Lipid compounds for gene delivery and uses thereof

Abstract

The invention discloses a lipid compound capable of being used for gene delivery, a preparation method of the lipid compound and application of the lipid compound in gene delivery. Also disclosed herein is a lipid nanoparticle comprising the lipid compound, a gene delivery composition comprising the lipid compound or the lipid nanoparticle. The lipid compounds, lipid nanoparticles and delivery systems herein enable efficient complexation, protection, intracellular and targeted delivery and release of biomolecules, such as oligonucleotides and nucleic acids, in vitro and in vivo tissues and organs.

Description

Cross-references to related applications

[0001] This application claims the benefit of PCT application PCT / CN2023 / 105072, filed on June 30, 2023. The entire contents of the foregoing application are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of gene loading and delivery, specifically relating to lipid compounds that can be used for gene delivery, methods for their preparation, and their use in gene delivery. This disclosure also relates to lipid nanoparticles comprising lipid compounds, and gene delivery compositions comprising lipid compounds or lipid nanoparticles. Background Technology

[0003] Currently, a large number of different types of nucleic acids are being developed as therapeutic agents for a variety of diseases. These nucleic acids include DNA and mRNA in gene therapy, plasmid-based interfering nucleic acids, small interfering nucleic acids (including siRNA and miRNA) for RNA interference (RNAi), antisense molecules, nucleases, and aptamers.

[0004] Effective nucleic acid drug delivery requires the intracellular delivery of therapeutic nucleic acid molecules to target cells. In living organisms, naked nucleic acid molecules are broken down and removed by numerous nucleases. Furthermore, due to the inherently negatively charged nature of nucleic acid molecules, they struggle to directly penetrate negatively charged cell membranes, necessitating specialized delivery systems to protect nucleic acid molecules and facilitate their entry into target cells. Lipid nanoparticles are currently the most efficient carrier form for nucleic acid drug delivery and have been validated in many commercially available drugs, including Covid-19 mRNA vaccines. Classical lipid nanoparticles are formed by encapsulating nucleic acid molecules with four classes of lipids (such as ionizable lipids, cholesterol, neutral phospholipids, and polyethylene glycol (PEG) lipids), with ionizable lipids determining the efficiency of nucleic acid molecule delivery in vitro and in vivo. Delivering therapeutic nucleic acid molecules to their targets is crucial for achieving therapeutic efficacy, but this process is often hampered by the limitations of ionizable lipids in reaching target cells and tissues. Expanding and optimizing the structure of ionizable lipids is essential for entry into targeted tissue cells. This disclosure relates to novel ionizable lipids that facilitate the targeted intracellular delivery of bioactive molecules.

[0005] Examples of bioactive molecules that cannot be effectively targeted to patient tissues include: (1) a variety of proteins, including immunoglobulins; (2) polynucleotides, such as genomic DNA, cDNA, or mRNA; (3) antisense polynucleotides; and (4) many small molecular weight compounds (whether synthetic or naturally occurring), such as peptide hormones and antibiotics.

[0006] A fundamental challenge facing healthcare practitioners today is the vast array of different types of nucleic acids being developed as therapeutics for a wide range of diseases. These nucleic acids include DNA in gene therapy, plasmid-based small interfering nucleic acids (iRNA) for RNA interference (RNAi), antisense molecules, nucleases, antagomir, microRNAs, and aptamers. With the development of these nucleic acids, there is a general need for lipid formulations that are easy to prepare and deliver to target tissues.

[0007] Despite progress in developing novel ionizable lipids that improve mRNA loading and delivery, significant challenges remain in designing novel lipid structures. Traditional ionizable lipid synthesis is characterized by a complex series of steps, including chemical protection and deprotection, catalyst use and removal, solvent exchange, and sophisticated purification processes. These time-consuming processes require customization for each synthetic reaction, limiting both throughput and the exploration of new lipid structures. Combinatorial chemistry holds particular value in the development of ionizable lipids, facilitating the creation of vast libraries through the systematic combination of multiple building blocks. This approach enables researchers to rapidly and efficiently generate diverse libraries of ionizable lipid structures, which can then be screened for their ability to encapsulate mRNA and efficiently deliver it to target cells. This high-throughput method significantly increases the likelihood of discovering more efficient and less toxic ionizable lipids. However, current methods using combinatorial chemistry are often limited in terms of the structural diversity of ionizable lipids generated due to the two-dimensional nature of the reactions employed. Combinatorial chemistry utilizing multi-component reactions (MCRs) is an effective strategy for high-throughput synthesis of ionizable lipids. Notable examples include Ugi-based three-component reactions (3CRs), which rapidly generate combinatorial libraries of ionizable lipids within 24 hours at room temperature, significantly enhancing the speed and diversity of lipid synthesis through their three-dimensional construction capabilities. However, integrating biodegradable bonds into lipid structures requires the custom synthesis of biodegradable lipid tails as Ugi-based 3CR building blocks, adding significant time and effort. Furthermore, this approach often results in unstable and low yields, complicating high-throughput screening. Although recent studies have improved the yield of this platform by incorporating non-toxic catalysts, there remains a pressing need to develop a simple, catalyst-free MCR platform that enables the rapid synthesis of high-yield, diverse biodegradable lipids. Finding suitable MCRs, besides Ugi reactions, that can meet the complex requirements for constructing ionizable lipid structures remains a major challenge in combinatorial chemistry. Summary of the Invention

[0008] To address the problems in the prior art, this disclosure aims to provide a lipid compound and lipid nanoparticles (LNPs) containing the lipid compound, as well as a modular platform for rapidly constructing large-scale, chemically diverse, biodegradable, ionizable lipid libraries using the Passerini reaction.

[0009] According to the technical solution of this disclosure, a novel modular system for synthesizing ionizable lipids using a catalyst-free P-3CR method is provided. This method is based on three key components: a head group containing an ionizable amine and two tail groups, which can combine under mild conditions to form ionizable lipids, thereby achieving high yield, diversity, efficiency, and biodegradability. This method has the advantage of simplifying the synthesis of ionizable lipids. Based on the classic Passellini three-component reaction and its variants, a series of ionizable lipid compounds with various topologies can be generated at higher throughput. The ionizable lipid compounds produced in this disclosure have asymmetric hydrophobic long-chain structures and more flexible molecular configurations, enabling more effective binding to target cell membranes, thereby achieving efficient intracellular delivery of nucleic acids to different target cells. This allows for the efficient intracellular delivery of nucleic acids to different target cells. The LNPs prepared from these lipid compounds have different particle sizes, and the delivery efficiency of nucleic acid molecules shows different trends for different cell types. The lipid compounds of this disclosure have α-acylamide binding characteristics, which enhances physiological biodegradability and reduces potential adverse reactions, thus making them suitable for multi-dose regimens.

[0010] In one aspect, this disclosure provides a lipid compound of formula (I): (I) Or its N-oxide, stereoisomer or pharmaceutically acceptable salt, wherein R I R II and R III Each is independently selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety, and (B) optionally substituted C6-C 25 A collection of aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

[0011] A lipid compound of formula (I'): (I') Or its N-oxide, stereoisomer or pharmaceutically acceptable salt, wherein R I R II and R III Each is independently selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety, and (B) optionally substituted C6-C 25A collection of aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, and R II' It is hydrogen or C1-C6 alkyl, such as C1-C4 alkyl, methyl, ethyl, propyl or butyl.

[0012] In some implementation schemes, R I R II R III One or both of them are selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety.

[0013] In some implementation schemes, R I R II R III Only one of them is selected from (A) a set of groups containing at least one ionizable tertiary amine moiety.

[0014] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia): (Ia) Where R Ia The C1-C6 alkylene group is optionally substituted, wherein the alkylene group is optionally replaced by 1, 2, or 3 ions selected from -oxo (=O), -OH, -SH, or -NR. Id R Id' The substituents are replaced by R, where R is a substituent. Id and R Id' Each is independently hydrogen or C1-C3 alkyl; R Ib and R Ic Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie R Ie' The substituents are replaced by R, where R is a substituent. Ie and R Ie' Each is independently hydrogen or C1-C3 alkyl, or R Ib and R Ic Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0015] In some implementation schemes, R Ib and R IcTogether with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib and R Ic Together with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib and R Ic Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0016] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia'): (Ia') Where Ar is C6-C that is optionally substituted. 10 arylene, for example, phenylene, wherein the arylene is optionally surrounded by 1, 2, 3 or 4 independently selected from halogen, C1-C6 alkyl, -OH, -SH or -NR Id'' R Id''' The substituents are replaced by R, where R is a substituent. Id'' and R Id''' Each is independently hydrogen or C1-C3 alkyl; R Ib' and R Ic' Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie'' R Ie''' The substituents are replaced by R, where R is a substituent. Ie'' and R Ie''' Each is independently hydrogen or C1-C3 alkyl, or R Ib' and R Ic'Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0017] In some implementation schemes, R Ib' and R Ic' Together with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib' and R Ic' Together with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib' and R Ic' Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0018] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in assembly (A) is a 5- to 12-membered heterocyclic group comprising one, two, or three heteroatoms independently selected from N, O, and S, wherein at least one heteroatom is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH. The 5- to 12-membered heterocyclic group may be saturated or unsaturated and may be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc.

[0019] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0020]

[0021] .

[0022] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0023]

[0024]

[0025] .

[0026] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety: .

[0027] In some implementations, set (B) includes C6-C that are optionally replaced. 25 Aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, wherein the aliphatic groups and heteroaliphatic groups optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 independently selected from -C=C-, -C≡C-, -NR m -、-NH-、-NH2、-OH、-OR n -O-, -C(O)-, -C(OR) o -, -C(O)O-, -SH, -SR p -S-, -C(S)-, -C(SR) q The group consisting of -, -C(S)O- and -P(O)- groups, wherein R m R n R o R p and R q Each independently represents the C1-C that are optionally replaced. 14 Aliphatic groups.

[0028] In some implementations, set (B) comprises the following groups:

[0029]

[0030]

[0031] .

[0032] In some implementations, set (B) comprises the following groups: .

[0033] In some implementation schemes, R I Selected from set (A), and R II and R III Selected from set (B), where R II and R III They can be the same or different.

[0034] In some implementation schemes, R II Selected from set (A), and R I and R III Selected from set (B); where R I and R III They can be the same or different.

[0035] In some implementation schemes, R III Selected from set (A), and R I and R II Selected from set (B), where R I and R II They can be the same or different.

[0036] In some implementation schemes, R I R II or R III Both of them are selected from set (B), each of which independently and optionally contains at least one degradable portion.

[0037] In some implementation schemes, R I R II or R III Both of them are selected from set (B), and at least one of them contains at least one degradable part.

[0038] In some implementation schemes, R I R II or R III The two in the set (B) are selected from the set, and only one of the two contains a degradable part.

[0039] In some embodiments, the lipid compounds disclosed herein are selected from the compounds listed in Tables 1, 2, 3 and / or 4.

[0040] In another aspect, this disclosure provides a method for preparing a lipid compound or its N-oxide, stereoisomer, or pharmaceutically acceptable salt thereof using the Passerini reaction, wherein the prepared lipid compound is used to prepare lipid nanoparticles for gene delivery.

[0041] In some implementations, the lipid compound is an ionizable lipid or a cationic lipid.

[0042] In another aspect, this disclosure provides a method for preparing the lipid compound of the present disclosure or its N-oxide, stereoisomer, or pharmaceutically acceptable salt, the method comprising the following synthetic route: , Where R I R II and R III As defined in this article.

[0043] This disclosure also provides a method for preparing the lipid compound of the present disclosure or its N-oxide, stereoisomer, or pharmaceutically acceptable salt thereof, the method comprising the following synthetic route: , Where R I R II R II' and R III The definition is as described in this article.

[0044] In another aspect, this disclosure provides the use of the lipid compounds of the disclosure or their N-oxides, stereoisomers or pharmaceutically acceptable salts in gene delivery. In another aspect, this disclosure provides the use of the lipid compounds of the disclosure or their N-oxides, stereoisomers or pharmaceutically acceptable salts in the preparation of lipid nanoparticles.

[0045] In another aspect, this disclosure provides a lipid nanoparticle comprising the lipid compound of the disclosure or its N-oxide, stereoisomer or pharmaceutically acceptable salt thereof, the lipid nanoparticle being assembled with one or more lipids selected from the group consisting of phospholipids, structural lipids and PEG lipids.

[0046] In another aspect, this disclosure provides a delivery system in which the lipid nanoparticles of this disclosure are used as a delivery medium in the delivery system. In some embodiments, the delivery system also includes an active pharmaceutical ingredient encapsulated in the lipid nanoparticles.

[0047] In some implementations, the active pharmaceutical ingredient is a nucleic acid.

[0048] In some implementations, the active pharmaceutical ingredient is a plasmid.

[0049] In another aspect, this disclosure provides a pharmaceutical composition comprising the lipid compound of the disclosure or its N-oxide, stereoisomer or pharmaceutically acceptable salt, or the lipid nanoparticle of the disclosure, or the delivery system of the disclosure, and a pharmaceutically acceptable carrier or excipient.

[0050] In one aspect, this disclosure relates to a method for preparing a library of lipid compounds. The method includes the following steps: i) Prepare a library of reactant components, including: a library of isonitriles, a library of aldehydes, and a library of carboxylic acids; ii) Select i compounds from the isonitrile compound library and label them as Iso-1, Iso-2, Iso-3...Iso-i, where i is an integer; Select j compounds from the aldehyde compound library and label them Alde-1, Alde-2, Alde-3...Alde-j, where j is an integer; Select k compounds from this carboxylic acid compound library and label them as Acid-1, Acid-2, Acid-3...Acid-k, where k is any integer; iii) Prepare a reaction vessel matrix, labeling each vial as vial-ijk, where i represents the sequence number of the isonitrile compound, j represents the sequence number of the aldehyde compound, and k represents the sequence number of the carboxylic acid compound; iv) Add the isonitriles, aldehydes, and carboxylic acid compounds to the corresponding numbered vials containing the solvent, according to their labels; v) Initiate the Passerini reaction, lasting from 1 hour to 48 hours; vi) After the reaction is complete, collect the products to obtain a compound in quantity equal to i. j k's lipid compound library.

[0051] In some implementations, the lipid compound is an ionizable lipid or a cationic lipid.

[0052] In some implementations, the reaction takes place for 1 to 48 hours, for example 12 to 36 hours, preferably 18 to 30 hours, more preferably 20 to 26 hours, for example 20, 21, 22, 23, 24, 25, 26 hours, or any value within the range described above.

[0053] In some embodiments, the solvent is selected from THF, DCM, toluene, EtOH, or mixtures thereof.

[0054] In some implementations, the reactant addition process can be performed manually or by automated equipment. Automated equipment can be programmed to add reactants automatically according to the manufacturer's instructions.

[0055] In some implementations, the ratio of isonitrile compound, aldehyde compound and carboxylic acid compound is 1:1:1.

[0056] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (A) as defined in this article.

[0057] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this article.

[0058] In some implementations, the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (A) as defined in this article.

[0059] In some implementations, the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (B) as defined in this article.

[0060] In some implementations, the carboxylic acid compound library is composed of general formula R III -COOH represents the library of compounds, where R III Selected from set (A) as defined in this article.

[0061] In some implementations, the carboxylic acid compound library is composed of general formula R III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0062] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I The aldehyde library is selected from the set (A) as defined in this paper; the aldehyde compound library is composed of the general formula R. II -CHO represents a library of compounds, where R... II Selected from set (B) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0063] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this paper; the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (A) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0064] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this paper; the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (B) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (A) as defined in this article.

[0065] In some implementations, the method further includes a step of purifying the product.

[0066] In one aspect, this disclosure relates to a library of lipid compounds prepared by the methods of this disclosure.

[0067] In some embodiments, the lipid compounds in the library have a structure as shown in formula (I): (I) Where R I R II and R III As defined in this article.

[0068] In some embodiments, the lipid compounds in this library have a structure as shown in formula (I'): (I') Where R I R II R II' and R III As defined in this article.

[0069] In one aspect, this disclosure relates to a method for screening lipid compounds, the method comprising the following steps: When preparing a lipid compound library using the method of this disclosure, step vi) of collecting the product after the reaction is complete is omitted. 1) Add non-cationic lipids (e.g., phospholipids), conjugated polymer lipids (e.g., PEG-lipids) to prevent particle aggregation, and optionally structural lipids (e.g., cholesterol) to a reaction vessel to formulate LNPs (lipid nanoparticles) with the prepared ionizable lipids. 2) Prepare an aqueous phase containing nucleic acids (e.g., mRNA); 3) Mix the LNP obtained in step (1) with an aqueous phase containing nucleic acids; 4) The obtained LNP-encapsulated nucleic acids were characterized in vitro or in vivo. Attached Figure Description

[0070] Figures 1 to 2 The relative in vitro transfection efficiencies of lipids 1 through 196 in A549 cells are shown. All data were normalized to reflect the luminescence signal from a control group treated with naked mRNA.

[0071] Figure 3 A classic formulation for preparing mRNA-LNP is shown.

[0072] Figure 4 A schematic diagram of an in vivo orthogonal batch test of LNPs from the P-3CR library via intramuscular (IM) injection is shown.

[0073] Figures 5 to 6 The results show that analysis was performed using batch 1 (0.32 mg / kg per mouse). -1 mLuc (a mixture of 16 LNPs, n=2) identified the best-performing head group.

[0074] Figure 7 The results showed that through batch 2 studies (0.08 mg / kg per mouse) -1 A best-performing lipid tail (A4) was identified in mLuc (a mixture of four LNPs, n=2) and was further evaluated in a batch 3 study (0.05 mg / kg per mouse). -1 mLuc, n=2) identifies the single lipid with the best transfection efficiency.

[0075] Figure 8 Histogram analysis of batches 2 and 3 is shown.

[0076] Figure 9 The results showed a comparison with the baseline MC3 (0.05 mg / kg per mouse). -1 The final comparison was performed using mLuc(n=3). Statistical significance was assessed using a two-tailed Student's t-test. =p value < 0.05, =p value < 0.01, =p value < 0.005. Data are expressed as mean ± SEM.

[0077] Figure 10 This demonstrates Cre-mediated gene delivery in Ai9 reporter mice. mCreLNP (0.5 mg / kg per mouse) was administered intravenously to Ai9 mice. -1 mCre (n=3). Liver cells were collected and analyzed by flow cytometry.

[0078] Figure 11 IVIS imaging of Ai9 cre reporter gene mice is shown. (a) Cg-Gt(ROSA)26Sor after intravenous injection of cre-mRNA LNP. tm9(CAG-tdTomato)Hze IVIS images of organs from Cre reporter mice (0.5 mg / kg) -1 (b) Comparison of relative fluorescence intensity in the liver after intravenous injection of cre-mRNA LNP. (tdTomato: Ex: 568nm, Em: 581nm).

[0079] Figure 12 Representative fluorescence images of liver sections are shown (channels: tdTomato and DAPI, scale bar: 250 μm).

[0080] Figure 13 Histogram analysis results of fluorescence images quantified using ImageJ are shown.

[0081] Figure 14 It shows tdTomato + Quantitative results of hepatocytes, endothelial cells, macrophages, dendritic cells, B cells, and T cells (n=3). Detailed Implementation

[0082] Reference will now be made in detail to certain embodiments, examples of which are shown in the appended detailed descriptions. While the enumerated embodiments will be described, it should be understood that they are not intended to limit this disclosure to those embodiments. Rather, this disclosure is intended to cover all alternatives, modifications, and equivalents that can be included within the scope of this disclosure as defined by the claims. Those skilled in the art will recognize many methods and materials similar to or equivalent to those described and materials herein, which can be used in the practice of this disclosure. This disclosure is by no means limited to the methods and materials described. In the event of any discrepancy or contradiction between this disclosure and one or more incorporated documents and similar materials, including but not limited to defined terminology, usage of terms, described techniques, etc., this disclosure shall prevail.

