Supramolecular ionizable lipid molecules with heteroatom tuning for nucleic acid delivery

Abstract

This application relates to ionizable lipids comprising ester functional groups. This application also relates to compositions for delivering ingredients such as nucleic acids and pharmaceuticals, and their uses.

Description

Cross-referencing

[0001] This application claims the rights and priorities of Luxembourg Application No. 505741, filed December 11, 2023, and U.S. Application No. 18 / 621,526, filed March 29, 2024, the entire contents of which are incorporated herein by reference. By referencing and incorporating into the sequence list

[0002] This application is accompanied by a sequence list submitted via EFS-WEB in .XML format, which is incorporated herein by reference in its entirety. The .XML copy was created on December 11, 2024, with the filename 2024-12-11 Sequence_Listing_ST26 062544-502001WO.xml and a size of 6,275 bytes. The electronically formatted information in the sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to ionizable lipid molecules comprising ester functional groups and compositions thereof. Furthermore, this application relates to nanoparticle compositions containing ionizable lipid molecules for delivering components such as nucleic acids and drugs. Background Technology

[0004] Lipid nanoparticles (LNPs) are used to deliver nucleic acids such as messenger RNA (mRNA) to regulate protein expression in vivo. LNPs are formed through the self-assembly of a group of lipid components comprising ionizable lipids. The ionizable lipids contain one or more tertiary amines that are positively charged under acidic conditions to bind nucleic acids and contain hydrophobic regions to facilitate self-assembly into nanoscale particles. Ionizable LNPs can be designed to be electroneutrally neutral in the blood and acquire a positive charge only in acidic compartments such as endosomes, thereby reducing the toxicity associated with permanently positively charged LNPs. The safety and efficacy of LNPs depend on the chemical structure of the ionizable lipids—factors such as carbon chain length, the variety of functional groups contained, and the overall biodegradability of the structure are important and require significant effort to design and optimize.

[0005] A class of ionizable lipids containing amide functional groups has been previously reported, with the general formula RC(=O)-NR'((Tilstra, Couture-Senécal et al., Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery , J. Am. Chem. Soc., 2023; Khan, Tilstra, Manning, Lau, Couture-Senécal, Multi-motif dendrons and their supramolecular structures and uses thereofInternational patent application SN PCT / CA2022 / 051745 describes ionizable lipids containing amides. There is currently a need for additional ionizable lipids for delivering components such as nucleic acids and drugs. Summary of the Invention

[0006] This application discloses a novel family of compounds based on ester functional groups, which exhibit higher potency and faster pharmacokinetic properties in intramuscular mRNA delivery compared to amide-containing lipid analogs. The disclosed compounds demonstrate improved physicochemical properties due to ionizable LNPs and the ability to efficiently deliver mRNA in vivo.

[0007] One aspect of this disclosure relates to compounds of formula I, or salts and / or solvates thereof:

[0008] (I) in: R 1 Selected from C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 1-20 alkylene aryl, C 1-20 alkylene heteroaryl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl, C 1-20 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or more OH, OC groups. 1-10 Alkyl, NR 6 R 6 'and (NR) 6 C 1-10 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or more OH, C, or C groups. 1-10 Alkyl, OC 1-10 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace; X 1 and X 2 They may be the same or different, and are selected from C(O)O and OC(O); R 2 R 3 R 4and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl, and alkylene group is optionally represented by one or more groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally separated by one or more OH and C groups. 1-20 Alkyl substitution; R 6 R 6 '、R 6 ''、R 7 R 7 '、R 8 R 8 'and R 9 Independently selected from H and C 1-10 alkyl; a, b, c, and d may be the same or different, and are selected from 2 to 6; and n is selected from 1 to 4.

[0009] Another aspect of this disclosure relates to nanoparticles comprising one or more compounds of this application, colloids comprising one or more compounds of this application, or supramolecular structures comprising one or more compounds of this application. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0010] Another aspect of this disclosure relates to compositions comprising one or more compounds of this application, and one or more nanoparticles, one or more colloids, and / or one or more supramolecular structures, each comprising one or more compounds of this application.

[0011] In some embodiments, the compositions of this application further comprise one or more components to be delivered to cells or subjects.

[0012] Another aspect of this disclosure relates to a method of delivering one or more ingredients to cells or a subject, the method comprising contacting the cells or subject with one or more compositions of the present application, wherein the contacting is performed under conditions that promote the uptake of the ingredients by the cells or subject.

[0013] Another aspect of this disclosure relates to a kit comprising one or more compounds of this application or one or more compositions of this application.

[0014] Other features and advantages of this application will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific examples point to embodiments of this application, they are given for illustrative purposes only, and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the entire specification. Attached Figure Description

[0015] Figure 1 The apparent pK of lipid nanoparticles (LNP) is shown. a A graph showing the relationship between the number of carbon atoms on both sides of the ester group in exemplary compounds I-1, I-2, I-3, I-4, I-5, I-6, and I-7.

[0016] Figure 2 The total luminous flux of the target region in mice injected intramuscularly with formulated firefly luciferase mRNA, captured by an in vivo imaging system (IVIS), is shown.

[0017] Figure 3 The intramuscular expression profile of firefly luciferase in an exemplary LNP formulation is shown in mice 6 to 96 hours after injection.

[0018] Figure 4 The intramuscular expression of firefly luciferase in an exemplary LNP formulation is shown in mice from 6 to 96 hours after injection, expressed as the area under the curve (AUC) of total luminous flux.

[0019] Figure 5 The table shows the (A, left panel) activity (%) and (B, right panel) of firefly luciferase expression in THP-1 cells 24 hours after transfection with firefly luciferase mRNA formulated with exemplary LNP.

[0020] Figure 6 The expression of firefly luciferase mRNA in (A) liver, (B) spleen, (C) lung, (D) heart and (E) kidney 6 hours after intravenous injection is shown, as well as the organ distribution of total flux in mice in (F).

[0021] Figure 7The following are shown: (A) the alkaline hydrolysis rate of the exemplary ionizable compounds of this application; (B) the concentration of the parent compound in mouse plasma after intramuscular injection of an LNP formulated with compound I-12 or exemplary compound I-6; (C) the concentration of the parent compound in mouse plasma after intramuscular injection of an LNP formulated with compound I-12 or exemplary compound I-6; and (D) the percentage of the remaining injected dose in all organs after intramuscular injection of an LNP formulated with compound I-12 or exemplary compound I-6.

[0022] Figure 8 The following data are presented: (A) mRNA uptake signals for each cell of compounds I-6, I-12, and SM102, and (B) fold change in mRNA uptake compared to SM102 at 6 hours.

[0023] Figure 9 The endosome escape events of (A) compounds I-6, I-12 and SM102 are shown, and the fold change of galactolectin 9+ spots is shown.

[0024] Figure 10 The neutralizing antibody response of (A) compounds I-6, I-12 and SM102 is shown, and the antigen-specific T cell response of (B) compounds I-6, I-12 and SM102 is shown. Detailed Implementation

[0025] I. Definition Unless otherwise stated, the definitions and embodiments described in this and other sections are intended to apply to all embodiments and aspects of this application described herein, provided that their applicability is understood by those skilled in the art.

[0026] All features disclosed in this specification (including the claims, abstract, and drawings), and all steps in any disclosed method or process, may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. Unless otherwise expressly stated, each feature disclosed in the specification (including the claims, abstract, and drawings) may be replaced by an alternative feature having the same, equivalent, or similar purpose.

[0027] As used in this application and claims, the terms “comprising” (and any form thereof), “having” (and any form thereof), “including” (and any form thereof), or “containing” (and any form thereof) are inclusive or open-ended expressions and do not exclude additional, undescribed elements or process steps.

[0028] As used herein, the term “composition” and its derivatives are intended as closed terms that specify the presence of stated features, elements, components, groups, integers and / or steps, and exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0029] As used herein, the term “substantially composed of” is intended to specify the presence of stated features, elements, components, groups, integers and / or steps, as well as those essential and novel characteristics that do not substantially affect those features, elements, components, groups, integers and / or steps.

[0030] As used herein, the terms “about,” “substantially,” and “approximately” mean a reasonable amount of deviation of the modified term such that the final result is not significantly altered. These degree terms should be interpreted to include at least ±5% deviation of the modified term unless such deviation does not negate the meaning of the word it modifies, or unless a person skilled in the art draws otherwise from the context.

[0031] As used herein, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. For example, embodiments including “compound” should be understood to mean that certain aspects of the invention have one compound, or two or more additional compounds.

[0032] In embodiments that include an “additional” or “second” component or effect (e.g., an additional or second compound), the second component, as used herein, is different from other compounds or the first compound. A “third” compound is different from other compounds, the first compound, and the second compound, and so are the further listed or “additional” compounds.

[0033] When referring to “one or more” molecules or materials (e.g., one or more compounds), it should be understood that this refers to the “type” or “identity” of that molecule or material. Therefore, a second molecule or material differs from the stated one or first molecule or material. Similarly, a “third” molecule or material differs from the stated one, first, or second molecule or material, and the same applies to further listed or “additional” molecules or materials.

[0034] As used herein, the term "and / or" means that the listed items are present or used individually or in combination. In practice, the term implies the use or presence of "at least one" or "one or more" of the listed items. With respect to enantiomers, prodrugs, salts, and / or solvates, the term "and / or" means that the compounds of this application may exist individually as enantiomers, prodrugs, salts, and hydrates, or as combinations of salts, such as solvates of the compounds of this application.

[0035] As used herein, the terms “compound of this application” or “compound of this application” mean the compounds disclosed herein, compounds of formula I, and their salts and / or solvates.

[0036] As used herein, the terms “composition of this application” or “composition of this application” mean a composition comprising one or more compounds of this application.

[0037] As used herein, the term “suitable” means that the choice of a particular compound or condition will depend on the specific synthetic operation to be performed, the identity of the molecule to be transformed, and / or the specific use of the compound, but that the choice is entirely within the capabilities of a person skilled in the art.

[0038] This specification involves many chemical terms and abbreviations used by those skilled in the art. Nevertheless, for clarity and consistency, definitions of the selected terms are provided.

[0039] As used herein, the terms “protecting group” or “PG” refer to a chemical moiety that protects or masks reactive portions of a molecule to prevent those reactive portions from undergoing side reactions when different portions of the molecule are manipulated or reacted. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not lead to degradation or decomposition of the rest of the molecule. The selection of a suitable protecting group can be made by someone skilled in the art. Many conventional protecting groups are known in the art, for example, as described in “Protective Groups in Organic Chemistry”, McOmie, JFW ed., Plenum Press, 1973; in Greene, TW and Wuts, PGM, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3rd ed., 1999; and in Kocienski, P. Protecting Groups, 3rd ed., 2003, Georg Thieme Verlag (The Americas).

[0040] As used herein, the term "inert organic solvent" refers to a solvent that is generally considered not to react with the functional groups present in the compounds to be combined in any given reaction, thus not interfering with or inhibiting the desired synthetic transformation. Organic solvents are typically nonpolar and capable of dissolving compounds insoluble in aqueous solutions.

[0041] As used herein, the term "alkyl," whether used alone or as part of another group, refers to a straight-chain or branched saturated alkyl group. The number of carbon atoms that may be contained in the alkyl group is indicated by the prefix "C".n1-n2 " indicates. For example, the term C" indicates... 1-10 Alkyl refers to an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Unless otherwise stated, all alkyl groups are optionally substituted with fluorine.

[0042] The term "alkylene," whether used alone or as part of another group, refers to a straight-chain or branched saturated alkylene, that is, a saturated carbon chain with substituents at both ends. The number of carbon atoms that may be contained in the alkylene mentioned is indicated by the prefix "C". n1-n2 " indicates. For example, the term C" indicates... 2-6 Alkylene refers to an alkylene having 2, 3, 4, 5, or 6 carbon atoms. Unless otherwise stated, all alkylenes are optionally substituted with fluorine.

[0043] As used herein, the term "alkenyl," whether used alone or as part of another group, refers to a straight-chain or branched unsaturated alkyl group containing at least one double bond. The number of carbon atoms that may be contained in the alkenyl group is indicated by the prefix "C". n1-n2 " indicates. For example, the term C" indicates... 2-6 An alkenyl group is defined as having 2, 3, 4, 5, or 6 carbon atoms and at least one double bond. Unless otherwise stated, all alkenyl groups are optionally substituted with fluorine.

[0044] As used herein, the term "alkynyl," whether used alone or as part of another group, refers to a straight-chain or branched unsaturated alkynyl group containing at least one triple bond. The number of carbon atoms that may be contained in the alkynyl group is indicated by the prefix "C". n1-n2 " indicates. For example, the term C" indicates... 2-6 The alkynyl group refers to an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0045] As used herein, the term "aryl," whether used alone or as part of another group, refers to a monovalent unsaturated aromatic group with an aromatic carbon atom as the linking point, said carbon atom forming part of one or more six-membered aromatic ring structures, wherein all ring atoms are carbon. An aryl group may contain six or more carbon atoms. Unless otherwise stated, all aryl groups are optionally substituted with fluorine.

[0046] As used herein, the term "cycloalkyl," whether used alone or as part of another group, refers to a saturated carbocyclic group containing one or more rings. The number of carbon atoms that may be contained in a cycloalkyl group is indicated by the numerical prefix "C". n1-n2 " indicates. For example, the term C" indicates... 3-10 Cycloalkyl refers to cycloalkyl groups having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0047] As used herein, the term "heteroaryl," whether used alone or as part of another group, refers to a cyclic group containing at least one heteroaryl ring, wherein one or more atoms are heteroatoms selected from O, S, and N. When a heteroaryl group includes the prefix C... n1-n2 In this case, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, where one or more (appropriately 1 to 5) ring atoms are replaced by heteroatoms as defined above.

[0048] As used herein, the term "heterocyclic alkyl," whether used alone or as part of another group, refers to a cyclic group containing at least one non-aromatic ring, wherein one or more atoms are heteroatoms selected from O, S, and N. Heterocyclic alkyl groups are either saturated or unsaturated (i.e., containing one or more double bonds). When a heterocyclic alkyl group contains the prefix C... n1-n2 In this case, the prefix indicates the number of carbon atoms in the corresponding carbocyclic group, where one or more (appropriately 1 to 5) ring atoms are replaced by heteroatoms as defined above.

[0049] The term "fluorine-substituted" means that one or more (including all) of the mentioned groups can be replaced by hydrogen with fluorine.

[0050] As used herein, the term “halogenated” or “halogen”, whether used alone or as part of another group, refers to a halogen atom and includes fluorine, chlorine, bromine, and iodine.

[0051] symbol" "When a link is perpendicular to a bond, it indicates the connection point of the group."

[0052] The term “available” (such as “available hydrogen atom” or “available atom”) refers to an atom that is known to those skilled in the art to be substituted by a substituent (e.g., a fluorine atom).

[0053] It should be clearly stated that, unless otherwise stated, all available hydrogen atoms in the compounds of this application and in all embodiments thereof are optionally replaced by fluorine atoms.

[0054] As used herein, the term “cell” refers to a single cell or multiple cells, and includes cells in cell cultures or subjects.

[0055] As used herein, the term "subject" refers to any target used to deliver one or more components using the compositions of this application. A subject can be a living subject, including all members of the animal and plant kingdoms, or an inanimate object. Therefore, the methods and uses of this application are suitable for human treatment, veterinary treatment, plant applications, and material handling.

[0056] The term "pharmaceuticalally acceptable" means that it is compatible with the treatment of the subject (e.g., human).

[0057] The term "pharmaceuticalally acceptable carrier" refers to a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with the active ingredient to form a pharmaceutical composition, i.e., a dosage form that can be administered to a subject.

[0058] The term “pharmaceutically acceptable salt” means an acid addition salt or base addition salt that is suitable for or compatible with the treatment of a subject.

[0059] As used herein, the term "solvent" refers to a compound, or a salt of a compound and / or a prodrug, wherein suitable solvent molecules are incorporated into a crystal lattice. Suitable solvents are physiologically tolerable at administered doses.

[0060] As used herein and as is well known in the art, the terms “treatment” and “method of treatment” mean a method of obtaining a beneficial or desired outcome (including clinical outcomes). Beneficial or desired clinical outcomes may include, but are not limited to: relief or improvement of one or more symptoms or conditions, reduction of disease extent, stabilization of the disease state (i.e., no worsening), prevention of disease spread, delay or slowing of disease progression, improvement or mitigation of the disease state, reduction of disease recurrence, and remission (partial or complete), whether detectable or undetectable. “Treatment” and “method of treatment” may also mean extended survival compared to expected survival without treatment. As used herein, “treatment” and “method of treatment” also include preventative treatment. For example, a subject with early-stage cancer may be treated to prevent progression, or alternatively, a subject in remission may be treated with a compound or composition of this application to prevent recurrence. Methods of treatment include administering a therapeutically effective amount of one or more compounds of this application to a subject, and optionally include a single administration, or alternatively, a series of administrations.

[0061] A “mitigated” disease or condition refers to a reduction in the severity and / or adverse clinical manifestations of the condition or disease state compared to an untreated condition, and / or a slowing or prolonging of its progression.

[0062] As used in this article, the term "prevention" or "preventive" or its synonyms refer to reducing the risk or probability of a patient developing a disease, symptom, or condition.

[0063] As used herein, the term "effective amount" or "therapeutic effective amount" means the amount of the compound of this application or one or more compounds that is effective at the dose and time period required to achieve the desired results.

[0064] "Inhibition, blocking and / or destruction" refers to any detectable inhibition, blocking and / or destruction in the presence of a compound, compared to other conditions under identical conditions but without the compound.

[0065] As used herein, the term “application” means applying a therapeutically effective amount of the compound, one or more compounds or compositions of this application to cells, tissues or organs, whether in vivo, in vitro or ex vivo.

[0066] The term "encapsulation efficiency" refers to the proportion of an ingredient that is encapsulated within particles (e.g., nanoparticles, confined within the nanoparticles).

[0067] As used herein, the term “nanoparticle” or variations thereof is intended to refer to particles whose size is measured at the nanoscale.

[0068] As used in this article, the term "colloid" refers to a mixture in which microscopically dispersed insoluble particles are suspended in another substance (usually a liquid).

[0069] As used in this article, the term "supramolecular structure" refers to a molecular complex that is bound together by non-covalent bonds.

[0070] The term "apparent pK" a "pK" refers to the experimentally determined pH value of nanoparticles. This value represents the pH of the system when the number of ionized (protonated) groups is equal to the number of unionized groups. a The value will depend on the measurement method used. The apparent pK values ​​of nanoparticles, including LNP, reported in this paper... a The value was determined using the 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) assay, for example, as described herein.

[0071] As used in this article, the term "lipid molecule pK" a "Refers to pK related to lipid molecules" a The calculated estimates are as follows. The lipid molecule pK values ​​reported in this paper... a The value was calculated using MarvinSketch version 22.22.

[0072] As used herein, the term "alkali hydrolysis rate" refers to a value determined experimentally for the compounds of this application. This value is the rate at which the compounds of this application degrade or form hydrolysis products in the presence and soluble state of an alkali. The alkali hydrolysis rate value will depend on the measurement method used. The rate values ​​reported herein were determined using the methods reported herein.

