CN117263882B

CN117263882B - Cationic lipid compound, composition containing cationic lipid compound and application of cationic lipid compound

Info

Publication number: CN117263882B
Application number: CN202311557594.1A
Authority: CN
Inventors: 胡勇; 余鹏程; 滕文琪; 欧阳立鹏
Original assignee: Wuhan Ruiji Biotechnology Co ltd; Shenzhen Ruiji Biotechnology Co ltd
Current assignee: Wuhan Ruiji Biotechnology Co ltd; Shenzhen Ruiji Biotechnology Co ltd
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2024-02-23
Anticipated expiration: 2043-11-21
Also published as: CN117263882A

Abstract

The invention provides a cationic lipid compound, a composition containing the same and application thereof. The invention provides a novel cationic lipid compound, which enriches the types of the cationic lipid compounds at present. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, high product yield, low instrument and equipment requirements and simple operation.(I)。

Description

Cationic lipid compound, composition containing cationic lipid compound and application of cationic lipid compound

Technical Field

The invention relates to the field of biological medicine, in particular to a cationic lipid compound, a composition containing the cationic lipid compound and application of the cationic lipid compound.

Background

Nucleic Acids (RNAs) have great potential by expressing proteins in vivo, show prophylactic and therapeutic effects according to different protein functions, but naked RNAs have short circulation time in vivo, low efficiency by cell internalization, and are very easily cleared by kidneys or degraded by in vivo rnases, thus the practical effect is greatly compromised. Cationic lipid compounds and related formulations are one of the key technologies for better delivery of nucleic acids to targets for enhancing their prophylactic and therapeutic effects.

LNP (lipid nanoparticles) acts as a current mainstream delivery vehicle, which can bind to mRNA to increase the in vivo cycle time of mRNA, control in vivo escape to increase its transfection efficiency, and increase protein expression. LNP is generally composed of four components, including: (1) a cationic lipid providing charge-entrapped mRNA molecules; (2) Supporting phospholipid, providing bilayer support, facilitating endosome escape; (3) Cholesterol, enhancing LNP stability, promoting membrane fusion; (4) PEG lipid, reduce LNP particle size, and improve in vivo circulation time.

In vivo, the cation is taken as one of key ingredients of the LNP, can have great influence on the instability and the action efficacy of the LNP preparation, and provides a novel high-efficiency and low-toxicity cationic compound.

Disclosure of Invention

It is an object of the present invention to provide a cationic lipid compound;

it is another object of the present invention to provide a liposome formulation;

it is a further object of the present invention to provide the use of said cationic lipid compounds.

To achieve the above object, in one aspect, the present invention provides a cationic lipid compound represented by the general formula (I):

(I)

Wherein,

ring A is a substituted or unsubstituted 4-10 membered N-containing heterocycle; the N-containing heterocyclic ring is other than with L ₁ The N atoms connected also contain 0, 1 or 2 hetero atoms selected from N, O or S; when substituted, the heterocycle is substituted with 1, 2, 3, 4, 5 or 6 substituents selected from halogen, hydroxy, cyano, nitro, carboxy, C1-C5 alkyl or C1-C5 alkoxy;

L ₁ a linear alkylene group of C1-C6;

L ₂ and L ₃ The same or different, each independently of the other is a C1-C12 alkylene group;

G ₁ and G ₂ The same or a different one of the above, each independently of the other is respectively- (c=o) O-, -O (c=o) -, -S (c=o) -, -O (c=s) -or- (c=o) S-;

R ₃ and R is ₄ Identical or different, each independently of the other is a C4-C17 linear alkyl radical, a C4-C17 linear alkenyl radical or a C8-C22 branched acyclic alkyl radical.

According to some embodiments of the invention, wherein ring a is a substituted or unsubstituted 4-10 membered N-containing heterocycle; the N-containing heterocyclic ring contains 1-3N atoms.

According to some embodiments of the invention, ring a is a substituted or unsubstituted 5-, 6-or 7-membered N-containing heterocycle.

