Lipids and uses thereof

Abstract

To provide ionizable amine-containing lipids.SOLUTION: The ionizable amine-containing lipids are able to deliver nucleic acids.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority information This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 512,852, filed July 10, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in .xml format and is incorporated by reference in its entirety. A copy of the .xml created on July 4, 2024 is entitled "JP1239_SeqListing.xml" and is 4 kilobytes in size.

[0003] Field of the Disclosure The present disclosure relates to lipids and the use of such lipids for active agent delivery. [Background technology]

[0004] 2. Description of Related Art The use of lipid nanoparticles (LNPs) composed of ionizable amine-containing lipids has emerged as a promising strategy for intracellular delivery of biologically active agents, particularly nucleotides, such as antisense oligonucleotides (ASOs), siRNAs, miRNAs, mRNAs, and guide RNAs (gRNAs). These LNPs incorporating ionizable amine-containing lipids have demonstrated their important role in facilitating the effective transport of oligonucleotide agents across cell membranes. Moreover, they provide a useful approach for introducing gene editing components and compositions into living cells, thereby propelling the field of genetic engineering forward.

[0005] Achieving successful cellular uptake of biologically active agents, such as proteins, nucleic acid-based drugs and their derivatives, presents significant challenges, especially for drugs that contain sizeable oligonucleotides, such as mRNA. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides ionizable amine-containing lipids / compositions that enhance the delivery of promising gene editing technologies, such as components of the CRISPR / Cas9 system, to cells. One exemplary ionizable amine-containing lipid / composition includes the incorporation of an mRNA encoding a nuclease that is linked to its corresponding guide RNA as a means to achieve effective delivery.

[0007] The present disclosure demonstrates the use of ionizable amine-containing lipids and their salts (including pharma- ceutically acceptable salts) to effectively deliver biologically active substances. These ionizable amine-containing lipids and salts are useful for intracellular delivery of such active agents, facilitating the creation of artificial cells. Moreover, the disclosed ionizable amine-containing lipids demonstrate their utility as lipids with ionizable potential in the development of lipid nanoparticle-based compositions.

[0008] In one aspect, the present disclosure provides a lipid of formula (I) or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof: [ka] [In the formula, n is 1 or 2; R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, R 2 and R 3 are each independently an alkyl, alkenyl, or alkynyl having 5 to 30 carbon atoms, and in some embodiments, R 2 and R 3are the same or different, and in another embodiment, the alkyl, alkenyl, or alkynyl may be linear or branched; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-.

[0009] In some embodiments, n is 1, R 1 is hydroxy, R 2 and R 3 are each independently -CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 ) or -(CH 2 ) m CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 wherein m is an integer of 1 to 3, and p and q are each independently an integer of 1 to 12; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-.

[0010] In some embodiments, n is 1, R 1 is hydroxy, R 2 and R 3 are each independently -CH((CH2 ) p CH 3 )((CH 2 ) q CH 3 ) or -CH 2 CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 where p and q are each independently an integer of 3 to 9; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 6 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-.

[0011] Examples of lipids include di(heptadecan-9-yl) 3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy)) dipropionate, di(heptadecan-9-yl) 3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) propionate, di(heptadecan-9-yl) 5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, di(heptadecan-9-yl ) 5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy)) bis(pentane-5,1-diyl)bis(2-hexyldecanoate), di(heptadecan-9-yl) 3,3'-(((3R, 4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate, (((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-di di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dihexanoate, di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy)) dihexanoate, di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate, (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(hexyldecanoate), (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate), bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate, bis(2-octyldodecyl)5,5'-(((3R,4S) -1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate, bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate, bis(2-butyloctyl)5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis( Bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate, and bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate.

[0012] In some embodiments, the pK of the protonated form of the lipid a is about 5.0 to about 9.0.

[0013] In another aspect, the present disclosure provides an active agent delivery vehicle comprising a lipid disclosed herein, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer, or isotopically enriched derivative thereof.

[0014] Examples of active agent delivery vehicles include, but are not limited to, liposomes, single emulsions, micelles, nanoemulsions, lipid nanoparticles (LNPs), nanostructured lipid carriers (NLCs), and self-emulsifying drug delivery systems (SEDDS).

[0015] In some embodiments of the present disclosure, the active agent delivery vehicle further comprises one or more lipids. Examples of lipids include, but are not limited to, neutral lipids, steroid lipids, and PEGylated lipids.

[0016] Examples of neutral lipids include 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine, 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylserine (DPPG ... These include, but are not limited to, stearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoleylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE), and combinations thereof.

[0017] Examples of steroid lipids include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol tomatidine, ursolic acid, and alpha-tocopherol, and combinations thereof.

[0018] Examples of PEGylated lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-cholesterol, PEG-diacylglycamide (PEG-DAG), and PEG-dialkyloxypropyl (PEG-DAA), and combinations thereof.

[0019] In another aspect, the present disclosure provides a composition comprising an active agent delivery vehicle disclosed herein and an active agent. In some embodiments of the present disclosure, the composition is a pharmaceutical composition.

[0020] In some embodiments of the present disclosure, the active agent is encapsulated in the active agent delivery vehicle.

[0021] In some embodiments of the present disclosure, the active agent comprises a nucleic acid. Examples of nucleic acids include, but are not limited to, antisense oligonucleotides, plasmids, interfering nucleic acids, aptamers, microRNAs (miRNAs), Dicer substrate RNAs (dsRNAs), short hairpin RNAs (shRNAs), mRNAs (messenger RNAs), guide RNAs (gRNAs), miRNA inhibitors (antagomirs), and ribozymes, and combinations thereof.

[0022] In some embodiments of the disclosure, the mRNA encodes an enzyme, an antigen, an antibody, or an antigen-binding fragment.

[0023] In some embodiments of the present disclosure, the composition further comprises one or more pharma- ceutically acceptable excipients or adjuvants.

[0024] In another aspect, the disclosure provides a method of treating or preventing a disease or disorder, comprising administering to a subject in need thereof a composition disclosed herein.

[0025] In another aspect, the disclosure provides a method of introducing a nucleic acid into or transfecting a target cell with a nucleic acid comprising contacting the target cell with a composition disclosed herein.

[0026] In another aspect, the disclosure provides a method of modulating expression of a target gene in a cell, the method comprising providing to the cell a composition disclosed herein.

[0027] The present invention is described in detail in the following sections. Other features, objects, and advantages of the invention can be found in the detailed description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description The present disclosure may be more easily understood by referring to the following detailed description of various embodiments of the present disclosure, examples, and chemical drawings and tables with their appropriate descriptions.Unless otherwise specifically indicated by the claims, the present disclosure is not limited to a specific preparation method, carrier or formulation, nor to a specific manner of formulating the extract of the present disclosure into a product or composition for topical, oral or parenteral administration, because such factors can of course vary as well recognized by those skilled in the relevant art.It should also be understood that the terms used herein are only for describing specific embodiments, and are not limiting.

[0029] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0030] Ranges are often expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, an embodiment includes the range from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the word "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to each other and independently of each other.

[0031] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, the phrase "optionally including an agent" means that the agent may or may not be present.

[0032] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0033] The term "subject" as used herein refers to any animal, preferably a mammal, more preferably a human. Examples of subjects include humans, non-human primates, rodents, guinea pigs, rabbits, sheep, pigs, goats, cows, horses, dogs and cats.