[0073] Compounds and Compositions This application includes compounds of formula I, or salts and / or solvates thereof:

[0074] (I) in: R 1 Selected from C 1-20 Alkyl, C2-20 alkenyl, C 2-20 alkynyl group, C 1-20 alkylene aryl, C 1-20 alkylene heteroaryl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl, C 1-20 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or more OH, OC groups. 1-10 Alkyl, NR 6 R 6 'and (NR) 6 C 1-10 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or more OH, C, or C groups. 1-10 Alkyl, OC 1-10 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace; X 1 and X 2 They may be the same or different, and are selected from C(O)O and OC(O); R 2 R 3 R 4 and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl, and alkylene group is optionally represented by one or more groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O) and C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally separated by one or more OH and C groups. 1-20 Alkyl substitution; R 6 R 6 '、R 6''、R 7 R 7 '、R 8 R 8 'and R 9 Independently selected from H and C 1-10 alkyl; a, b, c, and d may be the same or different, and are selected from 2 to 6; and n is selected from 1 to 4.

[0075] In some implementations, a, b, c, and d are different. In some implementations, a and b are the same, and c and d are the same. In some implementations, a, b, c, and d are the same.

[0076] In some implementations, a, b, c, and d are all 2. In some implementations, a + c ≥ 5 and b + d ≥ 5. In some implementations, a is 2 and c is 3, 4, 5, or 6, b is 2, and d is 3, 4, 5, or 6. In some implementations, a, b, c, and d are selected from 2 and 3. In some implementations, c is 2 and a is 3, 4, 5, or 6, d is 2, and b is 3, 4, 5, or 6. In some implementations, a, b, c, and d are selected from 3 and 4. In some implementations, a, b, c, and d are all 3. In some implementations, a is 3 and c is 4, 5, or 6, b is 3, and d is 4, 5, or 6. In some implementations, c is 3 and a is 4, 5, or 6, d is 3, and b is 4, 5, or 6. In some implementations, a, b, c, and d are all 4. In some implementations, a is 4 and c is 5 or 6, b is 4, and d is 5 or 6. In some implementations, c is 4 and a is 5 or 6, d is 4 and b is 5 or 6. In some implementations, a, b, c, and d are all 5. In some implementations, a is 5 and c is 6, b is 5, and d is 6. In some implementations, c is 5 and a is 6, d is 5, and b is 6.

[0077] In some implementation schemes, X 1 and X 2 Same. In some implementations, X 1 and X 2 It is C(O)O. In some implementations, X 1 and X 2 It is OC(O).

[0078] In some implementation schemes, R 1 Selected from C 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 alkylene aryl, C 1-10 alkylene heteroaryl, C 1-10 Alkylene C 3-8 Heterocyclic alkyl, C1-10 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one to four OH, OC 1-5 Alkyl, NR 6 R 6 'and (NR) 6 C 1-5 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one to four OH, C 1-5 Alkyl, OC 1-5 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace.

[0079] In some implementation schemes, R 1 Selected from C 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 alkylene phenyl, C 1-10 alkylene heteroaryl, C 1-10 Alkylene C 3-8 Heterocyclic alkyl, C 1-10 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or two OH, OC groups. 1-3 Alkyl, NR 6 R 6 'and (NR) 6 C 1-5 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or two OH, C, or OH groups. 1-5 Alkyl, OC 1-3 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'Replace.' In some implementations, R 6 R 6 '、R 6 ''、R7 R 7 '、R 8 and R 8 'Selected independently from H and C 1-5 Alkyl group. In some embodiments, n is 1 or 2.

[0080] In some implementation schemes, R 1 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0081] In some implementation schemes, R 1 yes .

[0082] In some implementation schemes, R 2 R 3 R 4 and R 5Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally composed of one to four groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl are optionally surrounded by one to four OH groups and C. 1-10 Alkyl substitution. In some embodiments, R 9 Independently selected from H and C 1-5 alkyl.

[0083] In some implementation schemes, R 2 R 3 R 4 and R 5 Different. In some implementations, R 2 and R 3 Same, and R 4 and R 5 The same. In some implementations, R 2 R 3 R 4 and R 5 same.

[0084] In some implementation schemes, R 2 R 3 R 4 and R 5 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , and , Furthermore, the alkyl, alkenyl, alkylene heterocyclic alkyl, and alkylene cycloalkyl groups are optionally surrounded by one or two OH groups and C atoms. 1-10 Alkyl substitution.

[0085] In some implementation schemes, R 2 R 3 R 4 and R 5 Selected from .

[0086] In some implementation schemes, all R 2 R 3 R 4 and R 5 yes .

[0087] In some embodiments, the compounds of formula I are selected from:

[0088] Or its salts and / or solvates.

[0089] In some implementations, the compounds are selected from the group consisting of:

[0090] In some embodiments, the alkaline hydrolysis rate of the compound of this application is greater than 0.001 min. -1 In some embodiments, the alkaline hydrolysis rate of the compound of this application is less than 1 hour. In some embodiments, the alkaline hydrolysis rate of the compound of this application is about 0.001 min. -1 approximately 1 minute -1 In some embodiments, the alkaline hydrolysis rate of the compounds of this application is from about 0.01 min to about 1 hour. It has been shown that the in vitro degradation rate depends on the distance between the ester group and nearby electron-withdrawing and sterically hindered groups. It has been shown that, compared to the gold standard lipids DLin-MC3-DMA, SM-102, and ALC-0315, the compounds of this application, containing multiple carbons in the linker between the ester group and the tertiary amine, exhibit an increased in vitro degradation rate. In some embodiments, the hydrolysis rate is measured by an alkaline (e.g., KOH) hydrolysis reaction in an alcoholic solvent (e.g., methanol) and using… 1 H NMR can be used for monitoring; however, any other method known in the art may also be used.

[0091] This disclosure also relates to the synthesis of novel polyamine-ionizable lipid molecules for the creation of a new class of delivery materials. The spatial arrangement of the amine, ester, and hydrophobic regions of these ionizable lipid molecules significantly influences the function of the LNPs formed from these materials. The orientation of the ester and the distance between heteroatoms (including amine and hydrophobic regions) cause dramatic changes in activity. These changes, in turn, lead to alterations in steric hindrance, thereby affecting the formation of supramolecular assemblies, charge shielding, inductive effects, and the structure of degradation products. Based on these insights, a new class of materials—supramolecular ionizable lipids tuned to heteroatoms—has been created, aiming to maximize performance and significantly improve current state-of-the-art technologies.

[0092] This application also includes compositions comprising one or more compounds of this application. In some embodiments, the compounds of this application are incorporated into nanoparticles in the composition. Therefore, this application also includes nanoparticles comprising one or more compounds of this application. This application also includes compositions comprising these nanoparticles. In some embodiments, the nanoparticles are formed by the self-assembly of one or more compounds. In some embodiments, the nanoparticles comprise two or more compounds of this application. In some embodiments, the nanoparticles are lipid nanoparticles (LNPs).

[0093] In some embodiments, the LNP includes one or more ionizable lipids (one or more compounds of this application), one or more lipids that form a bilayer, one or more structural lipids, and one or more lipid-conjugated polyethylene glycol (PEG-lipid).

[0094] In some embodiments, the maximum longest linear dimension (e.g., diameter) of the nanoparticles is 200 nm or greater. In some embodiments, the maximum longest linear dimension (e.g., diameter) of the nanoparticles is 200 nm or less. In some embodiments, the average diameter of the nanoparticles is about 150 nm, about 125 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm or less. In some embodiments, the average diameter of the nanoparticles is 50 nm or less. In some embodiments, the average diameter of the nanoparticles is 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less. Therefore, in some embodiments, the average diameter of the nanoparticles is about 1 nm to about 50 nm, about 5 nm to about 30 nm, about 10 nm to about 25 nm, about 10 nm to about 20 nm, or about 15 nm to about 20 nm. In some embodiments, the average diameter of the nanoparticles disclosed herein is about 150 nm, about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, about 15 nm, about 10 nm, or about 5 nm. In some embodiments, the average diameter is determined using dynamic light scattering intensity.

[0095] In some embodiments, one or more compounds of this application form a colloidal or supramolecular structure. Therefore, in some embodiments, this application also includes a colloid comprising one or more compounds of this application, or a supramolecular structure comprising one or more compounds of this application, and compositions comprising such colloids or supramolecular structures.

[0096] This application also includes compositions comprising one or more compounds of this application and one or more components to be delivered to cells or a subject. In some embodiments, the one or more components to be delivered to cells or a subject are selected from unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, staining agents, dyes, small molecule drugs, and other organic and inorganic fractions. In some embodiments, the one or more components to be delivered to cells or a subject are referred to as the payload.

[0097] This application also includes compositions comprising one or more compounds of this application and one or more therapeutic agents to be delivered to cells or a subject. In some embodiments, the one or more therapeutic agents to be delivered to cells or a subject are selected from unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, staining agents, dyes, small molecule drugs, and other organic and inorganic fractions. In some embodiments, the one or more therapeutic agents to be delivered to cells or a subject are referred to as a payload. In some embodiments, the one or more therapeutic agents to be delivered to cells or a subject include a payload.

[0098] In some implementations, one or more components to be delivered to cells or subjects have a total negative charge.

[0099] In some embodiments, one or more components to be delivered to cells or a subject are contained within a nanoparticle, colloidal, or supramolecular structure comprising one or more compounds of this application. "Contained within" should be understood as one or more components being encapsulated within a nanoparticle, colloidal, or supramolecular structure, and / or non-covalently bound to any portion of the one or more compounds constituting the nanoparticle, colloidal, or supramolecular structure.

[0100] In some implementations, the proteins and peptides are selected from endonucleases, a wide range of nucleases, proteases, and kinases.

[0101] In some embodiments, one or more components to be delivered to cells or a subject are one or more nucleic acids. In some embodiments, one or more nucleic acids are selected from short interfering RNA (e.g., small interfering RNA) (siRNA), circular RNA, cyclic RNA, long non-coding RNA (lncRNA), microRNA (miRNA), pri-miRNA, messenger RNA (mRNA), CRISPR-associated nucleic acids, single-stranded guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotides (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), exDNA, precursor RNA, single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). In some embodiments, one or more nucleic acids are selected from siRNA, tRNA, and nucleic acids used in the CRISPR process. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid used in the CRISPR process is a clustered regularly spaced short palindromic repeat (CRISPR)-associated nucleic acid, a single-stranded guide RNA (sgRNA), a CRISPR-RNA (crRNA), and / or a trans-activating crRNA (tracrRNA). In some embodiments, the nucleic acid is miRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is tRNA. In some embodiments, the nucleic acid is guide RNA. In some embodiments, the guide RNA is used in the CRISPR process. In some embodiments, the nucleic acid is pDNA.

[0102] In some embodiments, the compositions of this application are used for gene editing. In some embodiments, these compositions comprise Cas9 mRNA and one or more guide RNAs (gRNAs) designed to target a specific gene. In some embodiments, these compositions also comprise DNA, or are formulated for co-delivery with DNA for homology-directed repair.

[0103] In some embodiments, the compositions of this application are used for gene silencing. In some embodiments, these compositions comprise siRNA.

[0104] In some embodiments, the compositions of this application are used for gene regulation. In some embodiments, these compositions comprise non-coding RNA (ncRNA).

[0105] In some embodiments, the compositions of this application are used for gene expression upregulation or gene expression downregulation. In some embodiments, these compositions comprise unmodified or chemically modified messenger RNA (mRNA). In some embodiments, chemically modified mRNA refers to mRNA in which nucleotides are partially or completely replaced by chemically modified nucleotides.

[0106] In some embodiments, the compositions of this application are used for antiviral therapy. In some embodiments, these compositions comprise mRNA encoding the Ca9 protein and one or more sgRNAs. In some embodiments, one or more sgRNAs recognize the viral gene to be deleted.

[0107] In some embodiments, the compound to be delivered to cells or a subject and one or more components are present in a weight ratio of about 100:1 to about 1:5. In some embodiments, the weight ratio of the compound to be delivered to cells or a subject to one or more components is about 50:1 to about 2:1. In some embodiments, the weight ratio of the compound to be delivered to cells or a subject to one or more components is 20:1.

[0108] In some embodiments, the composition further comprises one or more lipids. In some embodiments, the one or more lipids are selected from steroids, steroid derivatives, PEG-lipids, and phospholipids, and mixtures thereof.

[0109] In some implementations, PEG-lipid is a compound containing one or more C atoms. 6-24 Alkyl or C 6-24 alkenyl or C 6-24 A fatty acid group is attached to a linker group having a PEG chain. Some non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines, PEG-modified 1,2-diacoxypropyl-3-amine, PEG-modified diacylglycerols and / or dialkylglycerols.

[0110] In some embodiments, the PEG-lipid is PEG-modified 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, and / or PEG-modified distearyl-rac-glycerol.

[0111] In some embodiments, PEG is measured by the molecular weight of the PEG component in the lipid. In some embodiments, the molecular weight of PEG is from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 10,000, from about 400 to about 8,000, from about 1,000 to about 6,000, or from about 2,000 to about 5,000. In some embodiments, the molecular weight of PEG is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that may be used herein can be found in U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0112] In some embodiments, the composition comprises a PEG-lipid in a molar ratio of about 1:1 to about 1:400 or 1:1 to about 1:250 to the compound. In some embodiments, the molar ratio is about 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:125, 1:150, 1:175, 1:200, 1:225 to about 1:250 or any range derived therefrom. In some embodiments, the PEG-lipid is present at 0 to 50 mol% of the total lipids in the composition, or any range derived therefrom. In some embodiments, the PEG-lipid is present at 0 mol%, 1.5 mol%, 3 mol%, 10 mol%, 15 mol%, 20 mol%, or 40 mol% or any range derived therefrom.

[0113] In some embodiments, the structural lipids are steroids or steroid derivatives. In some embodiments, the steroids or steroid derivatives are unmodified or modified cholesterol, phytosterols, cholecalciferol, dexamethasone, or any combination thereof. In some embodiments, the modified cholesterol is oxidized at the β-ring or hydrocarbon tail structure. In another embodiment, the phytosterols include, but are not limited to, β-sitosterol, stigmasterol, β-sitosterol, campesterol, sinasosterol, and their salts and esters. In some embodiments, the structural lipids and compounds are present in the composition in a molar ratio of 2:1 to 1:20 or any range from which they can be derived. In some embodiments, the structural lipids are present at 0 to 50 mol% of the total lipids in the composition, or any range from which they can be derived.

[0114] In some embodiments, the lipids forming the bilayer consist of naturally occurring lipids or lipids of synthetic origin, including phospholipids, sphingolipids, and glycolipids. Phospholipids include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE). In one embodiment, the sphingolipid is sphingomyelin. In some embodiments, the lipids and compounds forming the bilayer are present in the composition in a molar ratio of 2:1 to 1:20 or any range from which they can be derived. In some embodiments, the lipids forming the bilayer are present at 0 to 50 mol% of the total lipids, or at most 50 mol%, or any range from which they can be derived.

[0115] In some embodiments, this application includes a composition comprising one or more compounds of this application, one or more PEG-lipids, one or more phospholipids, and one or more sterols. In some embodiments, one or more compounds are present in an amount of about 45 mol% to about 55 mol%, one or more phospholipids are present in an amount of about 5 mol% to about 15 mol%, one or more PEG-lipids are present in an amount of about 0.5 mol% to about 2.5 mol%, and one or more sterols are present in an amount of about 28 mol% to about 48 mol%. In some embodiments, one or more compounds are present in an amount of about 50 mol%, one or more phospholipids are present in an amount of about 38.5 mol%, one or more PEG-lipids are present in an amount of about 1.5 mol%, and one or more sterols are present in an amount of about 10 mol%.

[0116] In some embodiments, this application includes a composition comprising one or more compounds of this application, one or more PEG-lipids, one or more phospholipids, one or more steroids, and one or more ingredients to be delivered. In some embodiments, the steroid is cholesterol. In some embodiments, the steroid is β-sitosterol.

[0117] In some embodiments, when lipids are present in the composition comprising one or more components to be delivered, the one or more components to be delivered to cells or a subject are present in a weight ratio of one or more compounds plus lipids to components of about 100:1 to 1:5, about 50:1 to 2:1, or about 25:1. In some embodiments, the molar percentage ratio of one or more compounds plus lipids to components is in the range of about 95 mol%:5 mol% to about 80 mol%:20 mol%.

[0118] In some embodiments, when one or more components to be delivered are RNA, the composition contains a lipid nitrogen to RNA phosphate ratio (N / P) of about 13 to 14.

[0119] This application also includes a pharmaceutical composition comprising: the composition or compound of this application and a pharmaceutically acceptable carrier.

[0120] In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated for administration by means of: oral, intrafacial, intra-articular, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravesical, intravitreal, liposome, topical, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, external, buccal, percutaneous, vaginal, cream, lipid composition, via catheter, via irrigation, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intra-arterial injection. In some embodiments, the pharmaceutical composition is formulated as a unit dosage form. In some embodiments, the pharmaceutical composition is formulated for intramuscular administration.

[0121] In some embodiments, the pH of the compositions and pharmaceutical compositions of this application is from about 2 to about 8.5. In some embodiments, the compositions are formulated in 10 to 100 mM sodium acetate at pH 3, 4, 5, or 6. It should be understood that the pH of the compositions of this application can be any suitable pH during preparation and storage. However, in order to deliver the compositions of this application to cells or subjects, the pH is adjusted to a physiologically acceptable range, such as about 7. In some embodiments, the polydispersity index (PDI) of the compositions and pharmaceutical compositions of this application is less than 0.2.

[0122] In some embodiments, the apparent pK of the compositions and pharmaceutical compositions of this application a The apparent pK is from about 4 to about 8. In some embodiments, the apparent pK of the compositions and pharmaceutical compositions of this application is... a The apparent pK is about 5 to about 7 or about 5.5 to about 7.5. In some embodiments, the apparent pK of the compositions and pharmaceutical compositions of this application is... a The apparent pK is approximately 6 to approximately 7. In some embodiments, the apparent pK of the compositions and pharmaceutical compositions of this application is... a It is approximately 6.5. In some implementations, the apparent pK a The value depends on the measurement method. In some implementations, apparent pK aThe value was determined using the 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) assay; however, any other method known in the art may also be used. If other methods are used to determine the apparent pK... a Then apparent pK a The value may differ from the value obtained using the TNS assay.

