CN117126071A

CN117126071A - Nitrogen-containing chain compound, preparation method thereof, composition containing nitrogen-containing chain compound and application of nitrogen-containing chain compound

Info

Publication number: CN117126071A
Application number: CN202311016686.9A
Authority: CN
Inventors: 林金钟; 卢静; 俞航; 姜婷; 张凡; 张博阳; 郭俊香; 陈林; 王冰
Original assignee: Shanghai Lanque Biomedical Co ltd
Current assignee: Shanghai Lanque Biomedical Co ltd
Priority date: 2022-08-12
Filing date: 2023-08-11
Publication date: 2023-11-28
Also published as: WO2024032753A1

Abstract

The invention discloses a nitrogen-containing chain compound, a preparation method thereof, a composition containing the nitrogen-containing chain compound and application of the nitrogen-containing chain compound. The invention provides a nitrogen-containing chain compound shown in a formula I or pharmaceutically acceptable salt thereof. The nitrogen-containing chain compound shown in the formula I can be used for preparing lipid carriers. The lipid carrier can be used for encapsulating nucleic acid medicaments, and can be used for delivering nucleic acid preventive and/or therapeutic agents to mammalian cells and organs and playing roles.

Description

Nitrogen-containing chain compound, preparation method thereof, composition containing nitrogen-containing chain compound and application of nitrogen-containing chain compound

Technical Field

The present invention relates to nitrogen-containing chain compounds, methods of making, compositions comprising and uses thereof.

Background

Nucleic acid pharmaceuticals are an important direction of current basic and applied research. The nucleic acid medicine can be used for preventing and/or treating viral and bacterial infectious diseases, tumors, metabolic diseases and the like, has lower production cost and shorter period, and is favorable for rapidly developing personalized medicine. However, nucleic acids are negatively charged macromolecules, which are difficult to permeate cell membranes, and have poor stability, so that instability of nucleic acid drugs can be overcome to a certain extent by developing various nucleic acid packaging and delivery systems, and delivery efficiency of nucleic acid drugs can be improved.

Lipid nanoparticles have proven useful as carriers for delivering biologically active substances (e.g., small molecule drugs, proteins, and nucleic acids) into cells and/or intracellular compartments. Optimizing nucleic acid drug delivery systems by designing and optimizing the types and amounts of components in lipid nanoparticles is important for improving the efficacy of nucleic acid drug prophylaxis and therapy, particularly lipid compounds useful for delivering RNA prophylactic and/or therapeutic agents, and related methods and compositions.

Disclosure of Invention

The present invention aims to provide a novel ionizable lipid compound useful for delivering nucleic acid drugs, to increase the variety of ionizable lipid compounds and to select nucleic acid prophylactic and/or therapeutic agent delivery vehicles. In order to solve the technical problems, the invention provides a nitrogenous chain compound, a preparation method thereof, a composition containing the nitrogenous chain compound and application of the nitrogenous chain compound. The compositions of the invention are useful for the efficient delivery of nucleic acid drugs.

The technical scheme of the invention is as follows:

the invention provides a nitrogen-containing chain compound shown in a formula I or pharmaceutically acceptable salt thereof,

x is C ₁ -C ₆ Alkylene or C of (2) ₃ -C ₆ Is a cycloalkylene group of (2);

y being unsubstituted or substituted by 1, 2 or 3Y ^1-1 Substitution C ₁ -C ₁₀ An alkylene group of (a);

each Y ^1-1 Independently hydroxy, halogen or C ₁ -C ₆ Alkyl of (a);

z is unsubstituted or substituted by 1, 2 or 3Z ^1-1 Substitution C ₁ -C ₁₀ An alkylene group of (a);

each Z is ^1-1 Independently hydroxy, halogen or C ₁ -C ₆ Alkyl of (a);

A ¹ and A ² Independently is

R ¹ Unsubstituted or substituted by 1, 2 or 3R ^1-1 Substituted C ₁ -C ₂₀ Is optionally substituted or substituted by 1, 2 or 3R ^1-2 Substituted C ₂ -C ₂₀ Alkenyl of (c);

each R is ^1-1 Independently is hydroxy, C ₁ -C ₁₀ Unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl group of (C),

R ^1-1-1 Is C ₁ -C ₆ Alkyl of (a); r is R ^1-1-2 Is C ₁ -C ₆ Alkyl of (a); r is R ^1-1-3 Is C ₁ -C ₆ Alkyl of (a); each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^1-2 Independently is hydroxy or C ₁ -C ₁₀ Alkyl of (a);

R ² unsubstituted or substituted by 1, 2 or 3R ^2-1 Substituted C ₁ -C ₂₀ Is optionally substituted or substituted by 1, 2 or 3R ^2-2 Substituted C ₂ -C ₂₀ Alkenyl of (c);

each R is ^2-1 Independently is hydroxy, C ₁ -C ₁₀ Unsubstituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl group of (C),

R ^2-1-1 Is C ₁ -C ₆ Alkyl of (a); r is R ^2-1-2 Is C ₁ -C ₆ Alkyl of (a); r is R ^2-1-3 Is C ₁ -C ₆ Alkyl of (a); each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-2 Independently is hydroxy or C ₁ -C ₁₀ Alkyl of (a);

when X is C ₁ -C ₆ R is, at alkylene group ¹ For C substituted by 1, 2 or 3 hydroxy groups ₁ -C ₂₀ Unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted cyclopentane, unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted cyclic 12 alkanes,

In a preferred embodiment, in the nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof, certain groups may be defined as described below, and other groups may be defined as described in any of the other embodiments (hereinafter referred to as "preferred embodiment"): x, the C ₁ -C ₆ The alkylene group of (C) ₁ -C ₄ Alkylene groups of (C), preferably For example

In a preferred embodiment, X is the same as C ₃ -C ₆ May be cycloalkylene group For example->

In a preferred embodiment, Y is the same as C ₁ -C ₁₀ Sub-group of (2)Alkyl can be C ₄ -C ₆ Alkylene groups of (C), preferably For example

In a preferred embodiment, each Y ^1-1 In (2), the halogen may be fluorine, chlorine, bromine or iodine.

In a preferred embodiment, each Y ^1-1 In the above, the C ₁ -C ₆ The alkylene group of (2) may be

In a preferred embodiment, Z is the same as C ₁ -C ₁₀ The alkylene group of (C) ₅ -C ₈ Alkylene groups of (C), preferably For example

In a preferred embodiment, each Z ^1-1 In (2), the halogen may be fluorine, chlorine, bromine or iodine.

In a preferred embodiment, each Z ^1-1 In,the C is ₁ -C ₆ The alkylene group of (2) may be

In a preferred embodiment, R ¹ In the above, the C ₁ -C ₂₀ The alkyl group of (2) may be C ₁ -C ₁₅ Preferably a straight chain alkyl group such as ethyl, propyl, hexyl, octyl, nonyl or dodecyl.

In a preferred embodiment, R ¹ In the above, the C ₂ -C ₂₀ Alkenyl of (2) may be C ₆ -C ₁₈ Alkenyl groups of (a), preferably straight chain alkenyl groups; the C is ₂ -C ₂₀ The alkenyl groups of (2) may contain 1 to 4 double bonds.

In a preferred embodiment, each R ^1-1 In the above, the C ₁ -C ₁₀ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl, for example methyl, ethyl, isopropyl, t-butyl or n-butyl.

In a preferred embodiment, each R ^1-1 In the above, the C ₃ -C ₁₅ Cycloalkyl of (C) may be C ₃ -C ₁₂ Cycloalkyl of (C) is preferred ₄ -C ₈ Cycloalkyl or cyclo12alkyl, such as cyclopentyl, cyclohexyl or cyclo12alkyl.

In a preferred embodiment, R ^1-1-1 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, R ^1-1-2 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, R ^1-1-3 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, each R ^1-1-4 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, each R ^1-2 In the above, the C ₁ -C ₁₀ The alkyl group of (2) may be C ₁ -C ₆ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, R ² In the above, the C ₁ -C ₂₀ The alkyl group of (2) may be C ₁ -C ₁₅ Preferably a straight chain alkyl group such as ethyl, propyl, hexyl, octyl, nonyl or dodecyl.

In a preferred embodiment, R ² In the above, the C ₂ -C ₂₀ Alkenyl of (2) may be C ₆ -C ₁₈ Alkenyl groups of (a), preferably straight chain alkenyl groups; the C is ₂ -C ₂₀ The alkenyl groups of (2) may contain 1 to 4 double bonds.

In a preferred embodiment, each R ^2-1 In the above, the C ₁ -C ₁₀ The alkyl group of (2) may be C ₁ -C ₈ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl or isooctyl, for example methyl, ethyl, isopropyl, tert-butyl, n-butyl or n-octyl.

In a preferred embodiment, each R ^2-1 In the above, the C ₃ -C ₁₅ Cycloalkyl of (C) may be C ₃ -C ₁₂ Cycloalkyl of (C) is preferred ₄ -C ₈ Cycloalkyl or cyclo12alkyl, such as cyclopentyl, cyclohexyl or cyclo12alkyl.

In a certain preferred embodiment，R ^2-1-1 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, R ^2-1-2 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, R ^2-1-3 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, each R ^2-1-4 In the above, the C ₁ -C ₆ The alkyl group of (2) may be C ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, each R ^2-2 In the above, the C ₁ -C ₁₀ The alkyl group of (2) may be C ₁ -C ₆ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

In a preferred embodiment, the nitrogen-containing chain compound of formula I is a compound of formula Ia, ib, ic, id or Ie

In a preferred embodiment, A ¹ Is thatWherein a and R ¹ B is connected with Y, or a is connected with Y, b is connected with R ¹ Are connected.

In a preferred embodiment, A ² Is thatWherein a and R ² B is attached to Z, or a is attached to Z, b is attached to R ² 。

In a preferred embodiment, R ₁ And R is R ₂ The same applies.

In a preferred embodiment, X is C ₁ -C ₄ Alkylene or C of (2) ₃ -C ₆ Is a cycloalkylene group of (2);

y is C ₁ -C ₁₀ An alkylene group of (a);

z is C ₁ -C ₁₀ An alkylene group of (a);

A ¹ and A ² Independently is

R ¹ Unsubstituted or substituted by 1, 2 or 3R ^1-1 Substituted C ₁ -C ₂₀ Alkyl of (a);

each R is ^1-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl of (c);

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

R ² unsubstituted or substituted by 1, 2 or 3R ^2-1 Substituted C ₁ -C ₂₀ Alkyl of (a);

each R is ^2-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl of (c);

each R is ^2-1-4 Independently C ₁ -C ₆ Is a hydrocarbon group.

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

Each R is ^1-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

when X is C ₁ -C ₄ R is, at alkylene group ¹ For C substituted by 1, 2 or 3 hydroxy groups ₁ -C ₂₀ Unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted cyclopentanes,

In a preferred embodiment of the present invention,x is C ₁ -C ₄ An alkylene group of (a);

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

R ¹ for C substituted by 1, 2 or 3 hydroxy or cyclopentane ₁ -C ₁₅ Alkyl group of (C),

R ² Is 1, 2 or 3R ^2-1 Substituted C ₁ -C ₁₅ Alkyl of (a);

each R is ^2-1 Independently C ₁ -C ₁₀ Is a hydrocarbon group.