[0034] The term "treat" or "treatment" as used herein refers to reversing, alleviating, inhibiting the progression of the disorder, disease or condition to which such term applies, or one or more symptoms of such disorder, disease or condition, or ameliorating such disorder, disease or condition, or one or more symptoms of such disorder, disease or condition.

[0035] The terms "prevent" or "prophylaxis" are art-recognized and when used in relation to a condition include administration of an agent prior to the onset of the condition that reduces the frequency or severity of a symptom of a pathological condition in a subject, or delays the onset of a symptom of such a pathological condition, compared to a subject not receiving the agent.

[0036] The term "pharmaceutical composition" refers to drugs and / or vaccines for treating and / or preventing one or more diseases or disorders in a subject in need of such treatment and / or prevention. Pharmaceutical compositions can act systemically or locally. For this purpose, they can be administered by suitable routes, such as injection (e.g., intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection, including drip infusion) and transdermal delivery, and can also be administered by oral, buccal, nasal, mucosal or topical routes, or in the form of ophthalmic preparations, or by inhalation. With respect to these routes of administration, the pharmaceutical compositions of the present invention can be administered in suitable dosage forms, including, but not limited to, tablets, capsules, lozenges, hard sugar tablets, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injections, elixirs, and syrups. The term "vaccine" refers to prophylactic or therapeutic materials that provide at least one antigen or antigenic function. An antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0037] The term "excipient" as used herein refers to any substance that is not a therapeutic agent itself, but is added to a formulation to improve its handling or storage properties, or to enable or facilitate the formation of a dosage unit of the composition into a separate article, such as a capsule or tablet suitable for oral administration. Suitable excipients are well known to those skilled in the art of manufacturing pharmaceutical formulations or food products. Excipients can include, by way of example and not limitation, buffers, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, flow agents, and substances added to mask or counteract unpleasant tastes or odors, flavors, dyes, fragrances, and substances added to improve the appearance of the composition. Acceptable excipients include citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, magnesium carbonate, talc, gelatin, gum acacia, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulosic materials (e.g., cellulose esters of alkanoic acids and cellulose alkyl esters), the low melting wax cocoa butter, amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), ethylenediaminetetraacetic acid (EDTA), dimethylsulfoxide (DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol or powders, polymers (e.g., polyvinyl-pyrrolidone, polyvinyl alcohol, and polyethylene glycol), and other pharma- ceutically acceptable materials.

[0038] The term "adjuvant" or "adjuvant component" is typically an agent or composition (e.g., pharmacological or immunological) that can modify (e.g., enhance) the effectiveness of other agents (e.g., drugs or vaccines). Conventionally, the term refers in the context of the present invention to a compound or composition that serves as an adjuvant for immunogens and / or other pharma- ceutical active compounds. It should be interpreted in a broad sense and refers to a wide range of substances that can increase the immunogenicity of the antigen incorporated in the adjuvant or co-administered with the adjuvant. In the context of the present invention, the adjuvant preferably enhances the specific immunogenic effect of the active agent of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be divided, for example, into immune enhancers, antigen delivery systems, or further combinations thereof.

[0039] The term "delivery" refers to the targeted delivery of an entity, e.g., the delivery of an active agent, drug, therapeutic agent and / or prophylactic agent to a subject, which may be human and / or other animal tissues and / or cells.

[0040] The term "delivery vehicle" refers to a substance that can be used with or without the agent to be delivered to administer or deliver one or more agents to a cell, tissue, or subject, particularly a human subject. A delivery vehicle can preferentially deliver an agent to a particular subset or type of cell. The selective or preferential delivery achieved by a delivery vehicle can be achieved by the properties of the vehicle or by a moiety that is conjugated to, attached to, or included in the delivery vehicle, which specifically or preferentially binds to a particular subset of cells. A delivery vehicle can also increase the in vivo half-life of the agent to be delivered, the efficiency of delivering the agent compared to delivery without the use of a delivery vehicle, and / or the bioavailability of the agent to be delivered.

[0041] As used herein, the term "nucleic acid" is meant to include any oligonucleotide or polynucleotide. Fragments containing up to 20 nucleotides are generally referred to as oligonucleotides, while longer fragments are called polynucleotides. In certain embodiments, the oligonucleotides of the invention are 20-50 nucleotides in length.

[0042] Within the context of this disclosure, the terms "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also encompass polymers or oligomers containing monomers of non-natural origin, or portions thereof which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms due to properties such as enhanced cellular uptake and increased stability in the presence of nucleases.

[0043] Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphate at the 5' and 3' carbons of this sugar to form an alternating unbranched polymer. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0044] The nucleic acid present in the lipid-nucleic acid particle according to the present disclosure includes any form of nucleic acid known in the art. The nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrid. Examples of double-stranded DNA include structural genes, genes including regulatory and termination regions, and self-replicating systems, such as virus or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides.

[0045] The term "RNA" refers to ribonucleic acid, which may be of natural or non-natural origin. For example, RNA may contain modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, and linkers. RNA may contain a cap structure, a chain terminating nucleoside, a stem loop, a polyA sequence, and / or a polyadenylation signal. RNA may have a nucleotide sequence that encodes a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA that encodes a particular polypeptide, for example in vivo translation of an mRNA inside a mammalian cell, may produce the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA, including cas9 mRNA), guide RNA (gRNA, also called single guide RNA (sgRNA)), miRNA inhibitors (antagomir), ribozymes, and mixtures thereof. Antisense nucleic acids and siRNAs can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0046] The term "modulating expression of a target gene" refers to the ability of a nucleic acid to silence, reduce or inhibit expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a sample of cells in culture expressing the target gene) is contacted with a nucleic acid that inhibits expression of the target gene. Expression of the target gene in the test sample or test animal is compared to expression of the target gene in a control sample (e.g., a sample of cells in culture expressing the target gene) that has not been contacted with or administered the nucleic acid. The expression of the target gene in the control sample may be assigned a value of 100%. In certain embodiments, modulating expression of a target gene is achieved when the level of target gene expression in the test sample or test mammal relative to the level of target gene expression in the control sample or control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%.

[0047] The term "transfect" or "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0048] The term "PEGylation" refers to the method of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, known in the pharmaceutical arts as macrogol) polymer chains to molecules and macrostructures, such as drugs, therapeutic proteins or vesicles, which are then referred to as "PEGylated."

[0049] The present disclosure presents amine-containing lipids that are highly useful for formulating lipid nanoparticle (LNP) compositions that confer advantageous properties for efficient delivery of nucleic acid cargo, such as CRISPR / Cas gene editing components, into cells.

[0050] The present disclosure relates to a lipid of formula (I) [ka] or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof. [In the formula, n is 1 or 2; R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, R 2 and R 3 each independently represents an alkyl, alkenyl, or alkynyl group having 5 to 30 carbon atoms; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-.

[0051] In some embodiments, R 2 and R 3 The alkyl, alkenyl and alkynyl may all be linear or branched and have 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 or 30 carbon atoms.

[0052] In some embodiments, L 1 may be alkyl having 2, 3, 4, 5 or 6 carbon atoms.

[0053] In some embodiments, L 2 and L 3 may be the same or different from each other. Alkyl may be straight or branched and may have 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms.

[0054] In some embodiments, X and Y can be the same or different from each other.

[0055] In some embodiments, n is 1 and R 1 is hydroxy and R 2 and R 3 are each independently -CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 ) or -(CH 2 ) m CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 wherein m is 1, 2, or 3, p and q are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and L 1 is an alkyl group having 2 to 4 carbon atoms; L 2 and L 3 are each independently alkyl having 3 to 6 carbon atoms, and X and Y are each independently -C(O)-O- or -OC(O)-.