[0123] In some implementation schemes, the apparent pK of LNP a The value is approximately 4 to approximately 8. In some implementations, the apparent pK of LNP is... a It is approximately 5 to approximately 7 or approximately 5.5 to approximately 7.5. In some embodiments, the apparent pK of LNP is... a It is approximately 6 to approximately 7. In some implementations, the apparent pK of LNP is... a It is approximately 6.5. As defined in this application, the apparent pK... a This makes LNP electrically neutral in the blood, while it carries a positive charge in acidic compartments (such as endosomes). In some embodiments, the pK a This advantageously reduces toxicity, for example, compared to LNPs with a permanently positive charge. In some embodiments, the apparent pK of LNPs is... a The value was determined using the 2-(p-toluidine)naphthalene-6-sulfonic acid (TNS) assay; however, any other method known in the art may also be used. If other methods are used to determine the apparent pK... a Then apparent pK a The values ​​may differ from those obtained using the TNS assay. Apparent pK values ​​for nanoparticles (e.g., LNP) a The zeta potential (surface charge) of the nanoparticles was determined, and it is a function of the nanoparticle pH (Carrasco, M. J et al., Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration. Commun Biology 4, 956, 2021). In some embodiments, the epigenetic pK of LNPs... a Related to its effectiveness (Jayaraman, M. et al., Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo). Angewandte Chemie Int Ed51, 8529-8533(2012); Tilstra, G. et al., Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery. J Am Chem Soc (2023)).

[0124] In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 2 and 3, and the apparent pK of the LNP is... a It ranges from approximately 5.5 to approximately 6.0.

[0125] In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 3 and 4, and the apparent pK of the LNP is... a It is approximately 6.0 to approximately 6.6.

[0126] In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 2 and 4, and the apparent pK of the LNP is... a The apparent pK is approximately 5.5 to approximately 6.6. In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 4, 5, and 6, and the apparent pK of the LNP is... a ≥ 6.6.

[0127] In some embodiments, the lipid molecule pK of the compound of this application a It is approximately 7 to approximately 11. In some embodiments, the lipid molecule pK of the compounds of this application a It is approximately 8 to approximately 11. In some embodiments, the lipid molecule pK a The value depends on the calculation method. In some implementations, lipid pK a The values ​​were calculated using MarvinSketch, but any other calculation method known in the art may also be used. If other methods are used to determine lipid pK... a Then the lipid molecule pK a The value may differ from the value obtained using MarvinSketch.

[0128] In some embodiments, the compositions and pharmaceutical compositions of this application comprise one or more compounds of this application, the lipid molecules of which are pK a The value is approximately 7 to approximately 11. In some embodiments, the compositions and pharmaceutical compositions of this application comprise one or more compounds of this application, the lipid molecules of which are pK a From approximately 8 to approximately 11.

[0129] In some embodiments, LNP comprises one or more compounds of this application, whose lipid molecules pK a The value is approximately 7 to approximately 11. In some embodiments, LNP comprises one or more compounds of this application, whose lipid molecules pK a The value is approximately 8 to approximately 11. In some embodiments, the lipid molecule pK a The value depends on the calculation method. In some implementations, lipid pK a The values ​​were calculated using MarvinSketch, but any other calculation method known in the art may also be used. If other methods are used to determine the pK of lipid molecules... a Then lipid pK a The value may differ from the value obtained using MarvinSketch.

[0130] In some embodiments, the lipid molecules pK of one or more compounds of this application (where a, b, c, and d are each selected from 2 and 3) a The value is approximately 8.3 to approximately 9.3. In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 2 and 3, and the lipid molecule pK of one or more compounds is... a It is approximately 8.3 to approximately 9.3.

[0131] In some embodiments, the lipid molecules pK of one or more compounds of this application (where a, b, c, and d are each selected from 3 and 4) a The value is approximately 8.5 to approximately 10. In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 3 and 4, and the lipid molecule pK of one or more compounds is... a It is approximately 8.5 to approximately 10.

[0132] In some embodiments, the lipid molecules pK of one or more compounds of this application (where a, b, c, and d are each selected from 4, 5, and 6) a The value is approximately ≥ 9.4. In some embodiments, this application includes an LNP comprising one or more compounds of this application, wherein a, b, c, and d are each selected from 4, 5, and 6, and the lipid molecule pK of one or more compounds is... a ≥9.4.

[0133] In some embodiments, the encapsulation efficiency of the compositions and pharmaceutical compositions of this application is greater than about 70%, greater than about 80%, or greater than about 90%. In some embodiments, the encapsulation efficiency is greater than about 93%. In some embodiments, the encapsulation efficiency is greater than about 95%.

[0134] The present invention also includes a kit. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit comprises compounds or compositions as described above.

[0135] Kits typically include at least one vial, test tube, flask, bottle, syringe, or other container in which one component is placed and optionally appropriately aliquoted. If the kit contains more than one component, it usually also includes a second, third, or other additional container in which the additional components are placed separately. However, various combinations of components can be contained in the containers. In some embodiments, all delivery components are combined in a single container. In other embodiments, some or all of the compound delivery components are provided in separate containers.

[0136] The kits of this application will also typically include packaging for tightly housing various containers for commercial sale. Such packaging may include cardboard or injection-molded or blow-molded plastic packaging in which the desired containers are retained. In some embodiments, the kit will also include instructions for using the kit components. These instructions may include implementable variations. In some embodiments, these instructions are for delivering one or more compounds or one or more compositions to a subject or cells.

[0137] Its methods and uses This application includes methods for delivering one or more ingredients to cells or a subject using the compounds and / or compositions of this application. Therefore, in some embodiments, this application includes a method for delivering one or more pharmaceutical agents to cells, comprising contacting the cells with one or more compounds or compositions or pharmaceutical compositions of this application under conditions that promote cellular uptake of the ingredients. In some embodiments, this application includes a method for delivering one or more ingredients to a subject, comprising contacting the subject with one or more compounds or compositions or pharmaceutical compositions of this application. In some embodiments, cell contact occurs in vitro. In some embodiments, cell contact occurs in vivo. In some embodiments, cell contact occurs ex vivo. In some embodiments, contact is for the treatment of a disease, symptom, or condition. In some embodiments, contact is performed by administering an effective amount of one or more compounds or compositions or pharmaceutical compositions of this application to the cells or subject in need.

[0138] In some embodiments, the method is a way of treating a disease, symptom, or condition. In some embodiments, the method is a way of treating a disease, symptom, or condition in a subject who requires treatment. In some embodiments, the subject is a rodent. In some embodiments, the subject is a human. In some embodiments, the method includes exposing the subject to one or more compounds or compositions or pharmaceutical compositions described herein.

[0139] In some embodiments, this application also includes the use of one or more compounds or compositions or pharmaceutical compositions of this application for diagnostic, preventive, or therapeutic applications. In some embodiments, the use is related to the payload delivered by the composition. In some embodiments, the payload includes polynucleotides, chemically modified polynucleotides, small molecules, biological agents, or other organic or inorganic portions. Diagnostic applications include the use of one or more compounds or compositions or pharmaceutical compositions of this application as a carrier of an identifiable marker (e.g., a dye or radiolabel), which can be detected after administration and indicate a disease, condition, or illness. Preventive applications include use as a vaccine, including vaccines against infectious diseases or other anticipated or potential illnesses in the subject. Therapeutic applications include treatment for any disease, condition, or illness, including but not limited to infectious diseases, autoimmune diseases, cancer, genetic diseases, chronic diseases, traumatic injuries, wound healing, traumatic brain injury, muscle diseases, neuromuscular diseases, and / or gastrointestinal diseases. Therapeutic applications can be performed in vivo or as ex vivo therapies, in which cells, tissues, or organs are processed ex vivo and implanted or transplanted into the subject.

[0140] In some embodiments, this application also includes using one or more compounds or compositions or pharmaceutical compositions of this application to deliver one or more ingredients to cells, whether in vitro, in vivo or ex vivo.

[0141] In some embodiments, one or more components to be delivered to cells are one or more nucleic acids. Therefore, this application also includes a method for regulating gene expression, comprising delivering one or more nucleic acids to cells, the method comprising contacting the cells with one or more compounds or compositions or pharmaceutical compositions of this application under conditions that promote the uptake of one or more nucleic acids by the cells. In some embodiments, the cells are contacted in vitro. In some embodiments, the cells are contacted in vivo. In some embodiments, the cells are contacted ex vivo. In some embodiments, the regulation of gene expression is sufficient to treat a disease, symptom, or condition.

[0142] In some implementations, the disease, symptom, or condition is, for example, but not limited to, infectious diseases, autoimmune diseases, cancer, genetic diseases, chronic diseases, traumatic injuries, wound healing, traumatic brain injury, neuromuscular diseases, and / or gastrointestinal diseases.

[0143] In some embodiments, this application also includes the use of one or more compounds or compositions or pharmaceutical compositions of this application for regulating gene expression. In some embodiments, gene expression is regulated by delivering one or more nucleic acids to cells (whether in vitro, in vivo, or ex vivo).

[0144] In some embodiments, when the compositions and pharmaceutical compositions of this application contain siRNA, miRNA, and mRNA as one or more components to be delivered to cells or a subject, the formulated delivery composition is designed to target bone marrow endothelial cells to inhibit, regulate, and induce genes that cause chronic inflammatory events. Currently, autoimmune diseases are a major source of chronic inflammation, affecting approximately two million Canadians. Due to their complexity, autoimmune diseases are not fully understood, often presenting challenges in discussing treatment options. Current treatments include immunosuppressants, corticosteroids, and pain management. These current treatments do not provide long-term or permanent effects and require optimization. Chronic and severe inflammation is caused by the overexpression and migration of monocytes and monocyte-derived macrophages to target areas in the body. Most monocytes and monocyte-derived macrophages are produced in the bone marrow and subsequently cross the endothelial barrier, reaching target areas via blood flow. Since the bone marrow is the primary source of monocyte production, directly targeting areas of high monocyte production can alter the processes of monocyte proliferation and entry into the bloodstream. In some embodiments, the silencing effect of siRNA encapsulated in nanoparticles can inhibit the proliferation of monocytes while simultaneously inhibiting their ability to leave the bone marrow. By not only reducing their ability to migrate freely but also reducing their total number in the body, the inflammatory response that leads to damaging effects in patients with chronic inflammation can be effectively reduced. Therefore, in some embodiments, this application includes a method of treating chronic inflammation comprising administering an effective amount of a composition to cells or a subject in need, the composition comprising one or more compounds of this application, siRNA, miRNA, and mRNA. It also includes the use of a composition comprising one or more compounds of this application, siRNA, miRNA, and mRNA for treating chronic inflammation. In some embodiments, the siRNA is siRNA targeting bone marrow endothelial genes required for monocyte attachment and entry into the bloodstream.

[0145] In some embodiments, the compositions of this application are used for gene editing. Therefore, in some embodiments, this application includes a method for editing the cellular genome, which includes contacting cells with one or more compositions of this application. It also includes the use of one or more compositions of this application for gene editing. In some embodiments, these compositions comprise Cas9 mRNA and one or more guide RNAs (gRNAs) designed to target specific genes, and one or more compositions are contacted with cells under conditions that induce Cas9 mRNA and one or more gRNAs to be taken up by the cells. After cell uptake, the Cas9 mRNA is translated into the Cas9 protein and binds to the gRNA to target a target sequence within the cell nucleus. DNA can also be co-delivered for homology-directed repair.

[0146] In some embodiments, the compositions of this application are used for gene silencing. Therefore, in some embodiments, this application includes a method for silencing genes in cells, comprising contacting cells with one or more compositions of this application. It also includes the use of one or more compositions of this application for gene silencing. In some embodiments, these compositions contain siRNA, which silences the gene corresponding to the siRNA upon delivery to the cell. Therefore, one or more compositions are contacted with cells under conditions that promote siRNA uptake by the cells.

[0147] In some embodiments, the compositions of this application are used for gene regulation. In some embodiments, these compositions comprise non-coding RNA (ncRNA), and one or more compositions are contacted with cells under conditions that induce ncRNA uptake by the cells, and after uptake, regulate genes associated with the ncRNA.

[0148] In some embodiments, the compositions of this application are used to upregulate or downregulate gene expression. Therefore, in some embodiments, this application includes a method for regulating gene expression in cells, comprising contacting cells with one or more compositions of this application. It also includes the use of one or more compositions of this application for regulating gene expression in cells. In some embodiments, these compositions comprise messenger RNA (mRNA), and one or more compositions are contacted with cells under conditions that induce mRNA uptake by the cells, and after uptake, protein expression occurs.

[0149] In some embodiments, the compositions of this application can be used to treat or prevent infectious diseases, autoimmune diseases, cancer, genetic diseases, chronic diseases, traumatic injuries, wound healing, traumatic brain injury, neuromuscular diseases and / or gastrointestinal diseases.

[0150] In some embodiments, the compositions of this application are used for antiviral therapy. Therefore, in some embodiments, this application includes a method of treating a viral infection, comprising administering one or more compositions of this application to cells or a subject in need. It also includes the use of one or more compositions of this application for treating a viral infection. In some embodiments, these compositions comprise mRNA encoding the Cas9 protein and one or more sgRNAs. In some embodiments, one or more sgRNAs recognize a viral gene to be deleted. In some embodiments, the virus is a DNA virus or an RNA virus.

[0151] In some embodiments, the compositions of this application are used for methods of delivering one or more proteins to cells, delivering one or more small molecule drugs to cells, or delivering one or more DNA molecules to cells.

[0152] In some embodiments, the compounds and compositions of this application achieve higher intramuscular expression than their amide analogs.

[0153] In some embodiments, intravenous injection of the compositions of this application achieves peak expression in the spleen. Therefore, the compounds and compositions of this application allow for controlled biodistribution.

[0154] In some embodiments, the compounds and compositions of this application achieve enhanced degradation rates. Therefore, the relevant compounds of this application are more readily hydrolyzed by various extracellular or intracellular enzymes than their amide analogs.

[0155] In some embodiments, the compositions of this application are used in cosmetic and / or personal care products, and one or more ingredients to be delivered to cells or subjects are any such ingredients. In some embodiments, one or more ingredients used in cosmetic and / or personal care products include, but are not limited to: hair moisturizers, hair growth agents, hair anti-frizz agents, skin moisturizers, anti-aging agents, and transient bioluminescent proteins.

[0156] In some embodiments, the compositions of this application are used in anti-counterfeiting methods, such as verifying that a product or packaging has not been tampered with or is in its original form. In these embodiments, one or more ingredients will contain a unique DNA or RNA sequence (barcode), which is placed in the product packaging / label and can be read to ensure that the product is genuine or has not been tampered with, and the compositions of this application are delivered to or inside the subject as packaged.

[0157] In some embodiments, the composition used in the method and for the purpose of application is a pharmaceutical composition as defined above.

[0158] In some embodiments, the cells used in the methods and uses of this application are in vitro. In some embodiments, the cells used in the methods and uses of this application are in vivo. In some embodiments, the cells used in the methods and uses of this application are ex vivo.

[0159] In some embodiments, the cell includes any cell having a cell membrane. Therefore, in some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a prokaryotic or eukaryotic cell selected from animal cells, insect cells, or plant cells.

[0160] Preparation of the compounds in this application The compounds of this application can be prepared by various synthetic methods. The choice of specific structural features and / or substituents may influence the choice of different methods. The selection of a specific method to prepare a given compound of formula (I) is within the knowledge of those skilled in the art. Some starting materials used to prepare the compounds of this application are available from commercial chemical sources. Other starting materials can be readily prepared from available starting materials using direct conversion methods well known in the art.

[0161] In some embodiments, compounds of formula I are assembled by linking the parts of a molecule together in a specific order, based on the functional groups present. Standard chemical reactions known in the art can be used to assemble compounds of formula I, including but not limited to nucleophilic substitution, cross-coupling, Michael reactions, and / or activating group strategies. Since many parts of the compound are known or based on known compounds, compounds that can be used to link the parts of the molecule are readily available from commercial sources or obtained using synthetic methods known in the art. For example, an amino alcohol undergoes an addition reaction with, for example, bromodecane (or any other suitable group) to form intermediates A, C, G, and N, which are subsequently esterified with a suitable acyl chloride (e.g., 3-bromopropionyl chloride) to form intermediates B, D, E, F, I, H, and J. Two different or two identical intermediates selected from B, D, E, F, I, H, and J undergo an addition reaction with a suitable amino alcohol to form a compound of formula I.

[0162] In an exemplary embodiment, in order to synthesize a compound of formula I (wherein R...) 2 and R 3 Or R 4 and R 5 Same (marked as R in the scheme) z), and z represents intermediates A, C, G and N of c / d as defined in Equation I, following the steps shown in Scheme 1.

[0163]

[0164] Option 1 In this addition reaction, 1 equivalent of an amino alcohol (e.g., ethanolamine, 3-amino-1-propanol, etc.) and 2.2 equivalents of LG-R are added. z (where LG is a suitable leaving group, such as a halogen (e.g., 1-bromodecane)) is dissolved in a suitable solvent (e.g., acetonitrile) and reacted under certain conditions in the presence of an excess base (e.g., K2CO3) (4.4 equivalents) and a halide salt (e.g., KI) (0.2 equivalents) to obtain intermediates A, C, G and N.

[0165] In an exemplary embodiment, in order to synthesize a compound of formula I (wherein R...) 2 and R 3 or R 4 and R 5 Same (marked as R in the scheme) z ), and z represents intermediates B and D of c / d as defined in Equation I, following the steps shown in Scheme 2.

[0166]

[0167] Option 2 Therefore, intermediates B and D are synthesized by reacting 2 equivalents of intermediate A or C with 1 equivalent of acyl chloride (e.g., 3-bromopropionyl chloride or the like) in a suitable solvent (e.g., DCM) and in the presence of 1.2 equivalents of any suitable alcohol.

[0168] In an exemplary embodiment, in order to synthesize a compound of formula I (wherein R...) 2 and R 3 or R 4 and R 5 Same (marked as R in the scheme) z (), z represents c / d as defined in Equation I, and z' represents a / b as defined in Equation I) intermediates E, F, I, H, J, follow the steps shown in Scheme 3.

[0169]

[0170] Option 3 Therefore, intermediates E, F, I, H and J are synthesized by reacting 2 equivalents of intermediate A, C or G with 1 equivalent of acyl chloride (e.g., 3-bromopropionyl chloride, 4-bromopropionyl chloride, 4-bromobutyryl chloride, etc.) in a suitable solvent (e.g., DCM) and in the presence of 1.2 equivalents of any suitable alcohol.

[0171] In an exemplary embodiment, in order to synthesize a compound of formula I (where z' represents a / b and is 3, z'' represents R) 1 The intermediate K (as defined in Formula I) follows the steps shown in Scheme 4.

[0172]

[0173] Option 4 Therefore, 1 equivalent of an amino alcohol (e.g., ethanolamine, 3-amino-1-propanol, etc.), 1.1 equivalent of tert-butyldimethylchlorosilane, and 2 equivalents of imidazole are dissolved in a suitable solvent (e.g., DCM) and then reacted with methyl acrylate and lithium aluminum hydride under certain conditions to obtain intermediate "K".

[0174] In an exemplary embodiment, in order to synthesize a compound of formula I (wherein R...) 2 and R 3 or R 4 and R 5 Same (marked as R in the scheme) z ), and z represents the intermediate L of c / d as defined in Equation I, following the steps shown in Scheme 5.

