CN114989182B - Lipid compound, composition containing lipid compound and application of lipid compound - Google Patents

Abstract

The present invention relates to lipid compounds, compositions comprising the same and uses thereof. The present invention provides compounds of formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof. The compound can be used for delivering nucleic acid medicines, gene vaccines, micromolecular medicines, polypeptides or protein medicines, enriches the types of cationic lipid compounds, and has important significance for the development and application of nucleic acid preventive and therapeutic agents.

Description

Lipid compound, composition containing lipid compound and application of lipid compound

Technical Field

The invention belongs to the technical fields of biological medicine and gene therapy, and in particular relates to a lipid compound, a lipid carrier containing the lipid compound, a nucleic acid lipid nanoparticle composition and a pharmaceutical preparation.

Background

The gene therapy technology is a hot spot of research in the field of modern biological medicine, and can prevent cancer, bacterial and viral infection, treat diseases with genetic etiology and the like by using nucleic acid medicines. Because nucleic acid drugs are easy to degrade and difficult to enter cells, and the like, the nucleic acid drugs need to be encapsulated by a carrier to be delivered to target cells, so that the development of safe and efficient delivery carriers becomes a precondition for clinical application of gene therapy.

Lipid nanoparticles (Lipid nanoparticle, LNP) are currently a research hotspot in the field of non-viral gene vectors. In 2018, the FDA approved LNP delivery patisiran (onpattro) for the treatment of hereditary transthyretin amyloidosis, since studies using LNP technology to deliver nucleic acid drugs have been shown to grow in bursts; in particular, at the end of 2020, the FDA approved new coronavirus vaccines against COVID-19 for Moderna and BioNtech & pyroxene, respectively, both of which delivered mRNA drugs using LNP technology, thus achieving prophylaxis against the COVID-19 virus.

LNP is typically composed of four lipid compounds, namely cationic lipids, neutral lipids, steroids, and polymer-bound lipids, where the cationic lipids are selected to have the greatest effect on LNP, such as affecting the encapsulation efficiency of nucleic acid drugs, the delivery efficiency of nucleic acid drugs in vivo, cytotoxicity, and the like.

However, the functioning of nucleic acid therapeutic drugs still faces challenges, mainly including low cell permeability and high sensitivity to degradation by certain nucleic acid molecules, including RNA. Thus, there remains a need to develop new lipid compounds that facilitate in vitro or in vivo delivery of nucleic acid molecules for therapeutic and/or prophylactic purposes.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to overcome the defects and provide a series of compounds, wherein the compounds can be used for preparing lipid carriers together with other lipid compounds, so that the delivery efficiency of nucleic acid medicaments in vivo is improved, and lipid compounds with specific structures can be selected as lipid carriers according to organs needing to be enriched by the nucleic acid medicaments.

The invention also provides a lipid carrier containing the compound.

The invention also provides nucleic acid lipid nanoparticle compositions comprising the above compounds or the above lipid carriers.

The invention also provides a pharmaceutical formulation comprising the above compound, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition.

The invention also provides application of the compound or a pharmaceutically acceptable form thereof or the lipid carrier or the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation in preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

The present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering to a subject in need thereof the nucleic acid lipid nanoparticle composition described above or the pharmaceutical formulation described above.

Solution for solving the problem

< first aspect >

The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof,

wherein the method comprises the steps of

n is selected from 1 or 2;

A. b are each independently selected from-R, -X ₁ or-X ₂ The method comprises the steps of carrying out a first treatment on the surface of the Provided that A and B are not both R;

r is selected from H, halogen, cyano, nitro, hydroxy, amino, -L-R _y Or (b)

X ₁ Is that

X ₂ Is that

R _x Selected from H, hydroxy, halogen, cyano, amino, C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl, said C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, C _1-6 Alkyl, C _1-6 Alkoxy, cyano, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or

R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from H, C _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _2-30 Alkynyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or-G ₉ -L-R ₆ The C is _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _2-30 Alkynyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl, 3-8 membered heterocyclyl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one or more of the following substituents: halogen, halogen,Cyano, oxo, C _1-6 Alkoxy, C _2-30 Alkenyl, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or

The C is _3-8 Cycloalkyl optionally substituted with one or more halogens or C _1-6 Alkyl substitution;

R ₆ selected from H, C _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _2-30 Alkynyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl, 3-8 membered heterocyclyl, C _6-10 Aryl or 5-10 membered heteroaryl; the C is _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _2-30 Alkynyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl, 3-8 membered heterocyclyl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one or more of the following substituents: halogen, cyano, oxo, C _1-6 Alkoxy, C _2-30 Alkenyl, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or

The C is _3-8 Cycloalkyl optionally substituted with one or more halogens or C _1-6 Alkyl substitution;

G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ and G ₉ Each independently selected from the group consisting of bond, C _1-30 Alkylene, C _2-30 Alkenylene, C _3-30 Cycloalkylene, C _3-30 A cycloalkenylene group or a 3-8 membered heterocyclylene group, said C _1-30 Alkylene, C _2-30 Alkenylene, C _3-30 Cycloalkylene, C _3-30 The cycloalkenyl or 3-8 membered heterocyclylene is optionally substituted with one or more of the following substituents: halogen, cyano, oxo, C _1-6 Alkoxy, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or-G ₉ -L-R ₆ ；

L、L ₁ 、L ₂ 、L ₃ And L ₄ Each independently selected from the group consisting of bond, -C (=O) NR ₅ -、-NR ₅ C(＝O)-、-C(＝O)O-、-OC(＝O)-、-OC(＝O)O-、-OC(＝O)NR ₅ -、-NR ₅ C(＝O)O-、-NR ₅ C(＝O)NR ₅ -、-NR ₅ C(＝NR ₅ )NR ₅ -、-C(＝S)NR ₅ -、-NR ₅ C(＝S)-、-C(＝S)O-、-OC(＝S)-、-OC(＝S)O-、-OC(＝S)NR ₅ -、-NR ₅ C(＝S)O-、-NR ₅ C(＝S)NR ₅ -、-C(＝O)S-、-SC(＝O)-、-OC(＝O)S-、-NR ₅ C(＝O)S-、-SC(＝O)NR ₅ -、-C(＝S)S-、-SC(＝S)-、-SC(＝S)O-、-NR ₅ C(＝S)S-、-SC(＝S)NR ₅ -、-O-N＝CR ₅ -、-CR ₅ =n-O-, -O-or-S-;

each R ₅ Independently selected from H, C _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl or 3-8 membered heterocyclyl, said C _1-6 Alkoxy, C _1-30 Alkyl, C _2-30 Alkenyl, C _3-30 Cycloalkyl, C _3-30 Cycloalkenyl or 3-8 membered heterocyclyl optionally substituted with one or more of the following substituents: halogen, cyano, C _1-6 Alkoxy, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl or 5-10 membered heteroaryl;

x is selected from-O-, -CH ₂ -、-NR _y -, -C (=O) -or-OCH ₂ -；

Y is selected from-CH ₂ -、-CH ₂ CH ₂ -, -C (=o) -or a bond;

u is selected from-CH ₂ -, -C (=o) -or a bond;

said-CH in Y and U ₂ -or-CH ₂ CH ₂ -optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, C _1-6 Alkyl, C _1-6 Alkoxy, cyano, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl or 5-10 membered heteroaryl.

In some embodiments, wherein n is 1; r is R _x H.

In some embodiments, wherein U is selected from the group consisting of-CH ₂ -or a bond; x is selected from-CH ₂ -or-O-.

In some embodiments, wherein U is-CH ₂ -; x is-NR _y -。

In some embodiments, wherein U is-C (=o) -; r is R _x Is H; y is-CH ₂ -; x is-NR _y -。

In some embodiments, wherein n is 1; x is-CH ₂ -; u is a bond; r is R _x Selected from H, hydroxy, halogen, cyano, amino, C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl, said C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, C _1-6 Alkyl, C _1-6 Alkoxy, cyano, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or

In some embodiments, wherein n is 2; u is-CH ₂ -; x is selected from-CH ₂ -or-O-; y is selected from-CH ₂ -or a bond; r is R _x Selected from H, hydroxy, halogen, cyano, amino, C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl, said C _1-6 Alkoxy, C _1-6 Alkyl or 3-8 membered heterocyclyl optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, C _1-6 Alkyl, C _1-6 Alkoxy, cyano, C _3-8 Cycloalkyl, 3-8 membered heterocyclyl, C _6-10 Aryl, 5-to 10-membered heteroaryl or

In some embodiments, A, B are each independently selected from-R or-X ₁ The method comprises the steps of carrying out a first treatment on the surface of the Provided that A and B are not both R; r is selected from H, halogen, cyano, nitro, hydroxyRadical, amino, -L-R _y Or (b)

In some preferred embodiments, A, B are each independently selected from-R or-X ₁ The method comprises the steps of carrying out a first treatment on the surface of the Provided that A and B are not both R; r is selected from H, hydroxy, -L-R _y Or (b)

In some embodiments, A is-X ₁ B is-R; more preferably, A is-X ₁ B is selected from H or hydroxy.