[0056] In some embodiments, n is 1 and R 1 is hydroxy and R 2 and R 3 are each independently -CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 ) or -CH 2 CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 where p and q are each independently 3, 4, 5, 6, 7, 8, or 9; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2and L 3 are each independently alkyl having 2 to 6 carbon atoms, and X and Y are each independently -C(O)-O- or -OC(O)-.

[0057] In some embodiments, the lipids of the present disclosure, or their pharma- ceutically acceptable salts, solvates, hydrates, polymorphs, tautomers, stereoisomers, or isotopically enriched derivatives, may be used as constituent lipids of lipid membrane structures for encapsulating active agents.For example, the lipids of the present disclosure may be used in liposomes or lipid nanoparticles (LNPs) for nucleic acid encapsulation and delivery.

[0058] In some embodiments, the lipids of the present disclosure can be ionizable. In some embodiments, the lipids of the present disclosure are ionizable cationic lipids that are positively charged at low pH (thus binding nucleic acids, e.g., RNA) and neutral at physiological pH, helping to reduce the toxicity of LNP-RNA complexes in vivo. The lipids can serve two main functions, including facilitating nucleic acid encapsulation into LNPs and mediating endosomal membrane disruption to allow nucleic acid release into the cytosol. In some embodiments, the lipids can enable endosomal uptake directly by interactions between their positive charge and negatively charged cell membranes or by binding to plasma proteins that aid in cellular uptake.

[0059] In some embodiments, the lipid may be deprotonated under neutral conditions and its acid dissociation constant (pK a ) may be positively charged. a The pK value appears to be high enough that at low pH the lipids become positively charged, allowing binding to negatively charged RNA molecules and the formation of LNPs. Thus, at low endosomal pH, the positive charge of the ionizable amine-containing lipids allows for interaction with endogenous anionic lipids, thereby leading to the disruption of endosomal structure and release of the LNP cargo into the cytoplasm. At the same time, the pK aThe pK value should be low enough so that at physiological pH, the surface charge of the LNPs remains relatively neutral. This allows for modulation of the toxicity and immunogenicity of the resulting LNPs as well as increasing their circulation time. In some embodiments, the pK value of the protonated form of the lipid a is about 5.0 to about 9.0, for example, about 5.5, about 6.0, about 6.5, about 7.0 or more, or about 8.5, about 8.0, about 7.5, about 7.0 or less. The pKa value of the lipid can be critical for efficient LNP delivery and transfection ability.

[0060] The present disclosure further provides an active agent delivery vehicle comprising a lipid, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer, or isotopically enriched derivative thereof.

[0061] In some embodiments, the active agent delivery vehicle can be a drug delivery system, such as a liposome, a single emulsion, a micelle, a nanoemulsion, a lipid nanoparticle (LNP), a nanostructured lipid carrier (NLC), and a self-emulsifying drug delivery system (SEDDS). In some embodiments, the active agent delivery vehicle can be prepared at an acidic pH (about 4 or 5) below the pKa value of the lipid, followed by a buffer / pH exchange step.

[0062] In some embodiments, the active agent delivery vehicle further comprises one or more neutral lipids, steroid lipids, and / or PEGylated lipids. In some embodiments, the active agent delivery vehicle comprises at least one neutral lipid, at least one steroid lipid, and at least one PEGylated lipid. These components can promote monodisperse nanoparticle formation, improve nanoparticle stability, enable efficient nucleic acid encapsulation, aid in cellular uptake, and promote endosomal escape of nucleic acid cargo.

[0063] In some embodiments, neutral lipids are also referred to as helper lipids or structural lipids. Neutral lipids can be any lipid substance that is uncharged or exists in a neutral zwitterionic form at a selected pH. In some embodiments, the neutral lipid is a phospholipid, such as 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine, 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine ... POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoleoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and combinations thereof. Neutral lipids may be synthetic or natural. In some embodiments, the preferred phospholipids for LNPs are DSPC and DOPE.

[0064] Steroid lipids can improve the cellular entry of active agents encapsulated in the delivery vehicle. Examples of steroid lipids include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and combinations thereof.

[0065] In some embodiments, the PEGylated lipid is a molecule that contains both lipid and PEG moieties, also called polyethylene glycol (PEG)-lipid conjugate. The PEGylated lipid can improve the stability and circulation time of the delivery vehicle by preventing serum protein binding. In some embodiments, the PEGylated lipid can be 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-cholesterol, PEG-diacylglycamide (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), and combinations thereof. Specifically, the PEGylated lipid can be one or more of PEG500-dipalmitoylphosphatidylcholine, PEG2000-dipalmitoylphosphatidylcholine, PEG500-stearylphosphatidylethanolamine, PEG2000-distearylphosphatidylethanolamine, PEG500-1,2-dioleoylphosphatidylethanolamine, PEG2000-1,2-dioleoylphosphatidylethanolamine, PEG2000-2,3-distearoylglycerol (PEG-DMG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000).

[0066] The present disclosure also provides compositions comprising an active agent delivery vehicle and an active agent encapsulated by the active agent delivery vehicle.

[0067] In some embodiments, the compositions are pharmaceutical compositions that can be used as therapeutic agents for any disease (e.g., cancer and malignant tumors), anti-infective agents, anti-viral agents, anti-fungal agents, and vaccines.

[0068] In some embodiments, the active agent is encapsulated in the active agent delivery vehicle. In some embodiments, the active agent comprises one or more nucleic acids. Examples of nucleic acids include DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA (messenger RNA), guide RNA (gRNA), miRNA inhibitor (antagomir), ribozyme, and combinations thereof. In some embodiments, the mRNA encodes an enzyme, an antigen, an antibody, or an antigen-binding fragment.

[0069] In some embodiments, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients or adjuvants. In some embodiments, the pharma- ceutically acceptable excipients may include deionized water, ultrapure water, phosphate buffer or saline, more preferably phosphate buffer or saline.

[0070] The present disclosure further provides a method of treating or preventing a disease or disorder, comprising administering the pharmaceutical composition to a subject in need thereof.

[0071] In some embodiments, the pharmaceutical composition according to the present disclosure is administered locally or systemically by any method known in the art, including but not limited to intramuscular, intradermal, intravenous, subcutaneous, intraperitoneal, intranasal, oral, mucosal or external routes. The appropriate route, formulation and administration schedule can be determined by those skilled in the art. In the present disclosure, the pharmaceutical composition can be formulated in various ways, such as liquid solutions, suspensions, emulsions, syrups, tablets, pills, capsules, sustained release formulations, powders, granules, ampoules, injections, infusions, kits, ointments, lotions, liniments, creams or combinations thereof, depending on the corresponding administration route. If necessary, it may be sterilized or mixed with any pharma- ceutically acceptable excipient, many of which are known to those skilled in the art.

[0072] As used herein, the external route is also known as topical administration and includes, but is not limited to, administration by insufflation and inhalation. Examples of different types of topical administration formulations include ointments, lotions, creams, gels, foams, formulations for delivery by transdermal patches, powders, sprays, aerosols, capsules, or cartridges for use in inhalers or insufflators, or drops (e.g., eye drops or nose drops), solutions / suspensions for nebulization, suppositories, vaginal suppositories, retention enemas, and chewable or inhalable tablets or pellets, or liposomal or microencapsulated formulations.

