CN114874107A

CN114874107A - Amino lipid and preparation method and application thereof

Info

Publication number: CN114874107A
Application number: CN202210812013.3A
Authority: CN
Inventors: 查高峰; 王静; 潘逸航
Original assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Current assignee: Shenzhen Hongxin Biotechnology Co ltd
Priority date: 2022-07-12
Filing date: 2022-07-12
Publication date: 2022-08-09
Anticipated expiration: 2042-07-12
Also published as: CN114874107B

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to an amino lipid and a preparation method and application thereof. In vitro, in vivo delivery studies, an excellent ability to deliver nucleic acids into cells was demonstrated. The amino lipidic compound has a plurality of degradable groups, the introduction of the groups not only improves the nucleic acid entrapment capability, but also obviously enhances the escape capability of the nucleic acid in an endosome/lysosome, greatly reduces the cytotoxicity, and is also beneficial to the release of target drugs or genes and other delivery targets, thereby improving the delivery efficiency. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements on instruments and equipment and simple operation.

Description

Amino lipid and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to amino lipid and a preparation method and application thereof.

Background

At present, nucleic acid drugs have extremely broad application prospects, and besides being used for cancers and infectious diseases, the nucleic acid drugs also have development potential in genetic diseases, cardiovascular diseases and other gene-related diseases. However, since RNA, DNA, siRNA, and the like are very easily degraded in vivo, and administered directly by oral or intravenous injection, bioavailability is extremely low, and thus delivery of a carrier is required.

Currently, mRNA vaccine delivery vectors mainly include two major types, viral vectors and non-viral vectors. The virus vector mainly comprises retrovirus, adenovirus, adeno-associated virus, vaccinia virus and the like. Although the viral vector has the advantages of high transfection efficiency (95%), difficult degradation in vivo and the like, the viral vector also has a plurality of defects, such as complex preparation process, limited size of the carried pDNA, lack of targeting property, random integration into host cells, easy causing of immunogenic reaction, large pathogenic and carcinogenic hidden danger and great limitation on clinical application. On the contrary, the non-viral vector has the advantages of higher biological safety, lower toxicity, lower immunogenicity, low exogenous gene integration probability, low cost, simple preparation process, convenient use, good reproducibility, no infectivity, easy preservation and inspection and the like, thereby becoming a research hotspot gradually.

Among them, Lipid Nanoparticles (LNP) are a more studied and mature delivery system, and mRNA is located in the core of LNP, and is not easily degraded by nuclease, and the lipophilicity of LNP makes it easily fused with host cell membrane, so as to deliver mRNA into cells by endocytosis. LNP thus has two duties: firstly, mRNA can be effectively wrapped and protected and can be stably maintained before reaching a target point, and secondly, the mRNA is released into cytoplasm before reaching lysosomes

LNPs typically comprise four components, consisting of ionizable cationic lipids, phospholipids, cholesterol, and pegylated lipids, and are structurally amphiphilic molecules with self-assembling properties. LNP has the advantages of determined structure of each component, good reproducibility, contribution to quality supervision, longer in-vivo circulation time, good biocompatibility and the like. After entering cells, the nanoparticles need to escape from endosomes/lysosomes, so that RNA can be released in cytoplasm and can be expressed to generate target proteins. However, the endosome/lysosome escape rate of LNP is generally low at present, and although DLin-MC3-DMA is the most efficient amino lipid at present, only 1% -4% of RNA escapes from the endosome/lysosome, so that endosome/lysosome escape has become a key step influencing nucleic acid delivery, and designing an amino lipid with good nucleic acid entrapment capability and high endosome/lysosome escape capability has great research significance and practical requirements for solving the problem of nucleic acid delivery.

Disclosure of Invention

Aiming at the technical problems of low transfection efficiency of cationic liposome, cytotoxicity caused by positive charge and the like in the prior art, the invention provides an amino lipid and a preparation method and application thereof.

The purpose of the invention is realized by the following technical scheme:

an amino lipid, the structure of which is shown in formula (I):

wherein R is ¹ And R ² Are the same or different from each other and are each independently selected from H, substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl; or R ¹ And R ² Are connected to form a 4-10 membered heterocyclic ring, wherein the heterocyclic ring contains 1-6 heteroatoms selected from nitrogen, sulfur or oxygen;

L ¹ 、L ² and L ³ Is substituted or unsubstituted C1-C12 alkylene, C1-C12 alkenylene, C1-C12 alkynylene, C3-C12 cycloalkylene, C3-C12 cycloalkenylene, and the substituent of the C1-C12 alkylene, C1-C12 alkenylene, C1-C12 alkynylene, C3-C12 cycloalkylene and C3-C12 cycloalkenylene is C1-C6 hydrocarbon;

X ¹ 、X ² and X ³ Are identical or different from each other and are each independently selected from C, N, O, S, S ═ O, S (═ O) ₂ And S-S, m, n and p are selected from 0, 1 or 2;

when X is present ¹ 、X ² And X ³ When C is selected as in (1), m, n and p are 2 correspondingly; x ¹ 、X ² And X ³ The two substituents attached are the same or different;

said X ¹ 、X ² And X ³ Attached substituent R ⁷ 、R ⁸ 、R ⁹ Are the same or different from each other and are each independently selected from H or C1-C12 alkyl;

G ¹ and G ² Identical or different from each other, G ¹ Selected from the group consisting of-O-C (= O) -, -O-C (= NH) -, -C (= O) -O-, -C (= NH) -, -CH ₂ -； G ² Selected from-O-C (= O) -, -O-C (= NH) -, -C (= O) -O-, -C (= NH) -;

R ³ and R ⁴ Are the same as or different from each other, andeach independently selected from H, substituted or unsubstituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 alkynyl, C1-C18 cycloalkyl, C1-C18 cycloalkenyl, C1-C18 cycloalkynyl, and the substituents of C1-C18 alkyl, C1-C18 alkenyl, C1-C18 alkynyl, C1-C18 cycloalkyl, C1-C18 cycloalkenyl, C1-C18 cycloalkynyl are selected from C1-C6 hydrocarbyl.

X is above ¹ 、X ² And X ³ Are identical or different from each other and are each independently selected from C, N, O, S, S ═ O, S (═ O) ₂ And S-S, wherein m, n and p are selected from 0, 1 or 2.

The values of m, n and p are according to C, N, O, S, S ═ O, S (═ O) ₂ And the bonding mode of S-S. When the value is C, the values of m, n and p are 2; when N is contained, the values of m, N and p are 1; when S, S ═ O, S (═ O) ₂ And when S-S is adopted, m, n and p take the values of 0.

Preferably, the amino lipid has a structure represented by formula (II) or formula (III):

preferably, L ² And L ³ Is substituted or unsubstituted C3-C12 alkylene, C3-C12 alkenylene, and the C3-C12 alkylene and C3-C12 alkenylene substituent is C1-C6 alkyl;

R ³ and R ⁴ The alkyl groups are the same or different from each other, and are respectively and independently selected from H, substituted or unsubstituted C1-C18 alkyl groups, C1-C18 alkenyl groups, and the substituents of the C1-C18 alkyl groups and the C1-C18 alkenyl groups are selected from C1-C6 hydrocarbon groups.