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-1 Independently is hydroxy, C ₁ -C ₈ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

In a preferred embodiment, X is C ₁ -C ₄ An alkylene group of (a);

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

R ² Is 1, 2 or 3R ^2-1 Substituted C ₁ -C ₁₅ Alkyl of (a);

each R is ^2-1 Independently C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

each R is ^2-1-4 Independently C ₁ -C ₆ Is a hydrocarbon group.

In a preferred embodiment, X is C ₁ -C ₄ Alkylene or C of (2) ₃ -C ₆ Is a cycloalkylene group of (d).

In a preferred embodiment, Y is C ₁ -C ₁₀ Alkylene groups of (a).

In a preferred embodiment, Z is C ₁ -C ₁₀ Alkylene groups of (a).

In a preferred embodiment, R ¹ Unsubstituted or substituted by 1, 2 or 3R ^1-1 Substituted C ₁ -C ₂₀ Is a hydrocarbon group.

In a preferred embodiment, each R ^1-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl groups of (a).

In a preferred embodiment, R ² Unsubstituted or substituted by 1, 2 or 3R ^2-1 Substituted C ₁ -C ₂₀ Alkyl of (2)。

In a preferred embodiment, each R ^2-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl groups of (a).

In a preferred embodiment, Y is C ₄ -C ₈ Alkylene groups of (a).

In a preferred embodiment, Z is C ₄ -C ₈ Alkylene groups of (a).

In a preferred embodiment, A ¹ Is thatWherein a and R ¹ And b is connected with Y.

In a preferred embodiment, A ² Is thatWherein a and R ² And b is connected with Z.

In a preferred embodiment, each R ^1-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₆ Cycloalkyl groups of (a).

In a preferred embodiment, each R ^2-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₆ Cycloalkyl groups of (a).

In a preferred embodiment, when X is C ₁ -C ₄ R is, at alkylene group ¹ For C substituted by 1, 2 or 3 hydroxy groups ₁ -C ₂₀ Unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted cyclopentanes,

One of themIn a preferred embodiment, X is

In a preferred embodiment, Y is

In a preferred embodiment, Z is

In a preferred embodiment, R ¹ Is that

In a preferred embodiment, R ² Is that

In a certain preferred embodiment, the nitrogen-containing chain compound shown in the formula I is any one of the following compounds:

/>

the invention also provides a preparation method of the nitrogen-containing chain compound shown in the formula I, which comprises the following steps: in a solvent, in the presence of alkali and iodized salt, carrying out a coupling reaction between a compound shown as a formula I-1 and a compound shown as a formula I-2;

m is halogen, X, Y, Z, A ¹ 、A ² 、R ¹ And R is ² As previously described.

In the coupling reaction, the halogen may be fluorine, chlorine, bromine or iodine, such as bromine.

In the coupling reaction, the molar ratio of the compound shown as the formula I-1 to the compound shown as the formula I-2 can be 1 (1-2), and the molar ratio is 1:1.2.

In the coupling reaction, the base is a conventional base in the art. The base may be a basic carbonate (the cation in the salt is an alkali metal ion and the anion is carbonate), e.g. K ₂ CO ₃ 。

In the coupling reaction, the molar ratio of the compound of formula I-1 to the base may be 1 (1-5), such as 1:3.5 or 1:2.

In the coupling reaction, the iodinated salt is a conventional iodinated salt in the art. The iodide salt may be a basic iodide salt, such as KI.

In the coupling reaction, the molar ratio of the compound of formula I-1 to the iodinated salt may be 1 (1-2), such as 1:1.2 or 1:1.

In the coupling reaction, the solvent is a solvent which is conventional in the art, and the solvent can be an ether solvent or/and a nitrile solvent. The ether solvent may be methyl tertiary butyl ether. The nitrile solvent may be acetonitrile. The volume ratio of the nitrile solvent to the ether solvent may be 1:1.

In the coupling reaction, the mass volume ratio of the compound shown as the formula I-1 to the solvent can be 10mg/mL-65mg/mL, such as 15mg/mL, 25mg/mL, 40mg/mL, 58.5mg/mL, 20mg/L, 22mg/L or 34mg/L.

In the coupling reaction, the reaction temperature of the coupling reaction is a reaction temperature conventional in the art, preferably 70℃to 90℃such as 80 ℃.

The invention also provides a lipid carrier, which comprises a substance Z, wherein the substance Z is a compound shown as a formula I or pharmaceutically acceptable salt thereof.

In a preferred embodiment, the lipid carrier further comprises a diluent.

In a preferred embodiment, the diluent may be phosphate buffer or Tris buffer.

In a preferred embodiment, the lipid carrier further comprises a phospholipid.

In a preferred embodiment, the phospholipid may be a phospholipid conventional in the art, which is an amphoteric helper molecule that aids in the fusion of the lipid particle and cell membrane. The phospholipids may be phospholipid molecules having charged polar and fatty chain non-polar ends, such as distearoyl phosphatidylcholine (DSPC), dimyristoyl phosphorylcholine (DMPC), dioleoyl phosphorylcholine (DOPC), palmitoyl phosphorylcholine (DPPC), 1, 2-distearoyl phosphorylcholine (DSPC), heneicosanoyl phosphorylcholine (DUPC), palmitoyl phosphorylcholine (POPC), or the like.

In a preferred embodiment, the lipid carrier further comprises a PEG lipid (polyethylene glycol modified lipid).

In a preferred embodiment, the PEG lipid may be a lipid molecule having a hydrophilic end modification of polyethylene glycol. The PEG lipid is preferably selected from one or more of PEG modified phosphatidylethanolamine, PEG modified phosphatidic acid, PEG modified ceramide, PEG modified dialkylamine, PEG modified diacylglycerol and PEG modified dialkylglycerol, such as PEG modified dimyristoylglycerol (DMG-PEG 2000) and the like.

In a preferred embodiment, the lipid carrier further comprises sterols.

In a preferred embodiment, the sterols may be sterols conventional in the art, including animal, vegetable, or fungus sterols. The sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid and alpha-tocopherol, such as cholesterol, etc.

In a preferred embodiment, the molar ratio of the substance Z to sterols in the lipid carrier is 0.5-5:1, preferably 0.5-3:1, for example 0.6-2:1.

In a preferred embodiment, the molar ratio of the substance Z to the phospholipid in the lipid carrier is 1-15:1, preferably 2-8:1, for example 3-6:1.

In a preferred embodiment, the molar ratio of the substance Z to PEG lipid in the lipid carrier is 20-130:1, preferably 20-80:1, for example 20-40:1.

In a preferred embodiment, the molar content of the substance Z is about 30mol% to 60mol%.

In the present invention, the molar content means that a content of a substance is a percentage of the total mass of the lipid carrier, and the sum of the molar contents of the components in the lipid carrier is not more than 100mol%. In a preferred embodiment, the phospholipid is present in an amount of about 0mol% to about 30mol%.

In a preferred embodiment, the sterol is present in an amount of about 15 mole% to about 55 mole%.

In a preferred embodiment, the PEG lipid is present in an amount of about 0mol% to about 10mol%.

In a preferred embodiment, said lipid carrier consists of said substance Z, said diluent, said phospholipid, said PEG lipid and said sterol.

The present invention also provides a lipid nanoparticle comprising a therapeutic and/or prophylactic agent and the aforementioned lipid carrier.

In a preferred embodiment, the therapeutic agent and/or prophylactic agent may be one or two or more nucleic acids. The nucleic acid may be a conventional nucleic acid in the art. The therapeutic and/or prophylactic agent may be single stranded deoxyribonucleic acid (DNA), double stranded DNA, small interfering RNA (siRNA), asymmetric double stranded small interfering RNA (aiRNA), microrna (miRNA), small hairpin RNA (shRNA), circular RNA (circRNA), transfer RNA (tRNA), messenger RNA (mRNA) and other forms of nucleic acid molecules known in the art, preferably mRNA, such as firefly luciferase (Fluc) mRNA or SARS-CoV-2 Spike protein (Spike) mRNA.

In a preferred embodiment, the ratio of nitrogen to phosphorus in the lipid nanoparticle may be from 2:1 to 30:1, and the ratio of nitrogen to phosphorus in the composition refers to the ratio of the moles of ionizable nitrogen atoms in the one or more ionizable lipid compounds to the moles of phosphate groups in the RNA. Preferably from 2:1 to 20:1, for example from 3:1 to 20:1, and also for example from 3:1 to 16:1.

In a preferred embodiment, the mass ratio of the lipid carrier to the therapeutic agent and/or prophylactic agent in the lipid nanoparticle may be 3 to 80:1, preferably 6 to 60:1.

In one embodiment, the lipid nanoparticle may have a particle size (average particle size) of 10 to 250nm, for example 40 to 250nm, further 50 to 250nm, or 40 to 150nm, or 60 to 150nm.

In a preferred embodiment, in the lipid nanoparticle, the lipid carrier encapsulates the therapeutic and/or prophylactic agent.

The present invention also provides a composition comprising a substance Z which is a compound of formula I as described above or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the composition further comprises one or more of a diluent, a phospholipid, a PEG lipid, a sterol, and a therapeutic and/or prophylactic agent.

In a preferred embodiment, the diluents, phospholipids, PEG lipids, sterols and therapeutic and/or prophylactic agents in the composition are as described above.

In a preferred embodiment, in the composition, the substance Z forms a lipid carrier with one or more of the diluents, phospholipids, PEG lipids and sterols as described above.

In a preferred embodiment, in the composition, the lipid carrier forms lipid nanoparticles as described above with the therapeutic and/or prophylactic agent. In a preferred embodiment, the encapsulation efficiency of the therapeutic and/or prophylactic agent in the composition is at least 50%, preferably at least 70%.

In a preferred embodiment, the composition has a polydispersity index of not more than 0.5, for example not more than 0.3.

Unless otherwise indicated, the terms used in the present invention have the following meanings:

the term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "pharmaceutically acceptable" refers to those compositions which are relatively non-toxic, safe, and suitable for use by a patient.

The term "pharmaceutically acceptable salt" refers to a salt of a compound that is obtained by reaction with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, the base addition salt may be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, bismuth salt, ammonium salt, and the like. When the compound contains a relatively basic functional group, the acid addition salt may be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to: hydrochloride, sulfate, mesylate, and the like. See for details Handbook of Pharmaceutical Salts Properties, selection, and Use (P.Heinrich Stahl, camill) G.Wermuth,2011,2ndRevisedEdition)。

In structural fragmentsIt is meant that the structural fragment is attached to the remainder of the molecule through this site. For example, a->Refers to cyclohexyl.

The "-" at the end of a group means that the group is attached to the remainder of the molecule through that site. For example, CH ₃ -C (=o) -means acetyl.

The term "alkyl" refers to a compound having a specified number of carbon atoms (e.g., C ₁ ～C ₆ ) Straight or branched, saturated monovalent hydrocarbon radicals. Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl and the like.

The term "alkylene" is a divalent group that is attached to the remainder of the molecule by two single bonds, the remainder being defined as the term "alkyl".

The term "cycloalkyl" refers to a compound having the indicated number of carbon atoms (e.g., C ₃ ～C ₆ ) Is composed of only carbon atomsA saturated monocyclic cyclic group. Cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

The term "alkenyl" refers to a straight or branched hydrocarbon group containing one or more carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds).