[0083] It should be understood that, for clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of this disclosure described in the context of a single embodiment may also be provided individually or in any suitable sub-combination.

[0084] definition Terms used herein but not defined have their usual meaning, and the meaning of such terms is independent each time they appear. However, unless otherwise stated, the following definitions apply throughout the specification and claims.

[0085] As used herein, unless the contrary is explicitly stated, the singular forms “a”, “an”, and “the” include plural references.

[0086] As used herein, the terms “comprising” and “including” are intended to indicate the presence of the stated feature, whole, component or step, but do not exclude the presence or addition of one or more other features, wholes, components, steps or combinations thereof.

[0087] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the CAS version of the periodic table of elements as presented on the inner cover of the 75th edition of the *Handbook of Chemistry and Physics*, and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are described in the following references: “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, “March's Advanced Organic Chemistry,” 5th edition, John Wiley & Sons, Inc., New York, 2001; Larock, “Comprehensive Organic Transformations,” VCH Publishers, Inc., New York, 1989; Carruthers, “Some Modern Methods of Organic Synthesis,” 3rd edition, Cambridge University Press, Cambridge, 1987.

[0088] Unless otherwise expressly stated, all scopes referenced in this article are inclusive. When listing a range of values, it is intended to cover every value within that range and its subranges. For example, "C 1-6 "Aims to cover C1, C2, C3, C4, C5, C6, C 1-‍6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 For example, a heteroaromatic ring described as containing "1 to 4 heteroatoms" means that the ring may contain 1, 2, 3, or 4 heteroatoms. It should also be understood that any scope cited herein includes all subscopes within that scope. Thus, for example, a heterocycle described as containing "1 to 4 heteroatoms" covers aspects including: heterocycles containing 2 to 4 heteroatoms, 3 or 4 heteroatoms, 1 to 3 heteroatoms, 2 or 3 heteroatoms, 1 or 2 heteroatoms, 1 heteroatom, 2 heteroatoms, 3 heteroatoms, or 4 heteroatoms.

[0089] When any variable appears more than once in any component, in formula (I), or in any other formula describing and depicting the compounds of this disclosure, the definition of that variable at each occurrence is independent of its definition at each other occurrence. Furthermore, combinations of substituents and / or variables are permitted only if such combinations produce stable compounds.

[0090] As used herein, the term "hydrocarbon group" refers to a chemical group containing hydrogen and carbon. Hydrocarbon groups can be substituted or unsubstituted. Hydrocarbon groups can be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic, and include alkyl, alkenyl, and alkynyl groups, etc. Hydrocarbon groups can be fully saturated, monounsaturated, or polyunsaturated, and can include groups having a specified number of carbon atoms (i.e., C1-C1). 10 Hydrocarbon groups (representing 1 to 10 carbon atoms, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbon atoms) are divalent and polyvalent groups. When a hydrocarbon group contains heteroatoms such as N, O, or S, it is also called a "heterohydrocarbon group".

[0091] As used herein, the term "alkyl" refers to a linear or branched saturated hydrocarbon group. The term "C"... i-j"Alkyl" refers to an alkyl group having i to j carbon atoms. Unless otherwise stated, an alkyl group may contain 1 to 10 carbon atoms. In some embodiments, the alkyl group contains 1 to 6 carbon atoms (C1 to J2). 1-6 ), such as 1 to 5 carbon atoms (C 1-5 ), 1 to 4 carbon atoms (C 1-4 ), 1 to 3 carbon atoms (C 1-3 ) or 1 to 2 carbon atoms (C 1-2 Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl, neopentyl, etc. Where valence allows, the alkyl group may optionally be substituted by one, two, three, or (in the case of an alkyl group having two or more carbons) four or more substituents (i.e., unsubstituted or substituted), which are independently selected from the group consisting of: amino; alkoxy; aryl; aryloxy; azide; cycloalkyl; cycloalkyloxy; cycloalkenyl; cycloalkynyl; halogen; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxyl; nitro; thiol; silyl; cyano; alkyl mercapto; alkylsulfonyl; alkylsulfinyl; alkylsulfenyl; =O; =S; -C(O)R or -SO2R, where R is amino; and =NR', where R' is H, alkyl, aryl, or heterocyclic. Each substituent may be unsubstituted, or, where valence allows, substituted with an unsubstituted substituent as defined herein for each respective group. In some embodiments, the alkyl group may optionally be selected from halogens, C... 1-4 Alkyloxy, C 1-4 Halogenated alkyloxy groups and C 1-4 It is substituted by one or more substituents of a haloalkyl mercapto group.

[0092] As used herein, the term "alkylene" refers to a divalent substituent, which is a monovalent alkyl group in which a hydrogen atom is replaced by a valence bond. Alkylene groups can be unsubstituted or substituted. Optionally substituted alkylenes are those that are optionally substituted as described herein with respect to alkyl groups.

[0093] As used herein, the term "alkenyl" refers to a linear or branched hydrocarbon radical having at least one (such as one, two, or three) carbon-carbon double bonds, which may optionally be independently substituted (i.e., unsubstituted or substituted) by one or more substituents as described herein, and includes radicals having "cis" and "trans" orientations or alternatively "E" and "Z" orientations. Unless otherwise stated, an alkenyl group may contain 2 to 10 carbon atoms. In some embodiments, an alkenyl group may contain 2 to 6 carbon atoms, such as 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. In some embodiments, an alkenyl group contains 2 carbon atoms. Non-limiting examples of alkenyl groups include ethylenyl, propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, etc. Optionally substituted alkenyl groups are those optionally substituted as described herein with respect to alkyl groups.

[0094] As used herein, the term "alkenyl" refers to a divalent substituent, which is a monovalent alkenyl group in which a hydrogen atom is replaced by a valence bond. Alkenyl groups can be unsubstituted or substituted. Optionally substituted alkenyl groups are those optionally substituted as described herein with respect to alkyl groups.

[0095] As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon radical having at least one (such as one, two, or three) carbon-carbon triple bonds, which may optionally be independently substituted (i.e., unsubstituted or substituted) by one or more substituents as described herein. Unless otherwise stated, an alkynyl group may contain 2 to 10 carbon atoms. In some embodiments, the alkynyl group may contain 2 to 6 carbon atoms, such as 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. In some embodiments, the alkynyl group contains 2 carbon atoms. Non-limiting examples of alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, etc. Optionally substituted alkynyl groups are those optionally substituted as described herein with respect to alkyl groups.

[0096] As used herein, the term "ynynyl" refers to a divalent substituent, which is a monovalent ynyl group in which a hydrogen atom is replaced by a valence bond. The ynynyl group can be unsubstituted or substituted. Optionally substituted ynynyl groups are those optionally substituted as described herein with respect to alkyl groups.

[0097] As used herein, the term "cycloalkyl" refers to a partially or fully saturated monocyclic or polycyclic carbon ring, which may include fused rings (where the cycloalkyl is bonded by non-aromatic ring atoms when fused with an aryl or heteroaryl ring), spirocyclic, or bridged ring systems. In some embodiments, the cycloalkyl is fully saturated. Unless otherwise stated, the cycloalkyl group may contain 3 to 10 cyclic carbon atoms. In some embodiments, the cycloalkyl group may contain 3 to 8 cyclic carbon atoms, such as 3 to 7 cyclic carbon atoms, 3 to 6 cyclic carbon atoms, 3 to 5 cyclic carbon atoms, 3 to 4 cyclic carbon atoms, 3 cyclic carbon atoms, 4 cyclic carbon atoms, 5 cyclic carbon atoms, 6 cyclic carbon atoms, 7 cyclic carbon atoms, 8 cyclic carbon atoms, etc. In particular, the cycloalkyl group may be monocyclic or bicyclic. Alternatively, bicyclic cycloalkyl groups may include fused, spirocyclic, and bridged cycloalkyl structures. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, 1-bicyclo[2.2.1.]heptyl, 2-bicyclo[2.2.1.]heptyl, 5-bicyclo[2.2.1.]heptyl, 7-bicyclo[2.2.1.]heptyl and decahydronaphthyl. The cycloalkyl group may optionally be replaced (i.e., unsubstituted or substituted) by one, two, three, four, or five independently selected substituents from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkyl mercapto; alkyl sulfinyl; alkyl thio; alkyl sulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkyloxy; cycloalkenyl; cycloalkynyl; halogen; heteroalkyl; heteroalkenyl; heteroalkynyl; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxyl; nitro; thiol; silyl; cyano; =O; =S; -SO2R, where R is an optionally substituted amino group; =NR', where R' is H, alkyl, aryl, or heterocyclic; and -CON( )2, of which each Independently H or alkyl, or both Together with the atoms to which they are attached, they form heterocyclic groups. Each substituent may be unsubstituted itself or substituted by an unsubstituted substituent as defined herein for each respective group. In some embodiments, the cycloalkyl groups may optionally be selected from C10. 1-4 Alkyl, halogen, C 1-4 Alkyloxy, C 1-4 Halogenated alkyloxy groups and C 1-4 It is substituted by one or more substituents of a haloalkyl mercapto group.

[0098] As used herein, the term "cycloalkylene" refers to a divalent substituent in a cycloalkyl group where a hydrogen atom is replaced by a valence bond. Cycloalkylene groups can be unsubstituted or substituted. Optionally substituted cycloalkylene groups are those that are optionally substituted as described herein with respect to cycloalkylene groups.

[0099] As used herein, the term "heterocyclic group" refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused, bridged, and / or spirocyclic 3- to 12-membered rings, which, unless otherwise specified, contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur as cyclic atoms. In some embodiments, the heterocyclic group may be 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered. In some embodiments, the heterocyclic group may be 3- to 9-membered, 3- to 8-membered, 3- to 6-membered, 4- to 10-membered, 4- to 8-membered, 4- to 6-membered, 5- to 12-membered, 5- to 10-membered, or 5- to 8-membered. In some embodiments, the heterocyclic group may contain one, two, or three heteroatoms. In some embodiments, the heterocyclic group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system having fused or bridged 5-, 6-, 7-, or 8-membered rings, the ring system containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The heterocyclic group can be aromatic or non-aromatic. In some embodiments, the heterocyclic group is non-aromatic. In some embodiments, the non-aromatic 5-membered heterocyclic group has zero or one double bond, the non-aromatic 6- and 7-membered heterocyclic groups have zero to two double bonds, and the non-aromatic 8-membered heterocyclic group has zero to two double bonds and / or zero or one carbon-carbon triple bond. In some embodiments, the heterocyclic group is a saturated ring. In some embodiments, the heterocyclic group can contain up to nine carbon atoms. Non-aromatic heterocyclic groups include pyrrolinyl, pyrrolylalkyl, pyrazolinyl, pyrazolylalkyl, imidazolinyl, imidazolinyl, piperidinyl, homopiperidinyl, piperazinyl, pyridazinyl, oxazolyl, isoxazolyl, morpholinyl, thiomorpholinyl, thiazolinyl, isothiazolyl, thiazolinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, dihydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyranyl, dihydropyranyl, dithiazolinyl, etc. If a heterocyclic ring system has at least one aromatic resonance structure or at least one aromatic tautomer, then this structure is an aromatic heterocyclic group (i.e., a heteroaryl). Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothiophene, benzooxazolyl, furanyl, imidazolyl, indolyl, isoinzolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purine, pyrroleyl, pyridyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, thiadiazolyl (e.g., 1,3,4-thiadiazolyl), thiazolyl, thiophene, triazolyl, tetrazolyl, etc. The term "heterocyclic group" also includes heterocyclic compounds having a bridging polycyclic structure, wherein one or more carbons and / or heteroatoms bridge two non-adjacent members of a monocyclic ring, such as quinine rings, tropane, or diazabicyclo[2.2.2]octane.The term "heterocyclic group" includes bicyclic, tricyclic, and tetracyclic groups, wherein any of the aforementioned heterocycles is fused with one, two, or three carbocyclic rings or another monocyclic heterocycle, such as aromatic rings, cyclohexane rings, cyclohexene rings, cyclopentane rings, and cyclopentene rings. Examples of fused heterocyclic groups include 1,2,3,5,8,8a-hexahydroindoleazine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, and 2,3-dihydrobenzothiophene. The heterocyclic group may be unsubstituted or substituted with one, two, three, four, or five substituents, which are independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halogen; heteroalkyl; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxyl; nitro; thiol; silyl; cyano; -C(O)R or -SO2R, wherein R is amino or alkyl; =O; =S; =NR', wherein R' is H, alkyl, aryl, or heterocyclic. Each substituent may itself be unsubstituted or substituted with an unsubstituted substituent as defined herein for each respective group. In some embodiments, the heterocyclic group may optionally be substituted with one or more substituents selected from 4- to 10-membered heterocyclic groups, 6- to 10-membered aryl groups, and 5- to 10-membered heteroaryl groups.

[0100] As used herein, the term "hypo-heterocyclic group" refers to a divalent substituent in which a hydrogen atom is replaced by a valence bond in a heterocyclic group. Hypo-heterocyclic groups can be unsubstituted or substituted. Optionally substituted hypo-heterocyclic groups are those that are optionally substituted as described herein with respect to heterocyclic groups.

[0101] As used herein, the term "aryl" refers to a monocyclic, bicyclic, or polycyclic carbocyclic ring system having at least one aromatic ring. Unless otherwise stated, aryl groups can be 6- to 10-membered. In some embodiments, the aryl group may contain six cyclic carbon atoms. All cyclic atoms within the carbocyclic aryl group are carbon atoms. Non-limiting examples of aryl groups include phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indene, indene, etc. In some embodiments, the aryl group is phenyl or naphthyl. In some embodiments, the aryl group is phenyl. In the context of this specification, the terms "aryl" and "aromatic ring" are used interchangeably. Aryl groups can be unsubstituted or substituted. The optionally substituted aryl group can be an aryl group optionally substituted with one, two, three, four, or five substituents, which are independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halogen; heteroalkyl; heteroalkenyl; heteroalkynyl; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxyl; nitro; thiol; silyl; -(CH2) n -C(O)OR'; -C(O)R; and -SO2R, where R is amino or alkyl, R' is H or alkyl, and n is 0 or 1. Each substituent may be unsubstituted or substituted by an unsubstituted substituent as defined herein for each respective group. In some embodiments, the aryl group may optionally be substituted by one or more substituents selected from 4- to 10-membered heterocyclic groups, 6- to 10-membered aryl groups, and 5- to 10-membered heteroaryl groups.

[0102] As used herein, the term "arylene" refers to a divalent substituent in which a hydrogen atom of an aryl group is replaced by a valence bond. Arylene groups can be unsubstituted or substituted. Optionally substituted arylene groups are those that are optionally substituted as described herein with respect to aryl groups.

[0103] As used herein, the term "heteroaryl" refers to a monocyclic ring system, or a fused or bridged bicyclic ring system, wherein the ring system contains one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur; and at least one ring is an aromatic ring. Unless otherwise stated, heteroaryl groups can be 5- to 10-membered. In some embodiments, the heteroaryl group can be a 5- to 6-membered heteroaryl ring having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, and sulfur; or an 8- to 10-membered bicyclic heteroaryl ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl group may contain one, two, or three heteroatoms. In some embodiments, the heteroaryl group may contain one or two heteroatoms. Non-limiting examples of heteroaryl groups include benzimidazolyl, benzofuranyl, benzothiazolyl, benzothiophene, benzooxazolyl, furanyl, imidazolyl, indolyl, isoindazolyl, isoquinolinyl, isothiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, purine, pyrrole, pyridyl, pyrazinyl, pyrimidinyl, quinazolinyl, quinolinyl, thiadiazolyl, thiazolyl, thiophene, triazolyl, tetrazolyl, dihydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc. A heteroaryl group comprises at least one ring having at least one heteroatom as described above and at least one aromatic ring. For example, the ring having at least one heteroatom may be fused with one, two, or three carbon rings or another monocyclic heterocycle, such as aryl rings, cyclohexane rings, cyclohexene rings, cyclopentane rings, and cyclopentene rings. Non-limiting examples of fused heteroaryl groups include 1,2,3,5,8,8a-hexahydroindoleazine, 2,3-dihydrobenzofuran, 2,3-dihydroindole, 2,3-dihydrobenzothiophene, etc. In the context of this disclosure, the terms "heteroaryl" and "heteroaryl ring" are used interchangeably. A heteroaryl group can be unsubstituted or substituted. Optionally substituted heteroaryl groups can be heteroaryl groups optionally substituted by one, two, three, four, or five substituents independently selected from the group consisting of: alkyl; alkenyl; alkynyl; alkoxy; alkylsulfinyl; alkylthio; alkylsulfonyl; amino; aryl; aryloxy; azide; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halogen; heteroalkyl; heteroalkenyl; heterokynyl; heterocyclic; (heterocyclic)oxy; heteroaryl; hydroxyl; nitro; thiol; silyl; -(CH2) n -C(O)OR'; -C(O)R; and -SO2R, where R is amino or alkyl, R' is H or alkyl, and n is 0 or 1. Each substituent may be unsubstituted or substituted by an unsubstituted substituent as defined herein for each respective group. In some embodiments, the heteroaryl group may optionally be substituted by one or more substituents selected from 4- to 10-membered heterocyclic groups, 6- to 10-membered aryl groups, and 5- to 10-membered heteroaryl groups.

[0104] As used herein, the term "heteroaryl" refers to a divalent substituent in which a hydrogen atom is replaced by a valence bond in a heteroaryl group. Heteroaryl groups can be unsubstituted or substituted. Optionally substituted heteroaryl groups are those that are optionally substituted as described herein with respect to heteroaryl groups.

[0105] As used herein, the term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, as well as any quaternized form of basic nitrogen.

[0106] As used in this article, the term "oxo" refers to a divalent oxygen atom, and the structure of an oxo atom can be represented as =O.

[0107] As used herein, the term "halogen" (or "halogenated") refers to fluorine, chlorine, bromine, and iodine. In some embodiments, non-limiting examples of halogens include fluorine, chlorine, and bromine. In some embodiments, the halogen is chlorine or bromine. In some embodiments, the halogen is fluorine.

[0108] As used herein, the term "haloalkyl" refers to an alkyl group as described herein, wherein one or more hydrogen atoms have been replaced by one or more halogen atoms selected from the group consisting of fluorine, chlorine, bromine, and iodine. Non-limiting examples of haloalkyl groups include -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, etc. In some embodiments, the haloalkyl group may be a perhaloalkyl group, such as a perfluoroalkyl group.

[0109] As used herein, the term "haloalkylene" refers to a divalent substituent in which a hydrogen atom is replaced by a valence bond in a haloalkyl group. Non-limiting examples of haloalkylene groups include -CH2CHF-, -CHFCHF-, etc. In some embodiments, the haloalkylene group may be a perhaloalkylene group, such as a perfluoroalkylene group. In some embodiments, the two valence bonds of the haloalkylene group are connected to the same atom of other parts (optionally ring parts) to form a double bond, such as =CHCH2F, =CHCHF2, and =CFCHF2.

[0110] As used herein, the term "substituted" when referring to a chemical group means that the chemical group has one or more hydrogen atoms that have been removed and replaced by substituents. As used herein, the term "substituent" has its usual meaning as known in the art and refers to a chemical moiety covalently connected to or (if appropriate) fused with a parent group. It should be understood that substitution at a given atom is limited by valence. It should be understood that substituents may be further substituted.

[0111] As used herein, the term "optionally substituted" means that a chemical group may have no substituents (i.e., unsubstituted) or may have one or more substituents (i.e., substituted). It should be understood that substitution at a given atom is restricted by valence.