[0175]

[0176] Option 5 Therefore, NH2(CH2) z C(O)OC4H9 (e.g., tert-butyl 4-aminobutyrate hydrochloride) (1 equivalent) and LG-R z (Where LG is a suitable leaving group, such as a halogen, like 1-bromodecane) (2.1 equivalents) is dissolved in a suitable solvent (e.g., a 1:1 mixture of acetonitrile and cyclopentylmethyl ether) and reacted in the presence of a base (e.g., K₂CO₃) (4 equivalents) and a halide salt (e.g., KI) (1.1 equivalents). The tert-butyl group is then removed, for example using a suitable acid, such as TFA, and the reaction is quenched with a suitable base to obtain intermediate "L". 。

[0177] In an exemplary embodiment, in order to synthesize compound I (where R... 2 and R 3 and / or R 4 and R 5Two different or two identical intermediates (1.8 equivalents) selected from B, D, E, F, H, I, and J are alkylated with an amino alcohol (e.g., ethanolamine, 3-amino-1-propanol, etc.) (1 equivalent) in a suitable solvent (e.g., a 1:1 mixture of acetonitrile:cyclopentylmethyl ether) in the presence of a base (e.g., K₂CO₃) (4 equivalents) and a halide salt (e.g., KI) (0.4 equivalents). In some embodiments, a non-nucleophilic base, such as 1,8-diazabicyclo(5.4.0)undec-7-ene, is added to the reactants.

[0178] Throughout the process, it should be understood that, where appropriate, suitable protecting groups will be added to various reactants and intermediates in a manner readily understood by those skilled in the art, and subsequently removed therefrom. Conventional procedures using such protecting groups, and examples of suitable protecting groups, are described, for example, in… Protective Groups in Organic Synthesis “, TW Green, PGM Wuts, Wiley-Interscience, New York, (1999). It should also be understood that the conversion of one group or substituent to another group or substituent by chemical manipulation can be carried out on any intermediate or final product along the synthetic pathway to the final product, wherein the types of conversions that may be carried out are limited only by the inherent incompatibility of other functional groups carried by the molecule at that stage with the conditions or reagents used for the conversion. Such inherent incompatibility, and the ways to circumvent them by performing appropriate conversions and synthetic steps in a suitable order, are readily understood by those skilled in the art. Examples of conversions are given herein, and it should be understood that the conversions described are not limited to the general groups or substituents exemplified by the conversions. References and descriptions of other suitable conversions can be found in “Comprehensive Organic Transformations - A Guide to Functional Group Preparations” RC Larock, VHCPublishers, Inc. (1989). References and descriptions of other suitable reactions can be found in organic chemistry textbooks, such as “ Advanced Organic Chemistry ", March, 4th edition. McGraw Hill (1992) or " Organic Synthesis ”, Smith, McGraw Hill, (1994).

[0179] Purification techniques for intermediates and final products include, for example, normal-phase and reversed-phase chromatography by column or rotating thin-layer plate, recrystallization, distillation, and liquid-liquid or solid-liquid extraction, which are readily understood by those skilled in the art.

[0180] The salts of the compounds in this application are typically formed by dissolving a neutral compound in an inert organic solvent, then adding the desired acid or base, and separating the resulting salt by filtration or other known methods.

[0181] The formation of solvates of the compounds in this application will vary depending on the compound and the solvate. Typically, solvates are formed by dissolving the compound in a suitable solvent and then separating the solvate by cooling or using an antisolvent. Solvates are typically dried or azeotropically treated under ambient conditions. Selecting suitable conditions to form a particular solvate can be done by those skilled in the art. Examples of suitable solvents are ethanol, water, etc. When water is used as the solvent, the molecule is referred to as a "hydrate". The following non-limiting examples are examples of this application.

[0182] The compositions of this application are prepared by combining the compounds of this application with optional excipients under conditions that form nanoparticles, colloids, and / or supramolecular structures, suitably containing one or more components to be delivered. In some embodiments, the conditions for forming nanoparticles, colloids, and / or supramolecular structures include first combining one or more compounds with any optional excipients (including one or more PEG-lipids, one or more phospholipids, and / or one or more sterols) in a suitable solvent (e.g., ethanol). In some embodiments, sonication and / or heating are used to remove any precipitates. Additionally, one or more components to be delivered are prepared in a suitable solvent (e.g., sodium acetate). The two solutions are then combined under certain conditions to form nanoparticles, colloids, and / or supramolecular structures. In some embodiments, the two solutions are combined under continuous flow conditions (e.g., using microfluidics). In some embodiments, the resulting nanoparticles, colloids, and / or supramolecular structures are treated by dialysis to adjust the pH. In some embodiments, the particles are stored at room temperature or -20°C for at least 48 hours without any noticeable changes.

[0183] Example General Synthesis Method General synthetic method for alkylation of A-amino alcohols The amino alcohol (1 equivalent) and 1-bromodecane (2.2 equivalents) were dissolved in acetonitrile in a round-bottom flask. K₂CO₃ (4.4 equivalents) and KI (0.2 equivalents) were added. The flask was then covered with aluminum foil, fitted with a condenser, and heated to 85°C for 18 hours. The reaction mixture was filtered through a diatomaceous earth stopper and washed thoroughly with ethyl acetate. The filtrate was concentrated to a crude oil and then purified by column chromatography using a 10–40% dichloromethane (DCM) gradient (22% MeOH, 3% NH₄OH in dichloromethane solution).

[0184] General synthetic method for esterification of β-acyl chlorides The alcohol (1.2 equivalents) and triethylamine (2 equivalents) were dissolved in dichloromethane (dried to a dryness of anhydrous molecular sieve) in a round-bottom flask and cooled to 0°C. With stirring, the acyl chloride (1 equivalent) was slowly added to the reaction mixture. The ice bath was removed, the flask was fitted with a condenser, and heated to 55°C for 2 hours. The reaction mixture was concentrated to a crude oil and then purified by column chromatography using a 0-60% ethyl acetate / hexane gradient.

[0185] General synthetic method for alkylation of C-ethanolamine core Ethanolamine (1 equivalent) and the halide substrate (1.8 equivalent) were dissolved in a 1:1 mixture of acetonitrile and cyclopentyl methyl ether in a glass vial. K₂CO₃ (4 equivalent) and KI (0.4 equivalent) were added. The vial was then covered with aluminum foil and heated to 85°C for 48 hours. The reaction mixture was filtered through a diatomaceous earth stopper and washed thoroughly with ethyl acetate. The filtrate was concentrated to a crude oil and then purified by column chromatography using a 10–40% dichloromethane gradient (dichloromethane solution of 22% MeOH and 3% NH₄OH).

[0186] Example 1: Synthesis of exemplary compounds of this application Synthesis of intermediates Intermediate A

[0187] According to general synthesis method A, intermediate A was synthesized using ethanolamine, and a pale yellow oily substance was obtained by separation. 1 H NMR (500 MHz, CDCl3) δ 3.56 (t, J = 5.6 Hz, 0H), 3.18 (s, 0H), 2.61 (t, J = 5.4Hz, 0H), 2.52 - 2.43 (m, 1H), 1.45 (p, J = 7.2 Hz, 1H), 1.32 - 1.21 (m, 5H), 0.88 (t, J = 7.2 Hz, 1H). 13 C NMR (126 MHz, CDCl3) δ 58.29, 55.70, 54.00,32.04, 29.77, 29.73, 29.69, 29.68, 29.46, 27.53, 27.03, 22.82, 14.25.

[0188] Intermediate B

[0189] Intermediate B was synthesized using intermediate A and 3-bromopropionyl chloride according to general synthetic method B. 1 H NMR (500 MHz, CDCl3) δ 6.39 (dd, J = 17.3, 1.5 Hz, 1H), 6.12 (dd, J = 17.4, 10.5 Hz, 1H),5.81 (dd, J = 10.4, 1.5 Hz, 1H), 4.20 (t, J = 6.2 Hz, 2H), 2.72 (t, J = 6.3Hz, 2H), 2.48 - 2.42 (m, 4H), 1.42 (p, J = 6.6 Hz, 4H), 1.26 (d, J = 5.2 Hz, 29H), 0.87 (t, J = 7.0 Hz, 6H). 13 C NMR (126 MHz, CDCl3) δ 166.36, 130.69,128.71, 63.09, 55.00, 52.30, 32.05, 29.82, 29.81, 29.76, 29.75, 29.48, 27.60,27.42, 22.83, 14.25.

[0190] Intermediate C

[0191] Intermediate C was synthesized using 3-amino-1-propanol according to general synthetic method A. 1 H NMR (500 MHz, cdcl3)δ 3.78 (t, J = 5.3 Hz, 2H), 2.69 (t, J = 5.8 Hz, 2H), 2.50 - 2.42 (m, 4H), 1.74 - 1.66 (m, 2H), 1.53 - 1.44 (m, 4H), 1.33 - 1.17 (m, 30H), 0.86 (t, J =7.4 Hz, 6H). 13C NMR (126 MHz, cdcl3) δ 64.29, 55.04, 54.11, 32.01, 29.71, 29.69, 29.65, 29.42, 27.68, 27.55, 26.47, 22.79, 14.22.

[0192] Intermediate D

[0193] Intermediate D was synthesized using intermediate C and 3-bromopropionyl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 6.38 (dd, J = 17.3, 1.5 Hz, 1H), 6.10 (dd, J = 17.3, 10.4 Hz, 1H),5.79 (dd, J = 10.4, 1.5 Hz, 1H), 4.19 (t, J = 6.5 Hz, 2H), 2.49 (t, J = 7.2Hz, 2H), 2.41 - 2.34 (m, 4H), 1.79 (p, J = 7.1 Hz, 2H), 1.45 - 1.35 (m, 4H), 1.34 - 1.23 (m, 27H), 0.92 - 0.80 (m, 6H). 13 C NMR (126 MHz, cdcl3) δ 166.34,130.52, 128.74, 63.20, 54.29, 50.51, 32.04, 29.79, 29.75, 29.74, 29.47,27.68, 27.19, 26.52, 22.81, 14.23.

[0194] Intermediate E

[0195] Intermediate E was synthesized using intermediate A and 4-bromobutyryl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 4.21 - 4.11 (m, 2H), 3.46 (t, J = 6.5 Hz, 1H), 2.70 (dt, J= 12.1,6.1 Hz, 2H), 2.55 - 2.39 (m, 5H), 2.21 - 2.12 (m, 1H), 1.51 - 1.37 (m, 4H),1.34 - 1.19 (m, 29H), 0.88 (t, J = 6.9 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ174.89, 172.50, 62.81, 54.78, 54.76, 54.64, 52.20, 52.14, 32.68, 32.47,31.90, 29.66, 29.65, 29.61, 29.60, 29.59, 29.59, 29.33, 27.76, 27.44, 27.44,27.12, 22.67, 14.10, 12.88, 8.39.

[0196] intermediate F

[0197] Intermediate F was synthesized using intermediate C and 4-bromobutyryl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 4.13 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 6.4 Hz, 2H), 2.50 (t, J = 7.1Hz, 4H), 2.46 - 2.39 (m, 4H), 2.22 - 2.09 (m, 2H), 1.84 - 1.75 (m, 2H), 1.49- 1.39 (m, 4H), 1.35 - 1.19 (m, 29H), 0.87 (t, J = 7.0 Hz, 6H). 13 C NMR (126MHz, cdcl3) δ 172.66, 63.21, 54.19, 50.55, 32.86, 32.59, 32.05, 29.80, 29.78,29.74, 29.73, 29.48, 27.89, 27.65, 22.83, 14.26.

[0198] intermediate G

[0199] Intermediate G was synthesized using 4-amino-1-butanol according to general synthetic method A. 1 H NMR (500 MHz, cdcl3)δ 3.57 (t, J = 5.4 Hz, 2H), 2.60 - 2.52 (m, 6H), 1.75 - 1.61 (m, 4H), 1.58 -1.48 (m, 4H), 1.33 - 1.17 (m, 28H), 0.87 (t, J = 6.9 Hz, 5H). 13 C NMR (126 MHz, cdcl3) δ 77.27, 77.02, 76.76, 62.32, 54.25, 53.36, 31.99, 31.86, 29.55, 29.52, 29.40, 29.27, 27.45, 25.18, 22.65, 14.08.

[0200] intermediate H

[0201] Intermediate H was synthesized using intermediate G and 4-bromobutyryl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 4.09 (t, J = 6.7 Hz, 2H), 3.46 (t, J = 6.5 Hz, 2H), 2.49 (t, J = 7.2Hz, 2H), 2.43 - 2.36 (m, 3H), 2.40 - 2.33 (m, 5H), 2.17 (p, J = 6.4 Hz, 2H), 1.63 (p, J = 7.9 Hz, 2H), 1.52 - 1.43 (m, 2H), 1.44 - 1.35 (m, 4H), 1.36 -1.18 (m, 29H), 0.87 (t, J = 7.1 Hz, 6H). 13C NMR (126 MHz, cdcl3) δ 172.73,64.84, 60.52, 54.34, 53.85, 32.84, 32.63, 32.06, 31.73, 29.83, 29.80, 29.76,29.49, 27.92, 27.79, 27.22, 26.83, 23.81, 22.83, 14.34, 14.26.

[0202] Intermediate I

[0203] Intermediate I was synthesized using intermediate C and 5-bromopentanoyl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 4.10 (t, J = 6.5 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 2.48 - 2.42 (m,2H), 2.35 (t, J = 7.4 Hz, 4H), 2.33 (t, J = 7.4 Hz, 2H), 1.93 - 1.85 (m, 2H), 1.82 - 1.69 (m, 4H), 1.39 (p, J = 7.1 Hz, 5H), 1.33 - 1.19 (m, 27H), 0.87 (t, J = 7.0 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ 173.25, 63.22, 54.33, 50.59, 33.45,33.09, 32.15, 32.05, 31.72, 29.82, 29.79, 29.76, 29.48, 27.71, 27.30, 26.65,23.67, 22.82, 22.78, 14.25.

[0204] Intermediate J

[0205] Intermediate J was synthesized using intermediate G and 5-bromopentanoyl chloride according to general synthetic method B. 1 H NMR (500 MHz, cdcl3) δ 4.08 (t, J= 6.7 Hz, 2H), 3.40 (t, J = 6.6 Hz, 2H), 2.40 (t, J = 7.5Hz, 2H), 2.38 - 2.35 (m, 4H), 2.33 (t, J = 6.9 Hz, 2H), 1.93 - 1.86 (m, 2H), 1.82 - 1.73 (m, 2H), 1.63 (p, J = 7.9 Hz, 2H), 1.51 - 1.44 (m, 2H), 1.40 (p, J = 6.9 Hz, 4H), 1.32 - 1.19 (m, 29H), 0.87 (t, J = 7.1 Hz, 6H). 13 C NMR (126MHz, cdcl3) δ 173.17, 64.53, 54.18, 53.71, 33.30, 32.96, 32.00, 31.90, 29.67,29.64, 29.60, 29.58, 29.33, 27.63, 27.07, 26.70, 23.67, 23.52, 22.67, 14.10.

[0206] intermediate K

[0207] Ethanolamine (0.305 g, 5 mmol, 1 equivalent), tert-butyldimethylchlorosilane (0.829 g, 5.5 mmol, 1.1 equivalent), and imidazole (0.681 g, 10 mmol, 2 equivalent) were dissolved in dichloromethane (40 mL) and stirred at ambient temperature for 3 hours. The reaction mixture was then diluted with water (60 mL) and extracted with dichloromethane (3 × 30 mL). The organic fractions were combined and concentrated to a clear oily solution of 2-((tert-butyldimethylsilyl)oxy)ethyl-1-amine (0.791 g, 90%). 1 H NMR (500 MHz, cdcl3) δ 3.59 (t, J = 5.4 Hz, 2H), 2.74 (t, J = 5.2 Hz, 2H), 0.86 (s, 9H), 0.03 (s, 6H). 13C NMR (126 MHz, cdcl3) δ 65.10, 44.22, 25.87, 25.74, 18.27, -3.52, -5.36.

[0208] 2-((tert-butyldimethylsilyl)oxy)ethyl-1-amine (2.63 g, 15 mmol, 1 equivalent) and methyl acrylate (3.87 g, 45 mmol, 3 equivalent) were dissolved in methanol (10 mL) and stirred at ambient temperature for 18 hours. Methanol and excess tert-butyl acrylate were removed under vacuum to give di-tert-butyl 3,3'-((2-((tert-butyldimethylsilyl)oxy)ethyl)azadiyl)dipropionate (5.21 g, quantified). 1 H NMR (400 MHz, CDCl3) δ 3.66 - 3.54 (m, 8H),2.78 (t, J = 6.9 Hz, 4H), 2.55 (t, J = 6.4 Hz, 2H), 2.40 (t, J = 7.2 Hz, 4H), 0.83 (s, 10H), -0.00 (s, 6H). 13 C NMR (101 MHz, CDCl3) δ 172.99, 61.82, 55.94, 51.50, 50.16, 32.85, 25.93, 25.74, 18.27, -3.51, -5.35.

[0209] A 1M lithium aluminum hydride (LAH) solution in tetrahydrofuran (13.0 mL, 13 mmol, 2.6 equivalents) was suspended in tetrahydrofuran (THF) (30 mL) at 0 °C. A THF (15 mL) solution of dimethyl 3,3'-((2-((tert-butyldimethylsilyl)oxy)ethyl)azadiyl)dipropionate (1.80 g, 5.5 mmol, 1 equivalent) was added dropwise to the LAH over 30 minutes. After the addition was complete, the combined solutions were stirred at ambient temperature for another 1 hour. The reaction mixture was then cooled back to 0 °C, and 1 g of ice was added directly to the flask and stirred for 10 minutes. Anhydrous Na₂SO₄ was added directly to the reaction mixture and stirred for 15 minutes. The reaction mixture was filtered through a diatomaceous earth stopper and washed with additional THF. The filtrate was concentrated and purified by column chromatography using a gradient of 20-30% dichloromethane (dichloromethane solution of 22% MeOH and 3% NH4OH). The resulting pale yellow oily substance was 3,3'-((2-((tert-butyldimethylsilyl)oxy)ethyl)azadiyl)bis(propane-1-ol) (0.340 g, 23%). 1H NMR (500 MHz, cdcl3) δ 3.73 - 3.64 (m, 6H), 2.64 (t, J = 6.4 Hz, 4H), 2.56 (t, J = 6.0 Hz, 2H), 1.68 (qd, J = 6.3, 5.4 Hz, 4H), 0.85 (s, 9H), 0.03 (s, 6H). 13 CNMR (126 MHz, cdcl3) δ 62.29, 61.01, 55.83, 53.28, 28.81, 25.98, 18.33, -5.37.