In some embodiments, R _x Selected from H, hydroxy, halogen, cyano, amino or C _1-6 Alkyl, said C _1-6 The alkyl group is optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, cyano, 3-to 8-membered heterocyclyl, 5-to 10-membered heteroaryl or

In some preferred embodiments, R _x Selected from H, hydroxy, halogen, cyano, amino or C _1-4 Alkyl, said C _1-4 The alkyl group is optionally substituted with one or more of the following substituents: halogen, nitro, hydroxy, cyano or

In some more preferred embodiments, R _x Selected from H or

Preferably, R _x Is H.

In some embodiments, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from H, C _1-30 Alkyl, C _2-30 Alkenyl, C _2-30 Alkynyl or-G ₉ -L-R ₆ The C is _1-30 Alkyl, C _2-30 Alkenyl or C _2-30 Alkynyl groups are optionally substituted with one or more of the following substituents: halogen, cyano, oxo, C _2-30 Alkenyl, C _3-8 Cycloalkyl or

The C is _3-8 Cycloalkyl optionally substituted with one or more halogens or C _1-6 Alkyl substitution.

In some preferred embodiments, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from H, C _1-24 Alkyl, C _2-24 Alkenyl, C _2-24 Alkynyl or-G ₉ -L-R ₆ The C is _1-24 Alkyl, C _2-24 Alkenyl or C _2-24 Alkynyl groups are optionally substituted with one or more of the following substituents: halogen, cyano, oxo, C _2-24 Alkenyl, C _3-6 Cycloalkyl or

The C is _3-6 Cycloalkyl optionally substituted with one or more halogens or C _1-4 Alkyl substitution.

In some preferred embodiments, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently is C _1-24 An alkyl group; preferably, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently is C _6-24 An alkyl group.

In some more preferred embodiments, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from H, methyl,

In some more preferred embodiments, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from H, methyl,

More preferably, R ₁ 、R ₂ 、R ₃ 、R ₄ And R is _y Each independently selected from->

In some embodiments, R ₆ Selected from H, C _1-30 Alkyl, C _2-30 Alkenyl or C _2-30 Alkynyl groups.

In some embodiments, R ₆ Selected from H, C _1-24 Alkyl, C _2-24 Alkenyl or C _2-24 Alkynyl groups.

In some embodiments, R ₆ Selected from the group consisting of

In some embodiments, G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from the group consisting of bond, C _1-30 Alkylene or C _2-30 Alkenylene group, the C _1-30 Alkylene or C _2-30 Alkenylene optionally substituted with one or more of the following substituents: halogen, cyano, oxo or-G ₉ -L-R ₆ 。

In some embodiments, G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from the group consisting of bond, C _1-24 Alkylene or C _2-24 Alkenylene group, the C _1-24 Alkylene or C _2-24 Alkenylene optionally substituted with one or more oxo groups or-G ₉ -L-R ₆ And (3) substitution.

In some embodiments, G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from a bond or C _1-24 An alkylene group; preferably G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from a bond or C _1-18 An alkylene group.

In some preferred embodiments, G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from the group consisting of bond, -CH ₂ -、-CH ₂ CH ₂ -、

Wherein G is ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Optionally substituted with one or more of the following substituents: oxo group,

In some preferred embodiments, G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ And G ₉ Each independently selected from the group consisting of bond, -CH ₂ -、-CH ₂ CH ₂ -、

In some embodiments, L, L ₁ 、L ₂ 、L ₃ And L ₄ Each independently selected from the group consisting of bond, -C (=O) NR ₅ -、-NR ₅ C (=o) -, -C (=o) O-, or-OC (=o) -.

In some embodiments, each R ₅ Independently selected from H, C _1-30 Alkyl or C _2-30 Alkenyl group, the C _1-30 Alkyl or C _2-30 Alkenyl groups are optionally substituted with one or more of the following substituents: halogen, cyano, C _1-6 Alkoxy, C _3-8 Cycloalkyl or 3-8 membered heterocyclyl.

In some embodiments, each R ₅ Independently selected from H, C _1-24 Alkyl or C _2-24 Alkenyl groups.

In some embodiments, each R ₅ Independently selected from H, methyl,

In some embodiments, L, L ₁ 、L ₂ 、L ₃ And L ₄ Each independently selected from the group consisting of bond, -C (=o) O-, or-OC (=o) -.

In some embodiments, X is selected from the group consisting of-O-or-CH ₂ -; preferably, X is-O-.

In some embodiments, Y is selected from the group consisting of-CH ₂ -or a bond; preferably Y is-CH ₂ -。

In some embodiments, U is selected from the group consisting of-CH ₂ -or a bond.

In some embodiments, the compound of formula (I) is selected from the following compounds:

wherein R is _x 、R _y 、G ₁ 、G ₂ A and B are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-1):

therein, R, R ₁ 、R ₂ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ 、L ₂ N, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-2):

therein, R, R ₁ 、R ₂ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ 、L ₂ N, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-3):

wherein,,R ₁ 、R ₂ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ 、L ₂ n, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-4):

therein, R, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ 、L ₁ 、L ₂ 、L ₃ 、L ₄ N, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-5):

therein, R, R ₁ 、R ₂ 、R ₃ 、R ₄ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ 、L ₁ 、L ₂ 、L ₃ 、L ₄ N, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (II-6):

/>

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、G ₅ 、G ₆ 、G ₇ 、G ₈ 、L ₁ 、L ₂ 、L ₃ 、L ₄ N, U, X and Y are as defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (III-1) or formula (III-2):

Therein, R, R ₁ 、R ₂ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ And L ₂ As defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (III-3) or formula (III-4):

therein, R, R ₁ 、R ₂ 、R _x 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ And L ₂ As defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (IV-1) or formula (IV-2):

therein, R, R ₁ 、R ₂ 、G ₁ 、G ₂ 、G ₃ 、G ₄ 、L ₁ And L ₂ As defined in formula (I).

In some embodiments, the compound of formula (I) has a structure represented by formula (V-1) or formula (V-2) or formula (V-3) or formula (V-4):

/>

wherein R is ₁ 、R ₂ 、G ₁ 、G ₃ 、G ₄ 、L ₁ And L ₂ As defined in formula (I).

The invention also provides the following compounds, or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs thereof:

TABLE 1

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< second aspect >

The present invention provides a lipid carrier comprising a compound according to < first aspect >, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof. The lipid carrier has high encapsulation efficiency on nucleic acid drugs, and greatly improves the delivery efficiency of the nucleic acid drugs in vivo.

In some embodiments, the lipid carrier comprises a first lipid compound comprising a compound according to < first aspect > or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof, and optionally other cationic lipids, and a second lipid compound comprising one or a combination of two or more of an anionic lipid, a neutral lipid, a steroid, and a polymer-bound lipid.

In some embodiments, the first lipid compound is any one of the compounds described above or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex, or prodrug thereof.

In some embodiments, the first lipid compound described above is any one of the compounds described above or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or combination of a prodrug and other cationic lipid thereof.

In some embodiments, the additional cationic lipids include one or more of 1, 2-dioleyloxy-N, N-dimethylaminopropane DLinDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane DODMA, DLin-MC2-MPZ, 2-dioleylene-4- (2-dimethylaminoethyl) - [1,3] -dioxolane DLin-KC2-DMA, 1, 2-dioleoyl-3-trimethylammonium-propane DOTAP, 1'- (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylaminon-dodecane-2-ol C12-200, 3β [ N-N' -dimethylaminoethane) -carbamoyl ] cholesterol DC-Chol, and N- [1- (2, 3-dioleoyl chloride) propyl ] -N, N-trimethylamine DOTMA.