[0112] The compounds provided herein are described with reference to both general formulas and specific compounds. Furthermore, the compounds of this disclosure may exist in a variety of different forms or derivatives, all of which are within the scope of this disclosure. These forms or derivatives include, for example, pharmaceutically acceptable salts, tautomers, stereoisomers, racemic mixtures, regioisomers, prodrugs, and active metabolites. In some embodiments, the compounds of this disclosure may contain rotationally blocked bonds, allowing two separate rotational isomers or transisomers to be separable and potentially possessing advantageous biological activity. This disclosure is intended to include all possible transisomers within its scope.

[0113] As used herein, the term "restricted rotation isomer" refers to a stereoisomer resulting from restricted rotation around a single bond, where the rotational barrier is high enough to allow the separation of isomers. Typically, rotation around the single bond is restricted or greatly slowed due to spatial interactions of other parts of the molecule and asymmetry of substituents at both ends of the single bond. As used herein, the terms “rich in…transisomer” or “transisomer enrichment” mean that the compound (i.e., a mixture of transisomers) contains a greater proportion or percentage of the specified transisomer of the compound relative to other transisomers, i.e., greater than 50 mol%, such as greater than 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, 95 mol%, 98 mol%, 99 mol%, etc. In some embodiments, transisomers other than the specified transisomer are undetectable. In some embodiments, the compound may contain close to 100 mol% or 100 mol% of the specified transisomer of the compound. In some embodiments, the compound is substantially transisomer-pure. As used herein, the term “substantially pure” means that the compound (i.e., a mixture of transisomers) contains at least 90 mol%, optionally at least 95 mol%, more optionally at least 98 mol%, and even more optionally at least 99 mol% of one transisomer. The term "substantially free" means that a compound contains less than 10 mol%, optionally less than 5 mol%, more optionally less than 2 mol%, and even more optionally less than 1 mol% of a transisomer. As used herein, unless otherwise stated, the term "pharmaceutically acceptable salt" includes salts that retain the biological efficacy of a particular compound in its free acid / base form without any adverse biological or other effects. Intended pharmaceutically acceptable salt forms include, but are not limited to, single, double, triple, tetrasalts, and so on. Pharmaceutically acceptable salts are non-toxic at the amount and concentration at which they are administered. The preparation of such salts can facilitate pharmacological applications by altering the physical properties of the compound without impairing its physiological effects. Useful alterations to physical properties may include, for example, increasing solubility to facilitate the administration of higher concentrations of the drug.

[0114] Pharmaceutically acceptable salts of compounds of formula (I) include acid addition salts and base salts. Suitable acid addition salts can be formed from acids that form non-toxic salts. Non-limiting examples may include acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, bisulfates / sulfates, borates, camphorsulfonates, citrates, cyclohexylamine sulfonates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, glucuronates, hexafluorophosphates, hymenates, hydrochlorides / chlorides, hydrobromates / bromines, hydroiodates / iodides, hydroxyethylsulfonates, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfates, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, dihydroxynaphthalates, phosphates / hydrogen phosphates / dihydrogen phosphates, pyroglutamates, glycosides, stearates, succinates, tannates, tartrates, toluenesulfonates, trifluoroacetates, 1,5-naphthalenedisulfonates, and sinetes. Suitable alkali salts are formed from bases that form non-toxic salts. Non-limiting examples may include aluminum salts, arginine salts, benzyl benzoate salts, calcium salts, choline salts, diethylamine salts, bis(2-hydroxyethyl)amine salts (diethanolamine salts), glycine salts, lysine salts, magnesium salts, meglumine salts, 2-aminoethanol salts (ethanolamine salts), potassium salts, sodium salts, 2-amino-2-(hydroxymethyl)propane-1,3-diol salts (tris salts or tromethamine salts), and zinc salts. Acids and bases may also form hemisalts, such as hemisulfates and hemicalcium salts. For a review of suitable salts, see Stahl and Wermuth, “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley-VCH, 2002).

[0115] Pharmaceutically acceptable salts of compounds of formula (I) can be prepared by one or more of three methods: (i) by reacting a compound of formula (I) with a desired acid or base; (ii) by removing an acid- or base-insecure protecting group from a suitable precursor of a compound of formula (I) or by ring-opening a suitable cyclic precursor (e.g., a lactone or lactam); or (iii) by reacting a compound of formula (I) with a suitable acid or base or by means of a suitable ion exchange column to convert one salt of a compound of formula (I) into another. These three reactions are generally carried out in solution. The resulting salt can be precipitated and collected by filtration, or recovered by evaporating the solvent. The degree of ionization in the resulting salt can vary from fully ionized to almost non-ionized. Compounds of formula (I) may have one or more chiral (asymmetric) centers. This disclosure covers all stereoisomers of compounds of formula (I). The asymmetric centers present in compounds of formula (I) may all independently possess ( R )or( S Configuration. When the bond connecting the chiral carbon is depicted as a straight line in the structural formula of this disclosure, or when the chiral carbon is not indicated in the compound name (…), the configuration is as follows. R )or( S When referring to chiral designation, it should be understood as the chiral carbon of each type. R )and( S The configuration, and therefore each enantiomer or diastereomer and mixture thereof, are included in the formula or the name. The production of a particular stereoisomer or mixture thereof may be determined in the embodiments in which such stereoisomers or mixtures are obtained, but this in no way limits the inclusion of all stereoisomers and mixtures thereof within the scope of this disclosure.

[0116] This disclosure includes all possible enantiomers and diastereomers, as well as mixtures of two or more stereoisomers, such as mixtures of enantiomers and / or diastereomers (in various proportions). Therefore, enantiomers are the subject of this disclosure, existing in the form of pure enantiomers, including both levorotatory and dextrorotatory enantiomers, as well as racemic mixtures and mixtures of two enantiomers (in various proportions). Unless otherwise stated, the structures described herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms; in other words, compounds in which one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Such compounds are referred to as “isotope variants.” This disclosure is intended to include all pharmaceutically acceptable isotope variants of compounds of formula (I). Examples of isotopes suitable for inclusion in compounds of this disclosure include, but are not limited to, isotopes of hydrogen, such as… 2 H and 3H; isotopes of carbon, such as 11 C 13 C and 14 C; isotopes of chlorine, such as 36 Cl; isotopes of fluorine, such as 18 F; isotopes of iodine, such as 123 I and 125 I; isotopes of nitrogen, such as 13 N and 15 N; isotopes of oxygen, such as 15 O、 17 O and 18 O; isotopes of phosphorus, such as 32 P; and isotopes of sulfur, such as 35 Certain isotopic variants of compounds of formula (I) (e.g., those incorporating radioactive isotopes) can be used for drug and / or substrate tissue distribution studies. In particular, compounds with the depicted structures differ only in the substitution of heavier isotopes (such as hydrogen replaced by deuterium). 2 Compounds of formula (H) substitution may offer certain therapeutic advantages, such as higher metabolic stability, prolonged in vivo half-life, or reduced dose requirements, and may therefore be used in certain specific situations. Isotopic variants of compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art, or by methods similar to those described in the appended examples and synthesis, using a suitable isotopically labeled reagent instead of a previously used unlabeled reagent. In some embodiments, the isotopic variants of the compounds disclosed herein are deuterated variants.

[0117] As used herein, the term “lipid” refers to a class of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three categories: (1) “simple lipids,” including fats and oils as well as waxes; (2) “complex lipids,” including phospholipids and glycolipids; and (3) “derived lipids,” such as steroids.

[0118] As used herein, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" composed of a phosphate group. These two components are most commonly linked together by a glycerol molecule; therefore, for the purposes of this disclosure, phospholipids are preferably glycerol-phospholipids. Furthermore, the phosphate group is typically modified with simple organic molecules such as choline (which produces phosphoric acid choline) or ethanolamine (which produces phosphoric acid ethanolamine). In some embodiments, the phospholipids may be selected from, but are not limited to, the group consisting of: 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-enocaenoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1,2-di- O -Octadecanyl-sn-choline glycerol-3-phosphate ester (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dimineralyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicyl-sn-glycerol-3-phosphate choline, 1,2-docohexanoyl-sn-glycerol-3-phosphate choline, 1,2-docohexanoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearate-sn-glycerol-3-phosphate Ethanolamine, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-disarachidanoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-docosahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingolipids and mixtures thereof.

[0119] As used herein, the term "structural lipid" refers to sterols, and also to lipids containing sterol moieties. In some embodiments, the structural lipid may be selected from the group consisting of, but not limited to, cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol.

[0120] As used herein, the term "polymer lipid" primarily includes PEG lipids, or alternatively, polyethylene glycol-modified lipids, which are any suitable lipids partially modified with PEG (polyethylene glycol). In some embodiments, the PEG lipid may be selected from the group consisting of, but not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. Specific examples of PEG lipids in some embodiments include, but are not limited to, C14-PEG2000 (1,2-dimyristoyl-rac-glycerol, methoxy polyethylene glycol-2000 (DMG-PEG2000)) and C18-PEG5000 (1,2-distearatel-rac-glycerol, methoxy polyethylene glycol-5000 (DSG-PEG5000)).

[0121] As used herein, the term "hydrophobic lipid" means a lipid compound having nonpolar groups, including (but not limited to) long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups are optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerols, dialkylglycerols, N / N-dialkylamines, 1,2-diaryloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0122] As used herein, the terms “cationic lipid” and “ionizable lipid” are used interchangeably and include those lipids and their salts having one, two, three or more fatty acid or aliphatic hydrocarbon chains and a pH-titrile amino head group (e.g., alkylamino or dialkylamino head group). Cationic lipids are typically protonated (i.e., positively charged) at pH conditions below their pKa and are substantially neutral at pH conditions above their pKa. The cationic lipids of this disclosure may also be referred to as titrizable cationic lipids. As used herein, the term "nucleic acid" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and altered RNA that differs from naturally occurring RNA by adding, deleting, substituting, and / or changing one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide substances to the ends or interior of interfering RNA (e.g., at one or more nucleotides of the RNA). Nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides (such as non-naturally occurring nucleotides or chemically synthesized nucleotides) or deoxyribonucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA. The term "lipid nanoparticles" refers to lipid-based formulations that can be used to deliver therapeutically active components (including nucleic acids, such as mRNA, plasmids) to target sites (e.g., cells, tissues, organs, etc.). In some embodiments, lipid nanoparticles are lipid particles encapsulating therapeutically active components, and these lipid particles are typically assembled from cationic lipids (e.g., lipid compounds of this disclosure), non-cationic lipids (e.g., phospholipids), conjugated polymer lipids that prevent particle aggregation (e.g., PEG-lipids), and optionally structural lipids (e.g., cholesterol). Typically, therapeutically active components (including nucleic acids, such as mRNA, plasmids) can be encapsulated in the lipid portion of lipid nanoparticles, thereby protecting them from enzymatic degradation.

[0123] lipid compounds The lipid compounds disclosed herein can be used for gene delivery, particularly in lipid nanoparticles, to deliver therapeutic and / or preventative agents (such as nucleic acids and plasmids) to cells or organs. When used to prepare lipid nanoparticles, the lipid compounds disclosed herein may also be referred to as "ionizable lipids" or "cationic lipids".

[0124] The lipid compounds disclosed herein are lipid compounds having the structure shown in formula (I) or their N-oxides, stereoisomers, or pharmaceutically acceptable salts. (I) in R I R II and R III Each is independently selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety, and (B) optionally substituted C6-C 25 A collection of aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

[0125] The lipid compounds disclosed herein are lipid compounds having the structure shown in formula (I') or their N-oxides, stereoisomers, or pharmaceutically acceptable salts. (I') in R I R II and R III Each is independently selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety, and (B) optionally substituted C6-C 25 A collection of aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, and R II' It is hydrogen or C1-C6 alkyl, such as C1-C4 alkyl, methyl, ethyl, propyl or butyl.

[0126] In some implementation schemes, R I R II R III One or both of them are selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety.

[0127] In some implementation schemes, R I R II R III Only one of them is selected from (A) a set of groups containing at least one ionizable tertiary amine moiety.

[0128] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia): (Ia) Where R Ia The C1-C6 alkylene group is optionally substituted, wherein the alkylene group is optionally replaced by 1, 2, or 3 ions selected from -oxo (=O), -OH, -SH, or -NR. Id R Id' The substituents are replaced by R, where R is a substituent. Id and R Id' Each is independently hydrogen or C1-C3 alkyl; R Ib and R Ic Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie R Ie' The substituents are replaced by R, where R is a substituent. Ie and R Ie' Each is independently hydrogen or C1-C3 alkyl, or R Ib and R Ic Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0129] In some implementation schemes, R Ib and R Ic Together with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib and R IcTogether with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib and R Ic Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0130] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia'): (Ia') Where Ar is C6-C that is optionally substituted. 10 arylene, for example, phenylene, wherein the arylene is optionally surrounded by 1, 2, 3 or 4 independently selected from halogen, C1-C6 alkyl, -OH, -SH or -NR Id'' R Id''' The substituents are replaced by R, where R is a substituent. Id'' and R Id''' Each is independently hydrogen or C1-C3 alkyl; R Ib' and R Ic' Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie'' R Ie''' The substituents are replaced by R, where R is a substituent. Ie'' and R Ie''' Each is independently hydrogen or C1-C3 alkyl, or R Ib' and R Ic' Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0131] In some implementation schemes, R Ib' and R Ic'Together with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib' and R Ic' Together with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib' and R Ic' Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0132] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in assembly (A) is a 5- to 12-membered heterocyclic group comprising one, two, or three heteroatoms independently selected from N, O, and S, wherein at least one heteroatom is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH. The 5- to 12-membered heterocyclic group may be saturated or unsaturated and may be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc.

[0133] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0134]

[0135]

[0136] .

[0137] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0138]

[0139] .

[0140] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety: .

[0141] In some implementations, set (B) includes C6-C that are optionally replaced. 25 Aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, wherein the aliphatic groups and heteroaliphatic groups optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 independently selected from -C=C-, -C≡C-, -NR m -、-NH-、-NH2、-OH、-OR n -O-, -C(O)-, -C(OR) o -, -C(O)O-, -SH, -SR p -S-, -C(S)-, -C(SR) q The group consisting of -, -C(S)O- and -P(O)- groups, wherein R m R n R o R p and R q Each independently represents the C1-C that are optionally replaced. 14 Aliphatic groups.

[0142] As used herein, the term "aliphatic group" refers to a substituted or unsubstituted straight-chain and / or branched, saturated or unsaturated hydrocarbon group, including straight-chain, branched or cyclic alkyl, alkenyl and alkynyl groups. In some embodiments, the term "aliphatic group" may be used interchangeably with "hydrocarbon group". In some embodiments, an aliphatic group includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) unsaturated carbon-carbon double bond (-C=C-), carbon-carbon triple bond (-C≡C-) groups, and / or any combination thereof.

[0143] As used herein, the term "heteroaliphatic group" refers to a substituted or unsubstituted straight-chain and / or branched, saturated or unsaturated hydrocarbon group containing a heteroatom selected from N, O, and S, including straight-chain, branched, or cyclic heteroalkyl, heteroalkenyl, and heteroynyl groups. In some embodiments, the term "heteroaliphatic group" may be used interchangeably with "heterohydrocarbon group." In some embodiments, the heteroatom contained in the heteroaliphatic group may form the backbone of the heteroaliphatic group together with a carbon atom, such as, but not limited to, group structures -CNC-, -COC-, -COOC, -CSC-, -CSSC, or any combination thereof. In some embodiments, the heteroatom contained in the heteroaliphatic group may be a substituent attached to a carbon atom, such as, but not limited to, substituted structures, such as -C≡N, -C=N-, -CN=, -C=O, -C-OH, -C=S, -C-SH, etc. In some embodiments, the heteroatom contained in the heteroaliphatic group may be any combination of the group structures listed above.

[0144] In some embodiments, the heteroaliphatic group comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) unsaturated carbon-carbon double bonds (-C=C-), carbon-carbon triple bonds (-C≡C-), or -NR-. m -、-NH-、-NH2、-OH、-OR n -O-, -C(O)-, -C(OR) o -, -C(O)O-, -SH, -SR p -S-, -C(S)-, C(SR) q -, -C(S)O- and -P(O)- groups, and / or any combination thereof, wherein R m R n R o R p and R q Each independently represents the C1-C that are optionally replaced. 14 Aliphatic groups, such as C1-C 12 C1-C 10 C1-C8, C1-C6, C1-C4 aliphatic groups. Optionally substituted C1-C 14 Non-limiting specific examples of aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethylene (vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, ethynyl, 1-propynyl, 2-propynyl, etc.

[0145] In some embodiments, the optionally substituted aliphatic groups of group (B) have 6 to 25 carbon atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 carbon atoms. In some embodiments, the optionally substituted heteroaliphatic groups of group (B) have 6 to 25 atoms (including C, N, O, and S), such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 atoms.

[0146] In some embodiments, the optionally substituted heteroaliphatic groups of set (B) comprise 1 to 8 heteroatoms independently selected from N, O, S, etc., such as 1, 2, 3, 4, 5, 6, 7, and 8 heteroatoms. In some embodiments, the optionally substituted heteroaliphatic groups of set (B) comprise, for example, 1 to 2 heteroatoms, 1 to 3 heteroatoms, 1 to 4 heteroatoms, 1 to 5 heteroatoms, or a plurality of heteroatoms, the number of which is in the range of any two values ​​between 1 and 8.

[0147] In some implementation schemes, R I R II or R III Both of the groups are selected from set (B), and each of the groups optionally independently contains at least one degradable group. Preferably, the lipid compounds of this disclosure contain degradable groups that break upon entry into cells, tissues, or organs, thereby partially or completely degrading the lipid compounds and reducing or completely eliminating the toxicity of the lipid compounds to cells. Introducing degradable groups can accelerate the metabolism of lipids in organs such as the liver, thereby reducing the accumulation of lipids in the body and reducing their potential toxicity.

[0148] In some embodiments, the degradable portion is selected from -C(O)O-, -OC(O)-, -OC(O)O-, -SS-, -C(O)NH-, -NHC(O)-, -NHC(O)O-, -NR 1 C(O)-、-C(O)NR 2 -、-NR 3 C(O)O-、-OP(O)OR 4 O-、-OCR 5 (OR 6 )O-、-CR 7 (OR 8 )O- and -CH(OR 9 )O-, where R 1 R 2 R 3 R 4 R5 R 6 R 7 R 8 and R 9 Each independently represents the C1-C that are optionally replaced. 14 Aliphatic groups, including straight-chain, branched, cyclic alkanes, alkenes, alkynes, or polyunsaturated hydrocarbon groups, such as C1-C... 12 C1-C 10 C1-C8, C1-C6, C1-C4 aliphatic groups. Optionally substituted C1-C 14 Non-limiting specific examples of aliphatic groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ethylene (vinyl), propenyl, butenyl, pentenyl, 1-methyl-2-buten-1-yl, 5-hexenyl, ethynyl, 1-propynyl, 2-propynyl, etc.

[0149] In some implementations, set (B) comprises the following groups:

[0150]

[0151]

[0152]

[0153] .

[0154] In some implementations, set (B) comprises the following groups:

[0155]

[0156]

[0157] .

[0158] In some implementations, set (B) comprises the following groups: .

[0159] In some implementation schemes, R I Selected from set (A), and R II and R III Selected from set (B), where R II and R III They can be the same or different.

[0160] In some implementation schemes, R II Selected from set (A), and R I and R III Selected from set (B); where R I and R III They can be the same or different.

[0161] In some implementation schemes, R III Selected from set (A), and R I and R II Selected from set (B), where R I and R II They can be the same or different.