Preferably, said X ¹ Is N, said R ¹ 、R ² 、L ¹ 、X ¹ Form R ₁ R ₂ -N-L ₁ -X ₁ Has the structure of

(ii) a The R is ¹ 、R ² Can be connected to form a 4-10 membered heterocyclic ring, wherein the heterocyclic ring contains 1-6 heteroatoms selected from nitrogen, sulfur or oxygen; and the knotIs selected from: one of D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20:

。

preferably, said X ¹ Is O, said R ¹ 、R ² 、L ¹ 、X ¹ Form R ₁ R ₂ -N-L ₁ -X ₁ Has the structure of

(ii) a The R is ¹ 、R ² Can be connected to form a 4-to 10-membered heterocyclic ring, wherein the heterocyclic ring contains 1 to 6 heteroatoms selected from nitrogen, sulfur or oxygen, and the structure is selected from one of O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15, O16:

。

more preferably, the amino lipids may be divided into a plurality of series, each series typically representing a compound having the formula:

。

the preparation method of the amino lipid of the formula (I) comprises the following steps:

s1 Compound SH-L ² -OH or SH-L ² -CH ₃ Reacting with cyanuric chloride under the catalysis of alkali at the temperature of minus 25 to minus 10 ℃ with stirring;

s2, adding SH-L into the reaction system in the step S1 ³ OH, adding alkali serving as a catalyst, and stirring for reaction;

s3 adding R into the reaction system in the step S2 ³ -COOH or R ⁴ -COOH, and adding a catalytic amount of DMAP to stir for reaction under the action of a condensing agent;

s4 adding R into the reaction system in the step S3 ¹ R ² -N-L ¹ -X ¹ H, stirring and reacting to obtain the catalyst.

The specific reaction process is as follows:

(1) compound SH-L at-20 deg.C ² -OH or SH-L ² -CH ₃ Carrying out a first-step reaction with cyanuric chloride under the catalysis of a catalyst DIPEA to obtain a first intermediate;

(2) adding SH-L into the reaction system of the step S1 at room temperature without separating the first intermediate ³ Carrying out a second-step reaction on the-OH and a catalyst DIPEA to obtain a second intermediate;

(3) isolating the second intermediate and adding R ³ -COOH or R ⁴ Carrying out a third step of reaction on-COOH, condensing agents EDC.HCl, DIPEA and catalytic amount DMAP at normal temperature to obtain a third intermediate;

(4) separating the third intermediate, adding R at room temperature ¹ R ² -N-L ¹ -X ¹ H, carrying out the fourth step reaction to obtain the amino lipid compound shown in the formula I.

When G is ¹ is-CH ₂ -when, the reaction formula is:

when G is ¹ And G ² In the case of-O-C (= O) -, the reaction formula is:

。

the use of said amino lipids and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof for the preparation of a medicament for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering with RNA drugs.

Preferably, the use in the manufacture of a medicament for the treatment of cancer or genetic disease.

Preferably, the application in preparing the medicine for treating lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymph cancer, leukemia, prostatic cancer, allergy and toxicity.

Preferably, the use in the manufacture of a medicament for the treatment of haemophilia, thalassemia or gaucher's disease.

Compared with the prior art, the invention has the following technical effects:

the amino lipid disclosed by the invention is ionizable amino lipid shown in a formula (I) or pharmaceutically available salt thereof, the reaction condition is mild in the process of constructing the amino lipid, protection and deprotection are not needed, and the atom economy is high. In vitro, in vivo delivery studies, an excellent ability to deliver nucleic acids into cells was demonstrated. The amino lipidic compound has a plurality of degradable groups, the introduction of the groups not only improves the nucleic acid entrapment capability, but also obviously enhances the escape capability of the nucleic acid in an endosome/lysosome, greatly reduces the cytotoxicity, and is also beneficial to the release of target drugs or genes and other delivery targets, thereby improving the delivery efficiency. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, good reaction selectivity, high reaction yield, low requirements on instruments and equipment and simple operation.

Drawings

FIG. 1 is a drawing of example 4 ¹ An H-NMR spectrum;

FIG. 2 is a drawing of example 4 ¹³ A C-NMR spectrum;

FIG. 3 is a drawing showing a modified example of example 7 ¹ An H-NMR spectrum;

FIG. 4 is a graph of tumor growth curves for tumor-bearing mice after intramuscular injection of OVA mRNA vaccines in example 8 and example 21;

FIG. 5 is a graph of tumor-bearing mouse survival curves following intramuscular injection of OVA mRNA vaccines in example 8 and example 21;

FIG. 6 is a graph of cell viability from a partial LNP cytotoxicity assay of example 35.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

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

The term "optionally substituted" as used herein means that one or more hydrogen atoms attached to an atom or group are independently unsubstituted or substituted with one or more, e.g., one, two, three or four, substituents. When an atom or group is substituted with a plurality of substituents, the plurality of substituents may be the same or different.

Abbreviations herein:

RNA ribonucleic acid

DSPC distearoyl phosphatidylcholine

DOPE dioleoyl phosphatidylethanolamine

DOPC dioleoylphosphatidylcholine

DSPE distearoyl phosphatidyl ethanolamine

PEG2000-DMG (1- (monomethoxypolyethylene glycol) -2, 3 dimyristoyl glycerol

kD kilodalton

PBS phosphate buffer solution.

In the following examples, amino lipids having the general structural formula shown in formula (I) are not specifically shown

In the amino lipid structure indicated by the number, O1-O16 and D1-D21 are defined as above

A group; as in example 4 of the formula

。

Example 1 when G ¹ And G ² Parallel synthesis and characterization of a library of amino-lipid compounds of one of the Ox series, identically selected from-O-C (= O) -

Cyanuric chloride (184 mg, 1 mmol) and 10mL of THF were added sequentially to a 50mL reaction flask at-20 ℃ to 3-mercapto-1-propanol (173. mu.L, 2 mmol), DIPEA (442. mu.L, 2.4 mmol), and the mixture was stirred at room temperature overnight to give a Step I solution (1 mmol/10 mL), lauric acid (481 mg, 2.4 mmol), EDC.HCl (460 mg, 2.4 mmol), DMAP (6 mg, 0.05 mmol) and DIPEA (442. mu.L, 2.4 mmol) and stirred at room temperature for 6 hours to give a 0.1M Step II solution (1 mmol/10 mL).

The Step II solution was transferred to 1.5 mL 96-well plates (0.1 mL each, 0.01 mmol) using a pipette, and a THF solution of an alcohol with a tertiary amine (0.1 mL, 0.02 mmol, 0.2M) was added to each well and stirred at room temperature for 6h, and TLC detected for the absence of the Step II starting material. After the reaction is finished, volatilizing at normal temperature until no solvent is basically generated, and obtaining 16 amino lipid compounds. Mass spectrometry was performed and the results are shown in Table 1 below.

Table 1: MW/z value of amino lipid compound library of one of Ox series

In Table 1, the structural general formulas of the compounds numbered 1 to 16 are

R groups of the compounds numbered 1-16 are respectively O1-O16.

Example 2 when G ¹ And G ² Are different from each other and are selected from-CH ₂ Parallel Synthesis and characterization of the amino-lipidic Compound library of one of the Ox series-and-O-C (= O) -

Cyanuric chloride (184 mg, 1 mmol) and 10mL of THF were added sequentially in a 50mL reaction flask, -hexadecylthiol (308. mu.L, 1 mmol) and DIPEA (221. mu.L, 1.2 mmol) were added at-20 ℃ and stirred for 30min, then 3-mercapto-1-propanol (87. mu.L, 1 mmol) and DIPEA (221. mu.L, 1.2 mmol) were added and stirred overnight at room temperature to give a 0.1M solution of Step I, nonanoic acid (210. mu.L, 2.4 mmol) EDC.HCl (230 mg, 1.2 mmol), DMAP (3 mg, 0.025 mmol) and DIPEA (221. mu.L, 1.2 mmol) were added and stirred for 6h at room temperature to give a 0.1M solution of Step II (1 mmol/10 mL).

The Step II solution was transferred to 1.5 mL 96-well plates (0.1 mL each, 0.01 mmol) using a pipette, and a THF solution of the alcohol with the tertiary amine (0.1 mL, 0.02 mmol, 0.2M) was added to each well and stirred at room temperature for 6h, and no Step II starting material was detected by TLC. After the reaction is finished, volatilizing at normal temperature until no solvent is basically generated, and obtaining 16 amino lipid compounds. Mass spectrometric measurements were carried out and the results are shown in Table 2 below.