In some embodiments, the anionic lipid comprises one or a combination of two or more of phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dioleoyl phosphatidylglycerol DOPG, 1, 2-dioleoyl-sn-glycerol-3-phosphatidylserine DOPS, and dimyristoyl phosphatidylglycerol.

In some embodiments, the neutral lipid comprises at least one of 1, 2-dioleoyl-sn-glycero-3-phosphatidylethanolamine DPPC, 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine DPPC, 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine DOPC, dipalmitoyl-phosphatidylglycerol DPPG, oleoyl phosphatidylcholine POPC, 1-palmitoyl-2-oleoyl phosphatidylethanolamine POPE, 1, 2-dipalmitoyl-sn-glycero-3-phosphate ethanolamine DPPE, 1, 2-dimyristoyl-sn-glycero-3-phosphate ethanolamine DMPE, distearoyl phosphatidylethanolamine DSPE, and 1-stearoyl-2-oleoyl phosphatidylethanolamine SOPE, or a lipid modified with an anionic or cationic group thereof. The anionic or cationic modifying group is not limited.

In some embodiments, the steroid comprises one or more of cholesterol, non-sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycosyline, ursolic acid, alpha-tocopherols, fecal sterols, and corticosteroids.

In some embodiments, the polymer-bound lipids include 1, 2-dimyristoyl-sn-glycerinomethoxy-polyethylene glycol PEG-DMG, dimyristoyl-polyethylene glycol PEG-C-DMG, polyethylene glycol-dimyristoyl glycerol PEG-C14, PEG-1, 2-dimyristoyloxy propyl-3-amine PEG-C-DMA, 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine-N- [ amino (polyethylene glycol) ] PEG-DSPE, pegylated phosphatidylethanolamine PEG-PE, PEG modified ceramides, PEG modified dialkylamines, PEG modified diacylglycerols, tween-20, tween-80, 1, 2-dipalmitoyl-sn-glycerol-methoxypolyethylene glycol PEG-DPG, 4-O- (2 ',3' -dimyristoyloxy) propyl-1-O- (ω -methoxy (polyethoxy) ethyl) succinate PEG-s-DMG, PEG-dialkoxypropyl PEG-DAA, one or a combination of two or more of mPEG2000-1, 2-di-O-alkyl-sn 3-carbamoyl glyceride PEG-c-DOMG and N-acetylgalactosamine ((R) -2, 3-bis (octadecyloxy) propyl-1- (methoxypoly (ethylene glycol) 2000) propyl carbamate)) GalNAc-PEG-DSG.

In some embodiments, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-65): 0-20): 5-25): 25-55): 0.3-15; illustratively, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid may be 20:20:5:50:5, 30:5:25:30:10, 20:5:5:55:15, 65:0:9.7:25:0.3, and the like; wherein, in the first lipid compound, the molar ratio of any one of the compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs thereof and other cationic lipids is (1-10): 0-10; illustratively, the molar ratio may be 1:1, 1:2, 1:5, 1:7.5, 1:10, 2:1, 5:1, 7.5:1, 10:1, etc.

In some embodiments, the molar ratio of the first lipid compound, the anionic lipid, the neutral lipid, the steroid, and the polymer-bound lipid in the lipid carrier is (20-55): 0-13): 5-25): 25-51.5): 0.5-15; wherein in the first lipid compound, the molar ratio of any one of the compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes or prodrugs thereof to other cationic lipids is (3-4): 0-5.

< third aspect >

The present invention provides a nucleic acid lipid nanoparticle composition comprising a compound according to < first aspect > or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof or a lipid carrier according to < second aspect >, and a nucleic acid drug.

In some embodiments, the nucleic acid drug comprises RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid, or PNA.

In some embodiments, the antisense nucleic acid is an antisense oligonucleotide.

In some embodiments, the RNA is mRNA, rRNA, circRNA, siRNA, saRNA, tRNA, snRNA, antagomir, a microrna inhibitor, a microrna activator, or a shRNA.

In some embodiments, the DNA comprises a plasmid.

In some embodiments, the mRNA includes a sequence encoding an RNA-guided DNA binding agent, more specifically, an mRNA including a Cas protein.

In some embodiments, the nucleic acid drug comprises a guide RNA, in particular, the guide RNA comprises a gRNA nucleic acid.

In some embodiments, the nucleic acid drug comprises mRNA and gRNA of a Cas protein.

In some embodiments, the gRNA is modified.

In some embodiments, the mass ratio of the nucleic acid agent to any of the above compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs thereof is 1 (3-40).

In some embodiments, the mass ratio of the nucleic acid agent to the lipid carrier is 1 (3-40).

Illustratively, the above mass ratio is 1:3, 1:5, 1:10, 1:15, 1:20, 1:30, etc.

< fourth aspect >

The present invention provides a pharmaceutical composition comprising a compound according to < first aspect >, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate, chelate, non-covalent complex or prodrug thereof, or a lipid carrier according to < second aspect >, or a nucleic acid lipid nanoparticle composition according to < third aspect >, and a pharmaceutically acceptable excipient, carrier or diluent.

< fifth aspect >

The present invention provides a pharmaceutical formulation comprising any of the above compounds or pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, chelates, non-covalent complexes, or prodrugs thereof, or the above lipid carrier, or the above nucleic acid lipid nanoparticle composition, and a pharmaceutically acceptable excipient, carrier, or diluent.

In some embodiments, the pharmaceutical formulation has a particle size of 30 to 500nm, and illustratively, the particle size may be 30nm, 50nm, 100nm, 150nm, 250nm, 350nm, 500nm, etc.

In some embodiments, the encapsulation efficiency of the nucleic acid drug in the pharmaceutical formulation is greater than 50%. Illustratively, the encapsulation efficiency may be 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 93%, 95%, etc.

< sixth aspect >

The invention also provides application of the compound or a pharmaceutically acceptable form thereof or the lipid carrier or the nucleic acid lipid nanoparticle composition or the pharmaceutical preparation in preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

The present invention also provides a method for delivering a nucleic acid drug in vivo, the method comprising administering to a subject in need thereof the nucleic acid lipid nanoparticle composition described above or the pharmaceutical formulation described above.

In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered by one of the following routes of administration: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, and intradermal. In some embodiments, the nucleic acid lipid nanoparticle composition or the pharmaceutical formulation described above is administered, for example, via an enteral or parenteral route of administration. In some embodiments, the nucleic acid lipid nanoparticle composition or pharmaceutical formulation is administered to the subject at a dose of about 0.001mg/kg to about 10 mg/kg.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention provides a series of compounds of formula (I) with novel structure, which can be used as cationic lipid to prepare lipid carriers together with other lipid compounds, and has controllable particle size, uniform distribution and high encapsulation rate for negatively charged drugs.

The compound can be used for delivering nucleic acid medicines, gene vaccines, micromolecular medicines, polypeptides or protein medicines, enriches the types of cationic lipid compounds, and has important significance for the development and application of nucleic acid preventive and therapeutic agents.

Drawings

Fig. 1 delivery strategy for PCSK9 gene editing efficiency detection of mouse liver cells.

Figure 2 PCSK9 gene editing efficiency of base editors encapsulated by different lipid compounds in mouse liver cells.

Detailed Description

For easier understanding of the present invention, certain technical and scientific terms are defined below in detail. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[ definition of terms ]

Unless otherwise indicated, the following terms have the following meanings:

the term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention which are substantially non-toxic to the organism. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed from the compounds of the present invention by reaction with pharmaceutically acceptable inorganic/organic acids or inorganic/organic bases, such salts also being referred to as acid addition salts or base addition salts. Common inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like, common organic acids include, but are not limited to, trifluoroacetic acid, citric acid, maleic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, oxalic acid, formic acid, acetic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like, common inorganic bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, barium hydroxide, and the like, and common organic bases include, but are not limited to, diethylamine, triethylamine, ethambutol, and the like.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. The present invention also includes stereoisomers and mixtures thereof, due to the presence of asymmetric centers and other chemical structures in the compounds of the present invention which may lead to stereoisomers. Since the compounds of the present invention and salts thereof include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, mixtures of enantiomers and diastereomers. Typically, these compounds can be prepared in the form of a racemic mixture. However, if desired, such compounds can be prepared or isolated to give pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in single stereoisomers (purity. Gtoreq.98%,. Gtoreq.95%,. Gtoreq.93%,. Gtoreq.90%,. Gtoreq.88%,. Gtoreq.85% or. Gtoreq.80%). The individual stereoisomers of the compounds are prepared synthetically from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, e.g., conversion to mixtures of diastereomers followed by separation or recrystallization, chromatography, use of chiral resolving agents, or direct separation of the enantiomers on chiral chromatographic columns. Starting compounds having specific stereochemistry are either commercially available or prepared according to the methods described below and resolved by methods well known in the art.