[0073] The present disclosure further provides a method of introducing a nucleic acid into or transfecting a target cell with a nucleic acid, comprising contacting a composition with the target cell. In some embodiments, an active agent delivery vehicle encapsulating a nucleic acid can be administered to a subject such that the nucleic acid introduction vehicle can contact the target cell of the subject.

[0074] In another aspect, the disclosure provides a method of modulating expression of a target gene in a cell, the method comprising providing to the cell a composition disclosed herein.

[0075] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure. EXAMPLES

[0076] Synthesis Examples General Procedure for the Synthesis of Ionizable Amine-Containing Lipids

[0077] [ka]

[0078] Scheme 1 Synthesis of tert-butyl (3S,4R)-3,4-dihydroxypyrrolidine-1-carboxylate (A-1)

[0079] [ka]

[0080] To a mixture of NMO (2188 mg; 18.68 mmol, 1.1 equiv.), osmium tetroxide (4% in water) (25 mg, 0.004 mmol) and water (30 mL) was added dropwise a solution of tert-butyl 2,5-dihydro-1H-pyrrole-1-carboxylate (2875 mg; 16.98 mmol) in acetone (30 mL) while maintaining the temperature below 25° C. The reaction mixture was then stirred at ambient temperature for 16 h. The acetone was evaporated and the organic phase was extracted with ethyl acetate. The combined organic phase was washed successively with water and saturated brine and dried over magnesium sulfate. The solvent was evaporated under vacuum and the residue was the crude product (A-1) (2865 mg, yield: 83%) which appeared as a brown oil and was utilized in the next reaction without further purification. 1 H NMR (600 MHz, CDCl 3 ) δ 4.30 - 4.21 (m, 2H), 3.60 (ddt, J = 15.8, 11.4, 5.3 Hz, 2H), 3.36 (ddd, J = 27.5, 11.6, 4.0 Hz, 2H), 2.77 (b, 1H), 1.67 (b, 1H),1.47 (d, J = 2.1 Hz, 9H).

[0081] Synthesis of diethyl 3,3'-(((3S,4R)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))(2E,2'E)-diacrylate (A-2)

[0082] [ka]

[0083] The crude product (A-1) (2070 mg, 10.08 mmol) and DABCO (340 mg, 3.02 mmol, 0.3 equiv.) were dissolved in 20 mL of dry DCM. This solution was then slowly added dropwise to a mixture of ethyl propiolate (5936 mg, 60.51 mmol, 6.0 equiv.) in 25 mL of dry DCM in an ice / water bath under nitrogen atmosphere. After the reaction was completed, NH 4 Cl (水性) Add EA and extract, remove the organic layer and dry with MgSO 4 The mixture was dried using hexane / EtOAc (3 / 2→2 / 3), concentrated and pumped to dryness. The dried product was purified by silica gel column chromatography using a gradient of hexane / EtOAc (3 / 2→2 / 3), and then concentrated in vacuum to give a pale yellow oily product (A-2) (1757 mg, yield: 43%). 1 H NMR (600 MHz, CDCl 3 ) δ 7.51 (d, J = 12.5 Hz, 2H), 5.33 (d, J = 12.6 Hz, 2H), 4.61 (h, J = 3.3, 2.8 Hz, 2H), 4.22 - 4.17 (m, 4H), 3.76 (dq, J = 11.6, 6.8, LCMS(ESI) :m / z C 19 H 29 NO 8 + H] + Calculated value: 399.44, measured value: 399.9.

[0084] Synthesis of 3,3'-(((3S,4R)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionic acid (A-3)

[0085] [ka]

[0086] A solution of (A-2) (1700 mg, 4.25 mmol) and 10% Pd-C (170 mg) in a solvent mixture of ethyl acetate and methanol (volume ratio 5:1) was added to a round-bottom bottle under hydrogen conditions and reacted at room temperature for 4 hours. The product was filtered through Celite, and the organic layer was taken and concentrated to dryness to obtain a colorless oily product.

[0087] The crude product was dissolved in MeOH / H 2 A solution of 2H2O (1 / 1, 40 mL) and 2M NaOH (6 mL) were added to a round-bottom bottle and reacted at room temperature for 16 h. After the reaction was complete, a portion of the solvent was drained off and the remaining solution was cooled in an ice bath and then adjusted to pH 4 with 2N HCl. The resulting solution was extracted twice with EtOAc and the combined organic layers were dried (MgSO 4 The dried product was filtered and concentrated in vacuo to give crude (A-3) as a viscous colorless oily product (1123 mg, yield: 76%). 1 H NMR (600 MHz, CDCl 3 ) δ 4.20 - 4.11 (m, 1H), 3.99 (dd, J = 22.2, 4.3 Hz, 2H), 3.82 (ddd, J = 13.9, 7.7, 3.2 Hz, 4H), 3.57 - 3.46 (m, 2H), 3.38 - 3.30 (m, 1H), 2.75 - 2.55 (m, 4H), 1.49 - 1.46 (m, 9H). LCMS(ESI):m / z C 19 H 33 NO 8 +H] + Calculated value 403.47; Found value 404.0 [M+H] + .

[0088] Synthesis of tert-butyl (3S,4R)-3,4-bis(4-cyanobutoxy)pyrrolidine-1-carboxylate (A-4)

[0089] [ka]

[0090] In a round-bottom bottle, the crude product of (A-1) (1900 mg, 9.35 mmol), 5-bromopentanenitrile (6057 mg, 37.39 mmol, 4.0 equiv.), and TBAB (605 mg, 1.87 mmol, 0.2 equiv.) were mixed in 40 wt.% NaOH (6 mL). The mixture was warmed to 65 °C and stirred for 24 h for the reaction. The mixture was cooled in an ice-water bath, diluted with water (20 mL), and then adjusted to pH = 5-6 with 12 N HCl. The resulting solution was extracted twice with EtOAc, and the combined organic layers were dried (MgSO 4 ). The dried product was filtered and concentrated in vacuum to give the crude product. The crude product was purified by CombiFlash MPLC using a gradient of hexane / EtOAc (EtOAc = 28-31%), then concentrated in vacuum to give a colorless oily product (A-4) (1776 mg, yield: 52%). 1 H NMR (600 MHz, CDCl 3 ) δ 3.92 (q, J = 4.8, 4.2 Hz, 2H), 3.63 - 3.46 (m, 6H), 3.44 (dd, J = 11.5, 3.8 Hz, 1H), 3.32 (dd, J = 10.8, 5.2 Hz, 1H), 2.51 - 2.35 (m, 4H), 1.78 (tq, J = 10.3, 6.0 Hz, 8H), 1.48 (d, J = 0.7 Hz, 9H); LCMS(ESI):m / z C 19 H 31 N 3 O+H] + Calculated value 365.47, Measured value 366.3 [M+H] + .