Table 2: MW/z value of amino lipid compound library of one of Ox series

In Table 2, the compounds numbered 1 to 16 have the general structural formulas

R groups of the compounds numbered 1-16 are respectively O1-O16.

Example 3: when G is ¹ And G ² Parallel Synthesis and characterization of the same library of amino-lipidic Compounds from one of the Dx series, when selected from-O-C (= O) -

Cyanuric chloride (184 mg, 1 mmol) and 10mL of THF were added sequentially to a 50mL reaction flask at-20 ℃ to 6-mercapto-1-hexanol (274. mu.L, 2 mmol), DIPEA (442. mu.L, 2.4 mmol), and the reaction was stirred overnight at room temperature to give a Step I solution (1 mmol/10 mL), decanoic acid (463. mu.L, 2.4 mmol), EDC.HCl (460 mg, 2.4 mmol), DMAP (6 mg, 0.05 mmol) and DIPEA (442. mu.L, 2.4 mmol) were added, and the mixture was stirred at room temperature for 6 hours to give a 0.1M Step II solution (1 mmol/10 mL).

The Step II solution was transferred to 1.5 mL 96-well plates (0.1 mL each, 0.01 mmol) using a pipette, and a THF solution with a tertiary amine (0.1 mL, 0.02 mmol, 0.2M) was added to each well and stirred at room temperature for 6h to check for the absence of Step II material by TLC. After the reaction is finished, volatilizing at normal temperature until no solvent exists basically, and obtaining 20 amino lipid compounds. Mass spectrometric measurements were carried out and the results are given in Table 3 below.

Table 3: MW/z value of one amino lipid compound library of Dx series

In Table 3, the compounds numbered 1 to 20 have the general structural formulas

R groups of the compounds numbered 1-20 are respectively as described in D1-D20.

Example 4: synthesis and characterization of representative amino lipid compounds

To a 10mL reaction tube were added 3-dimethylamino-1-propanol (71 μ L, 0.6 mmol), ((6-chloro-1, 3, 5-triazine-2, 4-diyl) bis (sulfonamido) bis (propane-3, 1-diyl) bisdodecanoate (330 mg, 0.5 mmol), 4mL thf, followed by stirring at room temperature for 3 h and purification by column chromatography (DCM: MeOH =10: 1) to give ((6- (3- (dimethylamino) propoxy) -1,3, 5-triazine-2, 4-diyl) bis (sulfonamido) bis (propane-3, 1-diyl) didecanoate (235 mg, 85%). ¹ H NMR (400 MHz，CDCl ₃ ): δ 0.88 (t, 6H), 1.25-1.35 (m, 32H), 1.62 (m, 4H), 1.96 (m, 2H), 2.07 (m, 4H), 2.27 (s, 6H), 2.32 (m, 6H), 3.17 (t, 4H), 4.19 (t, 4H), 4.42 (t, 2H) (FIG. 1). ¹³ C NMR (400 MHz，CDCl ₃ ): δ 13.61, 22.18, 24.46, 26.04, 26.46, 27.92, 28.68, 28.78, 28.83, 18.97, 29.1, 31.4, 33.78, 44.65, 55.43, 62.1, 65.97, 167.27, 173.27, 181.74 (fig. 2). ESI-MS calculated for C ₃₈ H ₇₀ S ₂ N ₄ O ₅ ⁺ [M+H] ⁺ 726.5, found727.6。

Example 5: ((6-chloro-1, 3, 5-triazine-2, 4-diyl) bis (sulfonamido) bis (hexane-6, 1-diyl) ditetradecanoate

Cyanuric chloride (1.84 g, 10 mmol) and 20mL of THF were added sequentially to a 50mL reaction flask, followed by 6-mercapto-1-hexanol (2.74 mL, 20 mmol), DIPEA (4.42 mL, 24 mmol) at-20 deg.C, stirred at room temperature overnight, and purified by column chromatography (hexane: EA =3:1 to 1: 2) to give 6,6' - ((6-chloro-1, 3, 5-triazine-2, 4-diacyl) bis (sulfonamido)) bis (hexane-1-ol) (3.0 g, 79%). This was followed by the addition of tetradecanoic acid (1.81 g, 7.9 mmol), EDC.HCl (1.51 g, 7.9 mmol), DMAP (48 mg, 0.395 mmol) and DIPEA (1.45 mL,7.9 mmol) in that order, stirring at room temperature for 6h, and purification by column chromatography (hexane: EA =10: 1) gave ((6-chloro-1, 3, 5-triazine-2, 4-diyl) bis (sulfanyl) bis (hexane-6, 1-diyl) bistetradecanoate (4.74 g, 75%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，40H），1.42（m，8H），1.60（m，4H），1.64（m，4H），1.66（m，4H），2.32（t，4H），3.15（t，4H），4.13（t，4H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，25.1，28.2，28.9，29.0，29.3，29.6，30.2，31.9，33.9，36.7，65.2，167.6，173.1，183.5。ESI-MS calculated for C ₄₃ H ₇₈ ClS ₂ N ₃ O ₄ ⁺ [M+H] ⁺ 800.5, found 800.7。

Example 6: ((6- (2- (pyrrolidin-1-yl) ethoxy) -1,3, 5-triazine-2, 4-diyl) bis (sulfonamido) bis (hexane-6, 1-diyl) bistetradecanoate

To a 25mL reaction tube were added N- (2-hydroxyethyl) -pyrrolidine (690. mu.L, 5.9 mmol), ((6-chloro-1, 3, 5-triazine-2, 4-diyl) bis (sulfonamido) bis (hexane-6, 1-diyl) ditetradecanoate (4.74 g, 5.9 mmol), 5mL THF in this order, and after stirring at room temperature for 3 hours, the compound was purified by column chromatography (DCM: MeOH =10: 1) to give ((6- (2- (pyrrolidin-1-yl) ethoxy) -1,3, 5-triazine-2, 4-diyl)Bis (sulfonamido) bis (hexane-6, 1-diyl) ditetradecanoate (4.1 g, 79%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，40H），1.42（m，8H），1.60（m，4H），1.64（m，4H），1.66（m，4H），1.68（m，4H），2.32（t，4H），2.51（t，4H），2.66（t，2H），3.15（t，4H），4.11（t，2H），4.13（t，4H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ14.1，22.7，23.6，25.0，25.1，28.2，28.9，29.0，29.3，29.6，30.2，31.9，33.9，36.7，56.5，56.9，65.2，66.8，173.1，172.8，181.0。ESI-MS calculated for C ₄₉ H ₉₀ S ₂ N ₄ O ₅ ⁺ [M+H] ⁺ 879.6, found880.7。

Example 7: 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, pentadecyl mercaptan (1443. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃ after stirring for 30min, 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to give a reaction solution of 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol, octanoic acid (951. mu.L, 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at room temperature for 6h, purification (hexane: EA =10: 1) is used to give 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.95 g, 68%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，32H），1.64（m，2H），1.71（m，2H），2.01（m，2H），2.32（t2H), 3.12 (t, 2H), 3.19 (t, 2H), 4.19 (t, 2H) (fig. 3). ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₂₉ H ₅₂ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 575.3, found 575.5。

Example 8: 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

To a 50mL reaction vial were added 1- (3-hydroxypropyl) -4-methylpiperazine (545 μ L, 3.4 mmol) and 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.95 g, 3.4 mmol), 10mL THF in sequence, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.42 g, 60%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，32H），1.64（m，2H），1.66（m，2H），1.82（m，2H），1.97（m，2H），2.14（s，3H），2.29（s，8H），2.32（t，2H），2.48（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.7，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，46.6，55.4，57.6，58.2，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₇ H ₆₉ S ₂ N ₅ O ₃ ⁺ [M+H] ⁺ 696.5, found697.6。