The term "tautomer" (or "tautomeric form") refers to structural isomers having different energies that can be converted to each other by a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or proton transfer tautomers) include, but are not limited to, interconversions by proton transfer, such as keto-enol isomerisation, imine-enamine isomerisation, amide-imine alcohol isomerisation, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "solvate" refers to a substance formed by the association of a compound of the invention, or a pharmaceutically acceptable salt thereof, with at least one solvent molecule by non-covalent intermolecular forces. Common solvates include, but are not limited to, hydrates, ethanolates, acetonates, and the like.

The term "chelate" is a complex having a cyclic structure, obtained by chelation of two or more ligands with the same metal ion to form a chelate ring.

The term "non-covalent complex" is formed by the interaction of a compound with another molecule, wherein no covalent bond is formed between the compound and the molecule. For example, recombination can occur by van der Waals interactions, hydrogen bonding, and electrostatic interactions (also known as ionic bonding).

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly after administration to a patient. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to a patient, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and the various prodrug forms are well known in the art.

The term "independently" means that at least two groups (or ring systems) present in the structure that are the same or similar in value range may have the same or different meanings in the particular case. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl; similarly, when the substituent Y is hydrogen, the substituent X may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl.

The terms "comprising" and "including" are used in their open, non-limiting sense.

The term "alkyl" refers to a monovalent, linear or branched alkyl group consisting of only carbon and hydrogen atoms, free of unsaturation, and attached to other moieties by a single bond, including, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, and the like. For example, "C _1-30 Alkyl "refers to a saturated monovalent straight or branched hydrocarbon radical containing 1 to 30 carbon atoms.

The term "sub-Alkyl "refers to a divalent straight or branched chain alkane group consisting of only carbon and hydrogen atoms, containing no saturation, and linked to other fragments by two single bonds, respectively, including, but not limited to, methylene, 1-ethylene, 1, 2-ethylene, and the like. For example, "C _1-30 Alkylene "refers to a saturated divalent straight or branched chain alkyl group containing from 1 to 30 carbon atoms.

The term "cycloalkyl" refers to a saturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkyl groups may include fused, bridged or spiro ring systems. For example, the term "C" as used in the present invention _3-6 Cycloalkyl "refers to cycloalkyl groups having 3 to 6 carbon atoms. For example, cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or bicyclo [2.2.1 ]Heptyl, and the like.

The term "cycloalkylene" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group as defined above, including, but not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, and the like. For example, "C _3-30 Cycloalkyl "refers to a divalent group obtained by removing a hydrogen atom from a cycloalkyl group containing 3 to 30 carbon atoms.

The term "branched alkyl" refers to an alkane radical that is attached to the parent molecule and itself forms at least two branched structures. For example

The term "alkenyl" refers to a monovalent, straight or branched chain, alkyl group consisting of only carbon and hydrogen atoms, containing at least one double bond, and attached to other moieties by a single bond, including, but not limited to, ethenyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups, and the like. For example "C _2-30 Alkenyl "means an alkenyl group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond [ ]>C＝C<) A monovalent straight or branched hydrocarbon group.

The term "alkenylene" refers to a divalent straight or branched chain alkane radical consisting of only carbonThe atomic and hydrogen atoms are composed of at least one double bond and are each linked to other fragments by two single bonds, including, but not limited to, vinylidene groups and the like. For example, "C _2-30 Alkenylene "means an alkylene group containing 2 to 30 carbon atoms and having at least 1 carbon-carbon double bond>C＝C<) A divalent straight or branched hydrocarbon group.

The term "alkynyl" refers to monovalent, straight or branched chain, alkanyl radicals consisting of only carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and attached to other moieties by a single bond, including, but not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. For example "C _2-30 Alkynyl "refers to a monovalent straight or branched hydrocarbon radical containing 2 to 30 carbon atoms and having at least 1 carbon-carbon triple bond.

The term "cycloalkenyl" refers to an unsaturated, monocyclic or polycyclic (e.g., bicyclic, tricyclic or tetracyclic) non-aromatic hydrocarbon group consisting of only carbon and hydrogen atoms. Cycloalkenyl groups may include fused, bridged or spiro ring systems. Such as cyclopropenyl and cyclobutenyl, and the like.

The term "cycloalkenyl" refers to a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group as defined above, including, but not limited to, cyclopropenyl, cyclobutenyl, and the like. For example, "C _3-30 The "cycloalkenylene group" means a divalent group obtained by removing a hydrogen atom from a cycloalkenyl group containing 3 to 30 carbon atoms.

The term "branched alkenyl" is an olefinic radical that is attached to the parent molecule and itself forms at least two branched structures. For example

The term "heterocyclyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g. fused, bridged or spiro) non-aromatic group, the ring atoms of which consist of carbon atoms and at least one heteroatom selected from N, O and S, wherein the S atom is optionally substituted to form S (=o), S (=o) ₂ Or S (=o) (=nr ^x )，R ^x Independently selected from H or C _1-4 An alkyl group. If it is full ofThe valency bond requires that the heterocyclic group may be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "3-8 membered heterocyclyl" as used herein refers to heterocyclyl groups having 3 to 8 ring atoms. For example, the heterocyclic group may be oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithianyl or trithianyl.

The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C" as used in the present invention _6-10 Aryl "refers to aryl groups having 6 to 10 carbon atoms. For example, aryl may be phenyl, naphthyl, anthracenyl, phenanthrenyl, acenaphthylenyl, azulenyl, fluorenyl, indenyl, pyrenyl, and the like.

The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system, the ring atoms of which are made up of carbon atoms and at least one heteroatom selected from N, O and S. If valence requirements are met, the heteroaryl group may be attached to the remainder of the molecule through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to heteroaryl groups having 5 to 10 ring atoms. For example, heteroaryl groups can be thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and its benzo derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purinyl, and the like.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "hydroxy" refers to-OH.

The term "cyano" refers to-CN.

The term "amino" refers to-NH ₂ 。

The term "nitro" refers to-NO ₂ 。

The term "oxo" refers to (=o).

[ preparation method ]

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.

In the present invention, "proper amount" means that the amount of the solvent or the amount of the drug to be added is large in adjustable range and less affects the synthesis result, and is not particularly limited.

In the examples described below, both solvents and drugs were used in analytical or chemical purity; redistilling the solvent before use; the anhydrous solvents were treated according to standard methods or literature methods.

Examples

Synthesis of intermediate 1 (((1S, 4R) -2-oxa-5-azabicyclo [2.2.1] hept-4-yl) methanolic hydrochloride)

(1S, 4R) -4- (hydroxymethyl) -2-oxa-5-azabicyclo [2.2.1] at room temperature]Heptane-5-carboxylic acid tert-butyl ester (3 g,13.09mmol, prepared as described in CN112778331A (2021) on pages 0207-0210) was added to concentrated hydrochloric acid (12M, 3mL,36.00 mmol) and tetrahydrofuran (30 mL) and stirred overnight at room temperature. The reaction solution was concentrated, absolute ethanol (30 mL) was added to the residue, the reaction solution was concentrated, absolute ethanol (30 mL) was further added to the residue, the reaction solution was concentrated to obtain a crude product, methylene chloride (30 mL) was added to the crude product, and 3g of potassium carbonate was further added and stirred at room temperature for 2 hours. The reaction solution was concentrated by filtration to give the title compound (1.69 g, yield 100%) as a white solid. MS: M/z [ M+H-tBu ] ⁺ ＝172。

Synthesis of intermediate 2 (((1R, 4S) -2-oxa-5-azabicyclo [2.2.1] hept-4-yl) methanolic hydrochloride)