[0091] Synthesis of 5,5'-(((3S,4R)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoic acid (A-5)

[0092] [ka]

[0093] In a round-bottom bottle, the product (A-4) (1300 mg, 3.55 mmol) and KOH (1944 mg, 35.54 mmol, 10 equiv.) were dissolved in EtOH / H 2 The mixture was mixed in 200 mL of 1:1 EtOAc (1 / 1, 20 mL). The mixture was warmed to 110 °C and stirred for 32 h for the reaction. The mixture was then cooled to room temperature and EtOH was evaporated. The resulting mixture was cooled in an ice-water bath, diluted with water (10 mL), and then adjusted to pH 4-5 with 36 N HCl. The resulting solution was extracted twice with EtOAc and the combined organic layers were dried (MgSO 4 The dried product was filtered and concentrated in vacuo to give a viscous colorless product (A-5) (1219 mg, yield: 85%). 1 H NMR (600 MHz, CDCl 3 ) δ 3.96 - 3.86 (m, 2H), 3.64 - 3.43 (m, 7H), 3.34 (dd, J = 11.0, 5.5 Hz, 1H), 2.41 (dq, J = 7.4, 4.1, 3.0 Hz, 4H), 1.76 (d, J = 15.0 Hz, 4H), 1.68 (dq, J = 12.5, 6.3 Hz, 4H), 1.47 (s, 9H). LCMS(ESI):m / z C 19 H 33 NO 8 +H] + Calculated value 403.47, Measured value 404.1 [M+H] + .

[0094] Synthesis of tert-butyl (3S,4S)-3,4-bis((5-hydroxypentyl)oxy)pyrrolidine-1-carboxylate (A-6)

[0095] [ka]

[0096] To a solution of (A-5) (600 mg, 1.49 mmol) in dry THF (10 mL) cooled in an ice-water bath under nitrogen, dimethylsulfide borane (2M in THF) (2.9 mL, 4.0 equiv.) was added over 5 min. The mixture was warmed to 65 °C and then stirred for 3 h. The mixture was then cooled in an ice-water bath under nitrogen and MeOH (10 mL) was carefully and slowly added to the mixture over 5 min. The mixture was again warmed to 65 °C and then stirred for 15 min. The mixture was then cooled to room temperature and the solvent was evaporated. The residue was extracted twice with EtOAc and the combined organic layers were dried (MgSO 4 The dried product was filtered and concentrated in vacuo to give a viscous colorless product (A-6) (497 mg, yield: 89%). 1 H NMR (600 MHz, CDCl 3 ) δ 3.85 (ddt, J = 4.8, 3.1, 1.8 Hz, 2H), 3.67 (tt, J = 6.7, 2.6 Hz, 4H), 3.56 - 3.42 (m, 7H), 3.38 - 3.31 (m, 1H), 1.60 (td, J = 7.1, 6.7, 2.9 Hz, 8H), 1.48 (s, 9H), 1.46 - 1.40 (m, 4H); LCMS(ESI):m / z C 19 H 37 NO 6 +H] + Calculated value 375.51, Measured value 376.0 [M+H] + .

[0097] Synthesis of di(heptadecan-9-yl)5,5'-(((3S,4R)-pyrrolidine-3,4-diyl)bis(oxy))dipentanoate (A-7)

[0098] [ka]

[0099] To a suspension of (A-5) (730 mg, 1.81 mmol) in dry DCM (10 mL) was added EDC-HCl (1038 mg, 5.43 mmol, 3.0 equiv), 9-heptadecanol (925 mg, 3.62 mmol, 2.0 equiv), DIPEA (1403 mg, 10.86 mmol, 6.0 equiv), and DMAP (88 mg, 0.72 mmol, 0.4 equiv) sequentially. The mixture was stirred under nitrogen at room temperature overnight, then poured into water and extracted twice with DCM. The organic layer was separated, washed with saturated brine, and purified by filtration with MgSO. 4 Drying using hexanes. The dried product was filtered and concentrated in vacuum to give the crude product. The crude product was purified by CombiFlash MPLC using a gradient of hexanes / EtOAc (EtOAc = 8-13%) and then concentrated in vacuum to give a colorless oily product. To a solution of the crude product (1140 mg, 1.29 mmol) in dry dichloromethane (15 mL) was added TFA (1.8 mL). The mixture was stirred at room temperature under nitrogen for 1.5 h. The mixture was cooled in an ice-water bath and diluted with DCM (10 mL) and then NaHCO 3(水性) The pH was adjusted to 8 with 50 ml of DCM. The resulting solution was extracted with DCM and the combined organic layers were dried (MgSO 4 The dried product was filtered and concentrated in vacuum to give a colorless oily product (A-7) (776 mg, yield: 55%). 1 H NMR (600 MHz, CDCl 3 ) δ 4.88 (p, J = 6.3 Hz, 2H), 4.07 (q, J = 3.9, 3.3 Hz, 2H), 3.61 (dt, J = 9.0, 6.1 Hz, 2H), 3.56 - 3.51 (m, 2H), 3.44 (dd, J = 11.6, 5.0 Hz, 2H), 3.31 (dd, J = 11.8, 4.7 Hz, 2H), 2.37 - 2.30 (m, 4H), 1.75 - 1.69 (m, 4H), 1.67 - 1.61 (m, 4H), 1.52 (d, J = 6.3 Hz, 8H), 1.33 - 1.24 (m, 48H), 0.90 (t, J = 7.0 Hz, 12H); LCMS(ESI):m / z C 48 H93 NO 6 +H] + Calculated value 780.27, Measured value 781.0 [M+H] + .

[0100] Synthesis of (((3S,4R)-pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate) (A-8)

[0101] [ka]

[0102] The synthesis steps are the same as those described in (A-7). 1 H NMR (600 MHz, CDCl 3 ) δ 4.09 (t, J = 6.8 Hz, 4H), 4.01 (q, J = 4.0 Hz, 2H), 3.58 (dt, J = 9.1, 6.5 Hz, 2H), 3.51 (dt, J = 9.0, 6.5 Hz, 2H), 3.33 (dd, J = 11.7, 5.0 Hz, 2H), 3.23 (dd, J = 11.8, 4.5 Hz, 2H), 2.33 (tt, J = 8.9, 5.3 Hz, 2H), 1.63 (ddd, J = 15.9, 10.6, 7.2 Hz, 12H), 1.49 - 1.41 (m, 8H), 1.33 - 1.23 (m, 40H), 0.90 (td, J = 7.0, 1.6 Hz, 12H); LCMS(ESI):m / z C 46 H 89 NO 6 +H] + Calculated value 752.22, Measured value 753.0 [M+H] + .

[0103] [Example 1] Di(heptadecan-9-yl) 3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate

[0104] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.94 - 4.85 (m, 2H), 3.97 (ddt, J = 7.8, 5.5, 2.8 Hz, 2H), 3.87 - 3.74 (m, 4H), 3.61 (dd, J = 6.2, 4.4 Hz, 2H), 3.07 - 3.00 (m, LCMS (ESI): 768.6[M+H] + .

[0105] [Example 2] Di(heptadecan-9-yl) 3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))propionate

[0106] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.96 - 4.83 (m, 2H), 3.93 (ddt, J = 8.0, 5.5, 2.8 Hz, 2H), 3.86 - 3.70 (m, 6H), 3.18 (dd, J = 10.0, 5.4 Hz, 2H), 2.77 (t, J = 5.8 Hz, 2H), 2.65 - 2.50 (m, 6H), 1.75 - 1.62 (m, 2H), 1.53 (dd, J = 10.4, 5.1 Hz, 8H), 1.36 - 1.21 (m, 48H), 0.90 (t, J = 7.0 Hz, 12H). LCMS (ESI): 783.4 [M+H]+ .