Example 9: 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, pentadecyl mercaptan (1443. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃ after stirring for 30min, 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to give a reaction solution of 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol, nonanoic acid (1048. mu.L 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at room temperature for 6h, purification (hexane: EA =10: 1) is used to give 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester (1.99 g, 68%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，34H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.19（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₀ H ₅₄ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 589.3, found 590.5。

Example 10: 3- ((4- (3- (dimethylamino) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester

3-dimethylamino-1-propanol was added to a 50mL reaction flask in sequence(402. mu.L, 3.4 mmol) and propyl 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.99 g, 3.4 mmol), 10mL of THF, stirring for 3 h at room temperature and purification by column chromatography (DCM: MeOH =10: 1) gave the compound propyl 3- ((4- (3- (dimethylamino) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.35 g, 61%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，34H），1.66（m，2H），1.80（m，2H），1.97（m，2H），2.15（t，6H），2.32（t，2H），2.34（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.1，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，47.0，57.9，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₅ H ₆₆ S ₂ N ₄ O ₃ ⁺ [M+H] ⁺ 655.5, found656.6。

Example 11: 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl decanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, pentadecyl mercaptan (1443. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃ after stirring for 30min, 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to give a reaction solution of 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol, octanoic acid (951. mu.L, 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at room temperature for 6h, purification (hexane: EA =10: 1) is used to give 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl decanoate (2.0 g, 68%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，36H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.19（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₁ H ₅₆ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 602.3, found 603.5。

Example 12: 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) decanoic acid propyl ester

To a 50mL reaction vial were added 1- (3-hydroxypropyl) -4-methylpiperazine (545 μ L, 3.4 mmol) and 3- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl decanoate (2.0 g, 3.4 mmol), 10mL of THF in sequence, and after stirring for 3 h at room temperature, purification was performed using column chromatography (DCM: MeOH =10: 1) to give the compound 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl decanoate (1.42 g, 60%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，36H），1.64（m，2H），1.66（m，2H），1.82（m，2H），1.97（m，2H），2.14（s，3H），2.29（s，8H），2.32（t，2H），2.48（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.7，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，46.6，55.4，57.6，58.2，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₇ H ₆₉ S ₂ N ₅ O ₃ ⁺ [M+H] ⁺ 724.5, found725.6。

Example 13: propyl 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, hexadecanethiol (1539. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃ after stirring for 30min, 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to give a reaction solution of 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol, nonanoic acid (1048. mu.L, 6 mmol), EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at room temperature for 6h, purification (hexane: EA =10: 1) is used to give 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester (2.36 g, 78%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，36H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₁ H ₅₆ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 602.3, found 603.5。

Example 14: 3- ((4- ((3- (dimethylamino) propyl) amino) -6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester

To a 50mL reaction vial were added N, N-dimethyl-1, 3-diaminopropane (492 μ L, 3.91 mmol) and propyl 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (2.36 g, 3.91 mmol), 10mL of THF in order, and after stirring for 3 h at room temperature, purification was performed using column chromatography (DCM: MeOH =10: 1) to give the compound propyl 3- ((4- ((3- (dimethylamino) propyl) amino) -6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.7 g, 65%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，36H），1.64（m，2H），1.66（m，2H），1.72（m，2H），1.97（m，2H），2.15（s，6H），2.32（t，2H），2.38（t，2H），3.08（t，2H），3.15（t，2H），3.35（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，26.5，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，40.0，47.0，55.5，64.1，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₃₆ H ₆₉ S ₂ N ₅ O ₂ ⁺ [M+H] ⁺ 668.5, found669.6。

Example 15: 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL THF were added sequentially in a 50mL reaction flask, -hexadecanethiol (1539. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) were added at 20 ℃ and stirred for 30min, then 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) were added and stirred overnight at room temperature to give a 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol reaction solution, octanoic acid (951. mu.L, 6 mmol), EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol), and usualAfter stirring for 6h, it was purified by column chromatography (hexane: EA =10: 1) to give propyl 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) octanoate (2.36 g, 80%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，34H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₁ H ₅₆ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 588.3, found 589.5。

Example 16: 3- ((4- (3- (dimethylamino) propoxy) -6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

To a 50mL reaction vial were added 3-dimethylamino-1-propanol (473 μ L, 3.4 mmol) followed by 3- ((4-chloro-6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propyl nonanoate (2.36 g, 4.0 mmol), 10mL THF in order, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound 3- ((4- (3- (dimethylamino) propoxy) -6- (hexadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.8 g, 68%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，34H），1.66（m，2H），1.80（m，2H），1.97（m，2H），2.15（t，6H），2.32（t，2H），2.34（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.1，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，47.0，57.9，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₅ H ₆₆ S ₂ N ₄ O ₃ ⁺ [M+H] ⁺ 655.5, found656.6。

Example 17: 4- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thiooctanoic acid butyl ester

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, octadecanethiol (1.43 mg, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃, stirring is carried out at 20 ℃ for 30min, 4-mercapto-1-butanol (516. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to obtain a 4- ((4-chloro-6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) butanol reaction solution, octanoic acid (951. mu.L, 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at normal temperature for 6h, purification by column chromatography (hexane: EA =10: 1) is carried out to obtain 4- ((4-chloro-6- (octadecylthio) -1, butyl 3, 5-triazin-2-yl) thiooctanoate (2.42 g, 76%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.6-1.66（m，8H），2.32（t，2H），3.15（t，4H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.4，27.8，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，33.9，36.4，36.7，64.5，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₃ H ₆₀ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 630.4, found 631.5。

Example 18: 4- (4- (3- (dimethylamino) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thiooctanoic acid butyl ester

3-diethyl ether was added to a 50mL reaction flask in sequenceAmino-1-propanol (570 μ L, 3.84 mmol) and butyl 4- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thiooctanoate (2.42 g, 3.84 mmol), 10mL THF, after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound butyl 4- (4- (3- (diethylamino) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thiooctanoate (1.76 g, 63%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.15（t，6H），1.25-1.35（m，38H），1.6-1.66（m，8H），1.82（m，2H），2.32（t，2H），2.48（t，2H），3.01（m，4H），3.15（t，4H），4.13（t，2H），4.2（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 13.3，14.1，22.7，25.0，26.4，27.7，27.8，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，33.9，36.4，36.7，49.9，58.2，64.5，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₈ H ₇₂ S ₂ N ₄ O ₃ ⁺ [M+H] ⁺ 697.5, found698.6。

Example 19: 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added sequentially in a 50mL reaction flask, octadecanethiol (1.43 mg, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃, stirring is carried out at-20 ℃ for 30min, then 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature, 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol reaction solution is obtained, nonanoic acid (1048. mu.L, 6 mmol), EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at normal temperature for 6h, purification (hexane: EA =10: 1) is used to obtain 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester (2.42 g, 76%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，40H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₃ H ₆₀ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 630.4, found 631.5。

Example 20: 3- ((4- ((2- (1-isopropylpiperidin-4-yl) ethyl) amino) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoic acid propyl ester

To a 50mL reaction vial were added 2- (1-isopropyl-piperidin-4-yl) -ethylamine (654 mg, 3.84 mmol), 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (2.42 g, 3.84 mmol) and 10mL THF in order, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound 3- ((4- ((2- (1-isopropylpiperidin-4-yl) ethyl) amino) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.55 g, 53%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.00（d，6H），1.25-1.35（m，40H），1.4（m，1H），1.5（m，2H），1.56（m，4H），1.6-1.66（m，4H），1.97（m，2H），2.32（t，2H），2.41-2.51（m，4H），2.69（m，1H），3.08（t，2H），3.15（t，2H），3.35（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，17.0，18.1，22.7，25.0，25.1，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，34.7，36.7，42.8，45.7，58.3，64.1，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₄₃ H ₈₁ S ₂ N ₅ O ₂ ⁺ [M+H] ⁺ 764.6, found765.8。