[0210] intermediate L

[0211] 4-Tetrabutyl 4-aminobutyrate hydrochloride (0.978 g, 5 mmol, 1 equivalent) and 1-bromodecane (2.32 g, 10.5 mmol, 2.1 equivalent) were dissolved in a 1:1 mixture of acetonitrile and cyclopentyl methyl ether (10 mL of each solvent). K₂CO₃ (2.76 g, 20 mmol, 4 equivalent) and KI (0.913 g, 5.5 mmol, 1.1 equivalent) were added to the reaction mixture. The flask was fitted with a condenser and stirred at 85 °C for 18 hours. The reaction mixture was filtered through a diatomaceous earth stopper and washed with ethyl acetate. The filtrate was concentrated and purified by column chromatography in a gradient of 0-60% ethyl acetate / hexane. 4-(dicepanoylamino)butyrate tert-butyl ester (1.011 g, 46%) was obtained as a pale yellow oil. 1 H NMR (500 MHz, cdcl3) δ 2.42 - 2.31 (m, 7H), 2.21 (t, J = 7.4 Hz, 2H), 1.68 (p, J = 7.4 Hz, 2H), 1.42 (s, 9H), 1.41 - 1.35 (m, 3H), 1.33 - 1.17 (m, 31H), 0.86 (t, J = 6.8 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ173.30, 79.99, 54.34, 53.32, 33.57, 32.04, 29.81, 29.78, 29.74, 29.47, 28.23,27.72, 27.34, 22.81, 22.78, 14.22.

[0212] 1.011 g (2.30 mmol) of tert-butyl 4-(decylamino)butyrate was dissolved in dichloromethane and cooled to 0 °C. Trifluoroacetic acid (2.30 mL) was added dropwise to the reaction mixture. The mixture was then stirred at ambient temperature for 18 hours, cooled to 0 °C, and quenched by adding saturated NaHCO3 (5 mL). The mixture was transferred to a separatory funnel and diluted with additional dichloromethane (5 mL). The organic layer was washed with saturated NaHCO3 (3 x 10 mL), dried over anhydrous salt, and concentrated to give 0.835 g (95%) of 4-(decylamino)butyric acid as a white powder. 1 H NMR (500 MHz, cdcl3) δ 2.82 - 2.76(m, 2H), 2.72 - 2.65 (m, 4H), 2.59 - 2.53 (m, 2H), 1.86 - 1.78 (m, 2H), 1.64- 1.56 (m, 4H), 1.34 - 1.18 (m, 30H), 0.86 (t, J = 7.0 Hz, 6H). 13 C NMR (126MHz, cdcl3) δ 176.30, 55.50, 53.42, 53.01, 37.40, 31.82, 29.45, 29.43, 29.22,27.55, 27.16, 24.30, 22.63, 21.20, 14.07.

[0213] intermediate M

[0214] 4-(decylamino)butyric acid (0.345 g, 0.9 mmol, 0.9 equivalents), 4-chloro-1-butanol (104.7 μL, 1 mmol, 1 equivalent, calculated based on industrial reagent purity), dichloroethane (EDC)·HCl (0.288 g, 2 mmol, 2 equivalents), 4-dimethylaminopyridine (DMAP) (0.024 g, 0.2 mmol, 0.2 equivalents), and N,N-diisopropylethylamine (DIPEA) (348 μL, 2 mmol, 2 equivalents) were dissolved in dichloromethane (10 mL) and stirred at ambient temperature for 3 hours. The reaction mixture was concentrated to a crude oil, which was then purified by column chromatography with a gradient of 0-60% ethyl acetate / hexane. Intermediate M (0.315 g, 74%), a pale yellow oil, was obtained. 1 H NMR (500 MHz, cdcl3) δ 4.10 (t,J = 6.2 Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 2.39 (t, J = 7.1 Hz, 2H), 2.37 - 2.34 (m, 4H), 2.32(t, J = 7.5 Hz, 2H), 1.88 - 1.76 (m, 4H), 1.73 (p, J = 7.5 Hz, 2H), 1.38 (p, J = 7.0 Hz, 4H), 1.33 - 1.18 (m, 28H), 0.87 (t, J = 7.1 Hz, 6H). 13 C NMR (126MHz, cdcl3) δ 173.97, 63.51, 54.29, 53.36, 44.58, 32.27, 32.05, 29.83, 29.80,29.76, 29.48, 29.32, 27.73, 27.30, 26.24, 22.83, 22.74, 14.25.

[0215] intermediate N

[0216] Intermediate N was synthesized using 5-amino-1-pentanol according to general synthetic method A. 1 H NMR (500 MHz, cdcl3)δ 3.60 (t, J = 6.5 Hz, 2H), 2.52 - 2.41 (m, 6H), 1.61 - 1.41 (m, 8H), 1.40 -1.32 (m, 2H), 1.32 - 1.18 (m, 28H), 0.86 (t, J = 6.9 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ 62.53, 53.97, 32.48, 32.00, 29.73, 29.67, 29.64, 29.42, 27.66, 26.23, 26.15, 23.77, 22.77, 14.20.

[0217] Intermediate O

[0218] Intermediate N (0.386 g, 1.00 mmol, 1 equivalent), 1-hydroxycyclohexylphenyl ketone (1.233 g, 6.04 mmol, 6 equivalent), and sodium hydroxide (0.256 g, 6.4 mmol, 6.4 equivalent) were added to a screw-capped vial containing 5 mL of 1,2-dimethoxyethane and heated to 80 °C overnight. The reaction was quenched by adding water and 6N hydrochloric acid (533 μL, 6.4 mmol). The mixture was transferred to a separatory funnel and extracted with ethyl acetate (3 x 20 mL). The combined organic fractions were washed with brine (1 × 40 mL), dried over anhydrous salt, and concentrated to a crude oil. The crude oil was purified by column chromatography with a gradient of 20–50% ultra / DCM to give a yellow oil (0.299 g, 75%). 1 H NMR (500 MHz, cdcl3) δ 2.95 - 2.72(m, 6H), 2.32 - 2.14 (m, 2H), 1.67 (d, J = 4.4 Hz, 2H), 1.58 (d, J = 4.0 Hz,6H), 1.25 (d, J = 21.7 Hz, 28H), 0.86 (t, J = 6.9 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ 179.46, 53.25, 50.61, 36.79, 31.96, 29.57, 29.55, 29.36, 29.28,27.11, 24.51, 23.67, 22.76, 22.62, 14.20.

[0219] intermediate P

[0220] Intermediate O (0.299 g, 0.75 mmol, 1 equivalent), 3-bromo-1-propanol (75.06 μL, 0.83 mmol, 1.1 equivalent), EDC·HCl (0.217 g, 1.13 mmol, 2 equivalent), DMAP (18.3 mg, 0.15 mmol, 0.2 equivalent), and DIPEA (261.3 μL, 1.5 mmol, 2 equivalent) were dissolved in dichloromethane (10 mL) and stirred overnight at ambient temperature. The reaction mixture was concentrated to a crude oil, which was then purified by column chromatography with a gradient of 0-60% ethyl acetate / hexane. Intermediate P (0.148 g, 38%) was isolated as a clear oil. 1 H NMR (500 MHz, cdcl3) δ 4.20 (t, J= 6.1 Hz, 2H), 3.45 (t, J = 6.6 Hz, 2H), 2.41 - 2.28 (m, 8H), 2.16 (p, J = 6.4 Hz, 2H), 1.61 (p, J = 7.5 Hz, 2H), 1.48 - 1.35 (m, 6H), 1.25 (s, 28H), 0.87 (t, J =6.8 Hz, 6H). 13 C NMR (126 MHz, cdcl3) δ 173.65, 62.09, 54.33, 53.84, 34.28,32.04, 31.84, 29.81, 29.78, 29.74, 29.48, 29.47, 27.78, 27.15, 26.77, 23.16,22.81, 14.24.

[0221] intermediate Q

[0222] Intermediate O (0.299 g, 0.75 mmol, 1 equivalent), 4-chloro-1-butanol (141.1 μL, 1.25 mmol, 1.2 equivalent, calculated at industrial reagent purity), EDC·HCl (0.290 g, 1.52 mmol, 1.5 equivalent), DMAP (25 mg, 0.21 mmol, 0.2 equivalent), and DIPEA (362 μL, 2.08 mmol, 2 equivalent) were dissolved in dichloromethane (10 mL) and stirred overnight at ambient temperature. The reaction mixture was concentrated to a crude oil, which was then purified by column chromatography with a gradient of 0-60% ethyl acetate / hexane. Intermediate Q (0.391 g, 77%) was isolated as a clear oil. 1 H NMR (400 MHz, CDCl3) δ 4.10 (t, J = 6.1 Hz, 2H), 3.56 (t, J = 6.2 Hz, 2H), 2.34 (m, 8H),1.89 - 1.74 (m, 5H), 1.61 (p, J = 7.4 Hz, 2H), 1.50 - 1.34 (m, 6H), 1.26 (m, 28H), 0.87 (t, J = 6.6 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 173.82, 63.52,54.36, 53.89, 44.56, 34.38, 32.05, 29.82, 29.79, 29.75, 29.48, 29.32, 27.79,27.19, 26.81, 26.25, 23.20, 22.82, 14.24.

[0223] Synthesis of the final compound Synthesis of compound I-1

[0224] Ethanolamine (9.17 μL, 0.15 mmol, 0.4 equivalents), intermediate B (0.150 g, 0.38 mmol, 1 equivalent), and 1,8-diazabicyclo(5.4.0)undec-7-ene (28.29 μL, 0.19 mmol, 0.5 equivalents) were dissolved in acetonitrile (0.5 mL) in a screw-cap vial and stirred at ambient temperature for 6 hours. The reaction mixture was concentrated and purified by column chromatography using a 10–40% dichloromethane gradient (dichloromethane solution of 22% MeOH and 3% NH4OH). The product (13 mg, 10%) was obtained as a clear oil. 1 H NMR (400 MHz, CDCl3) δ 4.13 (t, J = 6.2 Hz, 4H), 3.60 - 3.52 (m,2H), 2.78 (t, J = 6.8 Hz, 5H), 2.73 - 2.64 (m, 6H), 2.57 (t, J = 5.4 Hz, 2H),2.51 - 2.40 (m, 13H), 1.41 (p, J = 7.3 Hz, 9H), 1.34 - 1.17 (m, 70H), 0.86(t, J = 7.0 Hz, 14H). 13C NMR (101 MHz, CDCl3) δ 172.63, 59.27, 56.02, 54.79, 52.28, 49.21, 32.73, 32.02, 31.98, 29.79, 29.72, 29.64, 29.63, 29.45, 29.39, 27.57, 27.27, 27.08, 22.79, 22.77, 14.21, 14.20. MS (ESI+): m / z (MH+) 852.81, corresponding to C52H105N3O5.

[0225] Synthesis of compound I-2

[0226] Ethanolamine (12.1 μL, 0.20 mmol, 1 equivalent) and intermediate C (0.147 g, 0.36 mmol, 1.8 equivalent) were combined in a screw-cap vial and stirred at 70 °C for 8 hours. The reaction mixture was concentrated and purified by column chromatography using a 10–40% dichloromethane gradient (dichloromethane solution of 22% MeOH and 3% NH4OH). The product was separated to obtain a clear oily product (61 mg, 38% yield). 1 H NMR (400 MHz, CDCl3) δ 4.13 (t, J = 6.2 Hz, 4H), 3.60 -3.52 (m, 2H), 2.78 (t, J = 6.8 Hz, 5H), 2.73 - 2.64 (m, 6H), 2.57 (t, J = 5.4Hz, 2H), 2.51 - 2.40 (m, 13H), 1.41 (p, J = 7.3 Hz, 9H), 1.25 (d, J = 4.2 Hz, 70H), 0.86 (t, J = 7.0 Hz, 14H). 13C NMR (101 MHz, CDCl3) δ 172.63, 59.27, 56.02, 54.79, 52.28, 49.21, 32.73, 32.02, 31.98, 29.79, 29.72, 29.64, 29.63, 29.45, 29.39, 27.57, 27.27, 27.08, 22.79, 22.77, 14.21, 14.20. MS (ESI+): m / z (MH+) 880.84, corresponding to C54H109N3O5.

[0227] Synthesis of compound I-3

[0228] Compound 3 was synthesized using intermediate E according to general synthetic method C. The product was isolated as a clear oil (24.9 mg, 9% yield). 1 H NMR (500 MHz, cdcl3) δ 4.11 (t, J = 6.4 Hz, 4H), 3.53 (t,2H), 2.67 (t, J = 6.4 Hz, 4H), 2.58 (t, J = 5.4 Hz, 2H), 2.48 (t, J = 7.9 Hz, 4H), 2.43 (t, J = 8.1 Hz, 8H), 2.31 (t, J = 7.3 Hz, 3H), 1.76 (p, J = 7.3 Hz,4H), 1.46 - 1.35 (m, 8H), 1.32 - 1.20 (m, 56H), 0.87 (t, J = 6.9 Hz, 12H). 13 CNMR (126 MHz, cdcl3) δ 173.45, 62.72, 58.74, 55.71, 54.78, 52.80, 52.21, 31.91, 31.90, 29.67, 29.66, 29.64, 29.61, 29.60, 29.58, 29.33, 27.47, 27.16, 22.67, 22.21, 14.10. MS (ESI+): m / z (MH+) 880.84, corresponding to C54H109N3O5.

[0229] Synthesis of compound I-4

[0230] Compound 4 was synthesized using intermediate F according to general synthetic method C. The product (22 mg, 13%) was isolated as a clear oil. 1 H NMR (500 MHz, cdcl3) δ 4.10 (t, J = 6.5 Hz, 4H), 3.79 (t, J = 5.4Hz, 1H), 3.53 (t, J = 5.3 Hz, 2H), 2.70 (t, J = 5.7 Hz, 1H), 2.58 (t, J = 5.4Hz, 3H), 2.57 - 2.52 (m, 3H), 2.51 - 2.41 (m, 13H), 2.30 (t, J = 7.2 Hz, 4H),1.85 - 1.80 (m, 3H), 1.76 (p, J = 7.2 Hz, 5H), 1.72 - 1.67 (m, 1H), 1.45 (m, 9H), 1.34 - 1.19 (m, 72H), 0.95 - 0.77 (m, 15H). 13 C NMR (126 MHz, cdcl3) δ 173.56, 62.97, 58.86, 55.88, 53.99, 52.97, 50.57, 32.03, 32.01, 29.77, 29.71, 29.69, 29.68, 29.64, 29.45, 29.43, 27.59, 27.54, 22.80, 22.38, 14.24. MS (ESI+): m / z (MH+) 908.87, corresponding to C56H113N3O5.

[0231] Synthesis of compound I-5

[0232] Compound 5 was synthesized using intermediate H according to general synthetic method C. The product was isolated as a clear oil (78 mg, 18% yield). 1 H NMR (500 MHz, cdcl3) δ 4.08 (t, J = 6.6 Hz, 4H), 3.53 (t,J = 5.4 Hz, 2H), 2.58 (t, J = 5.4 Hz, 2H), 2.49 (t, J = 7.1 Hz, 5H), 2.46 -2.36 (m, 10H), 2.31 (t, J = 7.2 Hz, 4H), 1.76 (p, J = 7.3 Hz, 4H), 1.63 (p, J = 6.8 Hz, 4H), 1.51 (p, J = 8.2 Hz, 5H), 1.43 (p, J = 6.9 Hz, 8H), 1.34 -1.17 (m, 56H), 0.87 (t, J = 6.8 Hz, 12H). 13 C NMR (126 MHz, cdcl3) δ 173.71, 64.55, 58.92, 55.90, 54.16, 53.77, 53.01, 32.10, 32.05, 29.81, 29.77, 29.75, 29.74, 29.48, 27.74, 26.82, 22.83, 22.43, 14.26. MS (ESI+): m / z (MH+) 936.91, corresponding to C58H117N3O5.

[0233] Synthesis of compound I-6

[0234] Compound 6 was synthesized using intermediate I according to general synthetic method C. The product was isolated as a clear oil (36 mg, 8% yield). 1 H NMR (500 MHz, cdcl3) δ 4.10 (t, J = 6.5 Hz, 5H), 3.52 (t, J =5.4 Hz, 2H), 2.57 (t, J = 5.4 Hz, 2H), 2.48 (dt, J = 14.9, 7.4 Hz, 9H), 2.40(t, J = 7.6 Hz, 9H), 2.30 (t, J = 7.4 Hz, 5H), 1.77 (p, J= 6.7 Hz, 5H), 1.61(p, J = 7.4 Hz, 4H), 1.52 - 1.36 (m, 14H), 1.34 - 1.16 (m, 56H), 0.95 - 0.73 (m, 11H). 13 C NMR (126 MHz, cdcl3) δ 173.62, 63.01, 58.59, 55.69, 54.18, 53.65, 50.60, 34.20, 32.04, 29.80, 29.76, 29.75, 29.74, 29.47, 27.67, 26.97, 26.81, 26.38, 22.91, 22.82, 14.25. MS (ESI+): m / z (MH+) 936.90, corresponding to C58H117N3O5.

[0235] Synthesis of compound I-7

[0236] Compound 7 was synthesized using intermediate J according to general synthetic method C. The product was isolated as a clear oil (100 mg, 24% yield). 1 H NMR (500 MHz, cdcl3) δ 4.05 (t, J = 6.7 Hz, 4H), 3.51 (t, J = 5.4 Hz, 2H), 2.54 (t, J = 5.4 Hz, 2H), 2.47 - 2.34 (m, 16H), 2.29 (t, J =7.4 Hz, 4H), 1.66 - 1.55 (m, 8H), 1.52 - 1.35 (m, 16H), 1.27 - 1.22 (m, 46H), 0.93 - 0.80 (m, 12H). 13C NMR (126 MHz, cdcl3) δ 173.56, 64.33, 58.51, 55.56, 54.07, 53.65, 53.52, 50.54, 34.06, 31.88, 29.64, 29.60, 29.57, 29.56, 29.31, 27.60, 26.79, 26.69, 26.67, 23.48, 22.76, 22.65, 14.08. MS (ESI+): m / z (MH+) 964.94, corresponding to C60H121N3O5.

[0237] Synthesis of compound I-8

[0238] Intermediate K (0.032 g, 0.11 mmol, 1 equivalent), intermediate L (0.105 g, 0.27 mmol, 2.5 equivalent), and EDC were used. HCl (0.063 g, 0.33 mmol, 3 equivalents), DMAP (0.005 g, 0.04 mmol, 0.4 equivalents), and DIPEA (113 μL, 0.65 mmol, 6 equivalents) were dissolved in DCM (5 mL) and stirred at ambient temperature for 18 hours. The reactants were then concentrated to a crude oil and purified by column chromatography using a 10–30% dichloromethane gradient (dichloromethane solution of 22% MeOH and 3% NH4OH). A clear oily product ((2-((tert-butyldimethylsilyl)oxy)ethyl)azadiyl)bis(propane-3,1-diyl)bis(4-(decylamino)butyrate) (0.143 mg, 130% yield, with residual solvent impurities) was obtained. 1 HNMR (500 MHz, cdcl3) δ 4.09 (t, J = 6.6 Hz, 4H), 3.62 (t, J = 6.5 Hz, 2H),2.66 - 2.42 (m, 18H), 2.33 (t, J = 7.2 Hz, 4H), 1.85 - 1.77 (m, 4H), 1.73 (p, J = 7.1 Hz, 5H), 1.53 - 1.44 (m, 7H), 1.33 - 1.17 (m, 64H), 0.91 - 0.83 (m, 23H), 0.04 (s, 6H). 13C NMR (126 MHz, cdcl3) δ 173.58, 62.86, 61.91, 56.29, 53.87, 53.55, 51.42, 32.02, 29.75, 29.72, 29.70, 29.64, 29.44, 27.56, 26.95, 26.05, 22.80, 18.40, 14.23, -5.21. MS (ESI+): m / z (MH+) 1022.96, corresponding to C62H127N3O5Si.