[0162] In some implementation schemes, R I R II or R III Both of them are selected from set (B), each of which independently and optionally contains at least one degradable portion.

[0163] In some implementation schemes, R I R II or R III Both of them are selected from set (B), and at least one of them contains at least one degradable part.

[0164] In some implementation schemes, R I R II or R III The two in the set (B) are selected from the set, and only one of the two contains a degradable part.

[0165] In some implementation schemes, R I Selected from the following groups:

[0166]

[0167] .

[0168] In some implementation schemes, R I Selected from the following groups:

[0169]

[0170]

[0171] .

[0172] In some implementation schemes, R I Selected from the following groups: .

[0173] In some implementation schemes, R I Selected from the following groups: .

[0174] In some implementation schemes, R I Selected from the following groups: .

[0175] In some implementation schemes, R I Selected from the following groups: .

[0176] In some implementation schemes, R II Selected from the following groups:

[0177]

[0178] .

[0179] In some implementation schemes, R II Selected from the following groups:

[0180]

[0181]

[0182] .

[0183] In some implementation schemes, R II Selected from the following groups: .

[0184] In some implementation schemes, R II Selected from the following groups: .

[0185] In some implementation schemes, R II Selected from the following groups: .

[0186] In some implementation schemes, R II Selected from the following groups: .

[0187] In some implementation schemes, R III Selected from the following groups:

[0188]

[0189] .

[0190] In some implementation schemes, R III Selected from the following groups:

[0191]

[0192]

[0193] .

[0194] In some implementation schemes, R III Selected from the following groups: .

[0195] In some implementation schemes, R III Selected from the following groups: .

[0196] In some implementation schemes, R III Selected from the following groups: .

[0197] In some implementation schemes, R III Selected from the following groups: .

[0198] In some embodiments, the lipid compound is selected from the following compounds: Table 1 – Lipid Compounds

[0199] In some embodiments, the lipid compound is selected from the following compounds: Table 2 – Lipid Compounds

[0200] Table 3 – Lipid Compounds

[0201] Table 4 – Lipid Compounds

[0202] In some embodiments, the lipid compound of this disclosure has a molecular weight in the range of about 500 g / mol to about 1400 g / mol; preferably, the lipid compound has a molecular weight in the range of about 500 g / mol to about 1200 g / mol; more preferably, the lipid compound has a molecular weight in the range of about 600 g / mol to about 1000 g / mol; more preferably, the lipid compound has a molecular weight in the range of about 700 g / mol to about 1000 g / mol. In some embodiments, the lipid compound of this disclosure has a molecular weight of about 500 g / mol, 600 g / mol, 700 g / mol, 800 g / mol, 900 g / mol, 1000 g / mol, 1100 g / mol, 1200 g / mol, 1300 g / mol, or 1400 g / mol, or within any two of the above values.

[0203] The lipid compounds disclosed herein are used for the delivery of therapeutically active components, enabling the introduction of therapeutically active components (including, but not limited to, nucleic acids (e.g., DNA, RNA) and plasmids) into organelles, cells, tissues, or organisms.

[0204] Preparation of lipid compounds In one aspect, this disclosure provides a method for preparing a lipid compound or its N-oxide, stereoisomer, or pharmaceutically acceptable salt thereof using the Passerini reaction, wherein the prepared lipid compound is used to prepare lipid nanoparticles for gene delivery.

[0205] In some implementations, the lipid compound is an ionizable lipid or a cationic lipid.

[0206] In some implementations, the Passerini reaction requires three reactants: an isonitrile compound, an aldehyde compound, and a carboxylic acid compound.

[0207] In some implementations, the reaction takes place for 1 to 48 hours, for example 12 to 36 hours, preferably 18 to 30 hours, more preferably 20 to 26 hours, for example 20, 21, 22, 23, 24, 25, 26 hours, or any value within the range described above.

[0208] In some embodiments, the solvent is selected from THF, DCM, toluene, EtOH, or mixtures thereof.

[0209] In one aspect, this disclosure provides a method for preparing the lipid compound of the present disclosure or its N-oxide, stereoisomer, or pharmaceutically acceptable salt. The method comprises the following synthetic route: , This method also includes the following synthetic route: , Where R I R II R II' and R III The definition is as described in this article.

[0210] The multicomponent Passerini reaction, first reported in 1921, involves primarily one molecule of isonitrile, one molecule of aldehyde, and one molecule of carboxylic acid or alcohol, producing bifunctional α-acylamides or α-alkoxyamides. The Passerini reaction is characterized by mild reaction conditions, occurring at room temperature or below in aprotic solvents. This disclosure utilizes the Passerini reaction to facilitate the high-throughput preparation of the lipid compounds disclosed herein.

[0211] In some implementations, the reaction takes place for 1 to 48 hours, for example 12 to 36 hours, preferably 18 to 30 hours, more preferably 20 to 26 hours, for example 20, 21, 22, 23, 24, 25, 26 hours, or any value within the range described above.

[0212] In some embodiments, the solvent is selected from THF, DCM, toluene, EtOH, or mixtures thereof.

[0213] In some implementation schemes, R I R II and R III Each is independently selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety, and (B) optionally substituted C6-C 25A collection of aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups; The condition is R I R II R III One or both of them are selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety.

[0214] In some implementation schemes, R I R II R III Only one of them is selected from (A) a set of groups containing at least one ionizable tertiary amine moiety.

[0215] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia): (Ia) Where R Ia The C1-C6 alkylene group is optionally substituted, wherein the alkylene group is optionally replaced by 1, 2, or 3 ions selected from -oxo (=O), -OH, -SH, or -NR. Id R Id' The substituents are replaced by R, where R is a substituent. Id and R Id' Each is independently hydrogen or C1-C3 alkyl; R Ib and R Ic Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie R Ie' The substituents are replaced by R, where R is a substituent. Ie and R Ie' Each is independently hydrogen or C1-C3 alkyl, or R Ib and R Ic Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0216] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in assembly (A) is a 5- to 12-membered heterocyclic group comprising one, two, or three heteroatoms independently selected from N, O, and S, wherein at least one heteroatom is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH. The 5- to 12-membered heterocyclic group may be saturated or unsaturated and may be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc.

[0217] In some implementation schemes, R Ib and R Ic Together with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib and R Ic Together with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib and R Ic Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0218] In some embodiments, the group comprising at least one ionizable tertiary amine moiety in set (A) has 3 to 11 carbon atoms, and the group is as shown in formula (Ia'): (Ia') Where Ar is C6-C that is optionally substituted. 10 arylene, for example, phenylene, wherein the arylene is optionally surrounded by 1, 2, 3 or 4 independently selected from halogen, C1-C6 alkyl, -OH, -SH or -NR Id'' R Id''' The substituents are replaced by R, where R is a substituent. Id'' and R Id''' Each is independently hydrogen or C1-C3 alkyl; R Ib' and R Ic'Each of the following is independently a optionally substituted C1-C6 alkyl group, an optionally substituted C2-C6 alkenyl group, or an optionally substituted C2-C6 alkynyl group, wherein the alkyl, alkenyl, and alkynyl groups are optionally surrounded by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie'' R Ie''' The substituents are replaced by R, where R is a substituent. Ie'' and R Ie''' Each is independently hydrogen or C1-C3 alkyl, or R Ib' and R Ic' Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle containing 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of which is N, and the heterocycle is optionally substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

[0219] In some implementation schemes, R Ib' and R Ic' Together with the N atoms they are attached to, they form 5- to 12-membered heterocycles, which can be monocyclic, dense bicyclic, spirocyclic, or bridged bicyclic, etc. In some embodiments, R Ib' and R Ic' Together with the N atoms they are attached to, they form 5-, 6-, 7-, or 8-membered monocyclic heterocycles containing 1, 2, or 3 heteroatoms selected from N, O, and S, wherein at least one heteroatom is N. In some embodiments, R... Ib' and R Ic' Non-limiting embodiments of the heterocycles formed together with the N atoms they are attached to include, but are not limited to, the group consisting of: , , , , , , , , , and .

[0220] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0221]

[0222] .

[0223] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety:

[0224]

[0225]

[0226] .

[0227] In some embodiments, set (A) comprises groups containing at least one ionizable tertiary amine moiety: .

[0228] In some implementations, set (B) includes C6-C that are optionally replaced. 25 Aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, wherein the aliphatic groups and heteroaliphatic groups optionally comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 independently selected from -C=C-, -C≡C-, -NR m -、-NH-、-NH2、-OH、-OR n -O-, -C(O)-, -C(OR) o -, -C(O)O-, -SH, -SR p -S-, -C(S)-, -C(SR) q The group consisting of -, -C(S)O- and -P(O)- groups, wherein R m R n R o R p and R q Each independently represents the C1-C that are optionally replaced. 14 Aliphatic groups.

[0229] In some implementations, set (B) comprises the following groups:

[0230]

[0231]

[0232] .

[0233] In some implementations, set (B) comprises the following groups:

[0234]

[0235]

[0236]

[0237] .

[0238] In some implementations, set (B) comprises the following groups: .

[0239] In some embodiments, the isonitrile compound is selected from general formula R I -NC represents the library I of compounds, where R I Selected from set (A) as defined in this article.

[0240] In some embodiments, the isonitrile compound is selected from general formula R I -NC represents the library I of compounds, where R I Selected from set (B) as defined in this article.

[0241] In some implementation schemes, R I Selected from the following groups: .

[0242] In some implementation schemes, R I Selected from the following groups: .

[0243] In some implementation schemes, R I Selected from the following groups: .

[0244] In some implementations, the aldehyde compound is selected from general formula R II -CHO represents the composition of library II of compounds, where R II Selected from set (A) as defined in this article.

[0245] In some implementations, the aldehyde compound is selected from general formula R II -CHO represents the composition of library II of compounds, where R II Selected from set (B) as defined in this article.

[0246] In some implementation schemes, R II Selected from the following groups: .

[0247] In some implementation schemes, R II Selected from the following groups: .

[0248] In some implementation schemes, R II Selected from the following groups: .

[0249] In some implementations, the carboxylic acid compound is selected from general formula R III -COOH represents the composition of Library III, where R... III Selected from set (A) as defined in this article.

[0250] In some implementations, the carboxylic acid compound is selected from general formula R III -COOH represents the composition of Library III, where R... III Selected from set (B) as defined in this article.

[0251] In some implementation schemes, R III Selected from the following groups: .

[0252] In some implementation schemes, R III Selected from the following groups: .

[0253] In some implementation schemes, R III Selected from the following groups: .

[0254] In some implementations, the ketone compound is selected from general formula R II -CO-R II' Library IV represents the composition of compounds, where R II Selected from a set (A) as defined in this paper, and R II' It is selected from C1-C6 alkyl groups, such as C1-C4 alkyl groups, methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0255] In some implementation schemes, R II Selected from the following groups: .

[0256] In some embodiments, the isonitrile compound is selected from general formula R I -NC represents the library I' of compounds, where R I Selected from set (B).

[0257] In some implementation schemes, R I Selected from the following groups: .

[0258] In some implementation schemes, R I Selected from the following groups: .

[0259] In some implementation schemes, R I Selected from the following groups: .

[0260] In some implementations, the aldehyde compound is selected from general formula R II -CHO represents the composition of the compound library II', where R... II Selected from set (B): In some implementation schemes, R II Selected from the following groups: .

[0261] In some implementation schemes, R II Selected from the following groups: .

[0262] In some implementation schemes, R II Selected from the following groups: .

[0263] In some implementations, the carboxylic acid compound is selected from general formula R III -COOH represents the composition of the compound library III', where R... III Selected from set (A) as defined in this article.

[0264] In some embodiments, the isonitrile compound is selected from general formula R I -NC represents the library I'' of compounds, where R I Selected from set (B) as defined herein; aldehyde compounds selected from general formula R II-CHO represents the library II'' of compounds, where R II The compounds are selected from the set (A) as defined herein; and the carboxylic acid compounds are selected from the general formula R. III -COOH represents the composition of the library III'', where R III Selected from set (B) as defined in this article.

[0265] In some embodiments, the isonitrile compound is selected from general formula R I -NC represents the library I''' of compounds, where R I Selected from the set (A) as defined herein; ketone compounds are selected from general formula R. II -CH-R II' The library II''' represents the composition of compounds, where R II Selected from set (B) as defined in this paper, and R II' As defined herein; and carboxylic acid compounds are selected from general formula R. III -COOH represents the composition of the library III'', where R III Selected from set (B) as defined in this article.

[0266] Preparation of lipid compound libraries In one aspect, this disclosure relates to a method for preparing a library of lipid compounds. The method disclosed herein is a high-throughput combinatorial synthesis of lipid compounds.

[0267] P-3CR is used to synthesize a library of lipid compounds. This one-pot reaction combines carboxylic acids, isocyanates, and aldehydes. The P-3CR reaction provides an efficient and versatile process for the synthesis and purification of ionizable lipids, reducing the number of synthesis and purification steps, eliminating the need for catalysts or coupling agents, requiring only mild conditions (such as room temperature, short reaction times, and neutral pH levels), and exhibiting excellent functional group tolerance. Ionizable lipids often achieve rapid clearance through ester bond structures, aiming to improve tolerance during payload delivery. Ester-rich ionizable lipids maintain good stability under physiological pH conditions and endow them with the ability to undergo enzymatic hydrolysis in cells and tissues. Enhancing the in vivo metabolism and degradation of ionizable lipids is particularly important for therapeutic applications requiring repeated administration. The P-3CR reaction involves the condensation of isocyanate, aldehyde, and carboxylic acid groups, followed by the Mumm rearrangement, ultimately producing an α-acylamide scaffold. These α-acylamide scaffolds are characterized by hydrolyzable ester bonds, which impart the advantage of biodegradability, while eliminating the need for additional esterification reactions. This highlights the efficiency and simplicity of this method compared to alternative multicomponent reactions. The method includes the following steps: i) Prepare a library of reactant components, including: a library of isonitriles, a library of aldehydes, and a library of carboxylic acids; ii) Select i compounds from the isonitrile compound library and label them as Iso-1, Iso-2, Iso-3...Iso-i, where i is an integer; Select j compounds from the aldehyde compound library and label them Alde-1, Alde-2, Alde-3...Alde-j, where j is an integer; Select k compounds from this carboxylic acid compound library and label them as Acid-1, Acid-2, Acid-3...Acid-k, where k is any integer; iii) Prepare a reaction vessel matrix, labeling each vial as vial-ijk, where i represents the sequence number of the isonitrile compound, j represents the sequence number of the aldehyde compound, and k represents the sequence number of the carboxylic acid compound; iv) Add the isonitriles, aldehydes, and carboxylic acid compounds to the corresponding numbered vials containing the solvent, according to their labels; v) Initiate the Passerini reaction, lasting from 1 hour to 48 hours; vi) After the reaction is complete, collect the products to obtain a compound in quantity equal to i. j k's lipid compound library.

[0268] In some implementations, the lipid compound is an ionizable lipid or a cationic lipid.

[0269] In some implementations, the reaction takes place for 1 to 48 hours, for example 12 to 36 hours, preferably 18 to 30 hours, more preferably 20 to 26 hours, for example 20, 21, 22, 23, 24, 25, 26 hours, or any value within the range described above.

[0270] In some embodiments, the solvent is selected from THF, DCM, toluene, EtOH, or mixtures thereof.

[0271] In some implementations, the reactant addition process can be performed manually or by automated equipment. Automated equipment can be programmed to add reactants automatically according to the manufacturer's instructions.

[0272] In some implementations, the ratio of isonitrile compound, aldehyde compound and carboxylic acid compound is 1:1:1.

[0273] There are no particular limitations on the reaction vessels, as long as they can be easily numbered and are suitable for carrying out chemical reactions. In some embodiments, the reaction vessels are capped glass vials. In some embodiments, the reaction vessel matrix is ​​a 96-well plate, or any similar reaction plate used for chemical and biological experiments.

[0274] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (A) as defined in this article.

[0275] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this article.

[0276] In some implementation schemes, R I Selected from the following groups: .

[0277] In some implementation schemes, R I Selected from the following groups: .

[0278] In some implementation schemes, R I Selected from the following groups: .

[0279] In some implementations, the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (A) as defined in this article.

[0280] In some implementations, the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (B) as defined in this article.

[0281] In some implementation schemes, R II Selected from the following groups: .

[0282] In some implementation schemes, R II Selected from the following groups: .

[0283] In some implementation schemes, R II Selected from the following groups: .

[0284] In some implementations, the carboxylic acid compound library is composed of general formula R III -COOH represents the library of compounds, where R III Selected from set (A) as defined in this article.

[0285] In some implementations, the carboxylic acid compound library is composed of general formula R III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0286] In some implementation schemes, R III Selected from the following groups: .

[0287] In some implementation schemes, R III Selected from the following groups: .

[0288] In some implementation schemes, R III Selected from the following groups: .

[0289] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I The aldehyde library is selected from the set (A) as defined in this paper; the aldehyde compound library is composed of the general formula R. II -CHO represents a library of compounds, where R... II Selected from set (B) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0290] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this paper; the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (A) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (B) as defined in this article.

[0291] In some implementations, the isonitrile compound library is composed of general formula R I -NC represents a library of compounds, where R I Selected from set (B) as defined in this paper; the aldehyde compound library is composed of general formula R II -CHO represents a library of compounds, where R... II Selected from set (B) as defined herein; and the carboxylic acid compound library is composed of general formula R. III -COOH represents the library of compounds, where R III Selected from set (A) as defined in this article.

[0292] In some implementations, the method further includes a step of purifying the product.

[0293] In one aspect, this disclosure relates to a library of lipid compounds prepared by the methods of this disclosure.

[0294] In some embodiments, the lipid compounds in the library have a structure as shown in formula (I). (I) Where R I R II and R III As defined in this article.

[0295] In one aspect, this disclosure relates to a library of lipid compounds prepared by the methods of this disclosure.

[0296] In some embodiments, the lipid compounds in the library have a structure as shown in formula (I'). (I') Where R I R II R II' and R III As defined in this article.

[0297] In one aspect, this disclosure relates to a method for screening lipid compounds, the method comprising the following steps: When preparing a lipid compound library using the method of this disclosure, step vi) of collecting the product after the reaction is complete is omitted. 1) Add non-cationic lipids (e.g., phospholipids), conjugated polymer lipids (e.g., PEG-lipids) to prevent particle aggregation, and optionally structural lipids (e.g., cholesterol) to a reaction vessel to formulate LNPs (lipid nanoparticles) with the prepared ionizable lipids. 2) Prepare an aqueous phase containing nucleic acids (e.g., mRNA); 3) Mix the LNP obtained in step (1) with an aqueous phase containing nucleic acids; 4) The obtained LNP-encapsulated nucleic acids were characterized in vitro or in vivo.

[0298] lipid nanoparticles In one aspect, this disclosure relates to lipid nanoparticles. The lipid nanoparticles of this disclosure comprise the lipid compounds of this disclosure as described above. When used to prepare the lipid nanoparticles of this disclosure, the lipid compounds of this disclosure may also be referred to as cationic lipids or ionizable lipids.

[0299] In some embodiments, the lipid nanoparticles of this disclosure, based on the total molar concentration of the components constituting the lipid nanoparticles, contain a lipid compound comprising about 10 mol% to about 90 mol%, such as about 10 mol%, 15 mol%, 20 mol%, 25 mol%, 30 mol%, 40 mol%, 45 mol%, 50 mol%, 55 mol%, 60 mol%, 65 mol%, 70 mol%, 75 mol%, 80 mol%, 85 mol%, 90 mol%, or any two of the above molar percentages. Preferably, the lipid compound comprises about 10 mol% to about 70 mol%, more preferably, the lipid compound comprises about 20 mol% to about 50 mol.