Example 21: propyl 3- ((4- ((2- (4-methylpiperazin-1-yl) ethyl) amino) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate

4-methyl-1-piperazineethylamine (550 mg, 3.84 mmol), 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (2.42 g, 3.84 mmol) and 10mL THF were added sequentially to a 50mL reaction flask, and after stirring for 3 h at room temperature, purification was performed using column chromatography (DCM: MeOH =10: 1) to give the compound 3- ((4- ((2- (4-methylpiperazin-1-yl) ethyl) amino) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.66 g, 59%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，40H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.14（s，3H），2.29（s，8H），2.32（t，2H），2.50（t，2H），3.08（t，2H），3.15（t，2H），3.34（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，46.6，47.2，55.5，57.6，64.1，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₄₀ H ₇₆ S ₂ N ₆ O ₂ ⁺ [M+H] ⁺ 737.5, found738.7。

Example 22: propyl 3- ((4- (octadecylthio) -6- ((2- (pyrrolidin-1-yl) ethyl) amino) -1,3, 5-triazin-2-yl) thio) nonanoate

1- (2-aminoethyl) pyrrolidine (1) was added to a 50mL reaction flask in sequence228mg, 2 mmol), propyl 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) nonanoate (1.26 g, 2 mmol) and 10mL THF, after stirring for 3 h at rt, are purified using column chromatography (DCM: MeOH =10: 1) yielded the compound propyl 3- ((4- (octadecylthio) -6- ((2- (pyrrolidin-1-yl) ethyl) amino) -1,3, 5-triazin-2-yl) thio) nonanoate (1.08 g, 76%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，40H），1.64（m，2H），1.66（m，2H），1.68（m，4H），1.97（m，2H），2.32（t，2H），2.50（t，2H），2.51（t，4H），3.08（t，2H），3.15（t，2H）3.34（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，23.6，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，47.2，55.5，56.2，64.1，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₃₉ H ₇₃ S ₂ N ₅ O ₂ ⁺ [M+H] ⁺ 708.5, found709.7。

Example 23: 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask, octadecanethiol (1.43 mg, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃, stirring is carried out at-20 ℃ for 30min, then 3-mercapto-1-propanol (435. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to obtain a reaction solution of 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propan-1-ol, octanoic acid (951. mu.L, 6 mmol), EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at room temperature for 6h, purification is carried out using EA =10:1 to obtain 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (2.42 g, 78%)。 ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.64（m，2H），1.66（m，2H），1.97（m，2H），2.32（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，64.1，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₂ H ₅₈ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 616.4, found 617.5。

Example 24: 3- ((4- (3- (dimethylamino) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

To a 25mL reaction tube were added 3-dimethylamino-1-propanol (237 μ L, 2.0 mmol) and 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.21 g, 2.0 mmol), 5mL THF in order, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound 3- ((4- (3- (dimethylamino) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (0.83 g, 61%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.66（m，2H），1.80（m，2H），1.97（m，2H），2.15（t，6H），2.32（t，2H），2.34（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.1，28.5，28.9，29.0，29.3，29.6，30.2，31.9，32.9，33.9，36.7，47.0，57.9，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₇ H ₇₀ S ₂ N ₄ O ₃ ⁺ [M+H] ⁺ 683.5, found684.6。

Example 25: 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

To a 25mL reaction tube were added 1- (3-hydroxypropyl) -4-methylpiperazine (321 μ L, 3.4 mmol) and 3- ((4-chloro-6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (1.21 g, 2.0 mmol), 5mL THF in sequence, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave the compound 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (octadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate (0.88 g, 60%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.64（m，2H），1.66（m，2H），1.82（m，2H），1.97（m，2H），2.14（s，3H），2.29（s，8H），2.32（t，2H），2.48（t，2H），3.08（t，2H），3.15（t，2H），4.13（t，2H），4.20（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，26.3，27.7，28.5，28.9，29.0，29.3，29.6，30.2，31.8，31.9，32.9，33.9，36.7，46.6，55.4，57.6，58.2，64.1，66.4，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₄₀ H ₇₅ S ₂ N ₅ O ₃ ⁺ [M+H] ⁺ 738.5, found739.6。

Example 26: 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

Adding cyanuric chloride (920 mg, 5 mmol) and 15mL THF in sequence into a 50mL reaction flask, adding heptanethiol (784 uL, 5 mmol) and DIPEA (1105 uL, 6 mmol) at-20 deg.C, stirring at-20 deg.C for 30min, adding6-mercapto-1-hexanol (684. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) were stirred at room temperature overnight to give a reaction solution of 6- ((4-chloro-6- (heptanethiol) -1,3, 5-triazin-2-yl) thio) hexan-1-ol, tetradecanoic acid (1.38 g, 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) were added, stirred at room temperature for 6h, and purified by column chromatography (hexane: EA =10: 1) to give hexyl 6- ((4-chloro-6- (heptanethiol) -1,3, 5-triazin-2-yl) thio) tetradecanoate (1.77 g, 60%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，26H），1.4-1.45（m，6H），1.6（m，2H），1.64（m，4H），1.66（m，2H），2.32（t，2H），3.15（t，4H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，25.1，28.2，28.5，28.6，28.9，29.0，29.3，29.6，30.2，31.8，31.9，33.9，36.7，65.2，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₀ H ₅₄ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 588.3, found 589.5。

Example 27: 3- ((4- (3- (4-methylpiperazin-1-yl) propoxy) -6- (pentadecylthio) -1,3, 5-triazin-2-yl) thio) propyl octanoate

1- (3-hydroxypropyl) -4-methylpiperazine (481 μ L, 3 mmol) and 6- ((4-chloro-6- (heptanethiol) -1,3, 5-triazin-2-yl) thio) tetradecanoate (1.77 g, 3 mmol), 10mL of THF were added successively in a 50mL reaction flask, and after stirring for 3 h at room temperature, purification was performed by column chromatography (DCM: MeOH =10: 1) to obtain the compound 6- ((4- (heptanethiol) -6- (2- (4-methylpiperazin-1-yl) ethoxy) -1,3, 5-triazin-2-yl) thio) tetradecanoate (1.46 g, 70%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，26H），1.4-1.45（m，6H），1.6（m，2H），1.64（m，4H），1.66（m，2H），2.14（s，3H），2.29（s，8H），2.32（t，2H），2.66（t，2H），3.15（t，4H），4.11（t，2H）4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，25.1，28.2，28.5，28.6，29.0，29.3，29.6，30.2，31.8，31.9，33.9，36.7，46.6，56.9，57.6，57.9，65.2，66.8，172.8，173.1，178.9，183.2。ESI-MS calculated for C ₃₇ H ₆₉ S ₂ N ₅ O ₃ ⁺ [M+H] ⁺ 696.5, found697.6。

Example 28: octyl 8- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thiohexanoate

Cyanuric chloride (920 mg, 5 mmol) and 15mL THF are added in sequence in a 50mL reaction flask, decathiol (1058. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added at-20 ℃, stirring reaction is carried out at-20 ℃ for 30min, then 8-mercapto-1-octanol (873. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out overnight at room temperature to obtain a reaction solution of 8- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thio) octan-1-ol, hexanoic acid (752. mu.L, 6 mmol) EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirring is carried out at normal temperature for 6h, purification (hexane: EA =10: 1) is used to obtain 8- ((4-chloro-6- (decylthio) -1, octyl 3, 5-triazin-2-yl) thiocarbhexanoate (1.9 g, 69%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，24H），1.43（m，2H），1.6-1.66（m，8H），2.32（t，2H），3.15（t，4H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.4，22.7， 25.8，28.5，28.9，29.0，29.3，29.6，30.2，31.1，31.2，31.9， 33.9，36.7，65.2，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₂₇ H ₄₈ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 547.3, found 548.5。

