl)oxy)butanoyl)oxy)-2-(hydroxymethyl)propyl ester and 4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)decanoic acid for 4-(((2-(pyrrolidin-1-yl)ethyl)carbamoyl)oxy)decanoic acid. Isolated mass 150 mg, 33% yield. UPLC-MS: For UPLC, Method B2 was performed. Rt 6.88 min, MS calculated value: 1078.81 [M+H], MS experimental value 1079.43 s [M+H]. 1 H NMR(400MHz, CDCl3)δ0.84–0.88(m,6H),0.94(t,6H),1.02–1.31(m,36H), 1.33–1.43(m,...

Claims

1. A compound of formula I: or a pharmaceutically acceptable salt thereof, wherein: Each L 1 and L 1 ' is independently -C(O)- or -OC(O)-; Each L 2 and L 2 ' is independently an optionally substituted divalent saturated or unsaturated linear or branched C 1-12 hydrocarbon chain; L 3 is a covalent bond, -O-, -C(O)O-, -OC(O)- or -OC(O)O-; R 1 is optionally substituted C 1-20 Fatty Acids, L CyA is a covalent bond or an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 hydrocarbon chain; Cy A is an optionally substituted ring selected from a 3- to 7-membered saturated or partially unsaturated carbocyclic group, a 1-adamantyl group, a 2-adamantyl group, a steryl group and a phenyl group; L Ra is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 hydrocarbon chain; Every R a and R 1 ' is independently optionally substituted C 1-20 Fat; Y 1 is -C(O)- or -C(O)O-; Y 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 hydrocarbon chain; Y 3 is optionally substituted C 1-20 Fat; X 1 is a covalent bond, -O- or -NR-; X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-12 A hydrocarbon chain in which 1-3 methylene units are optionally and independently replaced by -O- or -NR-; X 3 is hydrogen or an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl; a phenyl group; a 3- to 7-membered heterocyclyl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; or a 5- to 6-membered heteroaryl group having 1 to 3 heteroatoms independently selected from nitrogen, oxygen and sulfur; and Each R is independently hydrogen or optionally substituted C 1-6 Fatty.

2. The compound according to claim 1, wherein the compound has formula II: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, wherein the compound has formula III: or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 or 3, wherein the compound has formula III-A: or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1 or 3, wherein the compound has formula III-B: or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1, 3 and 5, wherein the compound has the formula III-Bi: or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1, wherein the compound has formula IV: or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 1 or 7, wherein the compound has formula IV-A: or a pharmaceutically acceptable salt thereof.

9. The compound according to claim 1 or 7, wherein the compound has formula IV-B: or a pharmaceutically acceptable salt thereof.

10. The compound according to any one of claims 1, 7 and 9, wherein the compound has the formula IV-Bi: or a pharmaceutically acceptable salt thereof.

11. The compound of claim 1, wherein the compound has Formula V: or a pharmaceutically acceptable salt thereof.

12. The compound according to claim 1 or 11, wherein the compound has the formula VA: or a pharmaceutically acceptable salt thereof.

13. The compound according to claim 1 or 11, wherein the compound has the formula VB: or a pharmaceutically acceptable salt thereof.

14. The compound according to any one of claims 1, 11 and 13, wherein the compound has the formula VBi: or a pharmaceutically acceptable salt thereof.

15. The compound of claim 1, wherein the compound has Formula VI: or a pharmaceutically acceptable salt thereof.

16. The compound according to claim 1 or 15, wherein the compound has formula VI-A: or a pharmaceutically acceptable salt thereof.

17. The compound according to claim 1 or 15, wherein the compound has formula VI-B: or a pharmaceutically acceptable salt thereof.

18. The compound according to any one of claims 1, 15 and 17, wherein the compound has the formula VI-Bi: or a pharmaceutically acceptable salt thereof.

19. The compound of any one of claims 1, 2, 3 and 11, wherein the compound has Formula VII: or a pharmaceutically acceptable salt thereof.

20. The compound of any one of claims 1, 2, 3, 11 and 19, wherein the compound has Formula VII-A: or a pharmaceutically acceptable salt thereof.

21. The compound of any one of claims 1, 2, 3, 5, 11 and 19, wherein the compound has Formula VII-B: or a pharmaceutically acceptable salt thereof.