When any variable (e.g. group R ^1-1 ) In the definition of a compound, the definition of the compound is independent of each other and does not affect each other when the compound appears for a plurality of times. For example, by 3R ^1-1 Substituted C ₆ ～C ₁₀ Aryl means C ₆ ～C ₁₀ Aryl will be substituted with 3R ^1-1 Substituted, 3R ^1-1 Is independent of each other and does not affect each other.

The above preferred conditions can be arbitrarily combined to obtain the preferred examples of the present invention without departing from the common sense in the art

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: the invention provides a nitrogenous chain compound shown in a formula I, which has a novel structure and can be used for preparing lipid nano particles. Lipid nanoparticles comprising nitrogen-containing chain compounds of formula I have a low polydispersity index and allow efficient transport of mRNA.

Drawings

FIG. 1 is an electrophoresis chart of each of the Fluc-mRNA LNP gels prepared in example 23.

FIG. 2 shows the chemiluminescent intensities of 293FT cells in example 25 after 18-24 hours of co-cultivation with each of the Fluc-mRNA LNPs.

FIG. 3 shows the expression of Spike protein in example 25 after 24h co-culture of 293FT cells with each Spike-mRNA LNP, PBS group as a negative control.

Fig. 4 shows total in vivo bioluminescence (n=3) of mice in example 26 after various times of intravenous administration of each Fluc-mRNA LNP, PBS group as negative control (n=2).

Fig. 5 shows total in vivo antibody titers of mice of example 26 after intramuscular injection of Spike-mRNA LNP (1 week after the last dose, n=8).

Fig. 6 is the total in vivo bioluminescence (n=3) of example 27 following intravenous administration of each Fluc mRNA LNP for various times in mice.

Detailed Description

The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The reagent sources of the embodiment of the application are as follows:

8-bromooctanoic acid: purchased from Jiangsu Aikang, purity: 98 percent;

heptadec-9-ol: purchased from beijing foci source mill technologies, inc, purity: 98 percent;

DCC: dicyclohexylcarbodiimide, available from hadamard reagents limited, cat# 012041444, purity: RG,99%;

DMAP: 4-dimethylaminopyridine, available from adamas reagent limited under the designation: 01271081, purity: RG,99%;

DCM: dichloromethane, available from Shanghai Taitan technologies, inc., cat No.: 01111853, purity: AR is more than or equal to 99.5 percent;

ethanolamine: purchased from Shanghai Yi En chemical technologies Co., ltd., product number: r016710, purity: AR,99%;

acetonitrile: purchased from Shanghai Taitan technologies, inc., cat No.: 01111797, purity: AR is more than or equal to 99.0 percent;

6-bromohexanoic acid: available from hadamard reagents, inc., cat No.: 01073739, purity: RG,98% +;

3, 7-dimethyl-1-octanol: purchased from Annaiji chemistry, cat: a040472, purity: 98 percent;

K ₂ CO ₃ : purchased from Shanghai Yi En chemical technologies Co., ltd., product number: RH425011, purity: AR,99%;

KI: purchased from Shanghai Yi En chemical technologies Co., ltd., product number: RH432132, purity: AR,99%;

methyl tertiary butyl ether: purchased from Shanghai Taitan technologies, inc., cat No.: 01030342, purity: AR is more than or equal to 99.0 percent;

2-cyclohexylethanol: purchased from Shanghai Ala Biotechnology Co., ltd., product number: c153306, purity: GC, > 98.0%;

isodecyl alcohol: purchased from Shanghai Ala Biotechnology Co., ltd., product number: i298943, purity: 98 percent;

diatomaceous earth: purchased from Shanghai Taitan technologies, inc., cat No.: 01589000, purity: extra-high grade, more than or equal to 89.0 percent and 200 meshes;

ethyl acetate: purchased from Shanghai Taitan technologies, inc., cat No.: 01153552, purity: AR is more than or equal to 99.5 percent;

3, 5-trimethyl-1-hexanol: purchased from Shanghai Ala Biotechnology Co., ltd., product number: t162750, purity: GC, > 85.0%;

2-ethylhexanol: purchased from Shanghai Yi En chemical technologies Co., ltd., product number: r016621, purity: AR,99%;

anhydrous sodium sulfate: purchased from Shanghai Taitan technologies, inc., cat No.: 01224581, purity: AR is more than or equal to 99.0 percent;

ferric trichloride: purchased from Shanghai Meilin Biochemical technologies Co., ltd., product number: i811935, purity: AR,99%;

pyridine: purchased from Shanghai Meilin Biochemical technologies Co., ltd., product number: p816288, purity: AR,99%;

1, 2-epoxydodecane: purchased from Shanghai Meilin Biochemical technologies Co., ltd., product number: e808874, purity: 95%;

3-cyclopentyl-1-propanol: purchased from Shanghai Bi De medical science and technology Co., ltd., product number: BD86403, purity: 98 percent;

cyclohexyl methanol: purchased from Shanghai Yi En chemical technologies Co., ltd., product number: r014045, purity: 99 percent;

isononanol: purchased from guangdong Weng Jiang chemical company, inc;

8-methyl-1-decanol: purchased from south Beijing list biotechnology limited, purity: 97%;

3-butyl-1-heptanol: purity from meno pharmaceutical technologies, inc. In su zhou: 95%.

EXAMPLE 1 preparation of Compound LQ001

Step 1: preparation of LQ001-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.1eq	24.5g	110
Heptadec-9-ol	256	1eq	25.6g	100
					DCC	206	2eq	41.2g	200
DMAP	122	0.1eq	1.22g	10
					DCM	-	-	500mL	-

The operation process comprises the following steps:

8-bromooctanoic acid, heptadec-9-ol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction.

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 40g of a colorless oil with a yield of 85%.

¹ HNMR(500MHz，CDCl ₃ )δ：4.87(p，1H)，3.40(t，2H)，2.28(t，2H)，1.85(p，2H)，1.63(p，2H)，1.54-1.47(m，4H)，1.43(dt，2H)，1.33(dt，4H)，1.27(d，24H)，0.88(t，6H)。

Step 2: preparation of LQ001-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-1	460	1eq	30g	65.2
Ethanolamine	61	10eq	40g	652
					Acetonitrile	-	-	400mL	-

The operation process comprises the following steps:

LQ001-1, ethanolamine and acetonitrile are added into a reaction bottle, heated to 30 ℃ and stirred for reaction for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 500mL of ethyl acetate, washed with 500mL of water for 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by purification by column chromatography to give 25g of a colorless oil in 87% yield.

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，3.65-3.61(m，2H)，2.80-2.75(m，2H)，2.61(t，2H)，2.27(t，2H)，1.62(p，3H)，1.48(dt，7H)，1.32(s，7H)，1.27(d，22H)，0.87(t，6H)。

Step 3: preparation of LQ001-3

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
3, 7-dimethyl-1-octanol	158	1eq	1.58g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

6-bromohexanoic acid, 3, 7-dimethyl-1-octanol, DCC, DMAP and DCM are added into a reaction bottle, and the reaction is stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was filtered through celite and dried by spin-drying, followed by purification by column chromatography to give 2.8g of a colorless oil with a yield of 83.6%.

¹ HNMR(400MHz，CDCl ₃ )δ：4.16-4.05(m，2H)，3.41(t，2H)，2.31(t，2H)，1.96-1.84(m，4H)，1.73-1.60(m，4H)，1.53-1.42(m，4H)，1.35-1.23(m，6H)，1.17-1.12(m，2H)，0.88(dd，9H)。

Step 4: preparation of LQ001

The material ratio is as follows:

/>

the operation process comprises the following steps:

adding LQ001-2, LQ001-3 and K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed the reaction was complete.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 2g of a colorless oil in 77% yield.

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，4.15-4.04(m，2H)，3.64(t，2H)，2.71(t，2H)，2.59(q，4H)，2.29(dt，4H)，1.64(tt，5H)，1.53(dq，10H)，1.42(dq，2H)，1.28(d，35H)，1.16-1.09(m，3H)，0.88(dd，15H)。

LCMS：Rt：4.991min；MS m/z(ESI)：697.1[M+H] ⁺ 。

EXAMPLE 2 preparation of Compound LQ002

Step 1: preparation of LQ002-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.1eq	2.5g	11
3, 7-dimethyl-1-octanol	158	1eq	1.58g	10
					DCC	206	2eq	4.1g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

8-bromooctanoic acid, 3, 7-dimethyl-1-octanol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction.

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 2g of a colorless oil with a yield of 55%.

¹ HNMR(500MHz，CDCl ₃ )δ：4.17-4.03(m，2H)，3.40(t，2H)，2.29(t，2H)，1.85(dt，2H)，1.69-1.58(m，3H)，1.51(dt，2H)，1.42(dq，3H)，1.33(dt，4H)，1.30-1.19(m，3H)，1.17--1.07(m，3H)，0.88(dd，9H)。

Step 2: preparation of LQ002-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ002-1	363	1eq	1.4g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ002-1, ethanolamine and acetonitrile are added into a reaction bottle, and the reaction is stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 100mL of ethyl acetate, washed with 100mL of water for 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by purification by column chromatography to give 750mg of a colorless oil in a yield of 57.8%.

TLC case: DCM, meoh=10:1, rf=0.4.

Step 3: preparation of LQ002

The material ratio is as follows:

the operation process comprises the following steps:

adding LQ002-2, LQ001-3 and K into a reaction bottle ₂ CO ₃ Heating KI, methyl tertiary butyl ether and acetonitrile to 80 ℃ and stirring for reactionShould be 12h. TLC (DCM: meoh=10:1) showed the reaction was complete.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil in 76% yield.

¹ HNMR(400MHz，CDCl ₃ )δ：4.09(q，4H)，3.58(t，2H)，2.64(t，2H)，2.56-2.48(m，4H)，2.32-2.25(m，4H)，1.69-1.57(m，6H)，1.51(dt，7H)，1.42(dt，4H)，1.34-1.20(m，14H)，1.18-1.08(m，6H)，0.87(dd，18H)。

LCMS：Rt：2.429min；MS m/z(ESI)：598.9[M+H] ⁺ 。

EXAMPLE 3 preparation of Compound LQ004

Step 1: preparation of LQ004-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
2-Cyclohexylethanol	128	1eq	1.28g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

6-bromohexanoic acid, 2-cyclohexylethanol, DCC, DMAP and DCM were added to the reaction flask, and the reaction was stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 2g of a colorless oil.

TLC case: EA, pe=20:1, rf=0.6.

Step 2: preparation of LQ004

The material ratio is as follows:

Material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.6g	3.75
LQ004-1	305	1.2eq	1.35g	4.5
					K ₂ CO ₃	138	3.5eq	1.8g	13
KI	166	1.2eq	750mg	4.5
					Acetonitrile	-	-	20mL	-
Methyl tert-butyl ether	-	-	20mL	-

The operation process comprises the following steps:

adding LQ001-2, LQ004-1, K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed the reaction was complete.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 2g of a colorless oil in 80% yield.

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，4.10(t，2H)，3.64-3.58(m，2H)，2.67(s，2H)，2.55(s，4H)，2.29(dt，4H)，1.65(dddd，11H)，1.51(q，11H)，1.32-1.23(m，33H)，0.88(t，9H)。

LCMS：Rt：5.913min；MS m/z(ESI)：566.9[M+H] ⁺ 。

EXAMPLE 4 preparation of Compound LQ005

Step 1: preparation of LQ005-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.1eq	2.5g	11
2-Cyclohexylethanol	128	1eq	1.28g	10
					DCC	206	2eq	4.1g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

8-bromooctanoic acid, 2-cyclohexylethanol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction.