[0300] In some embodiments, the lipid nanoparticles of this disclosure also comprise phospholipids, structural lipids, PEG lipids, and nucleic acids.

[0301] In some embodiments, phospholipids account for about 0 mol% to about 20 mol% of the total molar amount of the components constituting the lipid nanoparticles, or in any range between about 0 mol% and about 20 mol%. Structured lipids account for about 30 mol% to about 50 mol% of the total molar amount of the components constituting the lipid nanoparticles, or in any range between about 30 mol% and about 50 mol%. PEG lipids account for about 0 mol% to about 10 mol% of the total molar amount of the components constituting the lipid nanoparticles, or in any range between about 0 mol% and about 10 mol%.

[0302] In some embodiments, the N / P ratio of the lipid compound to the nucleic acid is in the range of about 1.1:1 to 10:1, or any value between 1.1:1 and 10:1. For the purposes of this disclosure, the N / P ratio can be defined as the ratio of the number of nitrogen atoms (N atoms) of the ionizable groups contained in the lipid compound (also referred to as "cationic lipids" or "ionizable lipids") to the number of phosphoric acid atoms (P atoms) of the nucleic acid in the lipid nanoparticle containing the nucleic acid. The N / P ratio can be calculated based on the following: for example, 1 μg of RNA typically contains about 3 nmol of phosphate residues, provided that the RNA exhibits a statistical base distribution. The "N" value of the lipid compound can be calculated based on its molecular weight and the relative abundance of the cationic groups. If more than one lipid compound is present, the N value should be calculated based on all lipid compounds contained in the lipid nanoparticle.

[0303] In some embodiments, examples of nucleic acids include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. In some embodiments, examples of nucleic acids include any type of RNA, such as messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), CRISPR RNA (crRNA), trans-activating RNA (tracrRNA), plasmid DNA (pDNA), small circular DNA, genomic DNA (gNDA), and any fragments thereof.

[0304] Methods for preparing lipid nanoparticles are widely known in the art. The lipid nanoparticles disclosed herein can be prepared using conventional methods known to those skilled in the art.

[0305] Pharmaceutical Composition In one aspect, this disclosure relates to a pharmaceutical composition comprising the lipid compound of the disclosure or its N-oxide, stereoisomer or pharmaceutically acceptable salt, or the lipid nanoparticle of the disclosure, or the delivery system of the disclosure, and a pharmaceutically acceptable carrier or excipient.

[0306] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that can be used to prepare a pharmaceutical composition, which is generally safe and non-toxic, and is neither biologically undesirable nor otherwise undesirable, and includes carriers or excipients acceptable for veterinary and human pharmaceutical use. As used herein, a pharmaceutically acceptable carrier or excipient includes one or more such carriers or excipients. The specific carrier or excipient used will depend on the manner and purpose of administering the compounds disclosed herein. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in the following literature: for example, Ansel, Howard C et al., “Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems.”, Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., “Remington: The Science and Practice of Pharmacy.”, Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C., “Handbook of Pharmaceutical Excipients.”, Chicago, Pharmaceutical Press, 2005. The pharmaceutical compositions disclosed herein can be prepared by any well-known pharmaceutical technique, such as efficient formulation and administration procedures. The considerations above regarding efficient formulation and administration procedures are well known in the art and described in standard textbooks. Formulations of pharmaceutical products are discussed in the following literature: for example, Hoover, John E., “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al. (eds.), “Pharmaceutical Dosage Forms,” Marcel Decker, New York, NY, 1980; and Kibbe et al. (eds.), “Handbook of Pharmaceutical Excipients,” 3rd edition, American Pharmaceutical Association, Washington, 1999.

[0307] synthesis The compounds disclosed herein can be prepared using general and specific methods described below, which are well known to those skilled in the art of synthetic organic chemistry. Such general knowledge can be found in standard reference books such as Barton and Ollis (eds.), “Comprehensive Organic Chemistry,” Elsevier; Richard Larock, “Comprehensive Organic Transformations: A Guide to Functional Group Preparations,” John Wiley and Sons; and “Compendium of Organic Synthetic Methods,” Vol. I-XII, Wiley-Interscience. The raw materials used herein are commercially available or can be prepared using conventional methods known in the art.

[0308] The methods described below are intended to provide a general description of the methods used in preparing the compounds of this disclosure. Some compounds of this disclosure may contain stereochemical names of (…). R )or( S ( ) a single or multiple chiral centers. It will be apparent to those skilled in the art that all synthetic transformations can be carried out in a similar manner, regardless of whether the material is enantiomer-enriched or racemic. Furthermore, the resolution of the desired optically active material can be performed at any desired point in the procedure using well-known methods, such as those described herein and in the chemical literature.

[0309] Example To provide a fuller understanding of this disclosure, the following embodiments are illustrated. These embodiments are provided to illustrate the compounds, methods, and compositions provided herein and should not be construed as limiting the scope of this disclosure in any way. During the synthetic procedure, it may be necessary and / or desirable to protect any sensitive or reactive groups on the relevant molecules. This can be achieved with conventional protecting groups, such as those described in TW Greene and PGM Utts, “Protective Groups in Organic Synthesis,” 4th edition, John Wiley and Sons. Protecting groups are optionally removed at appropriate subsequent stages using methods well known in the art. The compounds of this disclosure can be readily prepared using readily available starting materials and conventional synthetic procedures, according to the reaction schemes and examples below or modifications thereof. Variations known to those skilled in the art but not described in detail may also be utilized in these reactions. Furthermore, other methods for preparing the compounds of this disclosure based on the reaction schemes and examples described herein will be apparent to those skilled in the art. Unless otherwise stated, all variables are as defined above. In general chemical procedures, all reagents and materials are either commercially available or readily prepared by those skilled in the art. As used herein, a head group refers to a group selected from set (A), and a tail group refers to a group selected from set (B).

[0310] Materials and methods The materials and methods commonly used in the embodiments are described below. Lipid library and ionizable lipid synthesis For the synthesis of ionizable lipid libraries, ionizable lipids were synthesized using a 3CR-Pacerini reaction with a carboxylic acid compound (-COOH), an aldehyde compound (-CHO), and an isocyanate compound (-NC). Briefly, the carboxylic acid, aldehyde, and isocyanate were dissolved in dichloromethane (DCM) in a 1:1:1 molar ratio in a single-necked round-bottom flask. The reaction was carried out at room temperature under a nitrogen atmosphere for 24 hours. All reactions were performed in 96-well plates. For in vivo high-throughput transfection studies, the solvent in the lipid mixture was removed in a vacuum-heated chamber. Subsequently, the lipids were resuspended by adding ethanol (EtOH). If purification was required, the lipids were purified using a preparative chromatography system (BUCHI) via rapid column chromatography. 1 The structure was confirmed by 1H NMR spectroscopy (400MHz or 500MHz, Bruker spectrometer) and Q Exactive HF-Orbitrap mass spectrometry (Thermo). LNP formulations LNPs were generated as follows: The lipid-containing ethanol phase and the aqueous phase of mRNA were mixed using a T-connector via a syringe pump and dialyzed against 1×PBS in a 10,000 MWCO box (Thermo Fisher) for 12 hours at 4°C to exchange the buffer. In short, the organic phase was prepared by dissolving a synthetic mixture of ionizable lipids, along with the helper phospholipids DSPC (Avanti), cholesterol (Avanti), and C14-PEG2000 (Avanti), in ethanol at a specific molar ratio of 50:10:38.5:1.5, similar to SM-102. MC3 and SM-102 were purchased from Echelon Bioscience. An aqueous phase containing mRNA was prepared using 10 mM citrate buffer (pH 4.0, Fisher) as the solvent. The final total mRNA concentration of the LNPs was adjusted to 0.1 μg / μL. -1 The mRNA variants studied included mLuc (firefly luciferase mRNA, TriLink), mCre (Cre-recombinase mRNA, 5-methoxyuridine (5 moU), TriLink), and hEPO (human erythropoietin mRNA, 5 moU, TriLink).

[0311] LNP characterization Particle size and PDI of LNPs were analyzed by dynamic light scattering (DLS). LNPs were first diluted in PBS (pH 7.4) to a total mRNA concentration of 0.8 ng / μL. -1 Up to 1.6 ng μL -1 The diluted LNP was then transferred to a polystyrene cuvette. Measurements were performed at 25°C using a Malvern Nano ZS Zetasizer with DLS. Analysis was conducted in PBS with a refractive index (RI) of 1.590, absorbance of 0.010, viscosity of 0.9073 cP, and a refractive index of 1.332. The measurement duration was set to 2 seconds, with the number of runs automatically determined. Each measurement was performed at a fixed position in the cuvette, which had a diameter of 4.65 mm. Automatic attenuation was applied during the measurement, and the diameter was reported as the Z-mean. A TNS binding assay was performed to determine the apparent pKa of LNPs. In this assay, LNPs were diluted and combined with TNS in a total volume of 150 μL of buffer solution to obtain final concentrations of (LNP, 75 μM) and (TNS, 6 μM). Buffer solutions with pH ranging from 3 to 10 were prepared containing 20 mM boric acid, 10 mM imidazole, 10 mM sodium acetate, 10 mM glycyl glycine, and 25 mM sodium chloride. After the addition of TNS, fluorescence was recorded at wavelengths Ex321 / Em445 using a Cytation 5 Cell Imaging Multimodal Reader (BioTek). The pH of each well was then determined. The fluorescence data were fitted to the Henderson-Hasselbalch equation using Mathematica (Wolfram Research) to derive the pKa. RNA concentration and encapsulation efficiency were quantified using the RiboGreen assay. The Quant-it RiboGreen assay from Invitrogen was used to determine RNA encapsulation efficiency and concentration. A standard curve was generated using serial dilutions derived from appropriate RNA stock solutions to quantitatively assess RNA in the LNP formulation. Standards and samples were diluted in 1×Tris-EDTA (TE) buffer (pH 8.0). The target final sample concentration in polystyrene cuvettes was 0.1 ng μL. -1 Fluorescence measurements were performed using a fluorescence spectrophotometer (Varian Cary Eclipse) set to 500 nm excitation and 525 nm emission. To calculate the standard curve, linear regression analysis was performed on fluorescence intensities plotted against standard sample concentrations. The encapsulation of RNA in the LNP samples was determined by comparing the signals of the fluorescent dye RiboGreen in the absence and presence of detergent (0.1% Triton X-100). In the absence of detergent, the signal was primarily generated by tightly associated (unencapsulated) RNA. In the presence of detergent, LNPs were disrupted, and the measured signal represented total RNA (encapsulated and unencapsulated). Encapsulation efficiency was calculated using the following formula:

[0312] Animal experiments All experimental procedures were ethically approved, and all animal studies were approved and conducted in accordance with the University Health Network Animal Resource Centre Guidelines (University of Toronto). Females and males C57BL / 6, BALB / C, and B6. Cg-Gt(ROSA)26Sor tm9（CAG-tdTomato）Hze / J mice (4 to 8 weeks old) were obtained from the Jackson Laboratory. I. Option I Synthesize the head groups (Iso-H1 to Iso-H6). Route A: (Iso-H1 to Iso-H3) Iso-H1

[0313] primary amine, N,N 0.4 mmol of diethylethane-1,2-diamine, 0.8 mmol of sodium dichlorofluoroacetate, and 0.8 mmol of K₂CO₃ were added to a 25 mL Schlenk tube, which had been dried in an oven and equipped with a magnetic stirrer. The Schlenk tube was then evacuated and refilled with dry nitrogen. Anhydrous DMF (5 mL) was added to the tube using a syringe. The contents of the tube were vigorously stirred at 100 °C for 12 hours using an oil bath. The reaction mixture was then allowed to cool to room temperature. Dichloromethane was used to extract the resulting mixture. The combined organic layers were washed four times with copious amounts of water, followed by washing with brine and drying over magnesium sulfate. The solvent was removed under vacuum, and the remaining residue was purified by silica gel column chromatography. The eluent used for purification was a mixture of petroleum ether (PE) and ethyl acetate (EA). This method yielded the purified isocyanate compound iso-H₁. 1 H NMR (400 MHz, CDCl3) δ 5.08 (s, 2H), 3.55 - 3.35 (m, 2H), 3.13 -2.64 (m, 4H), 1.08 (t, J = 6.8 Hz, 6H).

[0314] Iso-H2

[0315] The synthesis route is the same as that of Iso-H1. 1 H NMR (400 MHz, CDCl3) δ 3.80 (s, 3H), 3.39 (s,6H), 3.11 - 3.05 (m, 1H), 1.33 (t, J = 7.3 Hz, 2H).

[0316] Iso-H3

[0317] The synthesis route is the same as that of Iso-H1. 1H NMR (400 MHz, CDCl3) δ 3.44 - 3.27 (m, 2H), 2.58 - 2.44 (m, 6H), 1.66 (p, J = 6.1 Hz, 2H), 1.02 (td, J = 7.2, 4.9 Hz, 6H).

[0318] Route B: (Iso-H4 to Iso-H6) General program. The mixture was prepared by adding 30 mmol of methyl isocyanate to 30 mmol of amine, followed by stirring overnight at room temperature. If the product precipitated during the reaction, it was filtered, washed three times with cold diethyl ether, and then dried under vacuum overnight. If no precipitation was observed, cold diethyl ether was added to the reaction mixture, and the product was allowed to crystallize in a refrigerator at -20°C. In rare cases, if the product remained as an oil or did not crystallize, purification was performed using preparative chromatographic silica gel and ethyl acetate as eluents. In several cases, sonication was used to enhance the crystallization process.

[0319] Iso-H4

[0320] Same as the general procedure. 1 H NMR (400 MHz, CDCl3) δ 4.29 (s, 2H), 3.60 (p, J = 4.7Hz, 4H), 3.51 (p, J = 4.9 Hz, 2H), 3.28 (p, J = 4.9 Hz, 2H).

[0321] Iso-H5

[0322] Same as the general procedure. 1 H NMR (400 MHz, CDCl3) δ 4.48 (s, 2H), 3.70 - 3.65 (m,2H), 3.45 - 3.39 (m, 2H), 1.80 - 1.68 (m, 6H).

[0323] Iso-H6

[0324] Same as the general procedure. 1H NMR (400 MHz, CDCl3) δ 4.48 (s, 2H), 3.70 - 3.65 (m,2H), 3.45 - 3.39 (m, 2H), 1.80 - 1.68 (m, 6H).

[0325] Example 1. High-throughput combinatorial synthesis of lipid compounds In this embodiment, the isonitrile compound library consists of the following compounds: .

[0326] The aldehyde compound library consists of the following compounds: .

[0327] The carboxylic acid compound library consists of the following compounds: .

[0328] The reactants used in this embodiment were prepared according to the procedures described above or commercially available chemicals (e.g., from Sigma-Aldrich and TCI America).

[0329] Constructed from 6 3 A lipid library consisting of 144 lipid compounds was prepared and transfected in vitro using LNP.

[0330] For in vitro transfection, a lipid-mRNA mixture containing 0.1 μg mRNA was added to 96-well plates pre-seeded with HeLa and A549 cells. After overnight incubation, mLuc transfection efficiency was assessed using a One-Glo luciferase assay system (Promega) according to the manufacturer's instructions. Luminescence was measured using a Cytation imaging reader (BioTek).

[0331] II. Option Two Synthesis of carboxylic acid compounds (H1 to H20) Table 5 – List of Head Groups

[0332] H1 to H11 and H18 to H21 were all purchased from suppliers.

[0333] H12 to H17 were prepared according to conventional synthetic methods well known to those skilled in the art, and were identified.

[0334] H12: 3-Undecaneol (3.0025 g, 1 equivalent), N-diisopropylethylamine (3.4346 g, 2.5 equivalent), dicyclohexylcarbodiimide (2.1933 g, 1 equivalent), and 4-(dimethylamino)pyridine (0.1299 g, 0.1 equivalent) were dissolved in 100 mL of ultradry dichloromethane and purged three times with nitrogen. Succinic acid (1.2561 g, 1 equivalent) dissolved in 50 mL of ultradry dichloromethane was added through a constant-pressure dropping funnel. The reaction was allowed to proceed overnight at room temperature. The mixture was washed twice with saturated sodium bicarbonate solution, twice with saturated brine, and then dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was removed under vacuum, and the residue was purified by rapid column chromatography (PE solution of 0% to 20% EA).

[0335] 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 1H), 2.29 (m, 4H), 1.65 (m, 4H), 1.50–1.26 (m, 20H), 0.88 (m, 6H). LCMS (ESI): 327.5 m / z. Chemical formula: C 19 H 36 O4 H13: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 2H), 2.29 (m, 4H), 2.09 (m, 1H), 1.65 (m, 4H), 1.50–1.26 (m, 16H), 0.88 (t, 3H), 0.80 (t, 3H). LCMS (ESI): 313.5 m / z. Chemical formula: C 18 H 34 O4 H14: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.66 (s, 2H), 2.34 (m, 6H), 1.58 (m, 2H), 1.50–1.26 (m, 14H), 0.88 (t, 3H). LCMS (ESI): 295.2 m / z. Chemical formula: C 17 H 28 O4 H15: 1¹H NMR (500 MHz, CDCl₃, ppm): δ 4.08 (t, 2H), 2.30 (m, 3H), 1.66–1.25 (m, 32H), 0.88 (t, 6H). LCMS (ESI): 383.6 m / z. Chemical formula: C 23 H 44 O4 H16: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 1H), 2.29 (m, 4H), 1.65 (m, 4H), 1.50–1.26 (m, 32H), 0.88 (t, 6H). LCMS (ESI): 411.7 m / z. Chemical formula: C 25 H 48 O4 H17: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 3.96 (d, 2H), 2.33 (m, 4H), 1.65 (m, 5H), 1.50–1.26 (m, 32H), 0.88 (t, 6H). LCMS (ESI): 425.7 m / z. Chemical formula: C 26 H 50 O4 Synthesize isocyanate compounds (-NC) (A1 to A12) Table 6 – List of Isocyanide Compound A

[0336] A1: 1-Isocyanoundecane 1-Isocyanoundecane was synthesized using the same procedure as in our previous work. 1, 2In a 250 mL round-bottom flask, a mixture of 82.75 mL tetrahydrofuran (THF) and 2.2511 g solid sodium formate was stirred to prepare a suspension. Simultaneously, a solution of acetyl chloride (1.0 M) in dichloromethane (34.755 mL) was slowly added dropwise to the prepared suspension. The resulting mixture was stirred at room temperature for 12 hours to obtain reaction solution 1. Then, undecane-1-amine was dissolved in tetrahydrofuran (THF) and added dropwise to reaction solution 1, followed by stirring at room temperature for another 4 hours to obtain reaction solution 2. Reaction solution 2 was then diluted with 125 mL of deionized water, and the aqueous phase was extracted twice with 240 mL of ethyl acetate (120 mL each time). The resulting extract was dried over anhydrous magnesium sulfate (MgSO4) for 0.5 hours, followed by centrifugation to obtain crude... N -Undecylformamide. The crude... N A THF (50 mL) solution of the undecylformamide product was added to a 100 mL round-bottom flask, and the mixture was placed in an ice bath at 0 °C. After stirring for 10 min, triethylamine (TEA) was introduced, and the mixture was stirred again in an ice bath for 15 min to obtain reaction solution 1. In separate preparations, solution 1 was formed by combining THF (12.9 mL) and phosphorus oxychloride (3.519 g). Solution 1 was added dropwise to reaction solution 1 using a constant-pressure dropping funnel under ice bath conditions. The resulting reaction mixture was stirred for 1.5 h. The mixture was diluted and washed with 65 mL of added saturated sodium bicarbonate solution, and the washings were extracted twice with 130 mL of diethyl ether (65 mL each time). The combined extracts were treated with anhydrous MgSO4, stirred, and dried for 0.5 h. The solvent was then removed by rotary evaporation, and the crude product was purified using a column chromatography system with hexane / ethyl acetate to give compound A1 as a pale yellow oil (yield 83%). 1 H NMR(400 MHz, CDCl3) δ 3.42 – 3.27 (m, 2H), 1.63 – 1.52 (m, 2H), 1.31 – 1.24 (m,16H), 0.88 – 0.86 (m, 3H). MS ( m / z ):C 12 H 23 N (M + H + The calculated MW value is 181.18, and the measured value is 181.68.