22. The compound of any one of claims 1, 2, 3, 5, 11, 19 and 21, wherein the compound has the formula VII-Bi: or a pharmaceutically acceptable salt thereof.

23. The compound of any one of claims 1, 2, 7 and 15, wherein the compound has Formula VIII: or a pharmaceutically acceptable salt thereof.

24. The compound of any one of claims 1, 2, 7, 15 and 23, wherein the compound has Formula VIII-A: or a pharmaceutically acceptable salt thereof.

25. The compound of any one of claims 1, 2, 7, 15 and 23, wherein the compound has Formula VIII-B: or a pharmaceutically acceptable salt thereof.

26. The compound of any one of claims 1, 2, 7, 15, 23, and 25, wherein the compound has the formula VIII-Bi: or a pharmaceutically acceptable salt thereof.

27. The compound of any one of claims 1, 2, 3, 11 and 19, wherein the compound has Formula IX: or a pharmaceutically acceptable salt thereof.

28. The compound of any one of claims 1, 2, 3, 11, 19 and 27, wherein the compound has Formula IX-A: or a pharmaceutically acceptable salt thereof.

29. The compound of any one of claims 1, 2, 3, 11, 19 and 27, wherein the compound has Formula IX-B: or a pharmaceutically acceptable salt thereof.

30. The compound of any one of claims 1, 2, 3, 11, 19, 27 and 29, wherein the compound has the formula IX-Bi: or a pharmaceutically acceptable salt thereof.

31. The compound of any one of claims 1, 2, 7, 15 and 23, wherein the compound has formula X: or a pharmaceutically acceptable salt thereof.

32. The compound of any one of claims 1, 2, 7, 15, 23, and 31, wherein the compound has the formula XA: or a pharmaceutically acceptable salt thereof.

33. The compound of any one of claims 1, 2, 7, 15, 23 and 31, wherein the compound has the formula XB: or a pharmaceutically acceptable salt thereof.

34. The compound of any one of claims 1, 2, 7, 15, 23, 31 and 33, wherein the compound has the formula XBi: or a pharmaceutically acceptable salt thereof.

35. A compound according to any one of the preceding claims, wherein L 1 It is -C(O)-.

36. A compound according to any one of the preceding claims, wherein L 1 ' is -C(O)-.

37. A compound according to any one of claims 1 to 36, wherein L 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 Hydrocarbon chain.

38. A compound according to any one of claims 1 to 37, wherein L 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 Hydrocarbon chain.

39. The compound according to any one of claims 1 to 38, wherein L 2 A divalent saturated straight or branched chain C 4-8 Hydrocarbon chain.

40. The compound according to any one of claims 1 to 39, wherein L 2 It is -(CH2)6- or -(CH2)7-.

41. A compound according to any one of claims 1 to 39, wherein L 2 It is -(CH2)4- or -(CH2)5-.

42. The compound according to any one of claims 1 to 41, wherein L 2 ' is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 Hydrocarbon chain.

43. A compound according to any one of claims 1 to 42, wherein L 2 ' is an optionally substituted divalent saturated straight or branched chain C 1-6 Hydrocarbon chain.

44. The compound according to any one of claims 1 to 43, wherein L 2 ' is a divalent saturated straight or branched chain C 1-6 Hydrocarbon chain.

45. The compound according to any one of claims 1 to 44, wherein L 2 ' is -(CH2)2-.

46. ​​A compound according to any one of the preceding claims, wherein L 3 It is a covalent bond.

47. A compound according to any one of claims 1 to 45, wherein L 3 It is -C(O)O-.

48. A compound according to any one of claims 1 to 45, wherein L 3 It is -OC(O)-.

49. The compound according to any one of claims 1 to 48, wherein R 1 is optionally substituted C 1-20 Fatty.

50. The compound according to any one of claims 1 to 49, wherein R 1 is optionally substituted C 6-12 Fatty.

51. A compound according to any one of claims 1 to 49, wherein R 1 is optionally substituted C 12-20 Fatty.

52. The compound according to any one of claims 1 to 49, wherein R 1 for 53. A compound according to any one of the preceding claims, wherein -L 2 -L 3 -R 1 for 54. The compound according to any one of claims 1 to 53, wherein each R 1 ' is independently optionally substituted C 6-12 Fatty.