Post-treatment:

TLC case: EA, pe=20:1, rf=0.6.

Step 2: preparation of LQ005-2

/>

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ005-1	363	1eq	1.4g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ005-1, ethanolamine and acetonitrile are added into a reaction bottle, and the mixture is stirred at room temperature for reaction for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

The reaction mixture was dried by spin-drying, diluted with 100ml of ethyl acetate, washed with 100ml of water 2 times, and the organic phase was dried over anhydrous sodium sulfate, and purified by column chromatography to give 750mg of a colorless oil in a yield of 57.8%.

TLC case: DCM, meoh=10:1, rf=0.4.

Step 3: preparation of LQ005

The material ratio is as follows:

the operation process comprises the following steps:

adding LQ005-2, LQ004-1, K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(400MHz，CDCl ₃ )δ：4.09(t，4H)，3.67-3.60(m，2H)，2.75-2.68(m，2H)，2.65-2.54(m，4H)，2.29(q，4H)，1.73-1.58(m，15H)，1.51(q，6H)，1.40-1.08(m，18H)，0.97-0.86(m，4H)。

LCMS：Rt：0.086min；MS m/z(ESI)：538.7[M+H] ⁺ 。

EXAMPLE 5 preparation of Compound LQ007

Step 1: preparation of LQ007-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
Isobdecanol	158	1eq	1.58g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

6-Bromohexanoic acid, isodecanol, DCC, DMAP and DCM were added to the reaction flask and the reaction was stirred at room temperature for 12h. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

TLC case: EA, pe=20:1, product rf=0.6.

Step 2: preparation of LQ007

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.6g	3.75
LQ007-1	335	1.2eq	1.35g	4.5
					K ₂ CO ₃	138	3.5eq	1.8g	13
KI	166	1.2eq	750mg	4.5
					Acetonitrile	-	-	20mL	-
Methyl tert-butyl ether	-	-	20mL	-

The operation process comprises the following steps:

LQ001-2, LQ007-1 and K are added into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed the reaction was complete.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，4.14-4.02(m，2H)，3.56(t，2H)，2.61(t，2H)，2.48(q，4H)，2.32-2.25(m，4H)，1.68-1.58(m，6H)，1.56-1.40(m，10H)，1.36-1.21(m，38H)，1.19-1.03(m，4H)，0.87(t，12H)。

LCMS：Rt：3.83min；MS m/z(ESI)：696.7[M+H] ⁺ 。

EXAMPLE 6 preparation of Compound LQ008

Step 1: preparation of LQ008-1

The material ratio is as follows:

the operation process comprises the following steps:

the reaction flask was charged with 8-bromooctanoic acid, isodecyl alcohol, DCC, DMAP and DCM and stirred at room temperature for 12h, tlc (EA: pe=20:1) indicating completion of the reaction.

Post-treatment:

Step 2: preparation of LQ008-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding materialMeasuring amount	mmol
					LQ008-1	363	1eq	1.4g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ008-1, ethanolamine and acetonitrile are added into a reaction bottle, and the reaction is stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 100ml of ethyl acetate, washed with 100ml of water 2 times, and the organic phase was dried over anhydrous sodium sulfate, and purified by column chromatography to give 750mg of a colorless oil in 57.8% yield.

¹ HNMR(500MHz，CDCl ₃ )δ：4.16-3.99(m，2H)，3.69-3.60(m，2H)，2.85-2.73(m，2H)，2.63(t，2H)，2.28(td，2H)，2.06(s，4H)，1.66-1.57(m，3H)，1.49(q，2H)，1.35-1.24(m，2H)，1.16-1.05(m，2H)，0.85(tq，8H)。

Step 3: preparation of LQ008

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ008-2	343	1eq	750mg	2.2
LQ007-1	335	1.2eq	884mg	2.64
					K ₂ CO ₃	138	3.5eq	1.06g	7.7
KI	166	1.2eq	440mg	2.64
					Acetonitrile	-	-	15mL	-
Methyl tert-butyl ether	-	-	15mL	-

The operation process comprises the following steps:

LQ008-2, LQ007-1 and K are added into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.17-4.01(m，3H)，3.60(t，2H)，2.66(s，2H)，2.54(d，4H)，2.29(qd，4H)，1.75-1.44(m，13H)，1.30(d，18H)，1.19-0.97(m，5H)，0.94-0.71(m，18H)。

LCMS：Rt：4.452min；MS m/z(ESI)：598.9[M+H] ⁺ 。

EXAMPLE 7 preparation of Compound LQ010

Step 1: preparation of LQ010-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
3, 5-trimethyl-1-hexanol	144	1eq	1.44g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

6-bromohexanoic acid, 3, 5-trimethyl-1-hexanol, DCC, DMAP and DCM were added to the reaction flask, and the reaction was stirred at room temperature for 12h. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

TLC case: EA, pe=20:1, rf=0.6.

Step 2: preparation of LQ010

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.6g	3.75
LQ010-1	321	1.2eq	1.45g	4.5
					K ₂ CO ₃	138	3.5eq	1.8g	13
KI	166	1.2eq	750mg	4.5
					Acetonitrile	-	-	20mL	-
Methyl tert-butyl ether	-	-	20mL	-

The operation process comprises the following steps:

adding LQ001-2, LQ010-1 and K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.85(p，1H)，4.12-4.03(m，2H)，3.72(t，2H)，2.84-2.79(m，2H)，2.74-2.67(m，4H)，2.29(dt，4H)，1.67-1.57(m，10H)，1.47(dd，6H)，1.38-1.23(m，33H)，1.07(dd，1H)，0.94(d，3H)，0.88(t，15H)。

LCMS：Rt：3.83min；MS m/z(ESI)：682.7[M+H] ⁺ 。

Example 8 preparation of Compound LQ011

Step 1: preparation of LQ011-1

The material ratio is as follows:

the operation process comprises the following steps:

8-bromooctanoic acid, 3, 5-trimethyl-1-hexanol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction.

Post-treatment:

the reaction solution was filtered with celite and then dried by spin-drying, followed by purification by column chromatography to give 2g of a colorless oil.

TLC case: EA, pe=20:1, product rf=0.6.

Step 2: preparation of LQ011-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ011-1	349	1eq	1.4g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ011-1, ethanolamine and acetonitrile are added into a reaction bottle, and the mixture is stirred at room temperature for reaction for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.08(t，2H)，3.64(t，2H)，2.78(t，2H)，2.62(t，2H)，2.28(t，4H)，1.66-1.56(m，4H)，1.47(dt，3H)，1.31(s，6H)，1.22(dd，1H)，1.07(dd，1H)，0.94(d，3H)，0.88(s，9H)。

Step 3: preparation of LQ011

The material ratio is as follows:

The operation process comprises the following steps:

LQ008-2, LQ007-1 and K are added into a reaction bottle ₂ CO ₃ KI, methyl t-butylThe ether and acetonitrile are heated to 80 ℃ and stirred to react for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.07(t，4H)，3.60(t，2H)，2.67(t，2H)，2.54(q，4H)，2.28(q，4H)，1.61(ddd，8H)，1.54-1.43(m，6H)，1.30(q，8H)，1.22(dd，2H)，1.07(dd，2H)，0.93(d，6H)，0.88(t，18H)。

LCMS：Rt：1.609min；MS m/z(ESI)：570.5[M] ⁺ 。

EXAMPLE 9 preparation of Compound LQ013

Step 1: preparation of LQ013-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
2-ethylhexanol	130	1eq	1.3g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

6-bromohexanoic acid, 2-ethylhexanol, DCC, DMAP and DCM were added to the reaction flask, and the reaction was stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

TLC case: EA, pe=20:1, product rf=0.6.

Step 2: preparation of LQ013

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.6g	3.75
LQ013-1	307	1.2eq	1.35g	4.5
					K ₂ CO ₃	138	3.5eq	1.8g	13
KI	166	1.2eq	750mg	4.5
					Acetonitrile	--	-	20mL	-
Methyl tert-butyl ether	-	-	20mL	-

The operation process comprises the following steps:

adding LQ001-2, LQ013-1, K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.92-4.85(m，1H)，4.01(m，2H)，3.72(s，2H)，2.81(s，2H)，2.70(s，4H)，2.37-2.28(m，4H)，1.73-1.49(m，15H)，1.38-1.26(m，39H)，0.91(td，12H)。

EXAMPLE 10 preparation of Compound LQ014

Step 1: preparation of LQ014-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.1eq	2.5g	11
2-ethylhexanol	130	1eq	1.3g	10
					DCC	206	2eq	4.1g	20
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

the reaction flask was charged with 8-bromooctanoic acid, 2-ethylhexanol, DCC, DMAP and DCM and stirred at room temperature for 12h, tlc (EA: pe=20:1) indicated completion.

Post-treatment:

Step 2: preparation of LQ014-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ014-1	335	1eq	1.4g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ014-1, ethanolamine and acetonitrile were added to the reaction flask, and the mixture was stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 100ml of ethyl acetate, washed with 100ml of water 2 times, and the organic phase was dried over anhydrous sodium sulfate, and purified by column chromatography to give 750mg of a colorless oil in 55% yield.

TLC case: DCM, meoh=10:1, product rf=0.2.

Step 3: preparation of LQ014

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ014-2	315	1eq	750mg	2.2
LQ013-1	307	1.2eq	884mg	2.64
					K ₂ CO ₃	138	3.5eq	1.06g	7.7
KI	166	1.2eq	440mg	2.64
					Acetonitrile	-	-	15mL	-
Methyl tert-butyl ether	-	-	15mL	-

The operation process comprises the following steps:

adding LQ014-2, LQ013-1, K into a reaction flask ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil in 73% yield.

¹ HNMR(500MHz，CDCl ₃ )δ：4.03-3.93(m，4H)，3.65-3.55(m，2H)，2.66(s，2H)，2.53(s，4H)，2.30(td，4H)，1.69-1.47(m，11H)，1.39-1.26(m，24H)，0.94-0.85(m，12H)。

EXAMPLE 11 preparation of Compound LQ016

Step 1: preparation of LQ016-1

The material ratio is as follows:

the operation process comprises the following steps:

6-bromohexanoic acid, 1, 2-epoxydodecane, ferric trichloride and pyridine are added into a reaction bottle, and the mixture is stirred at room temperature for reaction for 12 hours. TLC (EA: pe=4:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was purified by column chromatography to give 1.4g of a product.

TLC case: EA, pe=4:1, product rf=0.5.

Step 2: preparation of LQ016

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.17g	2.64
LQ016-1	379	1.2eq	1.2g	3.16
					K ₂ CO ₃	138	3.5eq	1.28g	9.24
KI	166	1.2eq	440mg	2.64
					Acetonitrile	-	-	10mL	-
Methyl tert-butyl ether	-	-	10mL	-

The operation process comprises the following steps:

LQ001-2, LQ016-1 and K are added into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 900g of a colorless oil.