[0337] A2: 1-Isocyanododecane Compound A2 was obtained by the same method as A1, and was a pale yellow oil (yield 75%). 1H NMR (400MHz, CDCl3) δ 3.49 – 3.22 (m, 2H), 1.72 – 1.57 (m, 2H), 1.50 – 1.34 (m, 2H), 1.26 (d, J = 10.0 Hz, 16H), 0.89 – 0.84 (m, 3H). MS ( m / z ):C 13 H 25 N (M + H + The calculated MW value is 195.20, and the measured value is 195.35.

[0338] A3: 1-Isocyanohexadecane Compound A3 was obtained by the same method as A1, and was a pale yellow oil (yield 78%). 1 H NMR (400MHz, CDCl3) δ 3.45 – 3.26 (m, 2H), 1.66 (ddtt, J = 11.4, 6.7, 4.6, 2.3 Hz, 2H),1.46 – 1.38 (m, 2H), 1.26 (d, J = 11.1 Hz, 24H), 0.87 (t, J = 6.8 Hz, 3H). MS ( m / z ):C 17 H 33 N (M + H + The calculated MW value is 251.26, and the measured value is 251.67.

[0339] A4: (Z)-1-Isocyanooctadec-9-ene Compound A4 was obtained by the same method as A1, and was a pale yellow oil (yield 72%). 1 H NMR (400MHz, CDCl3) δ 5.41 – 5.21 (m, 2H), 3.45 – 3.29 (m, 2H), 1.99 (dq, J = 14.2, 6.0Hz, 3H), 1.66 (ddd, J = 8.4, 5.6, 3.4 Hz, 2H), 1.48 – 1.35 (m, 2H), 1.28 (dt, J =17.3, 6.2 Hz, 20H), 0.92 – 0.84 (m, 3H). MS ( m / z ):C19 H 35 N (M + H + The calculated MW value is 277.28, and the measured value is 277.47.

[0340] A5: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 3.38 (t, 2H), 1.65 (m, 2H), 1.29 (m, 22H), 0.88 (t, 3H). LCMS (ESI): 224.1 m / z. Chemical formula: C 15 H 29 N.

[0341] A6: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.08 (t, 2H), 3.40 (t, 2H), 2.30 (m, 1H), 1.80 (m, 2H), 1.66–1.25 (m, 32H), 0.88 (t, 6H). LCMS (ESI): 366.2 m / z. Chemical formula: C 23 H 43 NO2 A7: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 1H), 3.38 (t, 2H), 2.29 (t, 2H), 1.65 (m, 4H), 1.50–1.26 (m, 34H), 0.88 (t, 6H). LCMS (ESI): 408.3 m / z. Chemical formula: C 26 H 49 NO2 A8: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.21 (t, 2H), 3.51 (t, 2H), 2.25 (d, 2H), 2.02 (m, 2H), 1.83 (m, 1H), 1.42–1.14 (m, 32H), 0.88 (t, 6H). LCMS (ESI): 394.2 m / z. Chemical formula: C 25 H 47 NO2 A9: 1¹H NMR (500 MHz, CDCl₃, ppm): δ 4.10 (t, 2H), 3.38 (t, 2H), 2.31 (m, 2H), 1.67 (m, 5H), 1.44–1.20 (m, 16H), 0.88 (t, 6H). LCMS (ESI): 296.1 m / z. Chemical formula: C 18 H 33 NO2 A10: 1 ¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 2H), 3.38 (t, 2H), 2.29 (t, 2H), 2.09 (m, 1H), 1.65 (m, 4H), 1.50–1.26 (m, 18H), 0.88 (t, 3H), 0.80 (t, 3H). LCMS (ESI): 310.1 m / z. Chemical formula: C 19 H 35 NO2 A11: Boc-8-aminooctanoic acid (1.9179 g, 1.45 equivalents), 3-decyl alcohol (0.8788 g, 1 equivalent), dicyclohexylcarbodiimide (1.0523 g, 1 equivalent), and 4-(dimethylamino)pyridine (0.0623 g, 0.1 equivalent) were dissolved in a 250 mL single-end flask containing 50 mL of DCM and stirred thoroughly at room temperature for 18 hours. After the reaction was complete, the solvent was removed under vacuum, and the residue was purified by rapid column chromatography (PE solution of 0% to 10% EA). After column chromatography, the product was dissolved in 10 mL of DCM, and then 10 mL of LTA was slowly added while stirring thoroughly for 3 hours. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted three times with DCM. The extract was dried over anhydrous Na₂SO₄, filtered, and the solvent was removed by rotary evaporation to obtain crude 8-aminooctanoic acid heptadecane-9 ester. Add 3.1020 g (1 equivalent) of hepten-9-aminooctanoate, 1.4740 g (2 equivalents) of sodium dichlorofluoroacetate, and 2.1560 g (2 equivalents) of potassium carbonate to a 500 mL single-necked flask. Add 100 mL of DMF, purge with nitrogen, heat, and stir in an oil bath at 100 °C for 12 hours. Allow to cool to room temperature, add 100 mL of dichloromethane, wash four times with plenty of deionized water, wash once with saturated brine, and dry with anhydrous sodium sulfate. Remove the solvent under vacuum, and purify the residue by passing it through a rapid column (PE solution of 0% to 10% EA).

[0342] 1¹H NMR (500 MHz, CDCl₃, ppm): δ 4.47 (m, 1H), 3.38 (t, 2H), 2.29 (t, 2H), 1.65 (m, 4H), 1.50–1.26 (m, 22H), 0.88 (m, 6H). LCMS (ESI): 324.1 m / z. Chemical formula: C 20 H 37 NO2 A12: 1-Isocyanooctadecane 1 H NMR (400 MHz, CDCl3) δ 3.48 - 3.23 (m, 2H), 1.73 - 1.61 (m, 2H), 1.46 - 1.37 (m, 2H), 1.25 (s, 28H), 0.91 - 0.84 (m, 3H).

[0343] Synthetic aldehyde compounds (-CHO) (B1 to B12) Table 7 – List of Aldehyde Compounds B

[0344] B1: Decal To produce decanal, decanol was dissolved in DCM, and 1.2 times the volume of Des Martin periodane (DMP) was added, and the mixture was kept for 2 hours. After removing the solvent by vacuum rotary evaporation, the product was purified by column chromatography (hexane / EA = 80:20) to give compound B1 as a colorless oil (yield 85%). 1 H NMR (400 MHz, CDCl3) δ 9.76 (t, J = 1.9 Hz, 1H), 2.42(td, J = 7.4, 1.9 Hz, 2H), 1.62 (p, J = 7.2 Hz, 2H), 1.41 – 1.14 (m, 12H), 0.99 –0.68 (m, 3H). MS ( m / z ):C 10 H 20 O (M + H + The calculated MW value is 156.15, and the measured value is 156.22.

[0345] B2: Olealdehyde Compound B2 was obtained by the same method as B1, and was a colorless oil (yield 82%).

[0346] 1H NMR (400 MHz, CDCl3) δ 9.75 (t, J = 1.9 Hz, 1H), δ 5.51 – 5.20 (m,2H), 2.39 (dt, J = 36.8, 7.5 Hz, 2H), 2.19 – 1.86 (m, 4H), 1.62 (q, J = 7.5 Hz, 2H), 1.42 – 1.14 (m, 20H), 0.91 – 0.83 (m, 3H). MS ( m / z ):C 18 H 34 O (M + H + The calculated MW value is 266.26, and the measured value is 266.42.

[0347] B3: 6-Oxyhexyl dodecanoate Dodecanoic acid (2.0 g, 7.80 mmol), hexane-1,6-diol (1.84 g, 15.60 mmol), N,N'-dicyclohexylcarbodiimide (DCC) (2.41 g, 11.70 mmol), and 4-(dimethylamino)pyridine (DMAP) (95.29 mg, 779.94 μmol) were sequentially dissolved in a 100 mL round-bottom flask containing 50 mL of DCM and mixed thoroughly at room temperature for 24 hours. To generate the aldehyde, the intermediate was dissolved in DCM, and 1.2 times the volume of DMP was added, and the mixture was kept for 2 hours. After removing the solvent by vacuum rotary evaporation, the compound was purified by column chromatography (hexane / EA = 80:20) to give compound B3 as a colorless oil (78% yield).

[0348] 1 H NMR (400 MHz, CDCl3) δ 9.76 (t, J = 1.7 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.45 (td, J = 7.3, 1.7 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.75 – 1.54 (m,6H), 1.44 – 1.36 (m, 2H), 1.30 – 1.21 (m, 16H), 0.88 (dd, J = 6.7, 1.7 Hz, 3H). MS ( m / z ):C 18 H 34 O3(M + H+ The calculated MW value is 298.25, and the measured value is 298.33.

[0349] B4: 6-Oxyhexyl-2-hexyldecanoate Compound B4 was obtained by the same method as B3, as a colorless oil (yield 67%).

[0350] 1 H NMR (400 MHz, CDCl3) δ 9.76 (t, J = 1.7 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 2.44 (td, J = 7.3, 1.6 Hz, 2H), 2.30 (tt, J = 9.0, 5.3 Hz, 1H), 1.76 – 1.54 (m, 6H), 1.42 – 1.21 (m, 23H), 0.88 – 0.83 (m, 6H). MS ( m / z ):C 22 H 42 O3(M + H + The calculated MW value is 354.31, and the measured value is 354.58.

[0351] All other substances were obtained from suppliers.

[0352] Example 2. High-throughput combinatorial synthesis of lipid compounds In this embodiment, the isonitrile compound library consists of the following compounds:

[0353] The aldehyde compound library consists of the following compounds:

[0354] The carboxylic acid compound library consists of the following compounds:

[0355] The reactants used in this embodiment were prepared according to the procedures described above or commercially available chemicals (e.g., from Sigma-Aldrich and TCI America).

[0356] Build by 5 4 A lipid library consisting of 5=100 lipid compounds was prepared and transfected in vitro using LNP.

[0357] Example 3. High-throughput combinatorial synthesis of lipid compounds In this embodiment, the isonitrile compound library consists of the following compounds:

[0358] The aldehyde compound library consists of the following compounds:

[0359] The carboxylic acid compound library consists of the following compounds:

[0360] The reactants used in this embodiment were prepared according to the procedures described above or commercially available chemicals (e.g., from Sigma-Aldrich and TCI America).

[0361] Constructed from 6 5 A lipid library consisting of 3=90 lipid compounds was prepared and transfected in vitro using LNP.

[0362] The structure of each of these 90 lipids is shown in Table 2 for lipids 1 to 90, and is named in Table 8 as follows.

[0363] Table 8 – Names of Lipids 1 to 90

[0364] Example 4: High-throughput combinatorial synthesis of lipid compounds In this embodiment, the isonitrile compound library consists of the following compounds:

[0365] The aldehyde compound library consists of the following compounds:

[0366] The carboxylic acid compound library consists of the following compounds:

[0367] The reactants used in this embodiment were prepared according to the procedures described above or commercially available chemicals (e.g., from Sigma-Aldrich and TCI America).

[0368] Constructed from 4 4 A lipid library consisting of 9=144 lipid compounds was prepared and subjected to in vitro and in vivo experiments as described in Examples 12 to 13.

[0369] The structure of each of these 90 lipids is shown in Table 4. The ionizable lipid compounds and the LNPs formulated from them are characterized as shown in Table 9. Table 9 – Physicochemical properties and characterization data of ionizable lipids and corresponding LNPs

[0370] Example 5. Preparation of (Z)-6-((3-(dimethylamino)propionyl)oxy)-7-(octadecano-9-en-1-ylamino)-7-oxoheptyl dodecanoate (lipid H2A4B3)

[0371] The synthesis in Example 5 was the same as described above. At room temperature, a mixture of 3-(dimethylamino)propionic acid (30 mg, 256.09 μmol), (Z)-1-isocyanooctadec-9-ene (71.06 mg, 256.09 μmol), and 6-oxohexyl dodecanoate (76.43 mg, 256.09 μmol) in anhydrous solvent DCM (1 mL) was stirred for 12 hours in a capped glass vial. The mixture was purified using a gradient (1% ammonia, 10% MeOH, 89% DCM) to give the final product as a colorless oil (yield: 150 mg, 84%). Rf = 0.4 (ammonia / MeOH / DCM = 1:10:89). 1 H NMR (400 MHz, CDCl3) δ 5.34 (ddt, J = 5.5, 3.7, 1.8 Hz, 2H), 5.26 -5.08 (m, 1H), 4.05 (td, J = 4.1, 2.1 Hz, 2H), 3.34 - 2.99 (m, 4H), 2.68 - 2.42(m, 2H), 2.34 - 2.20 (m, 6H), 2.00 (q, J = 6.5 Hz, 7H), 1.15-1.55 (m, 50H), 0.87 - 0.86 (m, 6H). MS(m / z):C 42 H 81 N2O5 + [M+H] + Calculated value: 694.11, measured value: 694.80.

[0372] Example 6. Preparation of (Z)-6-((3-(dimethylamino)propionyl)oxy)-7-(octadecano-9-en-1-ylamino)-7-oxohepyl-2-hexyldecanoate (lipid H2A4B4)

[0373] Synthesis of Example 6 via 3CR-Pacerini reaction. At room temperature, a mixture of 3-(dimethylamine)propionic acid (30 mg, 256.09 μmol), (Z)-1-isocyanooctadec-9-ene (71.06 mg, 256.09 μmol), and 6-oxohexyl-2-hexyldecanoate (90.8 mg, 256.09 μmol) in anhydrous DCM (1 mL) was stirred for 12 hours in a capped glass vial. Further purification was achieved by rapid column chromatography on a Combiflash system, eluting with a gradient (1% ammonia, 10% MeOH, 89% DCM) to give the final product as a colorless oil (yield: 152 mg, 78%). Rf = 0.4 (ammonia / MeOH / DCM = 1:10:89). 1 H NMR (400 MHz, CDCl3) δ 5.32 (d, J = 16.3 Hz, 2H), 5.20 (dd, J = 7.9,3.7 Hz, 1H), 4.07 - 4.04 (m, 2H), 3.72 - 3.45 (m, 2H), 3.21 (dd, J = 45.0, 7.5Hz, 2H), 2.56 - 2.37 (m, 2H), 2.34 - 2.20 (m, 8H), 2.07 - 1.88 (m, 5H), 1.25(m, 49H), 0.92 - 0.83 (m, 9H). MS(m / z):C 46 H 88 N2O5 + [M+H] + Calculated value: 750.22, measured value: 750.55.

[0374] Example 7. Preparation of (Z)-6-((4-(dimethylamino)butyryl)oxy)-7-(octadecano-9-en-1-ylamino)-7-oxohepyl-2-hexyldecanoate (lipid H18A4B4)

[0375] The synthesis in Example 7 was the same as described above. At room temperature, a mixture of 5-(dimethylamino)valerate (30 mg, 206.6 μmol), (Z)-1-isocyanooctadec-9-ene (57.33 mg, 206.6 μmol), and 6-oxohexyl dodecanoate (73.26 mg, 206.6 μmol) in anhydrous solvents DCM or THF (1 mL) was stirred for 12 hours in a capped glass vial. The mixture was purified using a gradient (1% ammonia, 10% MeOH, 89% DCM) to give the final product as a colorless oil (yield: 136 mg, 84%). Rf = 0.4 (ammonia / MeOH / DCM = 1:10:89).

[0376] 1 H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 1.7 Hz, 1H), 5.40 (q, J = 7.4 Hz,3H), 4.23 - 3.66 (m, 2H), 3.30 (dq, J = 34.7, 6.8 Hz, 4H), 2.47 - 1.78 (m, 9H), 1.73 - 1.23 (m, 65H), 1.02 - 0.72 (m, 9H). MS(m / z):C 48 H 92 N2O5 + [M+H] + Calculated value: 777.70, measured value: 777.82.

[0377] Example 8. Preparation of (Z)-6-((5-(dimethylamino)pentanoyl)oxy)-7-(octadecano-9-en-1-ylamino)-7-oxohepyl-2-hexyldecanoate (lipid H19A4B4)

[0378] The synthesis in Example 8 was the same as described above. At room temperature, a mixture of 5-(dimethylamino)valerate (30 mg, 206.6 μmol), (Z)-1-isocyanooctadec-9-ene (57.33 mg, 206.6 μmol), and 6-oxohexyl dodecanoate (73.26 mg, 206.6 μmol) in anhydrous DCM (1 mL) was stirred for 12 hours in a capped glass vial. The mixture was purified using a gradient (1% ammonia, 10% MeOH, 89% DCM) to give the final product as a colorless oil (yield: 136 mg, 84%). Rf = 0.4 (ammonia / MeOH / DCM = 1:10:89).

[0379] 1 H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 1.7 Hz, 1H), 5.40 (q, J = 7.4 Hz,3H), 4.23 - 3.66 (m, 2H), 3.30 (dq, J = 34.7, 6.8 Hz, 4H), 2.47 - 1.78 (m, 9H), 1.73 - 1.23 (m, 65H), 1.02 - 0.72 (m, 9H). MS(m / z):C 48 H 92 N2O5 + [M+H] + Calculated value: 777.70, measured value: 777.82.

[0380] Example 9: Synthesis of lipid compounds lipids 91 to 196 Lipid 91 3-(dimethylamino)propionaldehyde (1.0 mmol), tetradecanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane solution, or 0% to 50% EA in PE solution).

[0381] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 3.27 (t,2H), 2.40 (m, 4H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 44H), 0.88 (t,6H).

[0382] Lipid 92 3-(dimethylamino)propionaldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0383] 1H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 38H), 0.88 (t, 6H).

[0384] Lipid 94 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0385] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.81 (t,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,46H), 0.88 (t, 9H).

[0386] Lipid 95 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0387] 1H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 3.95 (m,1H), 3.82 (m, 1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 42H), 0.91 (m, 3H), 0.88 (t, 6H).

[0388] Lipid 97 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0389] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.67 (s,1H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,38H), 0.88 (t, 6H).

[0390] Lipid 98 3-(dimethylamino)propionaldehyde (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane solution, or 0% to 50% EA in PE solution).

[0391] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,54H), 0.88 (t, 9H).

[0392] Lipid 99 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0393] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.86 (s,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,58H), 0.88 (t, 9H).

[0394] Lipid 100 3-(dimethylamino)propionaldehyde (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0395] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 3.98 (d,2H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,57H), 0.88 (t, 9H).

[0396] Lipid 101 3-(dimethylamino)propionaldehyde (1.0 mmol), myristic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0397] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.27 (t, 2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,44H), 0.88 (t, 6H).

[0398] Lipid 102 3-(dimethylamino)propionaldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0399] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 6H), 5.19 (s,1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 14H), 1.83 (m, 4H), 1.51-1.26 (m, 38H), 0.88 (t, 6H).

[0400] Lipid 104 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0401] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.81 (t, 1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 46H), 0.88 (t, 9H).

[0402] Lipid 105 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0403] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.95 (m, 1H), 3.82 (m, 1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 42H), 0.91 (m, 3H), 0.88 (t, 6H).

[0404] Lipid 107 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0405] 1H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.67 (s, 1H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 38H), 0.88 (t, 6H).