[0239] ((2-((tert-butyldimethylsilyl)oxy)ethyl)azadiyl)bis(propane-3,1-diyl)bis(4-(decylamino)butyrate) (0.120 g, 0.11 mmol, 1 equivalent) and a 1 M tetrabutylammonium fluoride THF solution (440 μL, 0.44 mmol, 4 equivalents) were dissolved in diethyl ether (5 mL) and stirred at ambient temperature for 18 hours. The reaction mixture was transferred to a separatory funnel and diluted with dichloromethane (5 mL). The organic layer was washed with saturated NH4OH solution containing 10% NaOH (4 × 15 mL), followed by washing with brine (1 × 50 mL), and then dried over anhydrous salt. The organic fraction was concentrated to a crude oil and purified by column chromatography using a 10–30% dichloromethane gradient (22% MeOH, 3% NH4OH in dichloromethane solution). Compound 8 (57 mg, 57%) was isolated as a clear oil. 1 H NMR (500 MHz, cdcl3) δ 4.10 (t, J = 6.4Hz, 4H), 3.54 (dd, J = 5.7, 4.9 Hz, 2H), 2.58 (t, J = 5.2 Hz, 2H), 2.53 (t, J = 7.5 Hz, 4H), 2.41 (t, J = 7.3 Hz, 4H), 2.39 - 2.35 (m, 8H), 2.31 (t, J =7.4 Hz, 4H), 1.83 - 1.66 (m, 8H), 1.49 - 1.35 (m, 11H), 1.33 - 1.19 (m, 64H), 0.91 (t, J = 7.3 Hz, 4H), 0.87 (t, J = 6.9 Hz, 12H). 13C NMR (126 MHz, cdcl3) δ 173.73, 62.28, 58.75, 55.82, 54.06, 53.68, 53.20, 50.31, 32.07, 31.90, 29.67, 29.63, 29.60, 29.33, 27.57, 27.01, 26.42, 22.67, 22.45, 20.71, 14.10, 14.02. MS (ESI+): m / z (MH+) 908.87, corresponding to C56H113N3O5.

[0240] Synthesis of compound I-9

[0241] Compound 9 was synthesized using intermediate M according to general synthetic method C. The product was isolated as a clear oil (59 mg, 10%). 1 H NMR (500 MHz, cdcl3) δ 4.04 (t, J = 6.6 Hz, 4H), 3.52 (t, J = 5.5Hz, 2H), 2.59 - 2.44 (m, 17H), 2.33 (t, J = 7.2 Hz, 4H), 1.81 (p, J = 7.3 Hz, 3H), 1.60 (p, J = 6.7 Hz, 5H), 1.53 - 1.43 (m, 11H), 1.31 - 1.16 (m, 58H), 0.85 (t, J = 6.9 Hz, 12H). 13 C NMR (126 MHz, cdcl3) δ 173.48, 64.29, 58.55,55.62, 53.73, 53.57, 52.95, 52.87, 31.96, 31.92, 31.86, 29.68, 29.65, 29.63,29.61, 29.55, 29.50, 29.45, 29.38, 29.37, 29.32, 29.31, 27.47, 27.44, 26.59,26.10, 23.68, 22.74, 21.58, 14.18. MS (ESI+): m / z (MH+) 936.90, corresponding to C58H117N3O5.

[0242] Synthesis of compound I-10

[0243] Compound 10 was synthesized using intermediate P according to general synthetic method C. The product was isolated as a clear oil (38 mg, 43%). 1 H NMR (500 MHz, cdcl3) δ 4.10 (t, J = 6.4 Hz, 4H), 3.53 (t, J = 5.4Hz, 2H), 2.57 (t, J = 5.4 Hz, 2H), 2.53 (t, J = 7.0 Hz, 3H), 2.40 - 2.33 (m,12H), 2.30 (t, J = 7.5 Hz, 4H), 1.76 (p, J = 6.6 Hz, 4H), 1.59 (p, J = 7.3Hz, 4H), 1.49 - 1.34 (m, 13H), 1.32 - 1.18 (m, 58H), 0.86 (t, J = 7.0 Hz, 12H). 13 C NMR (126 MHz, cdcl3) δ 173.76, 62.41, 58.93, 55.98, 54.26, 53.87, 53.52, 50.45, 34.32, 32.02, 29.79, 29.76, 29.72, 29.45, 27.77, 27.02, 26.77, 26.54, 23.16, 22.79, 14.22. MS (ESI+): m / z (MH+) 936.91, corresponding to C58H117N3O5.

[0244] Synthesis of compound I-11

[0245] Compound 11 was synthesized using intermediate Q according to general synthetic method C. The product was isolated as a clear oil (43 mg, 12%). 1 H NMR (500 MHz, cdcl3) δ 4.05 (t, J = 6.6 Hz, 4H), 3.52 (t, J = 5.5Hz, 2H), 2.56 (t, J= 5.5 Hz, 2H), 2.46 (t, J = 7.3 Hz, 4H), 2.41 - 2.33 (m,12H), 2.30 (t, J = 7.5 Hz, 4H), 1.65 - 1.55 (m, 8H), 1.52 - 1.35 (m, 16H), 1.24 (m, 58H), 0.91 - 0.81 (m, 12H). 13 C NMR (126 MHz, cdcl3) δ 173.84, 64.17, 58.66, 55.68, 54.26, 53.85, 53.61, 34.35, 32.02, 29.79, 29.76, 29.72, 29.45, 27.76, 27.02, 26.71, 26.68, 23.78, 23.17, 22.80, 14.23. MS (ESI+): m / z (MH+) 964.93, corresponding to C60H121N3O5.

[0246] Synthesis of compound I-12

[0247] 2-Amino-1-ethanol (0.916 g, 15 mmol, 1 equivalent) was added to a mixture of imidazole (2.042 g, 30 mmol, 2 equivalents) and tert-butyldimethylchlorosilane (2.487 g, 16.5 mmol, 1.1 equivalents) in dichloromethane (50 mL), and stirred at room temperature for 3 hours. The mixture was then diluted with water (60 mL) and extracted with dichloromethane (3 × 30 mL). The organic fraction was washed with brine (1 × 50 mL) and dried over anhydrous salt. The organic fraction was concentrated under vacuum to give 2-((tert-butyldimethylsilyl)oxy)ethyl-1-amine (2.96 g, 112%), which was ready for use without further purification. 1 H NMR (400 MHz, CDCl3) δ 3.58 (t, J = 5.3 Hz, 2H), 2.73 (t, 2H), 0.86 (s, 9H), 0.02 (s, 6H).

[0248] 2-((tert-butyldimethylsilyl)oxy)ethyl-1-amine (2.96 g, 16.9 mmol, 1 equivalent) was combined with methyl acrylate (3.87 g, 45 mmol, 3 equivalents) in methanol (10 mL). The mixture was purged with nitrogen, covered with aluminum foil, and stirred at room temperature for 3 days. The consumption of 2-amino-1-ethanol was monitored by TLC (20% MeOH / DCM). The solvent and excess methyl acrylate were removed under vacuum to give intermediate R (3.72 g, 71%), a clear, colorless oil. 1 H NMR (400 MHz, CDCl3) δ 3.59 (s, 7H), 2.76 (t, J = 7.2 Hz, 4H), 2.53 (t, J = 6.4 Hz, 2H), 2.38 (d, J = 14.3 Hz, 2H), 0.82 (s, 9H), -0.02 (s, 6H).

[0249]

[0250] Ethylenediamine (3.01 g, 50 mmol, 10 equivalents) and intermediate R (1.74 g, 5 mmol, 1 equivalent) were added together to methanol (10 mL). The mixture was purged with nitrogen, covered with aluminum foil, and stirred at room temperature for 3 days. The consumption of intermediate R was monitored by TLC (10% MeOH / DCM). The solvent and excess ethylenediamine were removed under reduced pressure. Residual ethylenediamine was removed by continuous washing with diethyl ether (5 x 30 mL). The residual diethyl ether was removed under vacuum to give intermediate S (0.510 g, 1.26 mmol, 25%), a clear, colorless, viscous oil. 1 H NMR (500 MHz, CDCl3) δ 7.57 (t, J = 5.8 Hz, 2H), 3.64 (t, J = 5.9 Hz, 2H), 3.24 (q, J = 5.8 Hz, 4H), 2.80 - 2.73 (m, 8H), 2.58(t, J = 5.9 Hz, 2H), 2.32 (t, J = 6.3 Hz, 4H), 0.84 (s, 9H), 0.01 (s, 6H). 13CNMR (500 MHz, CDCl3) δ 172.87, 61.11, 55.68, 50.67, 50.10, 41.83, 41.36, 34.09, 25.87, 18.25, -5.35.

[0251]

[0252] A mixture of 1-bromodecane (0.274 g, 1.239 mmol, 5 equivalents), DIPEA (0.216 mL, 1.239 mmol, 5 equivalents), and intermediate S (0.100 g, 0.248 mmol, 1 equivalent) was dissolved together in ethanol (3 mL) in an 8-fold screw-cap vial. The mixture was covered with aluminum foil and stirred overnight at 65 °C. The crude oil was purified by rapid chromatography using a 25 g silica gel column with a solvent gradient from 10% ULTRA / DCM to 40% ULTRA / DCM. The desired fractions were combined and concentrated under vacuum to give intermediate T (0.0675 g, 0.700 mmol, 28%), a pale yellow, clear, viscous oil. 1 H NMR (500 MHz, CDCl3) δ 7.12 (s, 2H), 3.66 (q, J = 5.9 Hz, 2H), 3.29 (p, J = 6.3 Hz, 4H), 2.80 (t, J = 6.7 Hz, 4H), 2.65 - 2.55 (m, 6H), 2.47 (t, J = 7.7 Hz, 7H), 2.32(t, J = 6.6 Hz, 4H), 1.43 (p, J = 7.3 Hz, 8H), 1.32 - 1.17 (m, 53H), 0.90 -0.82 (m, 23H), 0.03 (t, J = 1.4 Hz, 6H). 13 C NMR (500 MHz, CDCl3) δ 172.31,61.24, 53.97, 52.96, 50.70, 50.48, 33.91, 32.01, 32.00, 31.98, 29.80, 29.71,29.69, 29.44, 27.61, 26.02, 26.01, 22.78, 18.35, 14.21, -5.21.

[0253]

[0254] Intermediate T was dissolved in diethyl ether (50 mL) cooled to 0ºC, and a solution of tetrabutylammonium fluoride (TBAF) was added. The reaction mixture was brought to room temperature, covered with aluminum foil, and stirred for 18 hours. The consumption of the TBS-protected substance was monitored by TLC (35% ULTRA / DCM). The organic fraction was washed with saturated ammonium chloride (5 x 15 mL) containing 20% ​​1M sodium hydroxide and brine (1 x 50 mL), and then dried over anhydrous salt. The organic fraction was concentrated to a crude oil and purified by rapid chromatography using a 50 g silica gel column with a solvent gradient from 20% ULTRA / DCM to 50% ULTRA / DCM. The desired fraction was collected and concentrated under vacuum to give a clear, colorless, viscous oil (0.0270 g, 0.0317 mmol, 45%). 1 H NMR (500 MHz, CDCl3)δ 7.17 (s, 2H), 3.60 (t, J = 4.9 Hz, 1H), 3.28 (q, J = 5.7 Hz, 4H), 2.76 (t, J = 6.3 Hz, 4H), 2.60 - 2.52 (m, 5H), 2.44 (t, J = 7.5 Hz, 5H), 2.34 (t, J =6.3 Hz, 4H), 1.42 (p, J = 7.7 Hz, 7H), 1.38 - 1.17 (m, 52H), 0.92 (t, J = 7.4Hz, 2H), 0.87 (t, J = 7.1 Hz, 12H). 13 C NMR (500 MHz, CDCl3) δ 172.36, 59.16, 56.24, 53.89, 53.18, 50.08, 36.91, 33.96, 32.05, 29.84, 29.77, 29.74, 29.70, 29.48, 27.74, 26.32, 22.82, 20.80, 14.25, 14.12. MS (ESI+) m / z: [M+H]+, corresponding to C 52 H 107 N5O3, calculated value: 850.84; measured value: 850.84.

[0255] Example 2: mRNA-LNP reagents and characterization A. mRNA synthesis Transcription was performed on a linearized DNA template (Genscript USA, NJ, USA) containing a 101-nucleotide (nt) poly(A) tail using the HiScribe T7 mRNA kit containing CleanCap Reagent AG (New England Biolabs, MA, USA). In vitro transcription was performed using n1-methylpseudouridine (Trilink Biotechnologies, CA, USA.) to completely replace uridine. All mRNAs were purified by silica gel column chromatography followed by cellulose purification to remove dsRNA impurities (Baiersdörfer, M. et al., A Facile Method for the Removal of dsRNA Contaminants from...). In Vitro -Transcribed mRNA. Mol Ther - Nucleic Acids 15, 2019. The length and integrity of the mRNA samples were validated using the Agilent TapeStation system (Agilent Technologies, CA, USA) at the Center for Applied Genomics (Toronto, ON, CA). The A260 / A280 and A260 / A230 ratios were greater than 1.9 and 2.0, respectively.

[0256] Table 1. mRNA sequences

[0257] B. mRNA-LNP reagent The lipid components were mixed in ethanol at a specific molar ratio (50 mol% of the compound of this application or the compound in Table 2, 38.5 mol% cholesterol (VWR 97061-660), 10 mol% 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC) (VWR TCD3926) and 1.5 mol% 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (PEG-DMG) (Avanti PolarLipids 880151)) to a final lipid concentration of 16 mg / ml. SM-102, ALC-0315, and DLin-MC3-DMA were purchased from Cayman Chemicals. Compound I-12 was synthesized as previously described (Tilstra, G. et al., Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery. J Am Chem Soc, 2023, the entire contents of which are incorporated herein by reference). Messenger RNA was diluted to 0.27 mg / ml in 25 mM sodium acetate (pH 4.5). Formulations were prepared at a total lipid to mRNA weight ratio of 20:1, with 0.1 mg of mRNA and 2 mg of lipid per batch. Therefore, the molar ratio of the tertiary amine to mRNA phosphate (also known as the N / P ratio) of the compound varied between different formulations. The lipid phase and mRNA phase were rapidly injected into a chevron-shaped micromixer at flow rates of 4.5 ml / min and 1.5 ml / min, respectively, using a syringe pump. The prepared mRNA was directly collected into a dialysis unit (Thermo Scientific 66383) with a molecular weight cutoff of 10 kDa. The formulation was dialyzed twice at 4°C with 1000 volumes of 20 mM Tris-HCl (pH 7.5) solution. The first dialyze lasted 4 hours, followed by overnight dialyze. The obtained LNP suspension was removed from the dialysis apparatus and filtered through a 0.2 μm polyethersulfone syringe filter (Cytiva 99161302). The LNP solution was diluted with concentrated sucrose to a final concentration of 20 mM Tris-HCl and 8% sucrose. The LNP reagent in Tris-sucrose buffer was stored at a concentration of 0.03 to 0.05 mg / ml mRNA in 1.5 ml polypropylene tubes (Eppendorf 022431021) at -80°C. Aliquots were thawed overnight at 4°C and diluted with 20 mM Tris-HCl / 8% sucrose.

[0258] Table 2. Exemplary lipid compounds

[0259] Table 3. LNP formulations

[0260] C. mRNA-LNP characterization Size distribution and zeta potential The nanoparticle size (expressed as Z-mean hydrodynamic diameter), polydispersity index (PDI), and zeta potential (ZP) of LNP diluted to 2 ng / μl in 20 mM Tris-HCl pH 7.5 were determined using a Zetasizer (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

[0261] mRNA concentration and encapsulation efficiency For LNPs encapsulating mRNA, mRNA concentration and encapsulation efficiency were determined using a modified Quant-iT RiboGreen assay (Invitrogen). This assay was performed three times in 96-well plates. 12 μL of LNP sample or TE buffer (composed of 10 mM Tris-HCl containing 1 mM EDTA•Na2) (blank sample) was diluted with 300 μL of 1X TE buffer at pH 7.5. 50 μL of the diluted sample was transferred to six independent wells, and then 50 μL of TE buffer or 50 μL of TE buffer containing 4% Triton X-100 solution was added to each well. Two 6-point standard curves (5 mg / ml to 0.156 g / ml) were prepared on the same plate by diluting firefly luciferase mRNA to a final volume of 100 μL in 2% Triton X-100 or TE buffer. The plate was then incubated with gentle shaking at 40°C for 10 minutes. Dilute Ribogreen reagent 1:100 in TE buffer and add 100 μL of this solution to each well. Immediately transfer 180 μL of this mixture to a black 96-well plate. Incubate the plate in the dark with gentle shaking for 5 minutes. Measure the fluorescence intensity using a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 525 nm. Subtract the background fluorescence (blank sample) from the original fluorescence value. The concentrations of free mRNA and total mRNA were determined by linear interpolation using an appropriate standard curve. Encapsulation efficiency was determined by dividing the calculated encapsulated mRNA concentration by the total mRNA concentration in the LNP sample.

[0262] Apparent pK a Apparent pK of LNPs was determined using the TNS assay. aThe determination was performed. In short, a series of buffers were prepared by titrating a solution containing 10 mM citrate, 10 mM phosphate, 10 mM borate, and 150 mM NaCl with 1.0 M HCl. Aliquots of this combined buffer were taken to generate a total of 12 buffers, ranging in pH from 4.0 to 9.5 in increments of 0.5 pH. LNP (diluted in distilled water to a concentration of 100 μM ionizable lipids) and 6-(p-toluidine)-2-naphthalenesulfonic acid (TNS, Sigma Aldrich, 60 μM in distilled water) were diluted into these buffers to final concentrations of 9 µM and 5.45 µM, respectively. The plates were capped and equilibrated at room temperature for 20 minutes. Fluorescence intensity (325 nm excitation / 435 nm emission) was measured using a BioTek H1 Synergy microplate reader. The raw fluorescence values ​​were fitted using a GraphPad Prism 8 to determine pK. a This corresponds to the pH value at which 50% of the maximum fluorescence occurs.

[0263] lipid pK a Calculate the pK of lipid molecules using MarvinSketch version 22.22 a (that is, the pK) a (Related to lipid molecules, not to formulated LNPs). Import lipid structures into MarvinSketch and use the built-in pK... a Functional determination of the percentage of ionization of all amines in the pH range of 0-14 (calculation > protonation > pK) a Use the following settings: Mode=Macro, Acid / Base Prefix=Static, Minimum Basicity pK a =-2, maximum acidity pK a =16, Temperature (K) = 298, Correction Library = Selected, Consider Tautomerism / Resonance = Selected, Show Distribution Map = Selected). Output data for the percentage of ionization of each lipid relative to pH were fitted using GraphPad Prism 8 to determine the lipid pK. a This corresponds to the pH value at which ionization is 50%.