55. The compound according to any one of claims 1 to 54, wherein each R 1 ' is independently optionally substituted C 7-9 Fatty.

56. The compound according to any one of claims 1 to 55, wherein each R 1 'Independently 57. A compound according to any one of the preceding claims, wherein Y 1 It is -C(O)-.

58. A compound according to any one of claims 1 to 56, wherein Y 1 It is -C(O)O-.

59. The compound according to any one of claims 1 to 58, wherein Y 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-3 Hydrocarbon chain.

60. The compound according to any one of claims 1 to 59, wherein Y 2 is an optionally substituted divalent saturated straight or branched chain C 1-3 Hydrocarbon chain.

61. The compound according to any one of claims 1 to 60, wherein Y 2 A divalent saturated straight or branched chain C 1-3 Hydrocarbon chain.

62. The compound according to any one of claims 1 to 61, wherein Y 2 It is -CH2- or -(CH2)2-.

63. The compound according to any one of claims 1 to 62, wherein Y 3 is optionally substituted C 1-12 Fatty.

64. The compound according to any one of claims 1 to 63, wherein Y 3 is optionally substituted C 4-8 Fatty.

65. The compound according to any one of claims 1 to 64, wherein Y 3 It is -(CH2)5CH3.

66. The compound according to any one of claims 1 to 64, wherein Y 3 It is -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, -(CH2)4CH3, -(CH2)5CH3, -(CH2)6CH3 or -(CH2)7CH3.

67. A compound according to any one of the preceding claims, wherein X 1 It is a covalent bond.

68. The compound according to any one of claims 1 to 66, wherein X 1 For -O-.

69. The compound according to any one of claims 1 to 66, wherein X 1 It is -NR-.

70. The compound according to any one of claims 1 to 69, wherein X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 A hydrocarbon chain in which 1-2 methylene units are optionally and independently replaced by -O- or -NR-.

71. The compound according to any one of claims 1 to 70, wherein X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 1-6 Hydrocarbon chain.

72. The compound according to any one of claims 1 to 71, wherein X 2 is an optionally substituted divalent saturated straight or branched chain C 1-6 Hydrocarbon chain.

73. The compound according to any one of claims 1 to 72, wherein X 2 A divalent saturated straight or branched chain C 1-6 Hydrocarbon chain.

74. The compound according to any one of claims 1 to 69, wherein X 2 is an optionally substituted divalent saturated or unsaturated straight or branched chain C 4-8 A hydrocarbon chain in which 1-2 methylene units are independently replaced by -O- or -NR-.

75. The compound according to any one of claims 1 to 70 and 74, wherein X 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 A hydrocarbon chain in which 1-2 methylene units are independently replaced by -O- or -NR-.

76. The compound according to any one of claims 1 to 69, 74 and 75, wherein X 2 is an optionally substituted divalent saturated straight or branched chain C 4-8 A hydrocarbon chain in which one methylene unit is replaced by -NR-.

77. The compound according to any one of claims 1 to 76, wherein X 3 For hydrogen.

78. The compound according to any one of claims 1 to 76, wherein X 3 is an optionally substituted ring selected from: a 3- to 7-membered saturated or partially unsaturated carbocyclyl; a phenyl group; a 3- to 7-membered heterocyclyl group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur; or a 5- to 6-membered heteroaryl group having 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur.

79. The compound according to any one of claims 1 to 76 and 78, wherein X 3 is an optionally substituted 5- to 6-membered heterocyclic group having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur.

80. The compound according to any one of claims 1 to 76, 78, 79, wherein X 3 is optionally substituted pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl.

81. A compound according to any one of the preceding claims, wherein -X 2 -X 3 for 82. The compound of any one of claims 1 to 81, wherein each R is hydrogen.

83. The compound of any one of claims 1 to 81, wherein each R is independently an optionally substituted C 1-6 Fatty.

84. The compound of any one of claims 1 to 81, wherein each R is -CH3.

85. The compound according to any one of claims 1 to 84, wherein X 2 and X 3 At least one of the above comprises at least one ionizable nitrogen atom.

86. The compound of any one of claims 1-85, wherein the compound comprises an ionizable nitrogen atom, and wherein the pKa of the conjugate acid of the compound is between about 4 and about 12.