[0406] Lipid 108 3-(dimethylamino)propionaldehyde (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0407] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 54H), 0.88 (t, 9H).

[0408] Lipid 109 3-(dimethylamino)propionaldehyde (1.0 mmol), 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0409] 1H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.86 (s, 1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 58H), 0.88 (t, 9H).

[0410] Lipid 110 3-(dimethylamino)propionaldehyde (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0411] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.98 (d, 2H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 57H), 0.88 (t, 9H).

[0412] Lipid 111 3-(dimethylamino)propionaldehyde (1.0 mmol), myristic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0413] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,52H), 0.88 (t, 9H).

[0414] Lipid 112 3-(dimethylamino)propionaldehyde (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0415] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 46H), 0.88 (t, 9H).

[0416] Lipid 114 3-(dimethylamino)propionaldehyde (1.0 mmol), tetradecanoic acid (1.0 mmol), and heptadecan-9-yl-8-isocyanoctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0417] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.86 (m,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,58H), 0.88 (t, 9H).

[0418] Lipid 115 3-(dimethylamino)propionaldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and heptadecan-9-yl 8-isocyanooctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0419] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.86 (m, 1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 52H), 0.88 (t, 9H).

[0420] Lipid 117 3-(dimethylamino)propionaldehyde (1.0 mmol), myristic acid (1.0 mmol), and 3-isocyanopropyl 3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0421] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 7H), 1.83 (m, 4H), 1.51-1.26 (m,54H), 0.88 (t, 9H).

[0422] Lipid 118 3-(dimethylamino)propionaldehyde (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 3-isocyanopropyl-3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0423] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.72 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 11H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t, 9H).

[0424] Lipid 121 4-(dimethylamino)butyraldehyde (1.0 mmol), tetradecanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane solution, or 0% to 50% EA in PE solution).

[0425] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 3.27 (t,2H), 2.40 (m, 4H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 46H), 0.88 (t,6H).

[0426] Lipid 122 4-(dimethylamino)butyraldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0427] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 40H), 0.88 (t, 6H).

[0428] Lipid 124 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0429] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.81 (t,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,48H), 0.88 (t, 9H).

[0430] Lipid 125 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0431] 1H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 3.95 (m,1H), 3.82 (m, 1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 44H), 0.91 (m, 3H), 0.88 (t, 6H).

[0432] Lipid 127 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0433] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.67 (s,1H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,40H), 0.88 (t, 6H).

[0434] Lipid 128 4-(dimethylamino)butyraldehyde (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0435] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,56H), 0.88 (t, 9H).

[0436] Lipid 129 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0437] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.86 (s,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,60H), 0.88 (t, 9H).

[0438] Lipid 130 4-(dimethylamino)butyraldehyde (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0439] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 3.98 (d,2H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,59H), 0.88 (t,9H).

[0440] Lipid 131 4-(dimethylamino)butanal (1.0 mmol), myristic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0441] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.27 (t, 2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,46H), 0.88 (t, 6H).

[0442] Lipid 132 4-(dimethylamino)butyraldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0443] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 6H), 5.19 (s,1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 14H), 1.83 (m, 4H), 1.51-1.26 (m, 40H), 0.88 (t, 6H).

[0444] Lipid 134 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0445] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.81 (t, 1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t, 9H).

[0446] Lipid 135 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0447] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.95 (m, 1H), 3.82 (m, 1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 44H), 0.91 (m, 3H), 0.88 (t, 6H).

[0448] Lipid 137 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0449] 1H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.67 (s, 1H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 40H), 0.88 (t, 6H).

[0450] Lipid 138 4-(dimethylamino)butyraldehyde (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0451] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 56H), 0.88 (t, 9H).

[0452] Lipid 139 4-(dimethylamino)butyraldehyde (1.0 mmol), 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0453] 1H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 4.86 (s, 1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 60H), 0.88 (t, 9H).

[0454] Lipid 140 4-(dimethylamino)butanal (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0455] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 2H), 5.19 (s,1H), 3.98 (d, 2H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 59H), 0.88 (t, 9H).

[0456] Lipid 141 4-(dimethylamino)butyraldehyde (1.0 mmol), myristic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0457] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,54H), 0.88 (t, 9H).

[0458] Lipid 142 4-(dimethylamino)butyraldehyde (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0459] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t, 9H).

[0460] Lipid 144 4-(dimethylamino)butyraldehyde (1.0 mmol), myristic acid (1.0 mmol), and heptadecan-9-yl-8-isocyanoctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0461] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.86 (m,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,60H), 0.88 (t, 9H).

[0462] Lipid 145 4-(dimethylamino)butyraldehyde (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and heptadecan-9-yl 8-isocyanooctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0463] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.86 (m, 1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 54H), 0.88 (t, 9H).

[0464] Lipid 147 4-(dimethylamino)butyraldehyde (1.0 mmol), myristic acid (1.0 mmol), and 3-isocyanopropyl-3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0465] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 7H), 1.83 (m, 4H), 1.51-1.26 (m,56H), 0.88 (t, 9H).

[0466] Lipid 148 4-(dimethylamino)butyraldehyde (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 3-isocyanopropyl-3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0467] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.34 (m, 4H), 5.19 (s,1H), 4.05 (t, 2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 11H), 1.83 (m, 4H), 1.51-1.26 (m, 50H), 0.88 (t, 9H).

[0468] Lipid 151 4-(dimethylamino)but-2-one (1.0 mmol), myristic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0469] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 3.27 (t, 2H), 2.40 (m,4H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 47H), 0.88 (t, 6H).

[0470] Lipid 152 4-(dimethylamino)but-2-one (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0471] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 4H), 3.27 (t,2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,41H), 0.88 (t, 6H).

[0472] Lipid 154 4-(dimethylamino)but-2-one (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0473] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.81 (t, 1H), 3.27 (t,2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 49H), 0.88 (t,9H).

[0474] Lipid 155 4-(dimethylamino)but-2-one (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0475] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 3.95 (m, 1H), 3.82 (m,1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,45H), 0.91 (m, 3H), 0.88 (t, 6H).

[0476] Lipid 157 4-(dimethylamino)but-2-one (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0477] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.67 (s, 1H), 3.27 (t,2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 41H), 0.88 (t,6H).

[0478] Lipid 158 4-(dimethylamino)but-2-one (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane solution, or 0% to 50% EA in PE solution).

[0479] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.05 (t, 2H), 3.27 (t,2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 57H), 0.88 (t,9H).

[0480] Lipid 159 4-(dimethylamino)but-2-one (1.0 mmol), 8-(heptadecane-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0481] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.86 (s, 1H), 3.27 (t,2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 61H), 0.88 (t,9H).

[0482] Lipid 160 4-(dimethylamino)but-2-one (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0483] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 3.98 (d, 2H), 3.27 (t,2H), 2.40 (m, 8H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 60H), 0.88 (t,9H).

[0484] Lipid 161 4-(dimethylamino)but-2-one (1.0 mmol), myristic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0485] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 3.27 (t,2H), 2.40 (m, 4H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 47H), 0.88 (t,6H).

[0486] Lipid 162 4-(dimethylamino)but-2-one (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0487] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 6H), 3.27 (t,2H), 2.70 (t, 2H), 2.40 (m, 4H), 2.05 (m, 14H), 1.83 (m, 4H), 1.51-1.26 (m,41H), 0.88 (t, 6H).

[0488] Lipid 164 4-(dimethylamino)but-2-one (1.0 mmol), 8-oxo-8-(undecane-3-yloxy)octanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0489] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 4.81 (t,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,49H), 0.88 (t,9H).

[0490] Lipid 165 4-(dimethylamino)but-2-one (1.0 mmol), 8-((2-methylnonyl)oxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0491] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 3.95 (m,1H), 3.82 (m, 1H), 3.27 (t, 2H), 2.40 (m, 7H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 45H), 0.91 (m, 3H), 0.88 (t, 6H).

[0492] Lipid 167 4-(dimethylamino)but-2-one (1.0 mmol), 8-(non-2-yn-1-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0493] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 4.67 (s,1H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,41H), 0.88 (t, 6H).

[0494] Lipid 168 4-(dimethylamino)but-2-one (1.0 mmol), 7-((2-hexyldecanoyl)oxy)heptanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE solution).

[0495] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,57H), 0.88 (t, 9H).

[0496] Lipid 169 4-(dimethylamino)but-2-one (1.0 mmol), 8-(heptadecyl-9-yloxy)-8-oxooctanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0497] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 4.86 (s,1H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,61H), 0.88 (t, 9H).

[0498] Lipid 170 4-(dimethylamino)but-2-one (1.0 mmol), 6-((2-octyldodecyl)oxy)-6-oxohexanoic acid (1.0 mmol), and (Z)-1-isocyanooctadec-9-ene (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0499] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 2H), 3.98 (d,2H), 3.27 (t, 2H), 2.40 (m, 8H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m,60H), 0.88 (t, 9H).

[0500] Lipid 171 4-(dimethylamino)but-2-one (1.0 mmol), myristic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0501] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.05 (t, 2H), 3.27 (t,2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 55H), 0.88 (t,9H).

[0502] Lipid 172 4-(dimethylamino)but-2-one (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0503] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 4H), 4.05 (t,2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 5H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 49H), 0.88 (t, 9H).

[0504] Lipid 174 4-(dimethylamino)but-2-one (1.0 mmol), myristic acid (1.0 mmol), and heptadecan-9-yl-8-isocyanoctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0505] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.86 (m, 1H), 3.27 (t,2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 61H), 0.88 (t,9H).

[0506] Lipid 175 4-(dimethylamino)but-2-one (1.0 mmol), (9Z,12Z)-octadec-9,12-dienoic acid (1.0 mmol), and heptadecan-9-yl 8-isocyanooctanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0507] 1H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 4H), 4.86 (m,1H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 10H), 1.83 (m, 4H), 1.51-1.26 (m, 55H), 0.88 (t, 9H).

[0508] Lipid 177 4-(dimethylamino)but-2-one (1.0 mmol), myristic acid (1.0 mmol), and 3-isocyanopropyl-3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0509] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 4.05 (t, 2H), 3.27 (t,2H), 2.40 (m, 6H), 2.05 (m, 7H), 1.83 (m, 4H), 1.51-1.26 (m, 57H), 0.88 (t,9H).

[0510] Lipid 178 4-(dimethylamino)but-2-one (1.0 mmol), (9Z,12Z)-octadecano-9,12-dienoic acid (1.0 mmol), and 3-isocyanopropyl-3-octyltridecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0511] 1 H NMR (500 MHz, CDCl3, ppm): δ 5.91 (s, 1H), 5.34 (m, 4H), 4.05 (t,2H), 3.27 (t, 2H), 2.70 (t, 2H), 2.40 (m, 6H), 2.05 (m, 11H), 1.83 (m, 4H), 1.51-1.26 (m, 51H), 0.88 (t, 9H).

[0512] Lipid 181 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 3-(pyrrolidone-1-yl)propionic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0513] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,4H), 3.76 (m, 2H), 3.27 (t, 2H), 2.40 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,66H), 0.88 (t, 12H).

[0514] Lipid 182 Dextran (1.0 mmol), 3-(pyrrolidone-1-yl)propionic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0515] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.76 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 1.83 (m, 4H), 1.51-1.26 (m,56H), 0.88 (t, 9H).

[0516] Lipid 183 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 3-(pyrrolidone-1-yl)propionic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0517] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.76 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 1.83 (m, 4H), 1.51-1.26 (m,58H), 0.88 (t, 9H).

[0518] Lipid 184 Dextran (1.0 mmol), 3-(pyrrolidone-1-yl)propionic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0519] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 3.76 (m,2H), 3.27 (t, 2H), 2.40 (m, 4H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t,6H).

[0520] Lipid 185 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 1-methylpiperidine-4-carboxylic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0521] 1H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.17 (s, 1H), 4.05 (t,4H), 3.27 (t, 2H), 2.40 (m, 5H), 1.83 (m, 4H), 1.51-1.26 (m, 69H), 0.88 (t,12H).

[0522] Lipid 186 Dextran (1.0 mmol), 1-methylpiperidine-4-carboxylic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0523] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.17 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 4H), 1.83 (m, 4H), 1.51-1.26 (m, 59H), 0.88 (t,9H).

[0524] Lipid 187 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 1-methylpiperidin-4-carboxylic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0525] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.17 (s, 1H), 4.05 (t,2H), 3.27 (t, 2H), 2.40 (m, 4H), 1.83 (m, 4H), 1.51-1.26 (m, 61H), 0.88 (t,9H).

[0526] Lipid 188 Dextran (1.0 mmol), 1-methylpiperidin-4-carboxylic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0527] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 5.17 (s, 1H), 3.27 (t,2H), 2.40 (m, 4H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t, 6H).

[0528] Lipid 189 1.0 mmol of 7-oxohepyl-2-hexyldecanoate, 1.0 mmol of 4-(dimethylamino)butyric acid, and 1.0 mmol of 6-isocyanohexyl-2-hexyldecanoate were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0529] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,4H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 60H), 0.88 (t, 12H).

[0530] Lipid 190 Dextran (1.0 mmol), 4-(dimethylamino)butyric acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0531] 1H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 50H), 0.88 (t, 9H).

[0532] Lipid 191 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 4-(dimethylamino)butyric acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0533] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 52H), 0.88 (t, 9H).

[0534] Lipid 192 Dextran (1.0 mmol), 4-(dimethylamino)butyric acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0535] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 3.40 (m,2H), 3.27 (t, 2H), 2.40 (m, 4H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,42H), 0.88 (t, 6H).

[0536] Lipid 193 1.0 mmol of 7-oxohepyl-2-hexyldecanoate, 1.0 mmol of 3-(dimethylamino)propionic acid, and 1.0 mmol of 6-isocyanohexyl-2-hexyldecanoate were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0537] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,4H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 6H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 58H), 0.88 (t, 12H).

[0538] Lipid 194 Dextran (1.0 mmol), 3-(dimethylamino)propionic acid (1.0 mmol), and 6-isocyanohexyl-2-hexyldecanoate (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0539] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 48H), 0.88 (t, 9H).

[0540] Lipid 195 7-O-Hepyl-2-hexyldecanoate (1.0 mmol), 3-(dimethylamino)propionic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0541] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 4.05 (t,2H), 3.40 (m, 2H), 3.27 (t, 2H), 2.40 (m, 5H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m, 50H), 0.88 (t, 9H).

[0542] Lipid 196 Dextran (1.0 mmol), 3-(dimethylamino)propionic acid (1.0 mmol), and 1-isocyanotetradecane (1.0 mmol) were dissolved in dichloromethane or tetrahydrofuran (1 ml). The reaction mixture was stirred at room temperature under nitrogen for 18 hours. The reaction solvent was evaporated under vacuum, and the residue was purified by silica gel chromatography (0% to 10% methanol in dichloromethane, or 0% to 50% EA in PE).

[0543] 1 H NMR (500 MHz, CDCl3, ppm): δ 6.03 (s, 1H), 5.19 (s, 1H), 3.40 (m,2H), 3.27 (t, 2H), 2.40 (m, 4H), 2.05 (m, 6H), 1.83 (m, 4H), 1.51-1.26 (m,40H), 0.88 (t, 6H).

[0544] The structure of each of these 106 lipids is shown in lipids 91 to 196 in Table 3, and is named as follows in Table 10.

[0545] Table 10 – Names of lipids 91 to 196

[0546] Example 10. In vitro transfection The lipid compounds obtained in the above examples were subjected to the following in vitro assays: The organic phase was prepared by dissolving a mixture of cationic lipids, DOPE (Avanti), cholesterol (Chol, Sigma-Aldrich), and C14-PEG2000 (Avanti) in ethanol at a predetermined molar ratio. The aqueous phase was prepared using firefly luciferase mRNA (mLuc, Trilink) dissolved in 10 mM sodium citrate buffer (pH 4.0, Fisher). All mRNA samples were stored at -80°C and thawed on ice before use.

[0547] LNPs were prepared by vigorous mixing of an aqueous phase containing mRNA with an ethanol phase containing a lipid mixture. The weight ratio of ionizable lipids to mRNA was set at 10:1. An unpurified lipid library was used directly.

[0548] For in vitro transfection, a lipid-mRNA mixture containing 0.1 μg mRNA was added to 96-well plates pre-seeded with A549 cells. After overnight incubation, mLuc transfection efficiency was assessed using a One-Glo luciferase assay system (Promega) according to the manufacturer's instructions. Luminescence was measured using a Cytation imaging reader (BioTek).

[0549] In vitro assays showed that the synthesized ionizable lipid compounds were effective for preparing LNPs for in vitro transfection. Results are summarized in […]. Figures 1 to 2 middle.

[0550] Example 11. Identification of optimal ionizable lipids for mRNA delivery via in vivo batch testing The performance of the lipid compound library prepared in Example 4 in delivering mRNA was further evaluated. The resulting ionizable lipids were formulated into LNPs using a classic four-part fractionation system established by Moderna for SM-102. This formulation contained 1,2-distearatel-sn-glycerol-3-phosphocholine (DSPC) as an auxiliary lipid, cholesterol, 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (C14-PEG), and the synthesized ionizable lipids from Example 4. Figure 3 ).

[0551] In vivo batch testing For high-throughput batch testing in vivo, lipid-mRNA mixtures within the same taxonomic group were pooled and dialyzed, then administered to mice via intramuscular injection. Then, 0.25 mg / kg... -1 mLuc was used for the first round of batch-based LNP analysis, divided into 9 groups based on the head group, each containing a mixture of 16 LNPs. For the second round of batch-based LNP analysis, 0.8 mg / kg was administered to each mouse.-1 mFFL was divided into four groups based on the tail A group, each group consisting of a mixture of four LNPs. Additionally, for the third round of individual LNP analysis, each lipid group was injected with 0.05 mg / kg. -1 mLuc. Six hours after injection, to measure in vivo bioluminescence, 0.2 ml of XenoLight D-luciferin (10 mg / ml) was injected. -1 The luciferin was administered to mice via intraperitoneal injection using Dulbecco's phosphate-buffered saline (PerkinElmer) as the solvent. Mice were anesthetized in an oxygen anesthesia chamber containing 2.5% isoflurane, and imaging was performed using an in vivo imaging system (IVIS, PerkinElmer) 10 minutes after administration. Bioluminescence was quantified using LivingImage software (PerkinElmer, version 4.8.0).

[0552] To evaluate the ability of the synthesized lipids to deliver mRNA in vivo, LNPs were formulated with firefly luciferase mRNA (mLuc) using a microfluidic T-connector for in vivo batch testing and physical characterization. Figure 4 While P-3CR facilitates the rapid synthesis of a wide variety of ionizable lipid arrays, we still employed a batch-based assay approach to efficiently evaluate all 144 LNPs in vivo. First, the 144 LNPs were classified into nine groups (H1-H9) based on the head group structure of the ionizable lipids for subsequent characterization and batch assays. The head groups were further described as linear (H1-H3), cyclic (H4-H6), and aromatic (H7-H9). To preliminarily evaluate the delivery efficiency of 144 ionizable lipids, LNPs containing ionizable lipids sharing the same head group were pooled and administered via intramuscular (IM) injection. Figure 5 Notably, linear and cyclic alkane head groups achieved the highest transfection efficiencies, while aromatic head groups showed significantly weaker transfection efficiencies. Dimethylglycine (H1), 3-(dimethylamino)propionic acid (H2), and 3-(1-pyrrolyl)propionic acid (H6) lipids outperformed other head groups, with H2 achieving the highest mLuc expression. Figure 6 Therefore, H2-containing ionizable lipids were advanced to the next stage of batch testing, in which H2 LNPs were incorporated based on the shared tail A structure and further evaluated after IM administration. Results showed that (Z)-1-isocyanooctadec-9-ene (A4), containing an unsaturated bond, achieved the highest transfection rate. In the third round of testing, all four LNPs of ionizable lipids containing the head group H2 and the tail group A4 were tested in mice. Figures 7 to 8The first two lipid candidates, H2A4B3 and H2A4B4, were evaluated head-to-head with the FDA-approved ionizable lipid MC3. Interestingly, although lipids H2A4B3 and H2A4B4 differ only in one tail chain, the LNP containing H2A4B4 significantly outperformed both H2A4B3 and MC3. Figure 9 ).