[0264] Characterization of LNPs before freezing As shown in Table 4, the choice of compounds significantly affects size, PDI, mRNA encapsulation efficiency, and apparent pK. a The type of degradable functional groups also significantly affects the properties of nanoparticles. Replacing amides (compound I-12) directly with esters (compound I-1) while maintaining the same number of carbon atoms in the linker significantly reduced mRNA encapsulation efficiency and apparent pK. aCompounds I-3, I-5, I-6, and I-7 exhibited high efficiency (>90%) in encapsulating formulated mRNA. Compounds I-4, I-5, I-6, I-7, and I-8 possessed apparent pK values ​​of 6 to 7. a This range has been repeatedly demonstrated to be the optimal range for in vivo potency and tolerability of ionizable LNPs (Jayaraman, M. et al., Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing). In Vivo Angewandte Chemie Int Ed 51, 8529-8533 (2012). Hassett, KJ et al., Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol Ther - Nucleic Acids 15, 1-11 (2019). Tilstra, G. et al., Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery. J Am Chem Soc (2023). The number of carbon atoms on both sides of the esters of compounds I-1, I-2, I-3, I-4, I-5, I-6, and I-7 and their apparent pK a Directly related ( Figure 1 Increasing the number of carbon atoms between the ester and the tertiary amine may increase the pK of the ionizable compound. a .

[0265] Table 4. Characterization of LNPs before freezing

[0266] Table 5. Characterization of LNP after freeze-thaw cycle

[0267] Example 3: Overview of intramuscular expression in mice All procedures used in animal studies conducted at the University of Toronto were approved by the Institutional Animal Care and Use Committee (IACUC) under protocol number 20012621 and complied with all applicable local, provincial, and federal regulations. Female BALB / c mice (7–8 weeks old) were purchased from Charles River Laboratories and acclimatized for 1–2 weeks prior to the start of the studies.

[0268] Mice (n=4) were injected bilaterally into the quadriceps femoris muscle with 50 μL of 0.5 μg of firefly luciferase mRNA. At different time points (6, 24, 48, and 96 hours post-administration), mice were intraperitoneally injected with 200 μL of 1X PBS containing 3 mg D-luciferin (Thermo Scientific 88291). Five minutes later, the mice were anesthetized, and in vivo imaging (PerkinElmer, IVIS Spectrum) was used to measure the total bioluminescent flux (photons / second) of each hind limb. Figure 2 The area under the curve (AUC) of each hind limb was determined using GraphPad Prism 9.

[0269] result The ability of LNPs formulated with the compounds of this disclosure to functionally deliver firefly luciferase mRNA to muscle was tested and compared with compounds I-12, DLin-MC3-DMA, ALC-0315, and SM-102. Luminescent flux was measured to assess firefly luciferase protein expression. All compounds exhibited similar expression kinetics, peaking at 6 hours and rapidly declining within 96 hours. Figure 3 Replacing the amide (compound I-12) with an ester (compound I-1) significantly reduced intramuscular expression. Intramuscular expression of compounds I-3, I-4, I-5, I-6, I-7, and I-8 was higher than that of DLin-MC3-DMA. Among all the compounds tested, compound I-6 had the highest intramuscular expression. Figure 4 ).

[0270] Table 6. Total Flux AUC

[0271] Example 4: In vitro transfection of THP-1 monocytes THP-1 monocytes are human cells isolated from the peripheral blood of patients with acute monocytic leukemia. THP-1 monocytes were cultured in RPMI-1640 (ATCC modified) containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells were seeded at a density of 100,000 cells per well in 96-well plates and transfected quadruplicates with 500 ng / well of firefly luciferase mRNA formulated with lipid nanoparticles. After 24 hours, cell viability and firefly luciferase expression were measured using a microplate reader according to the manufacturer's protocol (Promega E7110).

[0272] The ability of LNPs formulated with the compounds of this disclosure to functionally deliver firefly luciferase mRNA to THP-1 monocytes was tested and compared with compounds I-12, DLin-MC3-DMA, ALC-0315, and SM-102. Compounds I-1 and I-3 caused significant cell death. Interestingly, replacing the amide (compound I-12) with an ester (compound I-1) increased toxicity. Luminescence (expression) was strongly correlated with cell viability. The expression of compounds I-4, I-5, I-6, I-7, and I-8 was similar to that of DLin-MC3-DMA and ALC-0315, but inferior to that of SM-102.

[0273] Example 5: Organ expression after intravenous injection in mice All procedures used in animal studies conducted at the University of Toronto were approved by the Institutional Animal Care and Use Committee (IACUC) under protocol number 20012621 and complied with all applicable local, provincial, and federal regulations. Female BALB / c mice (7–8 weeks old) were purchased from Charles River Laboratories and acclimatized for 1–2 weeks prior to the start of the studies.

[0274] Mice (n=3) were intravenously (tail vein) injected with 0.5 μg of firefly luciferase mRNA in 200 μL of solution. Six hours after administration, mice were intraperitoneally injected with 200 μL of 1X PBS containing 3 mg D-luciferin (Thermo Scientific 88291). Five minutes later, the mice were sacrificed, and major organs (liver, spleen, kidney, lung, and heart) were collected and immediately imaged using an in vivo imaging system (PerkinElmer, IVIS Spectrum) to measure total bioluminescence flux (photons / second). The ability of LNPs formulated with compounds I-2, I-4, I-5, I-6, and I-8 to functionally deliver firefly luciferase mRNA to major organs (liver, spleen, lung, heart, and kidney) after intravenous injection was tested and compared with compounds I-12 and SM-102.

[0275] For compounds I-2, I-4, I-8, and SM-102, the highest total flux was measured in the liver. For compounds I-5 and I-12, the highest total flux was measured in the spleen. Although luminescence was also detected in the lungs, heart, and kidneys, it accounted for less than 1% of the total luminescent flux in all organs. Among the compounds relevant to this disclosure, compound I-6 was expressed most highly in all organs. Interestingly, compound I-6 was expressed more in the spleen than SM-102, but this was not the case in the liver. The expression fractions of compounds I-2, I-4, I-5, I-6, and I-8 were all higher in the spleen than in SM-102. In summary, these results indicate that the relative performance of the compounds depends on the route of administration.

[0276] Example 6: In vitro degradation and pharmacokinetics in mice method The alkaline hydrolysis rate of the ester was determined by time-series 1H NMR. In a 3 mm NMR tube, ionizable lipids (2 μmol) containing the compounds described herein were hydrolyzed with 2.5 equivalents of KOH (200 μL, 0.025 M d4-methanol solution). 1H-NMR was acquired every 5 minutes for a total of 180 minutes using a 700 MHz Agilent DD2 spectrometer. Additionally, spectra of I-12, ALC-0315, and DLin-MC3-DMA were acquired at 24, 48, 72, 144, 192, and 240 hours. Data acquisition began approximately 10 minutes after the reaction started due to sample preparation and tuning / locking / shimming. Ester peaks were integrated using MestReNova and normalized to values ​​at t=0 minutes. The data sequences were fitted to a single exponential decay model following y = e -kt , where k is the hydrolysis rate.

[0277] Pharmacokinetics of compounds I-12 and I-6 after intramuscular injection of mRNA-LNP in mice Pharmacokinetic studies in mice were conducted according to Transpharmation Canada Ltd.'s standard operating procedures (SOPs) and recognized scientific practices, but not in accordance with Good Laboratory Practice (GLP) principles. Two studies were performed, and the results were combined for analysis. The aim was to determine the concentrations of each lipid in plasma, muscle, liver, spleen, urine, and feces following intramuscular (IM) administration of two ionizable lipids (compound I-12 and compound I-6) to female CD1 mice. All animals received an IM injection of one lipid formulated as a lipid nanoparticle (LNP) containing mRNA into the right quadriceps femoris muscle. The IM administration doses were 12.312 µg of compound I-12 and 12.764 µg of compound I-6, corresponding to 0.5 µg of mRNA formulated as LNPs. In Study 1, animals were housed in metabolic cages (n=3 per cage) and sacrificed at six time points (3, 6, 12, 24, 48, and 96 hours) within four days post-administration to collect blood, liver, spleen, and muscle samples (n=3 per time point). Additionally, a combined urine and fecal sample was collected from each cage at each appropriate time point. In Study 2, animals were housed in shoebox cages, and samples were collected at 6, 12, 24, 48, 96, and 168 hours post-administration (compounds I-12 and I-6) and on day 21 (I-12 only, n=3) (n=4 per time point). A combined fecal sample was collected from each cage upon cage change and weighed after terminal collection. Blood samples were aliquoted for bioanalytical and diagnostic testing. Data from both studies were combined for analysis. A tandem liquid chromatography-mass spectrometry (LC-MS / MS) method was established for the quantification of compounds I-12 and I-6 in mouse plasma, muscle, liver, spleen, urine, and feces. The validated method was used to determine the concentration of the analytes in each matrix. Pharmacokinetic (PK) parameters of compounds I-12 and I-6 were estimated using Phoenix® WinNonlin 8.3 software (Certara, Mountainview, CA) based on plasma, muscle, liver, and spleen concentration-time curves for each animal.

[0278] result To quantify the in vitro ester hydrolysis rate, the alkaline-catalyzed hydrolysis of ionizable lipids was monitored by time-series 1H NMR. Compounds I-1 to I-8 were subjected to this method. It was found that all novel ester compounds and SM-102 were completely degraded within 180 minutes. Figure 7A). The relative degradation rate depends on the distance between the ester and the tertiary amine. Furthermore, I-12, DLin-MC3-DMA, and ALC-0315 were found to degrade much more slowly, requiring 10 days to reach measurable hydrolysis levels. This data indicates that amides degrade significantly slower than their ester analogs, and that electron-withdrawing groups near the ester increase the degradation rate, while sterically hindered groups decrease it.

[0279] Following intramuscular injection of lipid-containing LNP, the plasma concentration of compound I-12 peaked 6 hours post-administration, with the corresponding maximum plasma concentration (C0) reaching its peak value. max The concentration was 348 ng / mL, while the plasma concentration of compound I-6 reached its peak earlier, 3 hours after administration. max The estimated apparent half-life (t) of compound I-12 in plasma is 108 ng / mL. 1 / 2 The sampling interval was 295 hours, but because the terminal sampling interval was less than 2×t 1 / 2 This estimate is considered inaccurate. The estimated value for compound I-6 is t... 1 / 2 Much shorter, at 15.3 hours. Total plasma exposure to compound I-12 (AUC) 0-tlast (13772 h) (ng / mL) compared to the total plasma exposure of compound I-6 (1033 h) (ng / mL) approximately 13 times.

[0280] Total tissue exposure to compound I-12 (AUC0-t) last The AUC values ​​of compound I-12 in muscle, liver, and spleen were 3244 µg / g, 694 µg / g, and 391 µg / g, respectively, with tissue / plasma AUC ratios of 236, 50, and 28, respectively. The tissue AUC values ​​of compound I-6 in muscle, liver, and spleen were 994 µg / g, 47.0 µg / g, and 61.1 µg / g, respectively, with tissue / plasma AUC ratios of 962, 46, and 59, respectively. At 21 days post-administration, approximately 18% of compound I-12 was excreted in feces, while a low percentage of compound I-6, varying from 0.1% to 3%, was excreted in feces. Neither compound I-12 nor compound I-6 was detected in urine.

[0281] in conclusion Novel polyamine-ionizable lipid molecules were synthesized to create a new class of delivery materials. The spatial arrangement of the amine, ester, and hydrophobic regions of these materials has been shown to significantly influence the function of the LNPs formed from them. The orientation of the ester and the distance between heteroatoms (including the amine and hydrophobic regions) induce dramatic changes in activity. These changes lead to alterations in steric hindrance, thereby affecting supramolecular assembly formation, charge shielding, inductive effects, and the structure of degradation products. Based on these insights, a new class of materials—heteroatom-tuned supramolecular ionizable lipids—was created to maximize performance and significantly improve upon current state-of-the-art technologies.

[0282] Example 7: Cellular uptake and Internal escape method Cellular uptake of mRNA-LNP Cellular uptake of mRNA-LNP was quantified by measuring the amount of mRNA in cells. SKOV3 cells were treated with mRNA-LNP formulations prepared with compounds I-6, I-12, or SM-102, respectively, to determine the cellular uptake of each formulation. Cells were seeded at a density of 80,000 cells per well on poly-D-lysine-treated coverslips in 500 μL of RPMI medium in 24-well plates. Cells were allowed to grow for 18 hours. The next day, the medium was replaced with 500 μL of fresh medium containing 0.5 μg / well of firefly luciferase mRNA-LNP prepared with specified ionizable lipids; cells were transfected for 1, 3, or 6 hours (n=3 replicates per treatment and time point). Cells were then fixed and treated with single-molecule fluorescence in situ hybridization (smFISH) probes to label firefly luciferase mRNA transcripts. The samples were then imaged using a laser scanning confocal microscope. The images were analyzed to identify and measure the amount of internalized mRNA.

[0283] Endosome escape of mRNA-LNP Endosomal escape of mRNA-LNP was quantified by measuring galactolectin 9, as its aggregation is caused by endosome rupture. SKOV3 cells stably integrated with mCherry-galactolectin 9 were treated with mRNA-LNP formulated with compounds I-6, I-12, or SM-102 to determine the number of endosome escape events generated by each formulation. Cells were seeded at a density of 80,000 cells per well on poly-D-lysine-treated coverslips in 500 μL of RPMI medium in 24-well plates. Cells were allowed to grow for 18 hours. The next day, the medium was replaced with 500 μL of fresh medium containing 0.5 μg / well of firefly luciferase mRNA-LNP prepared with specified ionizable lipids. Cells were transfected for 6 hours (n=3 replicates per treatment). Cells were then fixed and imaged using a laser scanning confocal microscope. Images were analyzed to identify and count small, bright areas of galactolectin 9 accumulation, which indicate endosome escape events.

[0284] result Successful mRNA delivery requires (1) uptake by the cell via endocytosis and (2) escape from the formed endosome into the cytoplasm, where the mRNA can be translated into protein. LNP formulations with enhanced cellular uptake and endosome escape capabilities directly improve their delivery efficiency. Compared to the industry gold standard and commercially available lipid SM-102, mRNA-LNPs formulated with compounds I-12 and I-6 achieved stronger mRNA signaling per cell within 1 to 6 hours. Figure 8 A). The greatest difference was observed at 6 hours, when cellular uptake of compound I-12 was 2.8 times that of SM-102, while cellular uptake of compound I-6 was 3.2 times that of SM-102. Figure 8 (B, Table 7). These results indicate that LNP formulations containing compounds I-12 and I-6 have higher cellular uptake compared to the industry gold standard.

[0285] Internalized LNPs become trapped in endosomes and must escape into the cytoplasm to avoid degradation. Galactolectin 9, a cytoplasmic protein, accumulates at the endosome rupture site, indicating a successful endosome escape event. Galactolectin 9 positive spots are generated from mRNA-LNP prepared with compound I-6. Figure 9 A) Significantly more than SM-102 (2.8 times) and compound I-12 (5.2 times) Figure 9 (B, Table 7). This disclosure demonstrates that compound I-6 achieves more efficient cytoplasmic delivery of mRNA than the industry gold standard.

[0286] Table 7. Relative differences in mRNA uptake and endosome escape compared to SM-102

[0287] Example 8: Immunogenicity of influenza mRNA vaccine in mice All procedures used in animal studies conducted at the University of Toronto were approved by the Institutional Animal Care and Use Committee (IACUC) under protocol number 20012621 and complied with all applicable local, provincial, and federal regulations. Female BALB / c mice (7–8 weeks old) were purchased from Charles River Laboratories and acclimatized for 1–2 weeks prior to the start of the studies.

[0288] On days 0 and 30, 0.5 μg of the formulated PR8 mRNA was injected into the left quadriceps femoris muscle of mice (n=8). On days 28–29 (4 weeks after primary immunization) and day 44 (2 weeks after booster immunization), 50–100 μL of blood was collected from the saphenous vein of the contralateral hind limb at the injection site.

[0289] HAI titer Blood was collected from the saphenous vein of the contralateral hind limb using capillary blood collection tubes 4 weeks after primary immunization (before booster immunization) and 2 weeks after booster immunization. Hemagglutination inhibition (HAI) is a measure of functional antibodies. HAI titers in serum samples were determined using a protocol adapted from the World Health Organization manual. First, serum was treated with receptor-destroying enzyme (Hardy Diagnostics, 370013) at 37°C for 18 hours to inactivate nonspecific inhibitors, followed by incubation at 56°C for 60 minutes to inactivate the receptor-destroying enzyme, according to the manufacturer's instructions. 25 µL of serum was serially diluted (1:2) in 96-well V-plates using DPBS, starting at a 1:20 dilution and going up to a 1:2560 dilution. 25 µL of H1N1 influenza A / Puerto Rico / 8 / 1934 virus (ATCC, VR-1469) containing 4 hemagglutination units was added to the diluted serum and incubated at room temperature for 30 minutes. Finally, 50 µL of 0.5% v / v turkey red blood cell solution (Rockland Immunochemicals, RLR408) was added to each well and incubated at room temperature for 45 minutes, then the plate was placed on its side for one minute. Agglutination inhibition was observed as blood formed “teardrops”. HAI titers are expressed as the reciprocal of the highest dilution from which red blood cells showed no complete agglutination. Each plate included a back titration to confirm the viral dose (4 HAU / 25 μl) and a negative control (serum from untreated mice).

[0290] IFN-γ ELISpot Three weeks after booster immunization, mouse spleens were collected in 5 mL RPMI 1640 solutions supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum. Spleens were dissociated in gentleMACS C tubes (Miltenyi Biotec, 130-093-237), filtered through a 70 µm filter, and treated with ammonium chloride-potassium chloride lysis buffer to remove erythrocytes. Splenic cells were seeded at 250,000 cells per well on pre-coated PVDF plates and cultured in serum-free CLT-Test medium (Cellular Technology Limited, CTLT-005) supplemented with 2 mM fresh L-glutamine and 1% penicillin-streptomycin. Samples were individually tested by in vitro stimulation with an overlapping 15-mer peptide (JPT Peptide Technologies) (1 µg / mL) covering the entire hemagglutinin protein of influenza A / PR / 8 / 34 virus. Cell stimulation with concanavalin A (Thermo Fisher, 00-4978-03) served as a positive control, while the test medium served as a negative control. Cells were incubated at 37°C and 5% CO2 for 18 hours. T cell ELISpot assays were performed using a mouse IFN-γ monochromatic kit, following the manufacturer's instructions (Cellular Technology Limited, mIFNg-1M). Spot-forming units (SFUs) were counted using an automated ELISpot reader (Cellular Technology Limited, S6 EntryM2).