87. The compound of claim 1, wherein the compound is selected from Table 1 or a pharmaceutically acceptable salt thereof.

88. A lipid nanoparticle (LNP) formulation comprising the ionizable lipid according to any one of claims 1 to 87.

89. A lipid nanoparticle (LNP) formulation comprising: The ionizable lipid according to any one of claims 1 to 87; Phospholipids; Cholesterol; and Conjugate linker lipid (eg, polyethylene glycol lipid).

90. The LNP formulation of claim 88 or 89, further comprising a therapeutic and / or prophylactic agent.

91. The LNP formulation of any one of claims 88 to 90, wherein the therapeutic and / or prophylactic agent is or comprises one or more nucleic acids.

92. The LNP formulation of any one of claims 88 to 91, wherein the one or more nucleic acids is or comprises RNA.

93. The LNP formulation of any one of claims 88 to 91, wherein the one or more nucleic acids is or comprises DNA.

94. The LNP formulation of any one of claims 88 to 91, wherein the LNP formulation is formulated to deliver the therapeutic and / or prophylactic agent to a target cell.

95. The LNP formulation of any one of claims 88 to 94, wherein the target cell is or comprises a spleen cell (e.g., a spleen B cell, a spleen T cell, a spleen monocyte), a liver cell (e.g., a hepatocyte), a bone marrow cell (e.g., a bone marrow mononuclear cell), an immune cell, a muscle cell (e.g., a myocyte), a heart cell (e.g., a cardiomyocyte), a kidney cell, or a cell in the central nervous system.

96. The LNP formulation of any one of claims 88 to 94, wherein the target cell is or comprises a hematopoietic stem cell.

97. The LNP formulation of any one of claims 88 to 95, wherein the target cell is or comprises a liver cell.

98. A pharmaceutical composition comprising the LNP formulation according to any one of claims 88 to 97 and a pharmaceutically acceptable excipient.

99. A method for administering a therapeutic and / or prophylactic agent to a subject in need thereof, the method comprising administering to the subject the LNP formulation of any one of claims 88 to 97.

100. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the LNP formulation of any one of claims 88 to 97, wherein the therapeutic and / or prophylactic agent is effective to treat the disease.

101. A method for delaying and / or preventing progression of a disease or condition in a subject in need thereof, the method comprising administering to the subject the LNP formulation of any one of claims 88 to 97, wherein the therapeutic and / or prophylactic agent is effective to treat the disease.

102. A method of delivering a therapeutic and / or prophylactic agent to mammalian cells derived from a subject, the method comprising contacting cells of the subject to which the LNP formulation of any one of claims 88 to 97 has been administered.

103. A method of producing a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with an LNP formulation according to any one of claims 88 to 97, wherein the therapeutic and / or prophylactic agent is or comprises mRNA, and wherein the mRNA encodes the polypeptide of interest, whereby the mRNA can be translated in the cell to produce the polypeptide of interest.

104. A method of inhibiting the production of a polypeptide of interest in a mammalian cell, the method comprising contacting the cell with the LNP formulation of any one of claims 88 to 97, wherein the therapeutic and / or prophylactic agent is or comprises RNA, whereby the RNA is capable of inhibiting the production of the polypeptide of interest.

105. A method for specifically delivering a therapeutic and / or prophylactic agent to a mammalian organ, the method comprising contacting the mammalian organ with the LNP formulation of any one of claims 88 to 97, thereby delivering the therapeutic and / or prophylactic agent to the organ.

106. The method of claim 105, comprising administering to the subject the LNP formulation of any one of claims 88 to 97.

107. A method of vaccination by administering the LNP formulation according to any one of claims 88 to 97.

108. A method of inducing an adaptive immune response in a subject, comprising administering to the subject an effective amount of a composition comprising at least one RNA; wherein the composition comprises an LNP formulation comprising a compound according to any one of claims 1 to 87, or a pharmaceutically acceptable salt thereof.

109. A method of base editing the genome of a cell, the method comprising contacting a cell of a subject to which the LNP formulation of any one of claims 88 to 97 has been administered.

110. A method for base editing the genome of a cell in a subject, the method comprising administering to the subject an effective amount of a composition comprising the LNP formulation of any one of claims 88 to 97.

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