[0553] The results of in vivo tests show that the lipid compounds prepared in the embodiments of this disclosure can be used to prepare LNPs for mRNA delivery and can achieve mRNA delivery efficiencies comparable to or even better than those of ionizable lipids (such as MC3) that are already widely used in the prior art.

[0554] Example 12. Delivery of Cre-recombinase mRNA and human EPO mRNA to mice via LNP prepared from an ionizable lipid compound. Gene delivery in the tdTomato Cre reporter gene mouse model On days 0 and 3, Cg-Gt(ROSA)26Sor was administered via the tail vein. tm9(CAG-tdTomato)Hze / J mice were intravenously (IV) injected with Cre mRNA-encapsulated LNPs (0.5 mg / kg). Mice were sacrificed on day 5 to collect tissue. For flow cytometry in Ai9 mice, mouse livers were harvested, hepatocytes were isolated and stained for later use. First, the liver tissue was minced using a blade and placed in 1 mL of digestion medium consisting of type IV collagenase (100 IU / μL) and DNase I (50 IU / μL). Then, the tissue mixture was transferred to a 15 mL centrifuge tube containing another 4 mL of digestion medium and incubated at 37 °C with shaking for 1 h. Next, the liver solution was filtered through a 70 μm filter and washed once with 1×PBS. The cell pellet was obtained by centrifugation at 500 g for 10 min at 4 °C. The supernatant was removed, and the cell pellet was resuspended in 4 mL of 1×RBC lysis buffer (BioLegend, 420301) and incubated at room temperature for 5 min. After incubation, 10 mL of PBS was added to terminate erythrocyte lysis. Then, the solution was centrifuged again at 500g for 10 minutes to obtain a cell pellet. Single cells were resuspended in cell staining buffer (2% FBS containing 1×PBS) to prepare a density of 5×10⁻⁶ cells / mL. 6Cell solutions were prepared at 100 μL / mL. 100 μL of cell solution was transferred to a new EP tube and incubated at room temperature for 30 minutes with anti-mouse CD45 antibody (FITC, BioLegend, 1:1000), anti-mouse CD31 antibody (Alexa Fluor® 594, BioLegend, 1:1000), anti-mouse F4 / 80 (PerCP / Cyanine5.5, BioLegend, 1:200), anti-mouse CD11c (PE / Cyanine7, BioLegend, 1:800), anti-mouse CD3 antibody (Brilliant Violet 605™, BioLegend, 1:50), and anti-mouse / human CD45R / B220 antibody (Brilliant Violet 421™, BioLegend, 1:50) to target hepatocytes (CD45R / mL). - CD31 - Cells), endothelial cells (CD45) - CD31 + Cells), macrophages (F4 / 80) + cells), dendritic cells (CD11c) + T cells (CD3 cells) + (cells) and B cells (B220) + Cells were stained (Table 11). Stained cells were washed twice with 1 mL of 1×PBS and then resuspended in 500 μL of 1×PBS for flow cytometry. The Zombie NIR™ kit (BioLegend, 423105) was used to distinguish live cells. The PBS-treated group (NC) served as a negative control. Flow cytometry data were analyzed using FlowJo software. For confocal fluorescence imaging, all collected tissues were fixed overnight in 4% buffered paraformaldehyde at 4°C, followed by dehydration overnight in 30% sucrose at 4°C. The tissues were then frozen and sectioned after freezing at the optimal cutting temperature (OCT). Cell nuclei were stained with DAPI (1:1000). A Zeiss LCSM700 was used to acquire and process the images.

[0555] Table 11 – Antibodies used in flow cytometry

[0556] In vivo therapeutic hEPO mRNA delivery SM-102, H18A4B4, and H19A4B4 LNPs were formulated into LNPs with hEPO mRNA. Subsequently, the LNPs were administered to mice via retroorbital vein injection (0.25 mg / kg per mouse, n=8) as described above. To assess hEPO protein kinetics, approximately 100 μl of blood was collected from the facial vein of each mouse and transferred to EDTA-coated tubes. The blood was centrifuged at 2,000 g for 20 minutes. The supernatant plasma was used as a sample for ELISA. The plasma was aliquoted and stored at -80°C until analysis. hEPO levels in mice were measured using the hEPO ELISA kit (Abcam, Ab274397) according to the manufacturer's instructions. For repeated-dose experiments, hEPO mRNA was formulated into H18A4B4 LNPs as described above and administered via retroorbital injection (0.25 mg / kg per mouse, n=2). Statistical analysis Statistical analysis was performed on the data using GraphPad Prism 9 (GraphPad Software). A two-tailed unpaired Student's t-test was performed to assess the significance of the comparisons shown. Data are expressed as mean ± standard deviation (SD). P-value < 0.05 ( ). P<0.01 P<0.001 ) and P<0.0001 ( The result indicates that it is statistically significant.

[0557] To evaluate the efficacy of LNPs prepared from the ionizable lipid compounds of this disclosure in mediating gene editing and cell tropism, genetically engineered Ai9 mice with the tdTomato reporter gene were used. The Ai9 reporter gene mice visualized gene editing mediated by Cre-recombinase mRNA (mCre). Upon translation of the mRNA, Cre-recombinase promotes the excision of a LoxP-side-attached terminator, thereby expressing the red fluorescent tdTomato protein (tdTomato protein) in the edited cells. Figure 10 Lipids H18A4B4 and H19A4B4 were formulated into LNPs with mCre and compared with SM-102 LNPs after intravenous (IV) injection. Following two administrations, IVIS imaging was used to visualize tdTomato fluorescence signals in various organs of Ai9 reporter mice. Figure 11The H18A4B4 LNP showed Cre-mediated gene editing efficiency comparable to that of the SM-102 LNP. To further validate these findings, confocal microscopy was used to analyze tissue sections to visualize tdTomato-positive cells. Figure 12 Following LNP administration, mCre-edited cells were uniformly distributed throughout the mouse liver. Notably, a significant increase in tdTomato-positive cells was observed after administration of H18A4B4 LNP compared to H19A4B4 and SM-102 LNP. The percentage of tdTomato-positive cells was quantified by flow cytometry. Figure 13 Notably, H18A4B4 LNP showed superior transfection efficacy in both hepatocytes and immune cells compared to SM-102 LNP. Compared to SM-102 LNP, H18A4B4 LNP demonstrated excellent gene-editing efficacy, achieving a higher percentage of gene-edited hepatocytes (56% vs. 48%) and total immune cells (34% vs. 28%). Furthermore, H18A4B4 LNP also exhibited comparable editing efficacy to SM-102 LNP in endothelial cells and specific immune cells. Figure 14 ). The foregoing description is intended to be merely illustrative of the principles of this disclosure. Furthermore, since many modifications and variations will be apparent to those skilled in the art, it is not intended to limit this disclosure to the exact construction and process described above. Therefore, all suitable modifications and equivalents are to be considered to fall within the scope of this disclosure as defined by the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A lipid compound of Formula (I): (I) or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein (I) 2. A lipid compound of Formula (I’): (I’) or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein R I , R II , and R III are each independently selected from the group consisting of (A) radicals comprising at least one ionizable tertiary amine moiety, (B) optionally substituted C6-C 25 aliphatic radicals and optionally substituted 6- to 25-membered heteroaliphatic radicals; Preferably, R I R II R III One or both of them are selected from (A) a collection of groups containing at least one ionizable tertiary amine moiety.

3. The lipid compound according to claim 1 or 2, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the group of the collection (A) comprising at least one ionizable tertiary amine moiety has 3 to 11 carbon atoms and the group is as shown in Formula (la): (I') 4. The lipid compound according to claim 1 or 2, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the group of the collection (A) comprising at least one ionizable tertiary amine moiety has 3 to 11 carbon atoms and the group is as shown in Formula (la’): R I , R II , and R III are each independently selected from the group consisting of (A) radicals comprising at least one ionizable tertiary amine moiety, (B) an optionally substituted C6-C 25 aliphatic group and an optionally substituted 6- to 25-membered heteroaliphatic group, and R II' is hydrogen or Ci-C6alkyl; Preferably, R I , R II , R III one or both of which is selected from the group of (A) radicals comprising at least one ionizable tertiary amine moiety.

5. The lipid compound according to any one of claims 1 to 4, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (A) comprises a group comprising at least one ionizable tertiary amine moiety as follows: (Ia) wherein R Ia is optionally substituted C1-C6alkylene, said alkylene being optionally substituted with 1, 2, or 3 substituents selected from the group consisting of -oxo (=0), -OH, -SH, or -NR Id R Id' Id and R Id' are each independently hydrogen or C1-C3alkyl; R Ib and R Ic are each independently optionally substituted C1-C6alkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6alkynyl, said alkyl, alkenyl, and alkynyl being optionally substituted with 1, 2, or 3 substituents selected from the group consisting of -oxo (=0), -OH, -SH, or -NR Ie R Ie' Ie and R Ie' are each independently hydrogen or C1-C3alkyl, or R Ib and R Ic together with the N atom to which they are attached form a 5- to 12-membered heterocyclic ring comprising 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of said heteroatoms being N, said heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of halogen, C1-C6alkyl, -NO2, and -OH.​​ 6. The lipid compound according to any one of claims 1 to 4, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (A) comprises a group comprising at least one ionizable tertiary amine moiety as follows: (Ia') Where Ar is C6-C that is optionally substituted. 10 arylene, wherein the arylene is optionally surrounded by 1, 2, 3 or 4 independently selected from halogen, C1-C6 alkyl, -OH, -SH or -NR. Id'' R Id''' The substituents are replaced by R, where R is a substituent. Id'' and R Id''' Each is independently hydrogen or C1-C3 alkyl; R Ib' and R Ic' Each of the following is independently a optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, wherein the alkyl, alkenyl, and alkynyl groups are optionally replaced by one, two, or three groups selected from -oxo (=O), -OH, -SH, or -NR. Ie'' R Ie''' The substituents are replaced by R, where R is a substituent. Ie'' and R Ie''' Each is independently hydrogen or C1-C3 alkyl, or R Ib' and R Ic' Together with the N atoms to which they are attached, they form a 5- to 12-membered heterocycle comprising 1, 2, or 3 heteroatoms independently selected from N, O, and S, at least one of the heteroatoms being N, and the heterocycle optionally being substituted by one or more substituents selected from the group consisting of halogens, C1-C6 alkyl groups, -NO2, and -OH.

7. The lipid compound according to any one of claims 1 to 4, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (A) comprises a group comprising at least one ionizable tertiary amine moiety as follows: 。 9. The lipid compound according to any one of claims 1 to 8, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the group of the collection (B) optionally comprises at least one degradable moiety. 。 11. The lipid compound according to any one of claims 1 to 10, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (B) comprises a group as follows: 。 8. The lipid compound of any one of claims 1 to 7, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (B) comprises an optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups, said aliphatic and heteroaliphatic groups optionally comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 groups independently selected from -C=C-, -CºC-, -NR m -, -NH-, -NH2, -OH, -OR n -, -O-, -C(O)-, -C(OR o )-, -C(O)O-, -SH, -SR p -, -S-, -C(S)-, -C(SR q )-, -C(S)O-, and -P(O)- groups, wherein R m , R n , R o , R p , and R q are each independently an optionally substituted C1-C 14 aliphatic group.

12. The lipid compound according to any one of claims 1 to 10, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (B) comprises a group as follows:

10. The lipid compound of any one of claims 1 to 9, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the degradable moiety is selected from -C(O)O-, -OC(O)-, -OC(O)O-, -S-S-, -C(O)NH-, -NHC(O)-, -NHC(O)O-, -NR 1 C(O)-, -C(O)NR 2 -, 3 -OC(O)O-, -S-S-, -C(O)NH-, -NHC(O)-, -NHC(O)O-, -NR 4 C(O)O-, -OP(O)OR 5 O-, -OCR 6 (OR 7 )O-, -CR 8 (OR 9 )O-, and -CH(OR 1 )O-, wherein R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently an optionally substituted C1-C 14 aliphatic group.

13. The lipid compound according to any one of claims 1 to 10, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the collection (B) comprises a group as follows: 。 15. The lipid compound according to claim 1, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the lipid compound is selected from the following compounds: 。 (Z)-6-((3-(dimethylamino)propionyl)oxy)-7-(octadec-9-en-1-ylamino)-7- oxoheptyl dodecanoate 。 14. The lipid compound of any one of claims 1 to 13, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein R I , R II , and R III satisfy the following conditions: R I selected from the set (A), and R II and R III selected from the set (B); or R II selected from the set (A), and R I and R III selected from the set (B); or R III selected from the set (A), and R I and R II selected from the set (B). (Z)-6-((3-(dimethylamino)propionyl)oxy)-7-(octadec-9-en-1-ylamino)-7- oxoheptyl 2-hexyldecanoate (Z)-6-((4-(dimethylamino)butanoyl)oxy)-7-(octadec-9-en-1-ylamino)-7- oxoheptyl 2-hexyldecanoate 、 ​ 、 ​ or (Z)-6-((5-(dimethylamino)pentanoyl)oxy)-7-(octadec-9-en-1-ylamino)-7- oxoheptyl 2-hexyldecanoate 。 16. The lipid compound of claim 1, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the lipid compound is selected from the compounds listed in Table 1, Table 2, Table 3, and / or Table 4.

17. A method of making the lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, comprising the following synthetic route: ， wherein R I , R II and R III are as defined in any one of claims 1 to 15; or the following synthetic route: ， wherein R I , R II , R II' and R III are as defined in any one of claims 1 to 15.

18. The lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, for use in gene delivery.

19. Use of the lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, in gene delivery.

20. Use of the lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, in the manufacture of a lipid nanoparticle.

21. A lipid nanoparticle comprising the lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, assembled with one or more lipids selected from the group consisting of a phospholipid, a structural lipid, and a PEG lipid.

22. A delivery system, wherein the lipid nanoparticle of claim 21 is used as a delivery vehicle in the delivery system.

23. The delivery system of claim 22, wherein the delivery system further comprises an active pharmaceutical ingredient encapsulated in the lipid nanoparticle.

24. The delivery system of claim 22 or 23, wherein the active pharmaceutical ingredient is a nucleic acid.

25. The delivery system of any one of claims 22 to 24, wherein the active pharmaceutical ingredient is a plasmid.

26. A pharmaceutical composition comprising the lipid compound of any one of claims 1 to 16, or an N-oxide, a stereoisomer, or a pharmaceutically acceptable salt thereof, or the lipid nanoparticle of claim 21, or the delivery system of any one of claims 22 to 25, and a pharmaceutically acceptable carrier or excipient.

27. A method of making a library of lipid compounds, the method comprising the steps of, i) preparing a library of reactant components, comprising: a library of isonitrile compounds, a library of aldehyde compounds, and a library of carboxylic acid compounds; ii) selecting i compounds from the library of isonitrile compounds, labeled Iso-1, Iso-2, Iso-3,... Iso-i, where i is an integer; selecting j compounds from the library of aldehyde compounds, labeled Alde-1, Alde-2, Alde-3,... Alde-j, where j is an integer; k compounds are selected from the library of carboxylic acid compounds, labeled as Acid-1, Acid-2, Acid-3... Acid-k, where k is any integer; iii) preparing a matrix of reaction vessels, labeling each vial as vial-ijk, where i represents the serial number of the isonitrile compound, j represents the serial number of the aldehyde compound, and k represents the serial number of the carboxylic acid compound; iv) adding the isonitrile, aldehyde, and carboxylic acid compounds into the corresponding numbered vials containing solvent according to their labels; v) performing the Passerini reaction for 1 hour to 48 hours; vi) collecting the product after the reaction is complete to obtain a library of lipid compounds in an amount equal to i j k.

28. The method of claim 27, wherein the lipid compound is an ionizable lipid or a cationic lipid.

29. The method of claim 27 or 28, wherein the solvent is selected from THF, DCM, toluene, EtOH, or a mixture thereof.

30. The method of any one of claims 27-29, wherein the ratio of the addition of the isonitrile compound, the aldehyde compound, and the carboxylic acid compound is 1:1:

1.

31. The method according to any one of claims 27 to 30, wherein the library of isonitrile compounds is a library consisting of compounds represented by the general formula R I -NC, wherein R I is selected from the set (A), i.e. a set of groups comprising at least one ionizable tertiary amine moiety; or the set (B), i.e. a set of optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

32. The method according to any one of claims 27 to 31, wherein the library of aldehyde compounds is a library consisting of compounds represented by the general formula R II -CHO, wherein R II is selected from the set (A), i.e. a set of groups comprising at least one ionizable tertiary amine moiety; or the set (B), i.e. a set of optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

33. The method according to any one of claims 27 to 32, wherein the library of carboxylic acid compounds is a library consisting of compounds represented by the general formula R III -COOH, wherein R III is selected from the set (A), i.e. a set of groups comprising at least one ionizable tertiary amine moiety; or the set (B), i.e. a set of optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

34. The method according to any one of claims 27 to 33, wherein the library of isonitrile compounds is a library consisting of compounds of the general formula R I -NC, wherein R I is selected from the set (A), i.e. a set of groups comprising at least one ionizable tertiary amine moiety; the library of aldehyde compounds is a library consisting of compounds of the general formula R II -CHO, wherein R II is selected from the set (B), i.e. a set of optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups; and the library of carboxylic acid compounds is a library consisting of compounds of the general formula R III -COOH, wherein R III is selected from the set (B), i.e. a set of optionally substituted C6-C 25 aliphatic groups and optionally substituted 6- to 25-membered heteroaliphatic groups.

35. The method according to any one of claims 27 to 33, wherein the library of isonitrile compounds is a library consisting of compounds of the general formula R -NC, wherein R is selected from the set (B), i.e. the set of optionally substituted C6-C20aromatic groups and optionally substituted 6- to 25-membered heteroaromatic groups; the library of aldehyde compounds is a library consisting of compounds of the general formula R -CHO, wherein R is selected from the set (A), i.e. the set of groups comprising at least one ionizable tertiary amine moiety; and the library of carboxylic acid compounds is a library consisting of compounds of the general formula R -COOH, wherein R is selected from the set (B), i.e. the set of optionally substituted C6-C20aromatic groups and optionally substituted 6- to 25-membered heteroaromatic groups. I I 25 II II III III 25 ​​​​​​​​ 36. The method according to any one of claims 27 to 33, wherein the library of isonitrile compounds is a library consisting of compounds of the general formula R -NC, wherein R is selected from the set (B), i.e. an optionally substituted C6-C24aliphatic group and an optionally substituted 6- to 25-membered heteroaliphatic group; the library of aldehyde compounds is a library consisting of compounds of the general formula R -CHO, wherein R is selected from the set (B), i.e. an optionally substituted C6-C24aliphatic group and an optionally substituted 6- to 25-membered heteroaliphatic group; and the library of carboxylic acid compounds is a library consisting of compounds of the general formula R -COOH, wherein R is selected from the set (A), i.e. a set of groups comprising at least one ionizable tertiary amine moiety. I I 25 II II 25 III III ​​​​​​​​ 37. A library of lipid compounds made by the method of any one of claims 27-36, wherein the lipid compounds of the library have a structure as shown in Formula (I): (I)， or a structure as shown in Formula (I’) (I')。

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