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，4.15(dd，1H)，3.94(dd，1H)，3.87-3.80(m，1H)，3.65-3.58(m，2H)，2.67(s，2H)，2.56(s，4H)，2.36(td，2H)，2.28(t，2H)，1.71-1.43(m，16H)，1.27(d，46H)，0.87(t，9H)。

LCMS：Rt：3.83min；MS m/z(ESI)：740.7[M] ⁺ 。

EXAMPLE 12 preparation of Compound LQ017

Step 1: preparation of LQ017-1

The material ratio is as follows:

Material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.05eq	2.53g	11.39
1, 2-epoxydodecane	184	1eq	2g	10.85
					Ferric trichloride	162	0.025eq	44mg	0.3
Pyridine compound	79	0.028eq	23mL	0.3

The operation process comprises the following steps:

8-bromooctanoic acid, 1, 2-epoxydodecane, ferric trichloride and pyridine were added into the reaction flask, and the reaction was stirred at room temperature for 12h, and TLC (EA: PE=4:1) showed that the reaction was complete.

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil in 25% yield.

¹ HNMR(400MHz，CDCl ₃ )δ：4.91(ddt，1H)，3.77-3.56(m，2H)，3.40(t，2H)，2.34(t，2H)，1.85(dt，3H)，1.68-1.52(m，6H)，1.43(dd，2H)，1.34(dt，5H)，1.30-1.23(m，13H)，0.88(t，3H)。

Step 2: preparation of LQ017-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ017-1	407	1eq	1.2g	3.86
Ethanolamine	61	10eq	2.4g	38.6
					Acetonitrile	-	-	40mL	-

The operation process comprises the following steps:

LQ017-1, ethanolamine and acetonitrile are added into a reaction bottle, and the reaction is stirred at room temperature for 2d. TLC (EA: pe=4:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 100mL of ethyl acetate, washed with 100mL of water for 2 times, and the organic phase was dried over anhydrous sodium sulfate, and purified by column chromatography to give 300mg of a colorless oil in a yield of 32.8%.

¹ HNMR(400MHz，CDCl ₃ )δ：4.15(dd，1H)，3.94(dd，1H)，3.87-3.79(m，1H)，3.71-3.66(m，2H)，2.85-2.79(m，2H)，2.66(t，2H)，2.35(t，2H)，1.70-1.40(m，8H)，1.29(d，22H)，0.88(t，3H)。

Step 3: preparation of LQ017

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ017-2	387	1eq	300mg	0.774
LQ016-1	379	1.2eq	355mg	0.93
					K ₂ CO ₃	138	3.5eq	374g	2.71
KI	166	1.2eq	154mg	0.93
					Acetonitrile	-	-	10mL	-
Methyl tert-butyl ether	-	-	10mL	-

The operation process comprises the following steps:

LQ017-2, LQ016-1 and K are added into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 160mg of a colorless oil in 76% yield.

¹ HNMR(400MHz，CDCl ₃ )δ：4.14(dd，2H)，4.07-4.02(m，2H)，3.96(ddd，2H)，3.89-3.81(m，2H)，3.25-3.19(m，2H)，3.17-3.09(m，4H)，2.38(dt，4H)，1.94-1.79(m，5H)，1.77-1.58(m，6H)，1.52-1.41(m，8H)，1.39-1.24(m，36H)，0.87(t，6H)。

EXAMPLE 13 preparation of Compound LQ025

Step 1: preparation of LQ025-1

Material proportioning

The operation process comprises the following steps:

6-bromohexanoic acid, 3-cyclopentyl-1-propanol, DCC, DMAP and DCM were added into the reaction flask, and the reaction was stirred at room temperature for 12h. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was filtered through celite and dried by spin-drying, followed by purification by column chromatography to give 2.8g of a colorless oil.

TLC case: EA, pe=20:1, product rf=0.6.

Step 2: preparation of LQ025

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.6g	3.75
LQ025-1	305	1.2eq	1.35g	4.5
					K ₂ CO ₃	138	3.5eq	1.8g	13
KI	166	1.2eq	750mg	4.5
					Acetonitrile	-	-	20mL	-
Methyl tert-butyl ether	-	-	20mL	-

The operation process comprises the following steps:

adding LQ001-2, LQ025-1 and K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction, product rf=0.4.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.86(p，1H)，4.05(t，2H)，3.58(t，2H)，2.64(t，2H)，2.55-2.46(m，4H)，2.29(dt，4H)，1.75(td，3H)，1.69-1.56(m，8H)，1.55-1.43(m，10H)，1.37-1.28(m，10H)，1.25(s，23H)，1.06(d，3H)，0.87(t，6H)。

LCMS：Rt：3.83min；MS m/z(ESI)：666.7[M+H] ⁺ 。

EXAMPLE 14 preparation of Compound LQ026

Step 1: preparation of LQ026-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	223	1.1eq	1.25g	5.5
3-cyclopentyl-1-propanol	128	1eq	0.64g	5
					DCC	206	2eq	2.1g	10
DMAP	122	0.1eq	122mg	1
					DCM	-	-	50mL	-

The operation process comprises the following steps:

8-bromooctanoic acid, 3-cyclopentyl-1-propanol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction.

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 1.2g of a colorless oil in 65% yield.

TLC case: EA, pe=20:1, product rf=0.6.

Step 2: preparation of LQ026-2

The material ratio is as follows:

the operation process comprises the following steps:

LQ026-1, ethanolamine and acetonitrile are added into a reaction bottle, and the mixture is stirred at room temperature for reaction for 12 hours. TLC (EA: pe=20:1) showed the reaction was complete.

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 100mL of ethyl acetate, washed with 100mL of water for 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil in a yield of 87.8%.

TLC case: DCM, meoh=10:1, product rf=0.2.

Step 3: preparation of LQ026

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ026-2	313	1eq	750mg	3.8
LQ025-1	305	1.2eq	884mg	4.56
					K ₂ CO ₃	138	3.5eq	1.06g	7.7
KI	166	1.2eq	440mg	2.64
					Acetonitrile	-	-	15mL	-
Methyl tert-butyl ether	-	-	15mL	-

The operation process comprises the following steps:

adding LQ026-2, LQ025-1, K into a reaction bottle ₂ CO ₃ KI, methyl tert-butyl ether and acetonitrile, heating to 80 ℃ and stirring for reaction for 12h. TLC (DCM: meoh=10:1) showed the reaction was complete.

Post-treatment:

¹ HNMR(500MHz，CDCl ₃ )δ：4.04(t，4H)，3.58(t，2H)，2.63(t，2H)，2.55-2.47(m，4H)，2.29(td，4H)，1.79-1.70(m，6H)，1.61(ddt，12H)，1.54-1.42(m，8H)，1.37-1.25(m，12H)，1.11-1.01(m，4H)。

LCMS：Rt：3.85min；MS m/z(ESI)：538.5[M+H] ⁺ 。

EXAMPLE 15 preparation of Compound LQ031

Step 1: preparation of LQ031-1

Material proportioning

Material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.1eq	8.6g	44
Cyclohexyl methanol	114	1eq	4.56g	40
					DCC	206	2eq	16.5g	80
DMAP	122	0.1eq	488mg	4
					DCM			150mL

The operation process comprises the following steps:

6-bromohexanoic acid, cyclohexylmethanol, DCC, DMAP and DCM were added to the reaction flask and stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed complete reaction (product Rf value of 0.5).

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 8.6g of a colorless oil in 69% yield.

H NMR(600MHz,CHCl ₃ -d)δ3.88(d,J＝6.6Hz,2H),3.40(t,J＝6.8Hz,2H),2.32(t,J＝7.5Hz,2H),1.88(p,J＝6.9Hz,2H),1.72(d,J＝10.4Hz,4H),1.66(dt,J＝15.5,7.6Hz,4H),1.48(p,J＝7.7Hz,2H),1.29–1.12(m,3H),1.00–0.92(m,2H).

Step 2: preparation of LQ031-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ031-1	291	1eq	8.6g	30
Ethanolamine	61	10eq	18g	300
					Acetonitrile			200mL

The operation process comprises the following steps:

LQ031-1, ethanolamine and acetonitrile are added into a reaction bottle, heated to 30 ℃ and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.3).

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 500ml of ethyl acetate, washed with 500ml of water 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by purification by column chromatography to give 5.6g of a colorless oil. The yield thereof was found to be 69%.

¹ H NMR(600MHz,CHCl ₃ -d)δ3.87(d,J＝6.6Hz,2H),3.65–3.62(m,2H),2.79–2.74(m,2H),2.65–2.60(m,2H),2.30(t,J＝7.5Hz,2H),1.71(d,J＝10.5Hz,4H),1.68–1.58(m,4H),1.51(p,J＝7.4Hz,2H),1.39–1.32(m,2H),1.28–1.11(m,3H),0.99–0.91(m,2H).

Step 3: preparation of LQ031

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ031-2	271	1eq	1g	3.69
LQ001-1	461	1.2eq	2g	4.4
					K ₂ CO ₃	138	2eq	1g	7.4
KI	166	1eq	610mg	3.69
					Acetonitrile			50mL

The operation process comprises the following steps:

adding LQ031-2, LQ001-1, K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.5).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1.55g of a colorless oil. The yield thereof was found to be 64%.

¹ H NMR(600MHz,CHCl ₃ -d)δ4.86(p,J＝6.8,6.3Hz,1H),3.88(d,J＝6.6Hz,2H),3.56(t,J＝4.9Hz,2H),2.64–2.58(m,2H),2.49(q,J＝6.9Hz,4H),2.29(dt,J＝19.0,7.5Hz,4H),1.72(d,J＝10.2Hz,4H),1.64(tt,J＝14.7,7.2Hz,6H),1.48(dd,J＝21.6,6.1Hz,8H),1.35–1.20(m,34H),1.20–1.14(m,1H),1.00–0.92(m,2H),0.87(t,J＝7.1Hz,6H).

EXAMPLE 16 preparation of Compound LQ032

Step 1: preparation of LQ032-1

The material ratio is as follows:

the operation process comprises the following steps:

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 9g of a colorless oil in 70% yield.

Step 2: preparation of LQ032

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ031-2	271	1eq	1g	3.69
LQ032-1	319	1.2eq	1.4g	4.4
					K ₂ CO ₃	138	2eq	1g	7.4
KI	166	1eq	610mg	3.69
					Acetonitrile			50mL

The operation process comprises the following steps:

adding LQ031-2, LQ032-1, K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.45).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil. The yield thereof was found to be 53%.

¹ H NMR(600MHz,CHCl ₃ -d)δ3.87(d,J＝6.6Hz,4H),3.70–3.64(m,2H),2.76(s,2H),2.69–2.60(m,4H),2.30(dt,J＝11.1,7.5Hz,4H),1.75–1.52(m,20H),1.37–1.13(m,15H),1.00–0.91(m,4H).

EXAMPLE 17 preparation of Compound LQ067

Step 1: preparation of LQ067-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
Isononol	144	1eq	1.44g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM			50mL

The operation process comprises the following steps:

6-bromohexanoic acid, isononanol, DCC, DMAP and DCM were added into the reaction flask, and the reaction was stirred at room temperature for 12h. TLC (EA: pe=20:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction mixture was filtered through celite and dried by spin-drying, followed by purification by column chromatography to give 2.4g of a colorless oil.

Step 2: preparation of LQ067

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.36g	3.08
LQ067-1	321	1.2eq	1.2g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			40mL

The operation process comprises the following steps:

adding LQ001-2, LQ067-1 and K into a reaction bottle ₂ CO ₃ KI andacetonitrile, heating to 80 ℃ and stirring to react for 12h. TLC (DCM: meoh=10:1) showed complete reaction (resulting in Rf value of 0.6).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1.5g of a colorless oil. The yield thereof was found to be 71%.