(1R, 4S) -4- (hydroxymethyl) -2-oxa-5-azabicyclo [2.2.1 at room temperature]Heptane-5-carboxylic acid tert-butyl ester (5 g,21.81mmol, prepared as described in CN112778331A (2021) on pages 0224-0227) was added to concentrated hydrochloric acid (12M, 5mL,60.00 mmol) and tetrahydrofuran (50 mL) and stirred overnight at room temperature. The reaction solution was concentrated, absolute ethanol (50 mL) was added to the residue, the reaction solution was concentrated, absolute ethanol (50 mL) was further added to the residue, the reaction solution was concentrated to obtain a crude product, methylene chloride (30 mL) was added to the crude product, and 5g of potassium carbonate was further added and stirred at room temperature for 2 hours. The reaction solution was concentrated by filtration to give the title compound (3.61 g, yield 100%) as a white solid. MS: M/z [ M+H-tBu] ⁺ ＝172。

EXAMPLE 1 Synthesis of Compound 1 (((2- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) ethyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate))

Step 1: synthesis of Compounds 1-2

2-hexyldecanoic acid (30 g,117mmoL.1.0 eq), 1, 6-hexanediol (41.48 g,351mmol,3.0 eq) and 4-dimethylaminopyridine DMAP (17.15 g,140.4mmol,1.2 eq) were added to dichloromethane DCM (100 mL) at room temperature and N, N' -dicyclohexylcarbodiimide DCC (26.55 g,128.70mmol,1.1 eq) was added with stirring. Stir at room temperature overnight. The reaction solution was filtered, the filter cake was washed with dichloromethane, the organic phase was concentrated, ethyl acetate EA (200 mL) was added to the residue, stirred at room temperature for 10min, petroleum ether PE (1L) was added, allowed to stand overnight, filtered, the filtrate was passed through a thin layer of silica gel (100 g of silica gel), the thin layer of silica gel was rinsed with PE: ea=5:1 mixed solvent (5L), and the filtrate was concentrated to give compound 1-2 (29 g, yield 69.51%) as a colorless oil. MS: M/z [ M+H ] ] ⁺ ＝357.3。

Step 2: synthesis of Compounds 1-3

Compounds 1-2 (15 g,42.07mmol,1.0 eq) and triethylamine TEA (12.77 g,126.21mmol,3.0 eq) were added to dichloromethane (200 mL) at room temperature followed by slow additionMethanesulfonic anhydride (10.99 g,63.11mmol,1.5 eq) was added and stirred overnight at room temperature. The extract was washed with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compounds 1 to 3 (16 g, yield 87.50%) as a colorless transparent oil. MS: M/z [ M+H ]] ⁺ ＝435.3。

Step 3: synthesis of Compounds 1-4

Ethanolamine (400 mg,6.65mmol,1.0 eq), compounds 1-3 (7.1 g,16.39mmol,2.5 eq), potassium carbonate (2.7 g,19.95mmol,3.0 eq), potassium iodide (108 mg,0.66mmol,0.1 eq) were added to acetonitrile ACN (15 mL) at room temperature, heated to 90℃under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 1-4 (871 mg, 18.03% yield) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝738.7。

Step 4: synthesis of Compounds 1-5

Compounds 1-4 (871 mg,1.18mmol,1.0 eq) and TEA (358.16 mg,3.54mmol,3.0 eq) were added to dichloromethane (20 mL) at room temperature, and methanesulfonic anhydride (308.2 mg,1.77mmol,1.5 eq) was slowly added and stirred overnight at room temperature. The extract was washed with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compounds 1 to 5 (842 mg, yield 87.50%) as a colorless transparent oil. MS: M/z [ M+H ] ] ⁺ ＝816.7。

Step 5: synthesis of Compound 1

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (90 mg,0.66mmol,1.0 eq), compounds 1-5 (539 mg,0.66mmol,1.0 eq), potassium carbonate (2793 mg,1.98mmol,3.0 eq), potassium iodide (11 mg,0.066mmol,0.1 eq) were added to ACN (15 mL), heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 1 (167 mg, 31.03% yield) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝819.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.04(t,J＝6.7Hz,4H),3.55(q,J＝7.0Hz,5H),3.46-3.41(m,2H),2.91-2.88(m,2H),2.58-2.48(m,3H),2.33–2.25(m,2H),1.90-1.81(m,5H),1.65–1.51(m,8H),1.46–1.35(m,17H),1.28-1.20(s,38H),0.86(t,J＝6.5Hz,12H)。

EXAMPLE 2 Synthesis of Compound 2 (((3- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) propyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate))

Step 1: synthesis of Compound 2-2

3-amino-1-propanol (1 g,13.32mmol,1.0 eq), compound 2-1 (14.46 g,33.30mmol,2.5 eq), potassium carbonate (5.52 g,39.96mmol,3.0 eq), potassium iodide (0.15 g,1.33mmol,0.1 eq) were added to ACN (30 mL) at room temperature, heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 2-2 (6.1 g, yield 50.46%) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝752.7。

Step 2: synthesis of Compound 2-3

Compound 2-2 (6.1 g,8.11mmol,1.0 eq) and TEA (2.46 g,24.34mmol,3.0 eq) were added to dichloromethane (60 mL) at room temperature, and methanesulfonic anhydride (2.12 g,12.17mmol,1.5 eq) was slowly added and stirred overnight at room temperature. The extract was washed with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compound 2-3 (4.8 g, yield 70.58%) as a yellow transparent oil. MS: M/z [ M+H ] ] ⁺ ＝830.7。

Step 3: synthesis of Compound 2

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (300 mg,3.03mmol,1.0 eq), compound 2-3 (2.5 g,3.03mmol,1.0 eq), potassium carbonate (1.26 g,9.09mmol,3.0 eq), potassium iodide (201 mg,1.21mmol,0.4 eq) were added to ACN (30 mL), heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 2 (790 mg, yield 31.34%) as a pale yellow solid. MS: M/z [ M/2+H ]] ⁺ ＝417.5. ¹ H NMR(400MHz,cdcl ₃ )δ4.42-4.38(m,1H),4.10–3.99(m,5H),3.63(dd,J＝7.7,1.2Hz,1H),3.51(s,1H),2.93(dd,J＝10,1.2Hz,1H),2.70–2.51(m,8H),2.35-2.28(m,2H),1.81(dd,J＝48.9,10.6Hz,3H),1.71-1.56(m,13H),1.46–1.19(m,53H),0.88(t,J＝6.7Hz,12H)。

EXAMPLE 3 Synthesis of Compound 3 ((4- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) butyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate))

Step 1: synthesis of Compound 3-2

4-amino-1-butanol (830 mg,9.32mmol,1.0 eq), compound 3-1 (10.12 g,23.28mmol,2.5 eq), potassium carbonate (3.86 g,27.93mmol,3.0 eq), potassium iodide (0.15 g,0.93mmol,0.1 eq) were added to ACN (30 mL) at room temperature and heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 3-2 (3.6 g, yield 50.46%) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝766.7。

Step 2: synthesis of Compound 3-3

Compound 3-2 (3.6 g,4.70mmol,1.0 eq) and TEA (1.43 g,14.10mmol,3.0 eq) were added to dichloromethane (20 mL) at room temperature, and methanesulfonic anhydride (1.23 g,7.05mmol,1.5 eq) was slowly added and stirred overnight at room temperature. The extract was washed with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compound 3-3 (2.8 g, yield 70.58%) as a colorless transparent oil. MS: M/z [ M+H ] ] ⁺ ＝844.7。

Step 3: synthesis of Compound 3

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (220 mg,2.22mmol,1.0 eq), compound 3-3 (1.37 g,1.62mmol,1.0 eq), potassium carbonate (0.67 g,4.86mmol,3.0 eq), potassium iodide (0.11 g,0.65mmol,0.4 eq) were added to ACN (10 mL), heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 3 (431 mg, 31.34% yield) as a pale yellow solid. MS: M/z [ M+H ]] ⁺ ＝847.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.04(t,J＝6.6Hz,4H),3.85-3.80(m,4H),3.47–3.39(m,4H),2.34-2.26(m,6H),1.81–1.14(m,72H),0.86(t,J＝6.5Hz,12H)。

EXAMPLE 4 Synthesis of Compound 4 ((5- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-ylpentyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate))

Step 1: synthesis of Compound 4-2

5-amino-1-pentanol (1 g,9.71mmol,1.0 eq), compound 4-1 (10.55 g,24.27mmol,2.5 eq), potassium carbonate (4.01 g,29.08mmol,3.0 eq), potassium iodide (640 mg,3.88mmol,0.4 eq) were added to ACN (30 mL) at room temperature, heated to 90℃under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 4-2 (3.8 g, 51% yield) as a pale yellow oil. MS: M/z [ m+h ] += 780.7.