[0107] [Example 3] Di(heptadecan-9-yl)5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0108] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.88 (p, J = 6.2 Hz, 2H), 3.88 (t, J = 4.3 Hz, 2H), 3.83 - 3.78 (m, 2H), 3.56 (dt, J = 9.2, 6.3 Hz, 2H), 3.47 (dt, J = 9.2, 6.3 Hz, 2H), 3.20 (s, 2H), 2.81 (s, 2H), 2.61 - 2.51 (m, 2H), 2.33 (t, J = 7.4 Hz, 4H), 1.72 (ddd, J = 9.0, 6.7, 4.5 Hz, 6H), 1.64 (dd, J = 8.5, 5.7Hz, 4H), 1.52 (d, J = 6.0 Hz, 8H), 1.35 - 1.22 (m, 48H), 0.90 (t, J = 7.0 Hz, 12H); LCMS(ESI): 838.9 [M+H] + .

[0109] [Example 4] Di(heptadecan-9-yl)5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0110] [ka] 1 H NMR (600 MHz, CDCl 3) δ 4.88 (p, J = 6.3 Hz, 2H), 3.87 - 3.79 (m, 2H), 3.63 (t, J = 5.4 Hz, 2H), 3.47 (ddt, J = 26.6, 9.1, 6.3 Hz, 4H), 2.93 (dd, J = 9.9, 6.0 Hz, 2H), 2.68 (dt, J = 12.3, 5.5 Hz, 1H), 2.60 (ddd, J = 10.0, 8.9, 4.6 Hz, 3H), 2.33 (t, J = 7.4 Hz, 4H), 1.71 (dtd, J = 9.1, 7.9, 7.2, 5.9Hz, 4H), 1.66 - 1.58 (m, 4H), 1.52 (d, J = 5.9 Hz, 8H), 1.36 - 1.22 (m, 48H), 0.90 (t, J = 7.0 Hz, 12H); LCMS (ESI): 825.1 [M+H] + .

[0111] [Example 5] (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0112] [ka] 1 H NMR (600 MHz, CDCl 3) δ 4.08 (t, J = 6.7 Hz, 4H), 3.87 (d, J = 5.0 Hz, 2H), 3.82 - 3.77 (m, 2H), 3.54 (q, J = 7.6 Hz, 2H), 3.46 (q, J = 7.5 Hz, 2H), 3.26 - 3.13 (m, 2H), 2.79 (t, J = 5.7 Hz, 2H), 2.54 (dd, J = 10.4, 4.7 Hz, 2H), 2.32 (ddd, J = 8.9, 6.0, 2.8 Hz, 2H), 1.72 (q, J = 5.6 Hz, 2H), 1.64 (ddq, J = 26.2, LCMS (ESI): 810.9 [M+H] + .

[0113] [Example 6] (((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0114] [ka] 1 H NMR (600 MHz, CDCl 3) δ 4.09 (t, J = 6.7 Hz, 4H), 3.91 (t, J = 4.3 Hz, 2H), 3.62 (t, J = 5.3 Hz, 2H), 3.55 (dt, J = 9.3, 6.6 Hz, 2H), 3.47 (dt, J = 9.2, 6.7 Hz, 2H), 3.08 (dd, J = 9.7, 5.3 Hz, 2H), 2.71 (t, J = 5.3 Hz, 2H), 2.64 (dd, J = 9.7, 4.6 Hz, 2H), 2.32 (td, J = 9.1, 4.6 Hz, 2H), 1.68 - 1.63 (m, 12H), 1.49 - 1.39 (m, 8H), 1.34 - 1.22 (m, 40H), 0.90 (t, J = 6.9 Hz, 12H); LCMS (ESI): 796.4 [M+H] + .

[0115] [Example 7] ジ(ヘプタデカン-9-イル)3,3'-(((3R,4S)-1-(4-ヒドロキシブチル)ピロリジン-3,4-ジイル)ビス(オキシ))ジプロピオネート

[0116]

change

[0117] [Example 8] (((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl)bis(2-hexyldecanoate)

[0118] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.11 (t, J = 6.5 Hz, 4H), 3.92 (t, J = 4.6 Hz, 2H), 3.83 - 3.78 (m, 2H), 3.59 (dt, J = 9.1, 6.2 Hz, 2H), 3.50 (dt, J = 9.1, 6.2 Hz, 2H), 3.29 (s, 2H), 2.87 (s, 2H), 2.63 (s, 2H), 2.33 (tt, J = 8.9, 5.4 Hz, 2H), 1.79 - 1.64 (m, 12H), 1.64 - 1.55 (m, 6H), 1.35 - 1.21 (m, 40H), 0.90 (td, J = 7.0, 1.8 Hz, 12H). LCMS (ESI): 782.8 [M+H] + .

[0119] [Example 9] (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0120] [ka] 1 H NM (600 MHz, CDCl 3) δ 4.09 (t, J = 6.7 Hz, 4H), 3.67 (t, J = 6.3 Hz, 2H), 3.60 (s, 2H), 3.49 (q, J = 7.2 Hz, 2H), 2.33 (tt, J = 9.0, 5.3 Hz, 2H), 1.69 - 1.58 (m, 24H), 1.44 (dt, J = 17.7, 6.7 Hz, 10H), 1.35 - 1.20 (m, 40H), 0.90 (td, J = 7.0, 1.5 Hz, 12H). LCMS (ESI): 839.0 [M+H] + .

[0121] [Example 10] (((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate)

[0122] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.08 (t, J = 6.7 Hz, 4H), 3.93 (s, 2H), 3.66 (t, J = 6.5 Hz, 2H), 3.61 - 3.52 (m, 2H), 3.47 (dt, J = 9.3, 6.7 Hz, 2H), 2.33 (tt, J LCMS (ESI): 853.2 [M+H] + .

[0123] [Example 11] Di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate

[0124] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.88 (p, J = 6.2 Hz, 2H), 3.89 (q, J = 4.9, 4.4 Hz, 2H), 3.85 - 3.75 (m, 2H), 3.54 (qd, J = 6.8, 3.3 Hz, 2H), 3.45 (td, J = 8.5, 7.9, 6.0 Hz, 2H), 3.33 - 3.15 (m, 2H), 2.83 (s, 2H), 2.64 - 2.48 (m, 2H), 2.30 (t, J = 7.6 Hz, 4H), 1.73 (p, J = 5.5 Hz, 2H), 1.64 (ddd, J = 20.2, 14.7, 7.3 Hz, 8H), 1.52 (q, J = 6.6, 6.0 Hz, 8H), 1.40 (tt, J = 9.9, 6.3 Hz, 4H), 1.33 - 1.24 (m, 48H), 0.90 (t, J = 7.0 Hz, 12H). LCMS (ESI): 867.1 [M+H] + .

[0125] [Example 12] Di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate

[0126] [ka] 1 H NMR (600 MHz, CDCl 3) δ 4.88 (p, J = 6.3 Hz, 2H), 3.97 (s, 2H), 3.67 (t, J = 6.3 Hz, 2H), 3.57 (d, J = 8.1 Hz, 2H), 3.47 (dt, J = 9.0, 6.6 Hz, 2H), 2.31 (t, J = 7.6 Hz, 4H), 1.71 - 1.58 (m, 19H), 1.54-1.51(dd, 8H), 1.44 - 1.36 (m, 4H), 1.28 (d, J = 9.8 Hz, 48H), 0.90 (t, J = 7.0 Hz, 12H). LCMS (ESI): 895.1 [M+H] + .