¹ H NMR(600MHz,CHCl ₃ -d)δ4.84(p,J＝6.3Hz,1H),4.06(t,J＝6.6Hz,2H),3.66(t,J＝5.1Hz,2H),2.76(t,J＝5.0Hz,2H),2.64(q,J＝7.8Hz,4H),2.27(dt,J＝17.6,7.4Hz,4H),1.55(ddtd,J＝53.6,28.1,14.1,13.1,7.0Hz,16H),1.36–1.17(m,35H),0.92(d,J＝6.5Hz,3H),0.88–0.84(m,14H).

EXAMPLE 18 preparation of Compound LQ068

Step 1: preparation of LQ068-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	222	1.1eq	9.77g	44
Isononol	144	1eq	5.76g	40
					DCC	206	2eq	16.5g	80
DMAP	122	0.1eq	488mg	4
					DCM			150mL

The operation process comprises the following steps:

8-bromooctanoic acid, isononanol, DCC, DMAP and DCM were added to the reaction flask and the reaction was stirred at room temperature for 12h, TLC (EA: PE=20:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 8.8g of a colorless oil in 63% yield.

Step 2: preparation of LQ068-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ068-1	349	1eq	8.8g	25
Ethanolamine	61	10eq	15g	250
					Acetonitrile			200mL

The operation process comprises the following steps:

LQ068-1, ethanolamine and acetonitrile are added into a reaction bottle, heated to 30 ℃ and stirred for reaction for 12 hours. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.3).

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 500mL of ethyl acetate, washed with 500mL of water 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by column chromatography to give 5.8g of a colorless oil. The yield thereof was found to be 70%.

Step 3: preparation of LQ068

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ068-2	329	1eq	1g	3.08
LQ067-1	321	1.2eq	1.2g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			50mL

The operation process comprises the following steps:

adding LQ068-2, LQ067-1 and K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1g of a colorless oil. The yield thereof was found to be 57%.

¹ H NMR(600MHz,CHCl ₃ -d)δ4.06(ddd,J＝6.9,4.0,2.5Hz,4H),3.63(t,J＝5.2Hz,2H),2.71(t,J＝5.2Hz,2H),2.59(q,J＝7.6Hz,4H),2.28(dt,J＝9.5,7.5Hz,4H),1.66–1.40(m,15H),1.36–1.19(m,13H),1.06(dd,J＝13.9,6.0Hz,2H),0.93(d,J＝6.5Hz,6H),0.87(s,15H).

EXAMPLE 19 preparation of Compound LQ073

Step 1: preparation of LQ073-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					6-Bromohexanoic acid	195	1.2eq	2.34g	12
8-methyl-1-decanol	173	1eq	1.73g	10
					DCC	206	2eq	4.12g	20
DMAP	122	0.1eq	122mg	1
					DCM			50mL

The operation process comprises the following steps:

6-bromohexanoic acid, 8-methyl-1-decanol, DCC, DMAP and DCM were added to the reaction flask and the reaction was stirred at room temperature for 12h. TLC (EA: pe=20:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 2.6g of a colorless oil.

Step 2: preparation of LQ073

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.36g	3.08
LQ073-1	349	1.2eq	1.3g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			40mL

The operation process comprises the following steps:

adding LQ001-2, LQ073-1 and K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1.2g of a colorless oil. The yield thereof was found to be 57%.

¹ H NMR(600MHz,CHCl ₃ -d)δ4.84(p,J＝6.2Hz,1H),4.04(t,J＝6.8Hz,2H),3.72(t,J＝5.1Hz,2H),2.82(t,J＝5.1Hz,2H),2.71(q,J＝7.9Hz,3H),2.28(dt,J＝20.3,7.4Hz,4H),1.61(th,J＝14.9,7.1Hz,9H),1.49(q,J＝6.2Hz,4H),1.34–1.21(m,42H),0.88–0.80(m,12H).

EXAMPLE 20 preparation of Compound LQ074

Step 1: preparation of LQ074-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	222	1.1eq	9.77g	44
8-methyl-1-decanol	172	1eq	6.9g	40
					DCC	206	2eq	16.5g	80
DMAP	122	0.1eq	488mg	4
					DCM			150mL

The operation process comprises the following steps:

the reaction flask was charged with 8-bromooctanoic acid, 8-methyl-1-decanol, DCC, DMAP, and DCM and stirred at room temperature for 12h, tlc (EA: pe=20:1) showed complete reaction (Rf value of product 0.6).

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 9.3g of a colorless oil in 62% yield.

Step 2: preparation of LQ074-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ074-1	377	1eq	9.3g	25
Ethanolamine	61	10eq	15g	250
					Acetonitrile			200mL

The operation process comprises the following steps:

LQ074-1, ethanolamine and acetonitrile are added into a reaction bottle, heated to 30 ℃ and stirred for reaction for 12 hours. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.3).

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 500mL of ethyl acetate, washed with 500mL of water 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by column chromatography to give 5.4g of a colorless oil. The yield thereof was found to be 60%.

Step 3: preparation of LQ074

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ074-2	358	1eq	1.1g	3.08
LQ073-1	349	1.2eq	1.3g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			50mL

The operation process comprises the following steps:

adding LQ074-2, LQ073-1 and K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

¹ H NMR(600MHz,CHCl ₃ -d)δ4.02(t,J＝6.7Hz,4H),3.61(t,J＝5.3Hz,2H),2.70(t,J＝5.3Hz,2H),2.58(q,J＝7.8Hz,4H),2.26(dt,J＝9.9,7.5Hz,4H),1.64–1.45(m,12H),1.34–1.17(m,31H),1.13–1.01(m,4H),0.84–0.78(m,12H).

EXAMPLE 21 preparation of Compound LQ076

Step 1: preparation of LQ076-1

The material ratio is as follows:

the operation process comprises the following steps:

6-bromohexanoic acid, 3-butyl-1-heptanol, DCC, DMAP and DCM were added to the reaction flask, and the reaction was stirred at room temperature for 12 hours. TLC (EA: pe=20:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

Step 2: preparation of LQ076

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ001-2	441	1eq	1.36g	3.08
LQ076-1	349	1.2eq	1.3g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			40mL

The operation process comprises the following steps:

adding LQ001-2, LQ076-1 and K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

¹ H NMR(600MHz,CHCl ₃ -d)δ4.84(p,J＝6.1Hz,1H),4.07(t,J＝7.1Hz,2H),3.66(t,J＝5.2Hz,2H),2.74(t,J＝5.2Hz,2H),2.63(q,J＝7.8Hz,4H),2.27(dt,J＝14.6,7.4Hz,4H),1.66–1.45(m,14H),1.33–1.18(m,43H),0.87(dt,J＝9.5,6.9Hz,12H).

EXAMPLE 22 preparation of Compound LQ077

Step 1: preparation of LQ077-1

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					8-Bromooctanoic acid	222	1.1eq	9.77g	44
3-butyl-1-heptanol	172	1eq	6.9g	40
					DCC	206	2eq	16.5g	80
DMAP	122	0.1eq	488mg	4
					DCM			150mL

The operation process comprises the following steps:

8-bromooctanoic acid, 3-butyl-1-heptanol, DCC, DMAP and DCM were added to the reaction flask and the reaction was stirred at room temperature for 12h, and TLC (EA: PE=20:1) showed completion of the reaction (product Rf value of 0.6).

Post-treatment:

the reaction mixture was filtered through celite and then dried by spin-drying, followed by purification by column chromatography to give 9g of a colorless oil in 61% yield.

Step 2: preparation of LQ077-2

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ077-1	377	1eq	9g	25
Ethanolamine	61	10eq	15g	250
					Acetonitrile			200mL

The operation process comprises the following steps:

Post-treatment:

the reaction mixture was dried by spin-drying, diluted with 500mL of ethyl acetate, washed with 500mL of water 2 times, and the organic phase was dried over anhydrous sodium sulfate and then dried by spin-drying, followed by column chromatography to obtain 5g of a colorless oil. The yield thereof was found to be 57%.

Step 3: preparation of LQ077

The material ratio is as follows:

material name	Molecular weight	Feed ratio	Feeding amount	mmol
					LQ077-2	358	1eq	1.1g	3.08
LQ076-1	349	1.2eq	1.3g	3.7
					K ₂ CO ₃	138	3.5eq	1.5g	11
KI	166	1.2eq	614mg	3.7
					Acetonitrile			50mL

The operation process comprises the following steps:

adding LQ077-2, LQ076-1 and K into a reaction bottle ₂ CO ₃ KI and acetonitrile, heated to 80℃and stirred for reaction for 12h. TLC (DCM: meoh=10:1) showed complete reaction (product Rf value 0.6).

Post-treatment:

the reaction solution was filtered and dried by spin-drying, followed by purification by column chromatography to give 1.1g of a colorless oil. The yield thereof was found to be 57%.

¹ H NMR(600MHz,CHCl ₃ -d)δ4.07(t,J＝7.1Hz,4H),3.56(dt,J＝10.7,5.3Hz,2H),2.61(dt,J＝13.5,5.3Hz,2H),2.50(p,J＝7.4Hz,4H),2.28(q,J＝7.4Hz,4H),1.66–1.54(m,8H),1.52–1.43(m,4H),1.38(q,J＝6.2Hz,2H),1.35–1.19(m,32H),0.88(t,J＝6.9Hz,12H).

Example 23 preparation and detection of Lipid Nanoparticles (LNPs)

A series of ionizable lipid compounds, distearoyl phosphatidylcholine (DSPC, japanese refining Co., ltd., cat# S01005), cholesterol (Japanese refining Co., ltd., cat# O01001) and dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000, guobang pharmaceutical industry, cat# O02005) were dissolved in ethanol solution, respectively, and then mixed in a certain molar ratio to prepare an ethanol solution of a mixed lipid (total lipid concentration: 12.5 mM). Firefly luciferase (Fluc) mRNA or SARS-CoV-2 Spike protein (Spike) mRNA (SARS-CoV-2 Spike protein mRNA see Tan, s. Et al, bioRxiv 2022.05.10.491301.) was diluted in 50mM citrate buffer (pH 4.0) to give an mRNA solution. Flow rate 12mL/min, volume ratio 1:3 mixing the lipid in ethanol with the mRNA solution in such a way that the ratio of ionizable lipid to mRNA nitrogen to phosphorus is 6:1 preparing lipid nanoparticles. Ethanol was removed by dialysis against 0.01M Phosphate Buffer (PBS) for 12-24 h. Finally, the lipid nanoparticle solution was filtered through a 0.22 μm sterile filter and concentrated by ultrafiltration (Amicon-Ultra, MWCO 10 KDa) to give LNP formulations using ionizable lipids/DSPC/cholesterol/DMG-PEG 2000 to encapsulate Fluc mRNA or Spike mRNA. SM102 ionizable lipid (Zhejiang Shenzhou pharmaceutical Co., ltd., lot number: W211-YB 211202) was used as a positive control, and its structure was as follows:

The molar ratio of ionizable lipid/DSPC/cholesterol/DMG-PEG 2000 is 50, without specific explanation: 10:38.5:1.5. particle size and polydispersity index (PDI) of each LNP were measured using Malvern Zetasizer Ultra (dynamic light scattering); quantitative determination of RNA Using Quant-it Ribogreen RNA kit (ThermoFisher Scientific, cat# R114)90 Determining the encapsulation efficiency of the LNP; the pKa of LNP was measured by a dye binding test of 6- (p-Toluidino) -2-naphthalene sulfonic acid sodium salt (TNS, nanjing Xze pharmaceutical technologies Co., ltd., product number: XZ 0743), and the mRNA integrity was examined by nucleic acid gel electrophoresis, and the test results are shown in Table 1 and FIG. 1 (LNP gel electrophoresis diagram of each Fluc-mRNA).