Step 2: synthesis of Compound 4-3

Compound 4-2 (3.8 g,4.87mmol,1.0 eq) and TEA (1.47 g,14.60mmol,3.0 eq) were added to dichloromethane (50 mL) at room temperature, and methanesulfonic anhydride (1.27 g,7.30mmol,1.5 eq) was slowly added and stirred overnight at room temperature. The extract was separated by washing with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compound 4-3 (3 g, yield 73%) as a colorless transparent oil. MS: M/z [ M+H ] ] ⁺ ＝858.7。

Step 3: synthesis of Compound 4

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (200 mg,2.02mmol,1.0 eq), compound 4-3 (1.73 g,2.02mmol,1.0 eq), potassium carbonate (837.64 g,6.06mmol,3.0 eq), potassium iodide (134.13 mg,0.81mmol,0.4 eq) were added to ACN (10 mL), heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 4 (522 mg, yield 30%) as a pale yellow solid. MS: M/z [ M+H ]] ⁺ ＝861.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.05(t,J＝6.7Hz,4H),3.75-3.70(m,4H),3.51–3.44(m,4H),2.33–2.26(m,2H),1.97-1.86(m,6H),1.73–1.52(m,18H),1.49-1.39(m,12H),1.32–1.15(m,42H),0.86(t,J＝6.6Hz,12H)。

EXAMPLE 5 Synthesis of Compound 5 ((2- (1S, 4R) -4-hydroxymethyl-2-oxa-5-azabicyclo [2.2.1] heptane-5-ylethyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate)

Intermediate 1 (100 mg,0.77mmol,1.0 eq), compounds 1-5 (630 mg,0.77mmol,1.0 eq), potassium carbonate (319 mg,2.31mmol,3.0 eq), potassium iodide (51 mg,0.31mmol,0.4 eq) were added to ACN (20 mL) at room temperature, heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 5 (229 mg, 35% yield) as a pale yellow oil. MS: M/z [ M/2+H ]] ⁺ ＝425.5。 ¹ H NMR(400MHz,CDCl ₃ )δ4.04(t,J＝6.6Hz,4H),3.55(dd,J＝14.1,6.8Hz,5H),3.45-3.39(m,2H),2.89-2.86(m,2H),2.49-2.46(m,3H),2.33-2.27(m,2H),2.06-1.92(m,4H),1.65–1.51(m,8H),1.42-1.35(m,16H),1.32-1.23(m,41H),0.86(t,J＝6.4Hz,12H)。

EXAMPLE 6 Synthesis of Compound 6 (4- ((1S, 4R) -4-hydroxymethyl-2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) butyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate))

Intermediate 1 (100 mg,0.77mmol,1.0 eq), compound 3-3 (650 mg,0.77mmol,1.0 eq), potassium carbonate (319 mg,2.31mmol,3.0 eq), potassium iodide (51 mg,0.31mmol,0.4 eq) were added to ACN (20 mL) at room temperature, heated to 90 degrees celsius under nitrogen and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give compound 6 (250 mg, yield 37%) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝877.8。EN0001-76(L50): ¹ H NMR(400MHz,CDCl ₃ )δ4.03(t,J＝6.7Hz,4H),3.84-3.80(m,4H),3.44–3.40(m,4H),2.33-2.25(m,6H),1.78–1.15(m,73H),0.85(t,J＝6.4Hz,12H)。

EXAMPLE 7 Synthesis of Compound 7 ((5- (1S, 4R) -4-hydroxymethyl-2-oxa-5-azabicyclo [2.2.1] heptan-5-ylpentyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate)

Intermediate 1 (91.0 mg,0.55 mmol), compound 4-3 (0.47 g,0.55 mmol), potassium carbonate K at RT ₂ CO ₃ (0.23 g,1.65 mmol) and potassium iodide KI (9 mg,0.055 mmol) were added to acetonitrile (10 mL), heated to 90℃under nitrogen, and stirred overnight. The reaction was washed with DCM, the organic phase was concentrated to dryness and purified by column chromatography to give the title compound 7 (0.23 g, 46.91%) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝891.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.05(t,J＝6.7Hz,4H),3.75-3.70(m,4H),3.47–3.43(m,4H),2.33-2.26(m,2H),2.05-1.85(m,12H),1.73–1.49(m,14H),1.49–1.24(m,56H),0.86(t,J＝6.7Hz,12H)。

EXAMPLE 8 Synthesis of Compound 8 ((3- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) propyl) azadiyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate))

Step 1: synthesis of Compound 8-2

Compound 8-1 (60 g,299.52 mmol), 1, 9-nonanediol (143.99 g,898.56 mmol) and 4-dimethylaminopyridine (47.57 g,389.38 mmol) were added to dichloromethane (600 mL) at room temperature and N, N' -dicyclohexylcarbodiimide (74.16 g,359.42 mmol) was added with stirring. Stir at room temperature overnight. The reaction solution was filtered, the filter cake was washed with dichloromethane, the organic phase was concentrated, and the organic phase was stirred for 30 minutes with PE/ea=5/1 mixed solvent and allowed to stand overnight. Filtration, concentration of the filtrate, column chromatography purification (PE/ea=100/1 to 10/1) gave compound 8-2 (76 g, yield 74)% of the total amount of the aqueous solution) was a colorless oil. MS: M/z [ M+H ]] ⁺ ＝343.3。

Step 2: synthesis of Compound 8-3

Compound 8-2 and triethylamine (44.31 g,437.88 mmol) were added to DCM (500 mL) at room temperature, methanesulfonic anhydride (38.14 g,218.94 mmol) was slowly added and stirred overnight at room temperature. The extract was washed with methylene chloride, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the residue was purified by column chromatography to give compound 8-3 (33 g, yield 53.75%) as a yellow oil. MS: M/z [ m+h ] =421.3.

Step 3: synthesis of Compound 8-4

3-amino-1-propanol (0.8 g,10.65 mmol), compound 8-3 (9.86 g,23.43 mmol), potassium carbonate (5.89 g,42.6 mmol) and potassium iodide (0.71 g,4.26 mmol) were added to acetonitrile (200 mL) at room temperature and stirred overnight at 90℃under nitrogen. The reaction solution was concentrated, water and methylene chloride were added to the residue to extract, and the organic phase was concentrated, and purified by flash chromatography to give compound 8-4 (5.84 g, yield 75.71%) as a pale yellow oil. MS: M/z [ M+H ] ] ⁺ ＝724.7。

Step 4: synthesis of Compound 8-5

Compound 8-4 (5.84 g,8.06 mmol) and triethylamine (2.45 g,24.18 mmol) were added to dichloromethane (50 mL) at room temperature, methanesulfonic anhydride (2.11 g,12.09 mmol) was slowly added, and stirred at room temperature for 5 hours. The reaction solution was poured into water, extracted with methylene chloride, and the organic phase was concentrated, and purified by flash preparative liquid chromatography to give compound 8-5 (1.91 g, yield 29.52%) as a yellow oil. MS: M/z [ M+H ]] ⁺ ＝802.7。

Step 5: synthesis of Compound 8

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (162.70 mg,1.20 mmol), compound 8-5 (0.96 g,1.2 mmol), potassium carbonate (0.67 g,4.8 mmol) and potassium iodide (0.080 g,0.48 mmol) were added to acetonitrile (15 mL) and stirred overnight at 90 ℃. The reaction solution was concentrated, poured into water, extracted with dichloromethane, and the organic phase was concentrated and purified by flash chromatography (DCM/meoh=0% -8%,50 column volumes) to give compound 8 (618 mg, 63.95% yield) as a yellow oil. MS: M/z [ M/2+H ]] ⁺ ＝403.5。 ¹ H NMR(400MHz,CDCl ₃ )δ4.36(s,1H),4.07–3.98(m,5H),3.58(d,J＝7.7Hz,1H),3.45(s,1H),2.89(d,J＝10.0Hz,1H),2.63–2.53(m,2H),2.53–2.35(m,8H),2.32–2.25(m,2H),1.76(dd,J＝51.0,9.7Hz,3H),1.65–1.52(m,10H),1.45-1.36(m,8H),1.31-1.20(m,42H),0.91–0.80(m,12H)。