[0127] [Example 13] ジ(ヘプタデカン-9-イル)6,6'-(((3R,4S)-1-(4-ヒドロキシブチル)ピロリジン-3,4-ジイル)ビス(オキシ))ジヘキサノエート

[0128]

change

[0129] [Example 14] (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(hexyldecanoate)

[0130] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 4.08 (t, J = 6.7 Hz, 4H), 3.90 (q, J = 4.9, 4.4 Hz, 2H), 3.81 (t, J = 5.3 Hz, 2H), 3.54 (dt, J = 9.0, 6.6 Hz, 2H), 3.46 (dt, J = 9.1, 6.6 Hz, 2H), 3.28 (s, 2H), 2.86 (s, 2H), 2.65 - 2.55 (m, 2H), 2.33 (tt, J = 9.0, 5.3 Hz, 2H), 1.75 (p, J = 5.5 Hz, 2H), 1.61 (dddd, J = 16.6, 12.9, 10.0, LCMS (ESI): 839.0 [M+H] + .

[0131] [Example 15] (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0132] [ka] 1H NMR (600 MHz, CDCl3) δ 4.08 (t, J = 6.7 Hz, 4H), 3.96 (s, 2H), 3.67 (t, J = 6.4 Hz, 2H), 3.57 (q, J = 7.2 Hz, 2H), 3.47 (dt, J = 9.1, 6.6 Hz, 2H), 2.33 (tt, J = 9.0, 5.3 Hz, 2H), 1.66 - 1.59 (m, 22H), 1.48 - 1.41 (m, 6H), 1.39 (p, J = 3.5 Hz, 8H), 1.33 - 1.22 (m, 40H), 0.90 (td, J = 7.0, 1.8 Hz, 12H). LCMS (ESI): 867.0 [M+H] + .

[0133] [Example 16] (((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate)

[0134] [ka] 1 H NMR (600 MHz, CDCl 3) δ 4.08 (t, J = 6.7 Hz, 4H), 3.92 (t, J = 4.4 Hz, 2H), 3.59 (d, J = 5.1 Hz, 2H), 3.54 (dt, J = 9.2, 6.6 Hz, 2H), 3.46 (dt, J = 9.1, 6.6 Hz, 2H), 3.26 (s, 2H), 2.63 (s, 2H), 2.59 - 2.48 (m, 2H), 2.33 (tt, J = 9.0, 5.3 Hz, 2H), 1.72 - 1.55 (m, 16H), 1.45 (dddt, J = 11.5, 9.8, 6.0, 3.0Hz, 4H), 1.39 (q, J = 3.7 Hz, 8H), 1.32 - 1.22 (m, 40H), 0.90 (td, J = 7.0, 1.8 Hz, 12H). LCMS (ESI): 853.2 [M+H] + .

[0135] [Example 17] Bis(2-octyldodecyl)5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dipentanoate

[0136] [ka] 1 H NMR (600 MHz, CDCl 3) δ 3.99 (d, J = 5.8 Hz, 4H), 3.90 (t, J = 4.4 Hz, 2H), 3.85 - 3.73 (m, 2H), 3.57 (dt, J = 9.2, 6.3 Hz, 2H), 3.48 (dt, J = 9.2, 6.3 Hz, 2H), 3.27 (s, 2H), 2.85 (t, J = 5.9 Hz, 2H), 2.60 (dd, J = 9.8, 4.9 Hz, 2H), 2.36 (t, J = 7.4 Hz, 4H), 1.78 - 1.68 (m, 6H), 1.65 (dt, J = 9.4, 6.3 Hz, 6H), 1.29 (d, J = 7.9 Hz, 64H), 0.91 (t, J = 7.0 Hz, 12H). LCMS (ESI): 923.2 [M+H] + .

[0137] [Example 18]

[0138] ビス(2-オクチルデシル)5,5'-(((3R,4S)-1-(5-ヒドロキシペンチル)ピロリジン-3,4-ジイル)ビス(オキシ))ジペンタノエート

[0139]

change

[0140] [Example 19] Bis(2-octyldodecyl) 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate

[0141] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 3.98 (d, J = 5.8 Hz, 4H), 3.67 (t, J = 6.5 Hz, 2H), 3.60 (d, J = 8.4 Hz, 2H), 3.54 - 3.41 (m, 2H), 2.35 (t, J = 7.4 Hz, 4H), 1.71 (p, J = 7.2 Hz, 4H), 1.62 (d, J = 18.8 Hz, 22H), 1.40 (s, 4H), 1.29 (d, J = 8.6 Hz, 60H), 0.90 (t, J = 6.9 Hz, 12H). LCMS (ESI): 965.4 [M+H] + .

[0142] [Example 20] Bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate

[0143] [ka] 1 H NMR (600 MHz, CDCl 3) δ 3.98 (d, J = 5.8 Hz, 4H), 3.67 (t, J = 6.5 Hz, 2H), 3.60 (d, J = 8.4 Hz, 2H), 3.54 - 3.41 (m, 2H), 2.35 (t, J = 7.4 Hz, 4H), 1.71 LCMS (ESI): 698.8 [M+H] + .

[0144] [Example 21] ビス(2-ブチルオクチル)5,5'-(((3R,4S)-1-(5-ヒドロキシペンチル)ピロリジン-3,4-ジイル)ビス(オキシ))ジペンタノエート

[0145]

change

[0146] [Example 22] Bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate

[0147] [ka] 1 H NMR (600 MHz, CDCl 3 ) δ 3.99 (d, J = 5.8 Hz, 4H), 3.66 (t, J = 6.5 Hz, 2H), 3.59 (d, J = 7.9 Hz, 2H), 3.49 (dt, J = 9.2, 6.2 Hz, 2H), 2.35 (t, J = 7.4 Hz, 4H), 1.75 - 1.68 (m, 4H), 1.61 (tq, J = 21.1, 6.3 Hz, 20H), 1.39 (d, J = 14.3 Hz, 4H), 1.34 - 1.25 (m, 30H), 0.91 (td, J = 6.7, 5.1 Hz, 12H); LCMS (ESI): 740.9 [M+H] + .

[0148] Lipid nanoparticle (LNP) preparation Firefly luciferase (FLuc) reporter mRNA loaded lipid nanoparticles (LNPs) were formulated by NANOASSEMBLER® IGNITE™. Briefly, a mixture of lipids, cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and 1,2-stearoyl-sn-glycerol-3-phosphocholine (DSPC) was prepared as an ethanolic solution with a molar ratio of 50:38.5:10:1.5. Three units of FLuc mRNA were dissolved in 25 mM sodium acetate (pH 5.0) buffer and rapidly combined with one unit of lipid mixture in a microfluidic mixing cartridge with an amine to phosphate (N / P) ratio constant of 6 and an instrument flow rate set at 12 mL / min. The mRNA-LNPs were diluted 40-fold with calcium and magnesium-free phosphate buffered saline (PBS) to reach pH neutrality. The LNPs were then concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit.

[0149] The mRNA (SEQ ID NO: 1) can be purchased from TRILINK BIOTECHNOLOGIES®, LLC.