Table 1 particle size, PDI, encapsulation efficiency, and pKa (n=3) of each LNP.

In the art, PDI less than 0.3 indicates that the nanoparticle size in the LNP formulation is relatively uniform; the encapsulation efficiency is used to indicate whether the LNP can effectively encapsulate mRNA, and if the encapsulation efficiency is higher than 70%, the LNP can effectively encapsulate mRNA; the single and bright bands in the agarose gel electrophoresis pattern indicate that the mRNA is structurally intact. Among them, the better the PDI tends to be 0, the better the encapsulation efficiency tends to be 100%. As can be seen from Table 1, the LNP particle size prepared in example 23 was between 60 and 150nm, PDI was less than 0.2, and the LNP pKa corresponding to each ionizable lipid was greater than 5.5. As can be seen from fig. 1, the mRNA bands corresponding to SM102, LQ001, LQ007, LQ013, LQ016, LQ025, and LQ017 were clear and bright after electrophoresis, indicating that they can effectively encapsulate mRNA and maintain the structural integrity of mRNA; the channels of the LNP corresponding to LQ109 were not brightly banded, indicating poor mRNA integrity in this group.

Example 24 preparation and detection of Lipid Nanoparticles (LNPs)

8 LNP formulations (entrapped Fluc mRNA) were prepared in the same manner as in example 23 and their particle size, PDI and entrapment efficiency were determined. Wherein, the ratio of the ionizable lipid to the mRNA is 6:1 preparation of lipid nanoparticles the molar ratio of ionizable lipid/DSPC/cholesterol/DMG-PEG 2000 was 50:10:38.5:1.5, the molar percentage content of each group of preparations is consistent based on 100% of the total molar number of the four components.

TABLE 2 particle size, PDI and encapsulation efficiency of LNPs

As can be seen from table 2, the particle size of each set of LNP formulations made from the different ionizable lipids described above was between 60 and 250 nm; PDI is less than 0.25, specifically between 0.038 and 0.234; the encapsulation efficiency is higher than 70 percent.

Example 25 in vitro cell experiments with LNP formulation

1 ten thousand 293FT cells per well were plated into 96-well plates and cultured overnight until cells adhered to the walls. The Fluc-mRNA LNP preparation prepared in example 23 (100 ng mRNA/well) was added separately to 96-well plate cell culture medium (manufacturer: gibco, cat# C11995500 BT) replaced with DMEM medium without antibiotics containing 10% Fetal Bovine Serum (FBS), cell supernatants were discarded after further incubation for 18h, 100 μl of cell lysate was lysed, D-fluorescein potassium salt (PerkinElmer, cat# 122799, final concentration 1 mM) and ATP (manufacturer: apexeo, cat# C6931, final concentration 2 mM) were added, and the chemiluminescent intensities were measured by a microplate reader, and the test results were shown in fig. 2 (chemiluminescent intensities after 293FT cells were co-cultured with each Fluc-mRNA LNP for 18-24 h. Statistical analysis by ANOVA. In vitro cell delivery and expression of LNP formulations of the application were verified by in vitro cell experiments, the test results are shown in figure 2.

In fig. 2, PBS is a negative control and the corresponding chemiluminescent intensity reading of this group can be considered as a background reading. The higher the chemiluminescent intensity reading, the higher the expression. As can be seen from FIG. 2, the Fluc-mRNA LNP corresponding to LQ001, LQ007, LQ013, LQ016 and LQ025 were each effective in delivering and expressing Fluc mRNA into cells.

40 ten thousand 293FT cells per well were plated into 12-well plates and cultured overnight until cells attached. The Spike-mRNA LNP prepared in example 23 (in 1. Mu.g/mL/well) was added to the cell culture broth of the 12-well plate (replaced with DMEM medium containing 10% Fetal Bovine Serum (FBS) without antibiotics) (manufacturer: gibco, cat# C11995500 BT) and cultured for 24 hours. Cells were lysed and total protein was extracted, and cell level expression of Spike protein was determined by Western blot, and the test results are shown in fig. 3 (Spike protein expression after 24h co-culture of 293FT cells with Spike-mRNA LNP, PBS group as negative control).

As can be seen from fig. 3, spike-mRNA LNP formulations corresponding to LQ001, LQ007, LQ013, LQ025 were each effective in delivering Spike mRNA into cells and expressing.

Example 26 in vivo animal studies of LNP formulations

The Fluc-mRNA-LNP prepared in example 23 was injected into 6-8 week old female Balb/C mice (velonto ritodv) by tail vein at a dose of 5 μg/dose, and D-fluorescein potassium salt was intraperitoneally injected at a specific time node (6, 24, 48, 72h in this example) after the administration, and then the radiation intensity (corresponding to bioluminescence expression intensity) of the protein expressed in the mice by mRNA carried by LNP was detected by IVIS Spectrum small animal in vivo imager, and the total luminescence intensity of the liver sites of the mice was counted. The test results are shown in fig. 4 (total in vivo bioluminescence (n=3) after various times of intravenous administration of each Fluc-mRNA LNP, PBS group as negative control (n=2)). At a given time point, after intraperitoneal injection of luciferin for 10min, luciferase activity was measured by bioluminescence imaging, and the total luminescence intensity of the liver region was counted by the live Image software (PerkinElmer). Further, the area under the curve (AUC, unit: p/s hr) was calculated by GraphPad software, i.e. the area under the curve connecting the measurement points of total luminous intensity from 4 hours or 6 hours after the drug (the same peak value from 3 to 6 hours after the drug was used in the experimental method of the present application) to 72 hours after the drug, and the larger AUC, the better the expression was.

As shown in FIG. 4, the Fluc-mRNA LNP formulations corresponding to LQ001, LQ007, LQ013, LQ016 and LQ025 were strongly expressed in mice, and luminescence intensity was observed to be more than 10 times higher than the background level (PBS group) up to 72 hours.

The Spike-mRNA-LNP formulation prepared in example 23 was intramuscular injected into 6-8 week old female Balb/C mice (velocin) at a dose of 2 μg/min, and the corresponding mRNA-LNP formulation was repeatedly injected 21 days after the first injection. And whole blood was collected from the mice at a specific time node after 2 doses (data in this example is one week after the second dose). Serum was isolated from whole blood by centrifugation at 2000g for 10min at 4℃and inactivated in a water bath at 56℃for 30min and stored at-80℃for analysis. SARS-CoV-2 (2019-nCoV)) Spike S1+S2 ECD-His Recombinant Protein (Yiqiaoshenzhou, cat: 40589-V08B 1) was coated with SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody Mouse Mab (Yinqiaoshenzhou, cat: 40591-MM 43), 2% bsa blocked, peroxidase AffiniPure Goat Anti-Mouse IgG (h+l) (Jackson ImmunoResearch, cat: 115-035-003) secondary antibody, TMB (Invitrogen, cat No.: 00-4201-56) was developed and analyzed by enzyme-linked immunosorbent assay (ELISA) to determine Spike antibody titer, and the test results are shown in fig. 5 (total antibody titer (n=8) in the serum of mice 1 week after the last administration). Statistical analysis was performed by ANOVA. * P < 0.01, p < 0.001, compared to PBS group).

The animals of the groups SM102, LQ001, LQ013, LQ025, LQ007, LQ010, LQ016 showed a significant increase in Spike protein total antibody titer compared to the animals of the PBS group, whereas no increase was seen in the groups LQ002, LQ014, LQ 109.

Example 27 in vivo animal studies of LNP formulation

Referring to the in vivo test method for mice in example 26, all LNP preparations prepared in example 24 were injected into female Balb/C mice of 6 to 8 weeks of age by tail vein at a dose of 5. Mu.g/dose, and D-potassium fluorescein salt was intraperitoneally injected at a specific time node (4, 24, 48 hours in this example) after administration, and then the total luminous intensity of the liver region was counted by the same method as in example 26, and the test results are shown in FIG. 6. The experimental results show that: of these, 5 (LQ 031, LQ067, LQ073, LQ076, LQ 077) LNP formulations were strongly expressed in mice, indicating better delivery capacity in vivo.

Claims

1. A nitrogen-containing chain compound shown in a formula I or pharmaceutically acceptable salt thereof is characterized in that,

each Y ^1-1 Independently hydroxy, halogen or C ₁ -C ₆ Alkyl of (a);

each Z is ^1-1 Independently hydroxy, halogen or C ₁ -C ₆ Alkyl of (a);

A ¹ and A ² Independently is

each R is ^1-2 Independently is hydroxy or C ₁ -C ₁₀ Alkyl of (a);

each R is ^2-2 Independently is hydroxy or C ₁ -C ₁₀ Alkyl of (c):

2. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the nitrogen-containing chain compound represented by formula I satisfies one or more of the following conditions:

(1) X, the C ₁ -C ₆ Alkylene of (C) ₁ -C ₄ Alkylene groups of (C), preferably For example->

(2) X, the C ₃ -C ₆ Is (1) cycloalkylene For example->

(3) Y, the C ₁ -C ₁₀ Alkylene of (C) ₄ -C ₆ Alkylene groups of (C), preferably For example->

(4) Each Y ^1-1 Wherein the halogen is fluorine, chlorine, bromine or iodine;

(5) Each Y ^1-1 In the above, the C ₁ -C ₆ Alkylene of (2)

(6) Z is the same as C ₁ -C ₁₀ Alkylene of (C) ₅ -C ₈ Alkylene groups of (C), preferably For example

(7) Each Z is ^1-1 Wherein the halogen is fluorine, chlorine, bromine or iodine;

(8) Each Z is ^1-1 In the above, the C ₁ -C ₆ Alkylene of (2)

(9)R ¹ In the above, the C ₁ -C ₂₀ Is C as alkyl ₁ -C ₁₅ Preferably a linear alkyl group such as ethyl, propyl, hexyl, octyl, nonyl or dodecyl;

(10)R ¹ in the above, the C ₂ -C ₂₀ Alkenyl of C ₆ -C ₁₈ Alkenyl groups of (2), preferably straight-chain alkenyl groups; the C is ₂ -C ₂₀ Alkenyl groups of (2) may contain 1 to 4 double bonds;

(11) Each R is ^1-1 In the above, the C ₁ -C ₁₀ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl, for example methyl, ethyl, isopropyl, t-butyl or n-butyl;

(12) Each R is ^1-1 In the above, the C ₃ -C ₁₅ Cycloalkyl of (C) ₃ -C ₁₂ Cycloalkyl of (C) is preferred ₄ -C ₈ Cycloalkyl or cyclo12alkyl, such as cyclopentyl, cyclohexyl or cyclo12alkyl;

(13)R ^1-1-1 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(14)R ^1-1-2 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(15)R ^1-1-3 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(16) Each R is ^1-1-4 In the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(17) Each R is ^1-2 In the above, the C ₁ -C ₁₀ Is C as alkyl ₁ -C ₆ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(18)R ² in the above, the C ₁ -C ₂₀ Is C as alkyl ₁ -C ₁₅ Preferably a linear alkyl group such as ethyl, propyl, hexyl, octyl, nonyl or dodecyl;

(19)R ² in the above, the C ₂ -C ₂₀ Alkenyl of C ₆ -C ₁₈ Alkenyl groups of (a), preferably straight chain alkenyl groups; the C is ₂ -C ₂₀ Alkenyl groups of (2) may contain 1 to 4 double bonds;

(20) Each R is ^2-1 In the above, the C ₁ -C ₁₀ Is C as alkyl ₁ -C ₈ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl or isooctyl, for example methyl, ethyl, isopropyl, t-butyl, n-butyl or n-octyl;

(21) Each R is ^2-1 In the above, the C ₃ -C ₁₅ Cycloalkyl of (C) ₃ -C ₁₂ Cycloalkyl of (C) is preferred ₄ -C ₈ Cycloalkyl or cyclo12alkyl, such as cyclopentyl, cyclohexyl or cyclo12alkyl;

(22)R ^2-1-1 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(23)R ^2-1-2 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(24)R ^2-1-3 in the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

(25) Each R is ^2-1-4 In the above, the C ₁ -C ₆ Is C as alkyl ₁ -C ₄ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl;

And (26) each R ^2-2 In the above, the C ₁ -C ₁₀ Is C as alkyl ₁ -C ₆ Preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl or sec-butyl.

3. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1,

the nitrogen-containing chain is shown as formula IThe compound is represented by formula Ia, ib, ic, id or Ie

4. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the nitrogen-containing chain compound represented by formula I is any one of scheme 1, scheme 2, scheme 3, scheme 4 or scheme 5:

scheme 1,

X is C ₁ -C ₄ Alkylene or C of (2) ₃ -C ₆ Is a cycloalkylene group of (2);

y is C ₁ -C ₁₀ An alkylene group of (a);

z is C ₁ -C ₁₀ An alkylene group of (a);

A ¹ and A ² Independently is

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

scheme 2,

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

Scheme 3,

X is C ₁ -C ₄ An alkylene group of (a);

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

R ¹ for C substituted by 1, 2 or 3 hydroxy or cyclopentyl groups ₁ -C ₁₅ Alkyl group of (C),

R ² Is 1, 2 or 3R ^2-1 Substituted C ₁ -C ₁₅ Alkyl of (a);

each R is ^2-1 Independently C ₁ -C ₁₀ Alkyl of (a);

scheme 4,

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

each R is ^1-1-4 Independently C ₁ -C ₆ Alkyl of (a);

each R is ^2-1-4 Independently C ₁ -C ₆ Alkyl of (a);

Scheme 5,

X is C ₁ -C ₄ An alkylene group of (a);

y is C ₄ -C ₈ An alkylene group of (a);

z is C ₄ -C ₈ An alkylene group of (a);

A ¹ is thatWherein a and R ¹ B is connected with Y;

A ² is thatWherein a and R ² B is connected with Z;

R ² Is 1, 2 or 3R ^2-1 Substituted C ₁ -C ₁₅ Alkyl of (a);

each R is ^2-1-4 Independently C ₁ -C ₆ Is a hydrocarbon group.

5. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the nitrogen-containing chain compound represented by formula I satisfies one or more of the following conditions:

(1) X is C ₁ -C ₄ Alkylene or C of (2) ₃ -C ₆ Is a cycloalkylene group of (2);

(2) Y is C ₁ -C ₁₀ An alkylene group of (a);

(3) Z is C ₁ -C ₁₀ An alkylene group of (a);

(4)R ¹ unsubstituted or substituted by 1, 2 or 3R ^1-1 Substituted C ₁ -C ₂₀ Alkyl of (a);

(5) Each R is ^1-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl of (c);

(6)R ² unsubstituted or substituted by 1, 2 or 3R ^2-1 Substituted C ₁ -C ₂₀ Alkyl of (a);

(7) Each R is ^2-1 Independently is hydroxy, C ₁ -C ₁₀ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₁₅ Cycloalkyl of (c);

and (8) R ₁ And R is R ₂ The same applies.

6. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 5, wherein the nitrogen-containing chain compound represented by formula I satisfies one or more of the following conditions:

(1) Y is C ₄ -C ₈ An alkylene group of (a);

(2) Z is C ₄ -C ₈ An alkylene group of (a);

(3) Each R is ^1-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^1-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

(4) Each R is ^2-1 Independently is hydroxy, C ₁ -C ₆ Is optionally substituted or substituted by 1, 2 or 3R ^2-1-4 Substituted C ₃ -C ₆ Cycloalkyl of (c);

and (5) when X is C ₁ -C ₄ Sub-group of (2)In the case of alkyl radicals, R ¹ For C substituted by 1, 2 or 3 hydroxy groups ₁ -C ₂₀ Unsubstituted or substituted by 1, 2 or 3R ^1-1-4 Substituted cyclopentanes,

7. The nitrogen-containing chain compound as set forth in claim 6, represented by formula I, or a pharmaceutically acceptable salt thereof, wherein the nitrogen-containing chain compound as set forth in formula I satisfies one or more of the following conditions:

(1) X is

(2) Y is

(3) Z is

(4)R ¹ Is that

And (5) R ² Is that

8. The nitrogen-containing chain compound represented by formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein the nitrogen-containing chain compound represented by formula I is any one of the following compounds:

/>

9. a method for preparing a nitrogen-containing chain compound shown in a formula I, which is characterized by comprising the following steps: in a solvent, in the presence of alkali and iodized salt, performing a coupling reaction between a compound shown as a formula I-1 and a compound shown as a formula I-2;

/>

M is halogen, X, Y, Z, A ¹ 、A ² 、R ¹ And R is ² The method of any one of claims 1-8;

preferably, the preparation method of the nitrogen-containing chain compound shown in the formula I meets one or more of the following conditions:

(1) In the coupling reaction, the halogen is fluorine, chlorine, bromine or iodine, such as bromine;

(2) In the coupling reaction, the molar ratio of the compound shown as the formula I-1 to the compound shown as the formula I-2 is 1 (1-2), such as 1:1.2;

(3) In the coupling reaction, the base is a basic carbonate, e.g. K ₂ CO ₃ ；

(4) In the coupling reaction, the molar ratio of the compound shown as the formula I-1 to the alkali is 1 (1-5), such as 1:3.5;

(5) In the coupling reaction, the iodinated salt is a basic iodinated salt, such as KI;

(6) In the coupling reaction, the molar ratio of the compound shown as the formula I-1 to the iodinated salt is 1 (1-2), such as 1:1.2;

(7) In the coupling reaction, the solvent is an ether solvent or/and a nitrile solvent; the ether solvent may be methyl tertiary butyl ether; the nitrile solvent may be acetonitrile; the volume ratio of the nitrile solvent to the ether solvent can be 1:1;

(8) In the coupling reaction, the mass volume ratio of the compound shown as the formula I-1 to the solvent is 10mg/mL-65mg/mL, such as 15mg/mL, 25mg/mL, 40mg/mL or 58.5mg/mL;

And (9) the coupling reaction, wherein the reaction temperature of the coupling reaction is 70 ℃ to 90 ℃, for example 80 ℃.

10. A lipid carrier comprising a substance Z which is a compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

11. The lipid carrier of claim 10, wherein the lipid carrier satisfies one or more of the following conditions:

(1) The lipid carrier further comprises a diluent;

(2) The lipid carrier further comprises a phospholipid;

(3) The lipid carrier further comprises a PEG lipid;

and (4) the lipid carrier further comprises a sterol.

12. The lipid carrier of claim 11, wherein the lipid carrier satisfies one or more of the following conditions:

(1) The diluent is phosphate buffer solution or Tris buffer solution;

(2) The phospholipid is a phospholipid molecule having a charged polar end and a fatty chain non-polar end, such as distearoyl phosphatidylcholine, dimyristoyl phosphorylcholine, dioleoyl phosphorylcholine, palmitoyl phosphorylcholine, 1, 2-distearoyl phosphorylcholine, heneicosanoyl phosphorylcholine or palmitoyl phosphorylcholine;

(3) The PEG lipid is a lipid molecule with polyethylene glycol hydrophilic end modification; preferably, the PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, for example, the PEG lipid is dimyristoylglycerol with PEG-modification;

(4) The sterols include animal, vegetable or fungus sterols; preferably, the sterol is selected from one or more of cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid and alpha-tocopherol, for example cholesterol;

(5) In the lipid carrier, the molar ratio of the substance Z to the sterol is 0.5-5:1, preferably 0.5-3:1, for example 0.6-2:1;

(6) In the lipid carrier, the molar ratio of the substance Z to the phospholipid is 1-15:1, preferably 2-8:1, such as 3-6:1;

(7) In the lipid carrier, the molar ratio of the substance Z to the PEG lipid is 20-130:1, preferably 20-80:1, such as 20-40:1;

(8) The molar content of the substance Z is 30mol% to 60mol%;

(9) The molar content of the phospholipid is 0mol% to 30mol%;

(10) The molar content of sterols is 15mol% to 55mol%;

and (11) the PEG lipid is present in a molar amount of 0mol% to 10mol%;

preferably, the lipid carrier consists of the substance Z, the diluent, the phospholipid, the PEG lipid and the sterol.

13. Lipid nanoparticle, characterized in that it comprises a therapeutic and/or prophylactic agent and a lipid carrier according to any one of claims 10-12.

14. The lipid nanoparticle of claim 13, wherein the lipid nanoparticle satisfies one or more of the following conditions:

(1) The therapeutic agent and/or prophylactic agent is one or two or more nucleic acids; preferably, the therapeutic and/or prophylactic agent is a single stranded deoxyribonucleic acid, double stranded DNA, small interfering RNA, asymmetric double stranded small interfering RNA, microrna, small hairpin RNA, circular RNA, transfer RNA or messenger RNA, preferably mRNA, such as firefly luciferase mRNA or SARS-CoV-2 spike protein mRNA;

(2) The ratio of nitrogen to phosphorus in the lipid nanoparticle is 2:1 to 30:1, preferably 2:1 to 20:1, for example 3:1 to 20:1, and also for example 3:1 to 16:1;

(3) In the lipid nanoparticle, the mass ratio of the lipid carrier to the therapeutic agent and/or the prophylactic agent is 3-80:1, preferably 6-60:1

(4) The lipid nanoparticle has a particle size of 10 to 250nm, for example 40 to 250nm, further 50 to 250nm, or 40 to 150nm, or 60 to 150nm;

and (5) in the lipid nanoparticle, the lipid carrier encapsulates the therapeutic and/or prophylactic agent.

15. A composition comprising a substance Z which is a compound of formula I according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

16. The composition of claim 15, further comprising one or more of a diluent, a phospholipid, a PEG lipid, a sterol, and a therapeutic and/or prophylactic agent;

preferably, in the composition, the diluent, phospholipid, PEG lipid and sterol are as described in claim 12; and/or, the therapeutic and/or prophylactic agent according to claim 14;

more preferably, in the composition, the substance Z forms a lipid carrier according to any one of claims 10-12 with one or more of the diluents, phospholipids, PEG lipids and sterols; and/or, in the composition, the therapeutic and/or prophylactic agent has an encapsulation efficiency of at least 50%, preferably at least 70%;

still preferably, in the composition, the lipid carrier forms a lipid nanoparticle according to claim 13 or 14 with the therapeutic and/or prophylactic agent; and/or, in the composition, the polydispersity index of the composition is not higher than 0.5, for example not higher than 0.3.