EXAMPLE 9 Synthesis of Compound 9 ((4- ((1S, 4S) -2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) butyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate))

Step 1: synthesis of Compound 9-2

4-amino-1-butanol (0.95 g,10.64 mmol), compound 8-3 (9.80 g,23.3 mmol), potassium carbonate (5.88 g,42.56 mmol) and potassium iodide (0.71 g,4.26 mmol) were added to acetonitrile (60 mL) at room temperature and stirred overnight at 90℃under nitrogen. The reaction solution was concentrated, water was then added to the residue, dichloromethane was extracted, and the organic phase was concentrated, followed by purification by column chromatography to give compound 9-2 (4.5 g, yield 57.20%) as a pale yellow oil. MS: M/z [ M+H ] ] ⁺ ＝738.7。

Step 2: synthesis of Compound 9-3

Compound 9-2 (3.5 g,4.74 mmol) and triethylamine (1.44 g,14.22 mmol) were added to dichloromethane (50 mL) at room temperature, and methanesulfonic anhydride (1.24 g,7.11 mmol) was slowly added and stirred overnight at room temperature. The reaction was poured into water, extracted with dichloromethane, and the organic phase concentrated and purified by flash preparative liquid chromatography (DCM/meoh=0% -8%) to give compound 9-3 (2.4 g, 62.01%) as a yellow waxy solid. MS: M/z [ M+H ]] ⁺ ＝817.3。

Step 3: synthesis of Compound 9

2-oxa-5-azabicyclo [2.2.1] s at room temperature]Heptane (200 mg,1.48 mmol), compound 9-3 (1.21 g,1.48 mmol), potassium carbonate (0.82 g,5.92 mmol) and potassium iodide (0.098 g,0.59 mmol) were added to acetonitrile (15 mL) and stirred overnight at 90 ℃. The reaction solution was concentrated to dryness, poured into water, extracted with dichloromethane, the organic phase concentrated and purified by flash preparative liquid chromatography (DCM/MeOH =0% -8%) to give compound 9 (0.831 g, yield 68.76%) as a yellow waxy solid. MS: M/z [ M+H ]] ⁺ ＝819.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.00(t,J＝6.7Hz,4H),3.80-3.74(m,4H),3.38–3.34(m,4H),2.30-2.22(m,6H),1.85–1.15(m,68H),0.85–0.79(m,12H)。

EXAMPLE 10 Synthesis of Compound 10 (4- (1S, 4R) -4-hydroxymethyl-2-oxa-5-azabicyclo [2.2.1] heptan-5-yl) butyl) azadiyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate))

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Intermediate 1 (245.10 mg,1.48 mmol), compound 9-3 (1.21 g,1.48 mmol), potassium carbonate (0.82 g,5.92 mmol) and potassium iodide (0.098 g,0.59 mmol) were added to acetonitrile (15 mL) at room temperature and stirred overnight at 90 ℃. The reaction was concentrated to dryness, poured into water, extracted with dichloromethane, and the organic phase concentrated and purified by flash chromatography (DCM/meoh=0% -8%) to give compound 10 (0.719 g, 57.20% yield) as a yellow waxy solid. MS: M/z [ M+H ] ] ⁺ ＝849.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.02(t,J＝6.6Hz,4H),3.81-3.77(m,4H),3.39–3.35(m,4H),2.32-2.22(m,8H),1.98-1.87(m,1H),1.73–1.12(m,66H),0.83(d,J＝7.1Hz,12H)。

EXAMPLE 11 Synthesis of Compound 11 ((5- (1-hydroxymethyl) -2-azabicyclo [2.1.1] hex-2-ylpentyl) azadiyl) bis (nonane-9, 1-diyl) bis (2-butyloctanoate))

Step 1: synthesis of Compound 11-2

5-amino-1-pentanol (1.04 g,10.10 mmol), compound 8-3 (9.33 g,22.18 mmol), potassium carbonate (5.58 g,40.4 mmol) and potassium iodide (0.67 g,4.03 mmol) were added to acetonitrile (60 mL) at room temperature and stirred overnight at 90℃under nitrogen. The reaction solution was concentrated, water was added to the residue, extracted with dichloromethane, and the organic phase was concentrated, followed by rapid reactionPurification by preparative liquid chromatography (DCM/meoh=0% -5%) afforded compound 11-2 (4.3 g, 56.70% yield) as a pale yellow oil. MS: M/z [ M+H ]] ⁺ ＝752.7。

Step 2: synthesis of Compound 11-3

Compound 11-2 (3.3 g,4.39 mmol) and triethylamine (1.33 g,13.17 mmol) were added to dichloromethane (50 mL) at room temperature, and methanesulfonic anhydride (1.15 g,6.58 mmol) was slowly added and stirred overnight at room temperature. The reaction was poured into water, extracted with dichloromethane, and the organic phase concentrated and purified by flash preparative liquid chromatography (DCM/meoh=0% -8%) to give compound 11-3 (1.82 g, yield 50.0%) as a pale yellow solid. MS: M/z [ M+H ]] ⁺ ＝830.7。

Step 3: synthesis of Compound 11

At room temperature, (2-azabicyclo [ 2.1.1)]Hexan-1-yl) methanol (100 mg,0.88 mmol), compound 11-3 (0.51 g,0.62 mmol), potassium carbonate (0.36 g,2.64 mmol) and potassium iodide (0.058 g,0.35 mmol) were added to acetonitrile (15 mL) and stirred overnight at 90 ℃. The reaction was concentrated to dryness, poured into water, extracted with dichloromethane, and the organic phase concentrated and purified by flash chromatography (DCM/meoh=0% -8%) to give compound 11 (0.719 g, 57.20% yield) as a yellow waxy solid. MS: M/z [ M+H ]] ⁺ ＝847.8。 ¹ H NMR(400MHz,CDCl ₃ )δ4.05(t,J＝6.7Hz,4H),3.85-3.80(m,4H),3.46–3.41(m,4H),2.32-2.26(m,2H),1.90(s,6H),1.68–1.52(m,16H),1.48–1.16(m,53H),0.86(t,J＝6.9Hz,12H)。

Example 12 preparation and characterization of lipid nanoparticles

1. Cationic lipids or the compounds of the invention/DSPC/cholesterol/PEG-lipids were prepared in a molar ratio of 50:10:38.5:1.5. Di-methylene methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA, commonly abbreviated as MC 3) and Compound 1-Compound 5 of the present invention were dissolved in absolute ethanol at the above molar ratios with DSPC, cholesterol, PEG-DMG, respectively.

Luciferase mRNA (L-6107,TriLink BioTechnologies,Inc.) was dissolved in 100mM enzyme-free citrate buffer pH 4 (mRNA concentration 0.2 mg/mL) such that the ethanol solutions of the different lipid carriers were mixed with the buffer of luciferase mRNA at 1:3 (volume/volume) (where the mass ratio of total lipid to mRNA was 40:1), and nucleic acid lipid nanoparticles A-F were obtained by the microfluidic nano-drug manufacturing system (NanoAssemblr Ignite, canada) at a flow rate of 12 mL/min.

The obtained nucleic acid lipid nanoparticles were immediately diluted 40-fold in 1 x DPBS buffer. The diluted nucleic acid lipid nanoparticle solution passes through an overspeed centrifuge tube and is concentrated to reach the target volume. After dilution, the particles were used for DLS particle size measurement and encapsulation efficiency detection.

2. The particle size and polydispersity index of the lipid nanoparticles were determined by dynamic light scattering in 173 ° back scattering detection mode using Malvern Zetasizer Nano ZS (Malvern UK), the test results are shown in table 2.

The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions and the test results are shown in table 2.

TABLE 2

Example 13 animal experiments

1. Luciferase mRNA animal in vivo delivery assay

C57BL/6 mice (purchased from Jiangsu Jiujiukang) of about 18-20g in weight, which were female for 6-8 weeks, were selected as subjects.

Nucleic acid lipid nanoparticles containing luciferase mRNA (luc mRNA) were obtained in the same manner as in example 12.