Example 29: octyl 8- ((4- (decylthio) -6- ((2-morpholinoethyl) amino) -1,3, 5-triazin-2-yl) thiohexanoate

To a 50mL reaction flask were added N- (2-aminoethyl) morpholine (453 μ L, 3.45 mmol), 8- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) octyl thiohexanoate (1.9 g, 3.45 mmol), and 10mL THF in order, and after stirring at room temperature for 3 h, purification was performed using column chromatography (DCM: MeOH =10: 1) to give the compound 8- ((4- (decylthio) -6- ((2-morpholinoethyl) amino) -1,3, 5-triazin-2-yl) octyl thiohexanoate (1.66 g, 75%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.45（m，26H），1.6-1.66（m，8H），2.32（t，2H），2.40（t，4H），2.50（t，2H），3.15（t，4H），3.34（t，2H），3.52（t，4H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.4，22.7，25.8，28.5，28.9，29.3，29.6，30.2，31.1，31.2，31.9，33.9，36.7， 47.2，55.8，65.2，66.7，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₃₃ H ₆₁ S ₂ N ₅ O ₃ ⁺ [M+H] ⁺ 640.4, found641.6。

Example 30: hexyl 6- ((4-chloro-6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) dodecanoate

Adding cyanuric chloride (920 mg, 5 mmol) and 15mL THF in sequence into a 50mL reaction flask, adding tetradecanethiol (1362 uL, 5 mmol) and DIPEA (1105 uL, 6 mmol) at-20 deg.C, stirring at 20 deg.C for 30min, adding 6-mercapto-1-hexanol (684 uL, 5 mmol) and DIPEA (1105 uL, 6 mmol), stirring at room temperature overnight to obtain 6- ((4-chloro-6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) hexan-1-ol reaction solution, adding lauric acid (lauric acid) (lauric acid: (lauric acid) (4-chloro-6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) hexane-1-ol1.2g, 6 mmol), edc.hcl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105 μ L, 6 mmol), stirred at room temperature for 6h, purified using column chromatography (hexane: EA =10: 1) to give hexyl 6- ((4-chloro-6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) dodecanoate (2.5 g, 76%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.43（m，4H），1.6-1.66（m，8H），2.32（t，2H），3.15（t，4H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，25.1，28.2，28.5，28.9，29.0，29.3，29.6，30.2，31.9，33.9，36.7，65.2，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₅ H ₆₄ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 658.4, found 659.7。

Example 31: hexyl 6- ((4- ((2- (piperidin-1-yl) ethyl) amino) -6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) dodecanoate

To a 50mL reaction vial were added 1- (2-aminoethyl) piperidine (542 μ L, 3.8 mmol), 6- ((4-chloro-6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) hexyl dodecanoate (2.5 g, 3.8 mmol) and 10mL THF in order, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave compound 6- ((4- ((2- (piperidin-1-yl) ethyl) amino) -6- (tetradecylthio) -1,3, 5-triazin-2-yl) thio) hexyl dodecanoate (1.71 g, 60%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，38H），1.37（m，2H），1.42（m，2H），1.43（m，2H），1.49（m，4H），1.6-1.66（m，8H），2.32（t，2H），2.42（t，4H），2.50（t，2H），3.15（t，4H），3.34（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，24.5，25.0，25.1，25.9，28.2，28.5，28.9，29.0，29.3，29.6，30.2，31.9，33.9，36.7，47.2，55.8，56.8，65.2，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₄₂ H ₇₉ S ₂ N ₅ O ₂ ⁺ [M+H] ⁺ 750.5, found751.7。

Example 32: 6- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thio) dodecanoic acid hexyl ester

Cyanuric chloride (920 mg, 5 mmol) and 15mL of THF are added in sequence in a 50mL reaction flask at-20 deg.C decathiol (1058. mu.L, 5 mmol), DIPEA (1105. mu.L, 6 mmol), stirred at 20 deg.C for 30min, then 6-mercapto-1-hexanol (684. mu.L, 5 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirred at room temperature overnight to give a 6- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thio) hexane-1-ol reaction solution, lauric acid (1.2 g, 6 mmol), EDC.HCl (1150 mg, 6 mmol), DMAP (30 mg, 0.25 mmol) and DIPEA (1105. mu.L, 6 mmol) are added, stirred at room temperature for 6h, purified using EA =10:1 to give 6- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thio) hexyl dodecanoate (2.2 g, 73%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.88 （t，6H），1.25-1.35（m，30H），1.43（m，4H），1.6-1.66（m，8H），2.32（t，2H），3.15（t，4H），4.13（t，2H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，22.7，25.0，25.1，28.2，28.5，28.9，29.0，29.3，29.6，30.2，31.9，33.9，36.7，65.2，167.6，173.1，182.2，184.9。ESI-MS calculated for C ₃₁ H ₅₆ ClS ₂ N ₃ O ₂ ⁺ [M+H] ⁺ 602.4, found 603.6。

Example 33: 6- ((4- (decylthio) -6- ((2- (4-methylpiperidin-1-yl) ethyl) amino) -1,3, 5-triazin-2-yl) thio) dodecanoic acid hexyl ester

To a 50mL reaction flask were added 2- (4-methyl-1-piperidinyl) ethylamine (520 mg, 3.65 mmol), 6- ((4-chloro-6- (decylthio) -1,3, 5-triazin-2-yl) thio) hexyl dodecanoate (2.2 g, 3.65 mmol), and 10mL THF in order, and after stirring for 3 h at room temperature, purification by column chromatography (DCM: MeOH =10: 1) gave compound 6- ((4- (decylthio) -6- ((2- (4-methylpiperidin-1-yl) ethyl) amino) -1,3, 5-triazin-2-yl) thio) hexyl dodecanoate (1.71 g, 66%). ¹ H NMR (400 MHz，CDCl ₃ )：δ 0.86（t，3H），0.88 （t，6H），1.25-1.35（m，30H），1.42（m，2H），1.43（m，2H），1.56（m，4H），1.6-1.66（m，9H），2.32（t，2H），2.4-2.5（m，6H），3.15（t，4H），3.34（t，2H），4.13（t，2H），7.01（s，1H）。 ¹³ C NMR (400 MHz，CDCl ₃ )：δ 14.1，20.4，22.7，25.0，25.1，28.2，28.5，28.9，29.0，29.3，29.6，30.2， 31.9，32.0，33.9，36.7，47.2，47.5，55.8，65.2，161.1，173.1，180.2，183.4。ESI-MS calculated for C ₃₉ H ₇₃ S ₂ N ₅ O ₂ ⁺ [M+H] ⁺ 708.5, found709.7。

Example 34: transfection of lipid nanoparticles prepared with amino-lipid compounds on BMDC primary cells

The preparation method comprises the following steps: the same as in example 23.

Animal preparation: female C57BL/6 mice of 6 weeks old are selected, the weight is about 20 g, the breeding environment is a SPF-grade breeding room, and animal experiments are carried out strictly according to the guidelines of the national health institution and the ethical requirements of animals.

Cell acquisition: killing C57BL/6 mouse by removing neck and socket, soaking in 75% alcohol for 5min for sterilization, dissecting to obtain thigh and calf tibia, removing attached muscle to expose bone, blowing out bone marrow in tibia with 1ml syringe with PBS, filtering out impurities with 50um filter screen, adding erythrocyte lysate into the filtrate(3-4 mL) and left for 5 minutes, then centrifuged at 800g and 5 minutes to remove the supernatant, the cells were resuspended in 1640 medium (containing 10% fetal calf serum, 20ng/mL GMCSF, 10ng/mL IL 4), seeded in a 6-well plate at a density of 100000 cells/mL medium, left at 37 ℃ and 5% CO ₂ In the cell culture box, half liquid change is carried out once every 2 days, suspension cells and loosely attached cells are collected on the seventh day and are inoculated to a 96-well holo-white enzyme standard plate, the inoculation density is 20000 cells per well, and the volume of a culture medium is 100 uL.