[0150] Characterization The mRNA-LNPs were characterized in terms of hydrodynamic size (z-average diameter), zeta potential, and polydispersity index by ZETASIZER™ PRO BLUE (MALVERN PANALYTICAL®). The QUANT-IT™ RIBOGREEN Assay Kit (THERMOFISHER®) was performed to identify the mRNA concentration and encapsulation efficiency. Briefly, the mRNA-LNPs were diluted in an equal volume of Tris / EDTA (TE) buffer or 2% Triton X-100 in TE buffer. Then, RIBOGREEN reagent was added to each sample and incubated for 15 minutes at 37° C. in the absence of light. The fluorescence intensity (Ex / Em 480 / 520 nm) was detected with a TECAN™ Microplate reader. The encapsulation efficiency (EE) can be calculated by the following formula: EE (%) = (total mRNA concentration - non-encapsulated mRNA concentration) / (total mRNA concentration) × 100%

[0151] Furthermore, the acid dissociation constant (pKa) of mRNA-LNPs was determined using 6-(p-toluidino)-2-naphthalenesulfonyl chloride (TNS) binding assay. Several pH range buffers (20 mM citrate buffer, pH 3.0-5.5 range; 20 mM sodium phosphate buffer, pH 6.0-8.0 range; 20 mM Tris buffer, pH 8.5-10.0 range) were prepared. LNPs (0.5 mM total lipid) were mixed with the above buffers and TNS reagent to achieve a final concentration of 6 μM TNS reagent in a total volume of 0.2 mL, and then incubated for 10 min. Fluorescence intensity values ​​were recorded by an Infinite M200 PRO (Ex / Em 321 / 447 nm).

[0152] [Table 1]

[0153] Transfection efficiency and cytotoxicity in HEPG2 cell line To investigate the potency and cytotoxicity of the mRNA-LNPs made from the novel lipids, FLuc mRNA (TRILINK®) was encapsulated in the LNPs and transfected into cell lines. HEPG2 cells were cultured at 37°C, 5% CO 2 The cells were maintained in DMEM medium supplemented with 10% fetal bovine serum at 4 × 10 4Cells / well were seeded and treated with FLuc mRNA-LNP for 18 h while the cells reached 70% confluence. To explore the transfection efficiency, HEPG2 cells were treated with D-luciferin, mixed thoroughly, and incubated at 37°C for another 5 min. Luminescence intensity was recorded using an M200 Microplate Reader (TECAN®). Cell viability was assessed by Cell Counting Kit-8 (SIGMA-ALDRICH®), and absorbance was measured at 450 nm. The results are shown in Table 2.

[0154] [Table 2]

[0155] Compounds 11 and 14 have a toxicity of less than 10%.

[0156] Although the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not intended to be limiting. Those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure, as defined in the appended claims. The illustrations may not necessarily be drawn to scale. Variations between the artistic interpretations in the present disclosure and the actual devices may exist due to manufacturing methods and tolerances. There may be other embodiments of the present disclosure that are not specifically illustrated. The specification and drawings should be considered illustrative rather than binding. Modifications may be made to adapt a particular situation, material, composition, method, or process to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. Although the methods disclosed herein have been described with reference to certain operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or rearranged to form equivalent methods without departing from the teachings of the present disclosure. Thus, unless specifically indicated herein, the order and grouping of the operations is not intended to be a limitation of the disclosure.

Claims

1. A lipid of formula (I) or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof. 【Chemistry 1】 [In the formula, n is 1 or 2; R 1 is amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, R 2 and R 3 are each independently an alkyl, alkenyl, or alkynyl having 5 to 30 carbon atoms; L 1 is an alkyl group having 2 to 6 carbon atoms; L 2 and L 3 are each independently an alkyl group having 2 to 9 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-.

2. n is 1, R 1 is hydroxy, R 2 and R 3 Each independently represents -CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 ) or -(CH 2 ) m CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 wherein m is an integer from 1 to 3, and p and q are each independently an integer from 1 to 12; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently alkyl having 2 to 6 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-; n is 1, R 1 is hydroxy, R 2 and R 3 Each independently represents -CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 ) or -CH 2 CH((CH 2 ) p CH 3 )((CH 2 ) q CH 3 wherein p and q are each independently an integer from 3 to 9; L 1 is alkyl having 2 to 6 carbon atoms; L 2 and L 3 are each independently alkyl having 2 to 6 carbon atoms; X and Y are each independently -C(O)-O- or -OC(O)-; 2. The lipid of claim 1, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof.

3. Lipids, Di(heptadecan-9-yl) 3,3'-(((3R,4S)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate, Di(heptadecan-9-yl) 3,3'-((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))propionate, di(heptadecan-9-yl) 5,5'-(((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, di(heptadecan-9-yl) 5,5'-(((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(2-hydroxyethyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), di(heptadecan-9-yl) 3,3'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dipropionate, (((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(butane-4,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy))bis(pentane-5,1-diyl)bis(2-hexyldecanoate), di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate, di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy)) dihexanoate, di(heptadecan-9-yl) 6,6'-(((3R,4S)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))dihexanoate, (((3S,4R)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(hexyldecanoate), (((3S,4R)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate), (((3S,4R)-1-(4-hydroxybutyl)pyrrolidine-3,4-diyl)bis(oxy))bis(hexane-6,1-diyl)bis(2-hexyldecanoate), Bis(2-octyldodecyl) 5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, bis(2-octyldodecyl) 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, Bis(2-octyldodecyl) 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(3-hydroxypropyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, Bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(5-hydroxypentyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate, and Bis(2-butyloctyl) 5,5'-(((3R,4S)-1-(6-hydroxyhexyl)pyrrolidine-3,4-diyl)bis(oxy)) dipentanoate 2. The lipid of claim 1, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof, selected from the group consisting of:

4. 2. The lipid of claim 1, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer, or isotopically enriched derivative thereof, wherein the protonated form of the lipid has a pKa of about 5.0 to about 9.

0.

5. 5. An active agent delivery vehicle comprising a lipid according to any one of claims 1 to 4, or a pharma- ceutically acceptable salt, solvate, hydrate, polymorph, tautomer, stereoisomer or isotopically enriched derivative thereof.

6. 6. The active agent delivery vehicle of claim 5, selected from the group consisting of liposomes, single emulsions, micelles, nanoemulsions, lipid nanoparticles (LNPs), nanostructured lipid carriers (NLCs), and self-emulsifying drug delivery systems (SEDDSs).

7. 6. The active agent delivery vehicle of claim 5, further comprising one or more lipids selected from the group consisting of neutral lipids, steroid lipids, and PEGylated lipids.

8. Neutral lipids were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-diundecanoyl-sn-glycero-3-phosphatidylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphatidylcholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphatidylcholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine, 1,2-diarachidonoyl-sn-glycero-3-phosphatidylcholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl 8. The active agent delivery vehicle of claim 7, wherein the active agent delivery vehicle is selected from the group consisting of phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoleoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE), and combinations thereof.

9. 8. The active agent delivery vehicle of claim 7, wherein the steroid lipid is selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol tomatidine, ursolic acid, and alpha-tocopherol, and combinations thereof.

10. 8. The active agent delivery vehicle of claim 7, wherein the PEGylated lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-cholesterol, PEG-diacylglycamide (PEG-DAG), and PEG-dialkyloxypropyl (PEG-DAA), and combinations thereof.

11. 6. A composition comprising the active agent delivery vehicle of claim 5 and an active agent.

12. 12. The composition of claim 11, wherein the active agent comprises a nucleic acid, and the nucleic acid is selected from the group consisting of an antisense oligonucleotide, a plasmid, an interfering nucleic acid, an aptamer, a microRNA (miRNA), a Dicer substrate RNA (dsRNA), a small hairpin RNA (shRNA), an mRNA (messenger RNA), a guide RNA (gRNA), an miRNA inhibitor (antagomir), and a ribozyme, and combinations thereof.

13. The composition according to claim 11 for treating or preventing a disease or disorder.

14. 12. A method for introducing a nucleic acid into or transfecting a target cell, comprising contacting the target cell with the composition of claim 11.

15. 12. A method for regulating expression of a target gene in a cell, comprising the step of supplying to the cell a composition according to claim 11.

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