[0291] result The immunogenicity of compounds I-6 and I-12 compared to the industry gold standard SM-102 was evaluated using a mouse influenza vaccine model. Serum hemagglutination inhibition (HAI) titers were measured 4 weeks after primary immunization (before booster immunization) and 2 weeks after booster immunization. HAI titer is a key clinically relevant indicator for influenza protection because it simply reflects the quality and quantity of neutralizing antibodies. All LNPs achieved an HAI titer of 40 after primary immunization. Figure 10 A). Following booster immunization, HAI titers increased by more than an order of magnitude, with some animals reaching titers as high as 1280. All LNPs induced equally strong neutralizing antibody responses. Three weeks post-boost immunization, HA-specific T cell responses were measured using an IFN-γ T cell ELISpot assay by stimulating isolated spleen cells with an overlapping peptide pool across the HA protein. All LNPs induced HA-specific T cells, determined by an increase in the mean number of IFN-γ spot-forming units (SFUs) compared to the initial mouse response. Figure 10(B) Both compounds I-6 and I-12 induced higher HA-specific T-cell responses than SM-102. Compound I-6 produced slightly more SFU than compound I-12 (1.7-fold, P = 0.0762) and significantly more than SM-102 (4.6-fold, P = 0.0002). This disclosure demonstrates that the chemical properties of ionizable lipids have a direct impact on cellular responses to mRNA vaccines.

[0292] Table 8. Fold increase in cellular response compared to SM-102

[0293] LNP apparent pK a Apparent pK of LNP formulations a It is one of the parameters that determines efficacy and safety in vivo. Apparent pK a Related to the pH value when LNP is positively charged. Apparent pK a The optimal range has been determined empirically – typically 6.2–6.5 for intravenous delivery and 6.6–6.9 for intramuscular administration – and depends on the individual pK of the ionizable lipids. a Value. The position of electron-withdrawing groups in the ionizable lipid structure relative to tertiary amines affects the apparent pK of LNP formulations. a .

[0294] The comparison of formulations F04 and F16 in Table 4 shows that directly replacing amides with esters will result in an increase in apparent pK. a Reduce by a whole unit. Figure 1 This shows that in order to achieve the best apparent pK a Ideally, multiple "flank" carbons should be placed in the linker region between the ester and the tertiary amine. The LNP formulated with the optimized ester lipid (F13) outperformed the industry gold standard lipids DLin-MC3-DMA, SM-102, and ALC-0315. Therefore, sufficient distance between heteroatoms is crucial for achieving the desired apparent pK. a Protein expression and transfected cell viability are essential.

[0295] Biodegradable Ionizable lipids are exogenous compounds that must be rapidly eliminated from the body to avoid toxic accumulation in cells and tissues. Elimination is crucial for applications requiring frequent administration to achieve therapeutic effects. To this end, a group of compounds related to this disclosure have replaced the previous amide groups with esters, which are more readily hydrolyzed by various extracellular or intracellular enzymes.

[0296] Example 6 demonstrates that, compared to amide lipids, ester-ionizable lipids exhibit significantly improved in vitro degradation and in vivo plasma clearance rates. Furthermore, it has been shown that the in vitro degradation rate depends on the distance between the ester and nearby electron-withdrawing and sterically hindered groups. Therefore, the combination of functional group selection (amide or ester) and heteroatom distance should be considered to optimize biodegradability.

[0297] Structural isomers based on ester orientation This new class of ionizable lipids includes structural isomers resulting from the reversal of ester group orientation. For example, compounds I-4 and I-8 have the same linker region (three carbons near the ethanolamine head group and three carbons near the alkyl tail), while the esters point in opposite directions. Changing the ester orientation is another way to alter the distance between adjacent heteroatoms, rather than directly increasing or decreasing the linker region. Figure 7 The effect of ester orientation on in vitro degradation rate is shown. For example, compound I-8 degrades approximately 50% faster than compound I-4. Furthermore, structural isomers with different ester orientations generate different metabolites after ester hydrolysis, which may affect their in vivo safety profile. Therefore, in the design of this type of ionizable lipid, both linker length and ester orientation should be considered, as they are both factors affecting the distance between heteroatoms.

[0298] List of implementation plans Listed embodiments: 1. A compound of formula I, or a salt and / or solvate thereof:

[0299] (I) in: R 1 Selected from C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 1-20 alkylene aryl, C 1-20 alkylene heteroaryl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl, C 1-20 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or more OH, OC groups. 1-10 Alkyl, NR 6 R 6 'and (NR) 6 C 1-10 Alkylene) n NR 6 'R 6The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or more OH, C, or C groups. 1-10 Alkyl, OC 1-10 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace; X 1 and X 2 They may be the same or different, and are selected from C(O)O and OC(O); R 2 R 3 R 4 and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl, and alkylene group is optionally represented by one or more groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally separated by one or more OH and C groups. 1-20 Alkyl substitution; R 6 R 6 '、R 6 ''、R 7 R 7 '、R 8 R 8 'and R 9 Independently selected from H and C 1-10 alkyl; a, b, c, and d may be the same or different, and are selected from 2 to 6; and n is selected from 1 to 4.

[0300] Scheme 2 is listed. The compound according to Scheme 1 is listed, wherein a, b, c and d are all 2.

[0301] Scheme 3 is listed. The compound according to Scheme 1 is listed, wherein a+c≥5 and b+d≥5.

[0302] Scheme 4. The compound according to Scheme 1, wherein a, b, c and d are selected from 2 and 3.

[0303] 5. The compound according to embodiment 1, wherein a, b, c and d are selected from 3 and 4.

[0304] 6. The compound according to any one of the listed embodiments 1 to 5, wherein X 1 and X 2 It is C(O)O.

[0305] 7. The compound according to any one of the listed embodiments 1 to 5, wherein X 1 and X 2 It is OC(O).

[0306] 8. The compound according to any one of the listed embodiments 1 to 7, wherein R 1 Selected from C 1-10 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 1-10 alkylene aryl, C 1-10 alkylene heteroaryl, C 1-10 Alkylene C 3-8 Heterocyclic alkyl, C 1-10 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one to four OH, OC 1-5 Alkyl, NR 6 R 6 'and (NR) 6 C 1-5 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one to four OH, C 1-5 Alkyl, OC 1-5 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace.

[0307] Listed Scheme 9. The compound according to listed Scheme 8, wherein R 1 Selected from C 1-10 Alkyl, C 2-10 alkenyl, C 2-10alkynyl group, C 1-10 alkylene phenyl, C 1-10 alkylene heteroaryl, C 1-10 Alkylene C 3-8 Heterocyclic alkyl, C 1-10 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or two OH, OC groups. 1-3 Alkyl, NR 6 R 6 'and (NR) 6 C 1-5 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or two OH, C, or OH groups. 1-5 Alkyl, OC 1-3 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace.

[0308] 10. The compound according to any one of the listed embodiments 1 to 9, wherein R 6 R 6 '、R 6 ''、R 7 R 7 '、R 8 and R 8 'Selected independently from H and C 1-5 alkyl.

[0309] 11. The compound according to any one of the listed embodiments 1 to 10, wherein R 1 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0310] Example 12. The compound according to example 11, wherein R 1 yes .

[0311] 13. The compound according to any one of the listed embodiments 1 to 12, wherein R 2 R 3 R 4 and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally composed of one to four groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl are optionally surrounded by one to four OH groups and C. 1-10 Alkyl substitution.

[0312] Listed Embodiment 14. The compound according to listed Embodiment 13, wherein R9 Independently selected from H and C 1-5 alkyl.

[0313] 15. The compound according to any one of the listed embodiments 1 to 14, wherein R 2 R 3 R 4 and R 5 same.

[0314] Example 16. The compound according to example 15, wherein R 2 R 3 R 4 and R 5 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , and , And the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally surrounded by one or two OH groups and C groups. 1-10 Alkyl substitution.

[0315] 17. The compound according to any one of the listed embodiments 1 to 16, wherein all R 2 R 3 R 4 and R 5 yes .

[0316] 18. An embodiment of the listed scheme. A compound selected from the group consisting of:

[0317] Or its salts and / or solvates.

[0318] Example 19. The compound according to any one of examples 1 to 18, wherein the alkaline hydrolysis rate of the compound is greater than 0.001 min. -1 .

[0319] Example 20. A nanoparticle comprising one or more compounds of any one of examples 1 to 19.

[0320] Example 21. A colloid comprising one or more compounds of any one of examples 1 to 19.

[0321] Example 22. A supramolecular structure comprising one or more compounds of any one of examples 1 to 19.

[0322] Example 23. A composition comprising one or more compounds of any one of Examples 1 to 19, one or more nanoparticles of Example 20, one or more colloids of Example 21, and / or one or more supramolecular structures of Example 22.

[0323] Example 24. The nanoparticles according to example 20, wherein the nanoparticles are lipid nanoparticles (LNP).

[0324] Example 25. The composition according to example 23 or 24 further comprises one or more components to be delivered to cells or a subject.

[0325] Example 26. The composition according to example 25, wherein one or more components to be delivered to cells or a subject are selected from unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, staining agents, dyes and small molecule drugs.

[0326] Example 27. The composition according to example 25 or 26, wherein one or more components to be delivered to cells or a subject have a total negative charge.

[0327] Example 28. The composition according to example 26 or 27, wherein the protein and peptide are selected from endonucleases, a wide range of nucleases, proteases and kinases.

[0328] Example 29. The composition according to example 25 or 26, wherein one or more components to be delivered to cells or a subject are one or more nucleic acids.

[0329] Example 30. The composition according to Example 29, wherein one or more nucleic acids are selected from short interfering RNA (e.g., small interfering RNA) (siRNA), circular RNA, cyclic RNA, long non-coding RNA (lncRNA), microRNA (miRNA), pri-miRNA, messenger RNA (mRNA), clustered regularly spaced short palindromic repeats (CRISPR) related nucleic acids, single-stranded guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), exDNA, precursor RNA, single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA).

[0330] Example 31. The composition according to example 29, wherein the nucleic acid is miRNA.

[0331] Example 32. The composition according to any one of the examples 23 to 31, wherein one or more components to be delivered to cells or a subject are present in a weight ratio of about 100:1 to about 1:5.

[0332] Example 33. The composition according to any one of the examples 23 to 31, wherein the composition further comprises one or more lipids.

[0333] Example 34. The composition according to example 33, wherein one or more lipids are selected from steroids, steroid derivatives, PEG-lipids and phospholipids, and mixtures thereof.

[0334] Example 35. The composition according to example 34, wherein the PEG-lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-ceramide conjugate, a PEG-modified dialkylamine, a PEG-modified 1,2-diacoxypropyl-3-amine, a PEG-modified diacylglycerol, and / or a dialkylglycerol.

[0335] Example 36. The composition according to example 34, wherein the PEG-lipid is PEG-modified 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, PEG-modified 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol and / or PEG-modified distearyl-rac-glycerol.

[0336] Example 37. The composition according to any one of examples 34 to 36, wherein the PEG-lipid is present at 0 to 50 mol% of the total lipids in the composition.

[0337] Example 38. The composition according to example 34, wherein the steroid is unmodified or modified cholesterol, phytosterol, cholecalciferol, dexamethasone or any combination thereof.

[0338] Example 39. The composition according to example 38, wherein the steroid is present at 0 to 50 mol% of the total lipids in the composition.

[0339] Example 40. The composition according to example 34, wherein the phospholipid is 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE).

[0340] Example 41. The composition according to example 34, wherein the phospholipids are present at 0 to 50 mol% or at most 50 mol% of the total lipids in the composition.

[0341] 42. The composition according to any one of the listed embodiments 23 to 41, wherein the apparent pK of the composition is... a It is approximately 5 to approximately 7.

[0342] Example 43. The composition according to any one of examples 23 to 42, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0343] Example 44. The composition according to example 43, wherein the pharmaceutically acceptable carrier is a solvent or solution.

[0344] Example 45. An LNP comprising one or more compounds of Example 1, wherein a, b, c, and d are each selected from 2 and 3, and the apparent pK of the LNP is... a It ranges from approximately 5.5 to approximately 6.0.

[0345] Example 46. An LNP comprising one or more compounds of Example 1, wherein a, b, c, and d are each selected from 3 and 4, and the apparent pK of the LNP is... a It is approximately 6.0 to approximately 6.6.

[0346] Example 47. An LNP comprising one or more compounds of Example 1, wherein a, b, c, and d are each selected from 2 and 4, and the apparent pK of the LNP is... a It is approximately 5.5 to approximately 6.6.

[0347] Example 48. An LNP comprising one or more compounds of Example 1, wherein a, b, c, and d are each selected from 4, 5, and 6, and the apparent pK of the LNP is... a ≥ 6.6.

[0348] Example 49. A method of delivering one or more ingredients to cells or a subject, comprising contacting the cells or subject with one or more compositions of any one of examples 23 to 44, or with one or more LNPs of any one of examples 45 to 48, wherein the contact is performed under conditions that promote the uptake of the ingredients by the cells or subject.

[0349] Example 50. The method according to example 49, wherein the cells are in contact in vitro.

[0350] Example 51. The method according to example 49, wherein the cells are in contact in vivo.

[0351] Example 52. The method according to example 49, wherein the cells are in vitro contact.

[0352] 53. The method according to any one of the listed embodiments 49 to 52, wherein the contact is used for the treatment or prevention of disease, symptom or condition.

[0353] Listed implementation scheme 54. The method according to listed implementation scheme 53, wherein the disease, symptom or condition is selected from infectious diseases, autoimmune diseases, cancer, genetic diseases, chronic diseases, traumatic injuries, wound healing, traumatic brain injury, neuromuscular diseases and gastrointestinal diseases.

[0354] Example 55. The method according to example 53 or 54, wherein contact is carried out by applying an effective amount of one or more compositions to the cells in need.

[0355] 56. The method according to any one of the listed embodiments 49 to 55, wherein the cell is a prokaryotic or eukaryotic cell selected from animal cells, insect cells, or plant cells.

[0356] 57. The method according to any one of the listed embodiments 49 to 56, wherein one or more components to be delivered to the cell are one or more nucleic acids.

[0357] 58. The method according to 57, wherein one or more ingredients are cosmetic ingredients.

[0358] Example 59. The method according to example 58, wherein one or more components include a unique DNA or RNA sequence (barcode) which is placed in the product packaging / label and can be read to ensure that the product is genuine or has not been tampered with.

[0359] 60. An example of an implementation scheme. A reagent kit comprising: One or more compounds as described in embodiment 1, or the composition of any one of embodiments 23 to 44, or one or more LNPs as described in any one of embodiments 45 to 48.

[0360] Example 61. The kit according to example 60 further includes at least one vial, test tube, flask, bottle, syringe and / or other container in which one component is placed and optionally aliquoted.

[0361] Example 62. The kit according to example 60 or 61 also includes instructions for use.

[0362] Example 63. The kit according to example 62, wherein the instructions are for delivering one or more compounds or one or more compositions to a subject or cells.

[0363] Equivalent implementation plan Detailed descriptions of one or more embodiments of this disclosure have been set forth in the foregoing appended description. While any methods and materials similar to or equivalent to those described herein may be used in implementing or testing this disclosure, preferred methods and materials are now described. Other features, objects, and advantages of this disclosure will be apparent from the specification and claims. In the specification and appended claims, the singular form also includes the plural referent, unless the context clearly specifies otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in this specification are incorporated herein by reference.

Claims

1. A compound of formula I, or a salt thereof and / or a solvate thereof: (I) in: R 1 Selected from C 1-20 Alkyl, C 2-20 alkenyl, C 2-20 alkynyl group, C 1-20 alkylene aryl, C 1-20 alkylene heteroaryl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl, C 1-20 Alkylene C 3-8 cycloalkyl, C 3-8 Heterocyclic alkyl and C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one or more OH, OC groups. 1-10 Alkyl, NR 6 R 6 'and (NR) 6 C 1-10 Alkylene) n NR 6 'R 6 The aryl, heteroaryl, heterocycloalkyl, and cycloalkyl groups are optionally replaced by one or more OH, C, or C groups. 1-10 Alkyl, OC 1-10 Alkyl, O and NR 7 R 7 'Substitution, and wherein the alkyl group is optionally further replaced by NR 8 R 8 'replace; X 1 and X 2 They may be the same or different, and are selected from C(O)O and OC(O); R 2 R 3 R 4 and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl, and alkylene group is optionally represented by one or more groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally separated by one or more OH and C groups. 1-20 Alkyl substitution; R 6 R 6 '、R 6 ''、R 7 R 7 '、R 8 R 8 'and R 9 Independently selected from H and C 1-10 alkyl; a, b, c, and d may be the same or different, and are selected from 2 to 6; and n is selected from 1 to 4.

2. The compound of claim 1, wherein a, b, c and d are selected from 2 and 3.

3. The compound according to any one of claims 1 to 2, wherein X 1 and X 2 It is C(O)O.

4. The compound according to any one of claims 1 to 2, wherein X 1 and X 2 It is OC(O).

5. The compound according to any one of claims 1 to 4, wherein R 1 Selected from: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

6. The compound according to any one of claims 1 to 5, wherein R 2 R 3 R 4 and R 5 Same or different, and selected from C 1-50 Alkyl, C 2-50 alkenyl, C 1-20 Alkylene C 3-8 Heterocyclic alkyl and C 1-20 Alkylene C 3-8 Cycloalkyl, wherein each alkyl, alkenyl and alkylene group is optionally surrounded by one to four groups selected from SS, C(O), OC(O), C(O)O, OC(O)O, NR 9 C(O)O、OC(O)NR 9 C(O)S, SC(O), NR 9 C(O), C(O)NR 9 The group is interrupted, and the alkyl, alkenyl, alkylene heterocyclic alkyl and alkylene cycloalkyl groups are optionally surrounded by one to four OH groups and C groups. 1-10 Alkyl substitution.

7. The compound according to any one of claims 1 to 6, wherein the compound of formula I is selected from: Or its salts and / or solvates.

8. A nanoparticle comprising one or more compounds according to any one of claims 1 to 7.

9. A colloid comprising one or more compounds according to any one of claims 1 to 7.

10. A supramolecular structure comprising one or more compounds according to any one of claims 1 to 7.

11. A composition comprising one or more compounds according to any one of claims 1 to 7, one or more nanoparticles according to claim 8, one or more colloids according to claim 9, and / or one or more supramolecular structures according to claim 10.

12. The composition of claim 11, wherein one or more components to be delivered to cells or a subject are selected from unmodified or modified nucleic acids, mitochondria, plasmids, PolyIC and related adjuvants, ribonucleoproteins, proteins, peptides, cells, staining agents, dyes and small molecule drugs, optionally wherein the one or more components to be delivered to cells or a subject have a total negative charge, or optionally wherein the one or more components to be delivered to cells are one or more nucleic acids.

13. An LNP comprising one or more compounds according to claim 1, wherein a, b, c and d are each selected from 2 and 4, and the apparent pK of the LNP is... a It is approximately 5.5 to approximately 6.

6.

14. The composition of claim 11, used for treating or preventing a disease, symptom, or condition, wherein the disease, symptom, or condition is selected from infectious diseases, autoimmune diseases, cancer, genetic diseases, chronic diseases, traumatic injuries, wound healing, traumatic brain injury, neuromuscular diseases, and gastrointestinal diseases.

15. A reagent kit comprising: One or more compounds according to claim 1, or one or more compositions according to any one of claims 11, 12 or 14, or one or more LNPs according to claim 13.

Images