Cell transfection: adding lipid nanoparticles wrapping luciferase mRNA into a 96-well total white enzyme standard plate paved with primary cells, controlling the addition volume of the mRNA lipid nanoparticles in each well to be 10 mu l, and adding RNA content to be 0.1 mu g/well. Then placed at 37 ℃ with 5% CO ₂ Concentration incubator for 16 hours.

And (3) detecting the transfection efficiency: add 20 uL of the substrate ONE-Glo to each well of a 96-well holo-white enzyme standard ^TM Luciferase, 1min later, was detected with a multifunctional microplate reader (Biorek SynergyH 1). The expression intensity of representative amino lipid compounds transfected LucmRNA on BMDC is shown in Table 4. DLin-MC3 served as controls, and many of the amino lipids were similar in expression intensity to MC3 and were significantly better than the positive control.

TABLE 4 expression intensity of partial amino lipid compounds transfection on BMDC

In Table 4, Compound 1 has the formula

(ii) a The structural general formula of the compound 2-11 is

(ii) a The general structural formula of the compounds 12-18 is

(ii) a The structural general formula of the compounds 19 to 476 is

(ii) a The structural general formula of the compounds 477-563 is

In the above formula, R ₂ And R ₃ Is a saturated alkyl radical, in Table 4, R ₁ The values of (A) refer to the corresponding substituents in the substituents O1 to O16, D1 to D20, R, as defined above ₂ And R ₃ The value of (b) is the number of carbon atoms in the carbon chain.

Example 35: evaluation of luciferase mRNA in vivo delivery Performance of lipid nanoparticles prepared from amino lipid Compound

1. Preparation of lipid nanoparticles

Mixing the amino lipid compound with neutral lipid (such as DSPC, DOPE, cholesterol) and polyethylene glycol lipid (such as PEG2000-DMG, PEG 2000-DSPE) at optimized molar ratio, and dissolving in anhydrous ethanol. The resulting ethanol solution and sodium acetate solution (25 mM, pH = 5.0) or sodium citrate solution (25 mM, pH = 4.0 or pH = 5.0) dissolved with Luc-mrna (trilink) were mixed in a 1:3 volume ratio in a microfluidic chip using a microfluidic preparation system to make a crude solution of lipid nanoparticles, which was then diluted 8-fold or more with 1X PBS and ultrafiltered three times with 15ml or 50ml ultrafiltration centrifuge tubes (Millipore, 100K) at controlled temperature 4 ℃ at 1.5rcf rpm for 15 min. The mass ratio of aminolipid compound to luciferase mrna (luc mrna) was about 5:1.

Characterization of lipid nanoparticles:

characterization of particle size: the particle size and PDI of the prepared lipid nanoparticles were determined by Nano-ZSZEN3600 (Malvern). Particle size measurements were taken at 60uL (RNA concentration 100. mu.g/ml) of LNP solution and cycled three times for 30s each.

And (3) determining the encapsulation efficiency: the assay was performed according to the standard protocol of the Quant-iT RiboGreen RNA kit.

Table 5: characterization data for LNP prepared using representative amino lipidic compounds

Note: in the above table:

the general formula of LNP-1-LNP-5 amino lipid is

(ii) a The general formula of LNP-6-LNP-68 amino lipid is

(ii) a The general formula of LNP-69-LNP-88 amino lipid is

(ii) a In the above formula, R ₂ And R ₃ Is a saturated alkyl radical, in Table 5, R ₁ The values of (A) refer to the corresponding substituents in the substituents O1 to O16, D1 to D20, R, as defined above ₂ And R ₃ The value of (b) is the number of carbon atoms of the carbon chain;

LNP-1-LNP-9, LNP-89 has a lipid formulation: amino lipid DSPC cholesterol PEG2000-DMG = 50:10:38.5: 1.5; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

the lipid formula of LNP-10-LNP-18 is as follows: amino lipid DSPC cholesterol PEG2000-DMG = 50:10:38.5: 1.5; the aqueous phase used was a sodium citrate solution (25 mM, pH = 4.0) with Luc-mrna (trilink) dissolved;

the lipid formula of LNP-19-LNP-27 is as follows: amino lipid DSPC cholesterol PEG2000-DMG = 50:10:38.5: 1.5; the aqueous phase used was a sodium citrate solution (25 mM, pH = 5.0) with Luc-mrna (trilink) dissolved;

the lipid formula of LNP-28-LNP-36 is as follows: amino lipids DOPE cholesterol PEG2000-DMG = 45:10:43: 2; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

the lipid formula of LNP-37-LNP-45 is as follows: amino lipids DOPC cholesterol PEG2000-DMG = 45:5:48.5: 1.5; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

the lipid formulations of LNP-46 and LNP-54 are: amino lipids DSPC cholesterol PEG2000-DMG =42:9:47.5: 1.5; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

the lipid formulations of LNP-55 and LNP-63 are: amino lipids DSPC cholesterol PEG2000-DMG =42:9:47.5: 1.5; the aqueous phase used was a sodium citrate solution (25 mM, pH = 4.0) with Luc-mrna (trilink) dissolved;

the lipid formula of LNP-64-LNP-72 is as follows: amino lipids DSPC cholesterol PEG2000-DMG =42:9:47.5: 1.5; the aqueous phase used was a sodium citrate solution (25 mM, pH = 5.0) with Luc-mrna (trilink) dissolved;

the lipid formula of LNP-73-LNP-81 is as follows: amino lipids DSPC cholesterol PEG2000-DMG =36:9:52: 1; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

the lipid formula of LNP-82-LNP-88 is as follows: amino lipids DSPC cholesterol PEG2000-DMG =46:6:46.2: 1.8; the aqueous phase used was a sodium acetate solution (25 mM, pH = 5.0) in which Luc-mrna (trilink) was dissolved;

2. animal experiments

Animal preparation: female C57BL/6 mice 6 weeks old, weighing approximately 20 g, were selected and housed in SPF-rated housing. Animal experiments were performed strictly according to the guidelines of the national health authorities and the ethical requirements of animals.

In vivo delivery: according to the above table, 9C 57BL/6 mice were randomly selected from each group, and the lipid nanoparticle solution was injected using three administration modes of subcutaneous, intramuscular, and tail vein injection (3 mice per administration mode) at an amount of 0.5 mg/kg mRNA, respectively. After 12 hours, 200. mu.L of 10 mg/mL potassium D-luciferin was injected into each mouse via the tail vein, and after 10 minutes, the mice were placed under an in vivo imaging system (IVIS-200, Xenogen), and the total fluorescence intensity of each mouse was observed and recorded by photographing. The expression intensities of Fluc mRNA delivered by 3 administration modalities for representative amino lipid compounds are shown in tables 6-8. DLin-MC3 served as a control.

Table 6: expression intensity of Luc mRNA delivered by subcutaneous administration of representative amino lipid compound LNP

Table 7: expression intensity of Luc mRNA delivered by intramuscular administration of representative amino lipid compound LNP

Table 8: expression intensity of Luc mRNA delivered by tail vein administration of representative amino lipid compound LNP

Example 36: evaluation of in vivo Immunity and tumor treatment Effect of lipid nanoparticles prepared from amino lipid Compound

The preparation method comprises the following steps: the mol ratio of the amino lipidic compound to DSPC, cholesterol and PEG2000-DMG is 50:10:38.5:1.5 in the absolute ethanol. Ovalbumin mrna (ova mrna) was dissolved in sodium citrate solution (25 mM, pH = 5.0). The resulting ethanol solution and sodium citrate solution (25 mM, pH = 5.0) with Luc-mrna (trilink) dissolved therein were mixed in a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to prepare lipid nanoparticles, which were then diluted 8-fold or more with 1X PBS and ultrafiltered three times with 15ml or 50ml ultrafiltration centrifuge tubes (Millipore, 100K) at a temperature controlled 4 ℃ at a rotation speed of 1.5rcf for 15 min. The mass ratio of aminolipid compound to luciferase mrna (luc mrna) was about 5:1.