The nucleic acid lipid nanoparticles obtained were administered by tail vein injection (dose 0.2 mg/kg) and mice were sacrificed 4 hours after administration. Livers were collected in pre-weighed tubes, weighed, cut to about 50mg, and luciferase expression levels (units: ng/g (luciferase/mouse liver)) in mouse livers were measured, and the expression results are shown in Table 3 below.

TABLE 3 Table 3

Lipid nanoparticles	Cationic lipid compounds	ng/g (luciferase/mouse liver)
			LNP A	MC3	4120±719
LNP B	Compound 1	8556±1517
			LNP D	Compound 3	20117±2365
LNP E	Compound 4	11386±2206
			LNP F	Compound 5	6102±1078

2. mRNA and sgRNA delivery experiments targeting the base editor ABE8e of PCSK9

Cholesterol in blood is mainly synthesized by the liver, which is also a main organ for decomposing excessive cholesterol, and a low-density lipoprotein LDL receptor (LDLR) exists on the surface of the liver, which can be combined with cholesterol circulating back to the liver, so that the cholesterol is decomposed into bile acid, and is excreted outside the body through the intestinal tract; PCSK9 is a liver-synthesized protease capable of binding to LDL receptors, promoting their entry into hepatocytes, leading to degradation of LDL receptors by lysosomes, and a reduction in the number of LDL receptors; thus, inhibiting the activity of PSCK9, the amount of LDLR can be increased, and thus the uptake and decomposition ability of cholesterol can be enhanced. Basic and clinical studies show that the PCSK9 gene is an effective target for treating hyperlipidemia and atherosclerosis. FIG. 1 shows the changes in the number of LDL receptors and ultimately the changes in cholesterol metabolism caused before and after editing of a specific site of the PCSK9 gene.

The strategy of PCSK9 gene editing and delivery of the mouse liver cells is shown in figure 1, and the main flow is as follows: the prepared lipid nanoparticle is utilized to target and deliver mRNA and sgRNA encoding ABE8e to mouse liver cells by intravenous injection, mutation is introduced into PCSK9 genes under the action of the ABE8e and the sgRNA, the mutation of bases A to G is realized at a specific site, and the editing efficiency is calculated by sequencing.

The specific experimental design is as follows:

(1) Selecting appropriate mutation sites and editing design

The single base editor ABE8e realizes accurate base substitution from a to G without a donor template and without DSB, based on which the first exon of the PCSK9 gene is selected as the screening mutation site. mRNA encoding a single-base editing tool ABE8e and sgRNA are delivered into an animal body together through lipid nanoparticles, the mRNA encoding the base editor ABE8e is translated into protein in cytoplasm, the protein forms a complex with the sgRNA and enters a cell nucleus, the base editor ABE8e targets a first exon splice donor site of a PCSK9 gene under the guidance of the sgRNA, adenine (A) on a first exon template strand is deaminated into inosine (I), I can be regarded as G at DNA level for reading and copying, and finally, replacement from A to G is realized, so that the splice donor site is destroyed so that a PCSK9 gene reading frame is terminated in advance.

(2) mRNA and sgRNA preparation of base editor ABE8e

The sequence of the first exon and the first intron of the mouse PCSK9 Gene (NCBI Gene ID: 100102) is selected as a targeting region, and a target sequence PCSK9-sgRNA for single-base editing of the PCSK9 Gene is determined.

By analyzing the sequence of the PCSK9 gene across the first exon and the intron, the sgrnas of the targeting region were designed: PCSK9-sgRNA (synthesized by auresli, south kyo) having the sequence:

PCSK9-sgRNA：5’-CCCATACCTTGGAGCAACGG-3’(SEQ ID NO：1)；

The base editor ABE8E used in this experiment was the one evolved by David R.Liu team, and was the one developed by Richter MF, zhao KT, eton E, lapinaite A, newby GA, thuronyi BW, wilson C, koblan LW, zeng J, bauer DE, doudna JA, liu DR.Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.Nat Biotechnol.2020Jul;38 (7): 883-891.doi:10.1038/s41587-020-0453-z.Epub 2020Mar 16.Erratum in:Nat Biotechnol.2020May 20;: PMID:32433547; PMCID: PMC 7357821). mRNA from ABE8e was synthesized in the laboratory for later use.

(3) Animal study

Lipid nanoparticles comprising a compound of the invention (see table 2) encapsulating mRNA and sgRNA encoding base editor ABE8e were systemically administered to 6-7 week old C57BL/6 female mice (purchased from Jiangsu Jieqiangkang corporation) by tail vein injection at a dose of 0.5 mg/kg.

Lipid nanoparticles comprising dioleylmethylene-4-dimethylaminobutyrate (DLin-MC 3-DMA, abbreviated as MC 3) encapsulating mRNA and sgRNA of base editor ABE8e were applied in a similar manner to a comparable group of mice of both week-old and sex-old as positive controls. In addition, PBS buffer was also used as a negative control for mice of comparable age and sex in a similar manner to tail vein injection.

(4) Editing efficiency detection

Editing efficiency detection was performed one week after mice were dosed, liver tissue was taken after mice were sacrificed, genome was extracted after lysis, and efficiency analysis was performed by deep sequencing.

The deep sequencing procedure was as follows:

SEQ ID NO:	sequence object	Sequence(s)
			2	PCSK9-F2	5’-ACCAGACGGCTAGATGAGCA-3’
3	PCSK9-R2	5’-CCCAGGACGAGGATGGAGATTA-3’

The PCR procedure was as follows:

after the PCR is finished, gel electrophoresis is used for verification, a single band with proper size is selected, the amplified product is determined to be correct, and the obtained PCR product is sent to Nanjing gold Style company for sequencing. And analyzing the reading of the deep sequencing result through the crispress software, and analyzing the specific site to realize the calculation of the editing efficiency, wherein the calculation result is shown in figure 2.

As shown in table 2, table 3 and fig. 2, the cationic lipid compounds employed in the present invention are capable of effectively delivering drugs such as nucleic acid molecules, small molecule compounds and the like; by contrast, the lipid nanoparticle has the advantages of better particle size distribution, high encapsulation efficiency and obviously better delivery effect than the lipid nanoparticle contrast, and can meet the in-vivo delivery requirement.

The description of the exemplary embodiments presented above is merely illustrative of the technical solution of the present invention and is not intended to be exhaustive or to limit the invention to the precise form described. Obviously, many modifications and variations are possible in light of the above teaching to those of ordinary skill in the art. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable others skilled in the art to understand, make and utilize the invention in various exemplary embodiments and with various alternatives and modifications. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Claims (8)

1. A compound represented by the formula (V-1) or the formula (V-2) or a pharmaceutically acceptable form thereof,

wherein,,

R ₁ and R is ₂ Each independently selected from

G ₁ 、G ₃ And G ₄ Each independently selected from the group consisting of bond, -CH ₂ -、-CH ₂ CH ₂ -、

L ₁ And L ₂ Each independently selected from-C (=o) O-or-OC (=o) -;

the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts or stereoisomers.

2. A compound or pharmaceutically acceptable form thereof, wherein the compound is selected from the group consisting of:

the pharmaceutically acceptable form is selected from pharmaceutically acceptable salts or stereoisomers.

3. A lipid carrier comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof.

4. A lipid carrier according to claim 3, comprising a first lipid compound comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof and optionally further cationic lipids and a second lipid compound comprising one or a combination of more than two of anionic lipids, neutral lipids, steroids and polymer-bound lipids.

5. A nucleic acid lipid nanoparticle composition comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 3 or 4, and a nucleic acid drug.

6. The nucleic acid lipid nanoparticle composition of claim 5, wherein the nucleic acid drug is selected from the group consisting of RNA, DNA, antisense nucleic acid, aptamer, ribozyme, immunostimulatory nucleic acid, and PNA.

7. A pharmaceutical formulation comprising a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 3 or 4 or a nucleic acid lipid nanoparticle composition according to claim 5 or 6, and a pharmaceutically acceptable excipient, carrier or diluent.

8. Use of a compound according to claim 1 or 2 or a pharmaceutically acceptable form thereof or a lipid carrier according to claim 3 or 4 or a nucleic acid lipid nanoparticle composition according to claim 5 or 6 or a pharmaceutical formulation according to claim 7 in the preparation of a nucleic acid drug, a genetic vaccine, a small molecule drug, a polypeptide or a protein drug.

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