Animal preparation: selecting 5-6 week-old female C57BL/6 mice, weighing about 18-20 g, and feeding in SPF-level feeding room, wherein animal test is carried out strictly according to the national health institution guide and animal ethical requirements.

In vivo delivery: B16-OVA melanoma cells (1.5X 10) ⁵ ) Injected subcutaneously into the outer thigh of mice. When the tumor size is equal to 50 mm ³ Vaccination was started (approximately day 6 or 7 after tumor vaccination). Animals were immunized twice by intramuscular injection of LNP preparation containing 1 μ g OVA-mRNA, with 7 days intervals for the second needle. Tumor growth was measured 3 times per week using digital caliper, and the formula was 0.5 x length x width. When the tumor volume reaches 1500 mm ³ Mice were euthanized at time. Tumor growth rates of LNP-23 and LNP-61 were significantly slower than those of the MC3 group (as shown in FIG. 4), and complete remission was achieved in 40% (LNP-23 group) and 70% (LNP-61 group), respectively, mice significantly better than the MC3 group (as shown in FIG. 5).

Example 37: evaluation of cytotoxicity of lipid nanoparticles prepared from amino lipid compounds

Cell treatment: hela cells were plated in 96-well plates at 5000 cells per well, and six duplicate wells were set, and a background group without cells and a blank control group with PBS were set. Cells were allowed to adhere completely after 24 hours of culture.

The preparation method comprises the following steps: the mol ratio of the amino lipidic compound to DSPC, cholesterol and PEG2000-DMG is 50:10:38.5:1.5 in the absolute ethanol. The resulting ethanol solution and sodium citrate solution (25 mM, pH = 5.0) were mixed in a microfluidic chip at a volume ratio of 1:3 using a microfluidic preparation system to prepare lipid nanoparticles without any RNA, which were then diluted more than 8-fold with 1X PBS and ultrafiltered three times with 15ml or 50ml ultrafiltration centrifuge tubes (Millipore, 100K) at a temperature controlled 4 ℃ at a rotation speed of 1.5rcf for 15 min. The mass ratio of aminolipid compound to luciferase mrna (luc mrna) was about 5:1. After ultrafiltration, the cells were diluted with 1X PBS to different concentrations and added. Then placed at 37 ℃ with 5% CO ₂ The culture was carried out in a concentration incubator for 16 hours.

And (3) toxicity detection: adding 10 uL CCK-8 reagent into each well of a 96-well plate, and adding 5% CO at 37 DEG C ₂ After incubation for 30min-1h in an incubator with a concentration, absorbance at 450nm was measured with a multifunctional microplate reader (Biorek SynergyH 1), and cytotoxicity was calculated by the following formula:

cell viability (%) = [ a (medicated) -a (blank) ]/[ a (0 medicated) -a (blank) ] × 100

A (dosing): OD value of well having cells, CCK-8 solution and drug solution

A (0 dosing): OD of wells with cells, CCK-8 solution and no drug solution

A (blank): OD value of wells without cells

Part of the results are shown in FIG. 6, and under the same conditions, the toxicity of the amino lipid on the cells is close to 0 compared with the anticancer drug cisplatin.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. An amino lipid, characterized in that it has the structure shown in formula (I):

L ¹ 、L ² and L ³ Is substituted or unsubstituted C1-C16 alkylene, C1-C16 alkenylene, C1-C16 alkynylene, C3-C16 cycloalkylene, C3-C16 cycloalkenylene, and the substituent of the C1-C16 alkylene, C1-C16 alkenylene, C1-C16 alkynylene, C3-C16 cycloalkylene and C3-C16 cycloalkenylene is C1-C6 hydrocarbon;

when X is ¹ 、X ² And X ³ When C is selected as in (1), m, n and p are 2 correspondingly; x ¹ 、X ² And X ³ The two substituents attached are the same or different;

G ¹ and G ² Identical or different from each other, G ¹ Selected from the group consisting of-O-C (= O) -, -NH-C (= O) -, -C (= O) -O-, -C (= O) -NH-, -CH ₂ -； G ² Selected from-O-C (= O) -, -NH-C (= O) -, -C (= O) -O-, -C (= O) -NH-;

R ³ and R ⁴ Are identical to or different from each other and are each independently selected from H, substituted or unsubstitutedSubstituted C1-C18 alkyl, C1-C18 alkenyl, C1-C18 alkynyl, C1-C18 cycloalkyl, C1-C18 cycloalkenyl and C1-C18 cycloalkynyl, wherein the substituent of the C1-C18 alkyl, C1-C18 alkenyl, C1-C18 alkynyl, C1-C18 cycloalkyl, C1-C18 cycloalkenyl and C1-C18 cycloalkynyl is selected from C1-C6 hydrocarbyl.

2. The amino lipid according to claim 1, wherein L is L ² And L ³ Is substituted or unsubstituted C3-C16 alkylene, C3-C16 alkenylene, and the C3-C16 alkylene and C3-C16 alkenylene substituent is C1-C6 alkyl;

3. The amino lipid of claim 1, wherein G is ¹ And G ² Identical or different from each other, G ¹ Selected from the group consisting of-O-C (= O) -, -C (= O) -O-, -CH ₂ -； G ² Selected from-O-C (= O) -, -C (= O) -O-; L ² And L ³ Is substituted or unsubstituted C3-C16 alkylene, and the C3-C16 alkylene substituent is C1-C6 alkyl.

4. The amino lipid according to claim 1, wherein X is ¹ Is N, said R ¹ 、R ² 、L ¹ 、X ¹ Form R ₁ R ₂ -N-L ₁ -X ₁ Has the structure of

；

Wherein, R is ¹ 、R ² Can be connected to form a 4-10 membered heterocyclic ring, wherein the heterocyclic ring contains 1-6 heteroatoms selected from nitrogen, sulfur or oxygen, and the structure is selected from one of D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19 and D20:

。

5. the amino lipid according to claim 1, wherein X is ¹ Is O, said R ¹ 、R ² 、L ¹ 、X ¹ Form R ₁ R ₂ -N-L ₁ -X ₁ Has the structure of

；

Wherein, R is ¹ 、R ² Can be connected to form a 4-10 membered heterocyclic ring, wherein the heterocyclic ring contains 1-6 heteroatoms selected from nitrogen, sulfur or oxygen, and the structure is selected from one of O1, O2, O3, O4, O5, O6, O7, O8, O9, O10, O11, O12, O13, O14, O15 and O16:

。

6. a process for the preparation of an amino lipid according to any of claims 1 to 5, comprising the steps of:

7. Use of an amino lipid according to any one of claims 1 to 5, and pharmaceutically acceptable salts, prodrugs or stereoisomers thereof, for the manufacture of a medicament for use in gene therapy, gene vaccination, antisense therapy or therapy by interfering RNA drugs.

8. Use according to claim 7, for the preparation of a medicament for the treatment of cancer or genetic diseases.

9. Use according to claim 8, for the preparation of a medicament for the treatment of lung cancer, stomach cancer, liver cancer, oesophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic cancer, blood cancer, prostate cancer, allergies, toxicity.

10. Use according to claim 8, for the preparation of a medicament for the treatment of haemophilia, thalassemia or gaucher's disease.