According to some embodiments of the invention, ring a is a substituted or unsubstituted 5-, 6-or 7-membered N-containing heterocycle, wherein the heteroatoms in the N-containing heterocycle are all N atoms.

According to some embodiments of the invention, wherein L ₁ Is a C2-C4 linear alkylene group.

According to some embodiments of the invention, wherein L ₂ And L ₃ At least one of which is a C1-C6 alkylene group.

According to some embodiments of the invention, wherein L ₂ And L ₃ Each independently of the others is C1-Alkylene of C6.

According to some embodiments of the invention, ring a is methylpiperazinyl, hydrogenated pyrrolyl, piperidinyl or cyclohexylimino.

According to some embodiments of the invention, wherein ring a is:

。

according to some embodiments of the invention, wherein G ₁ And G ₂ Is- (c=o) O-.

According to some embodiments of the invention, wherein G ₁ And G ₂ Is- (C=O) O-, and G ₁ And G ₂ Through which-O-is respectively with R ₃ And R is ₄ And (5) connection.

According to some embodiments of the invention, wherein R ₃ And R is ₄ Each independent is respectively:

R ₅ and R is ₆ Identical or different, each independently of the other is a C3-C11 straight-chain alkyl radical.

Wherein it is understood that R ₅ And R is ₆ The sum of the numbers of carbon atoms should satisfy the previously defined range of C8-C22, e.g. when R ₅ And R is ₆ When one is a C3 linear alkyl group, the other is at least a C5 linear alkyl group.

According to some embodiments of the invention, wherein R ₅ And R is ₆ Identical or different, each independently of the other is a C4-C8 straight-chain alkyl radical.

。

according to some embodiments of the invention, wherein the cationic lipid compound is selected from one or more of the following structures:

in another aspect, the present invention also provides a liposome formulation comprising a cationic lipid compound of any one of the present invention, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and a prophylactic or therapeutic nucleic acid.

According to some embodiments of the invention, wherein the molar ratio of the cationic lipid compound to the nucleic acid is 20:1 to 1:1.

according to some embodiments of the invention, wherein the liposome formulation has an average particle size of 50nm to 200 nm.

According to some embodiments of the invention, wherein the liposome formulation further comprises one or more additional lipid components, including neutral lipids, steroids, and polymer conjugated lipids.

According to some embodiments of the invention, wherein the steroid is β -sitosterol, stigmasterol, ergosterol, cholesterol, or dihydrocholesterol; among them, cholesterol is preferable.

According to some embodiments of the invention, the molar ratio of the steroid to the cationic lipid compound is (0.5-1): 1.

according to some embodiments of the invention, wherein the polymer in the polymer conjugated lipid is polyethylene glycol.

According to some embodiments of the invention, the cationic lipid compound is present in a molar ratio to the polymer conjugated lipid of 100:1 to 20:1.

According to some embodiments of the invention, wherein the polyethylene glycol conjugated lipid is PEG2k-DSG, PEG2k-DMG, PEG2k-DPPE, PEG2k-DSPE, PEG2k-cer, PEG2k-DMG or ALC-0159; PEG2k-DMG is preferred.

According to some embodiments of the invention, wherein the neutral lipid is selected from one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), sodium 1, 2-dipalmitoyl-sn-glycero-phosphate (DPPG-Na), sphingomyelin (SM), ceramide and sterol.

According to some embodiments of the invention, the cationic lipid compound is present in a molar ratio to the neutral lipid of from 2:1 to 8:1.

According to some embodiments of the invention, wherein the nucleic acid is selected from mRNA, siRNA, miRNA or ASO; the nucleic acid is preferably mRNA.

In a further aspect, the present invention also provides the use of a cationic lipid compound according to the invention or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof or a liposome preparation according to the invention for the manufacture of a medicament for inducing protein expression in a subject.

In summary, the present invention provides a cationic lipid compound, a composition comprising the same and applications thereof. The cationic lipid compound of the present invention has the following advantages:

the invention provides a novel cationic lipid compound, which enriches the types of the cationic lipid compounds at present. The preparation method of the amino lipid compound has the advantages of easily available raw materials, mild reaction conditions, high product yield, low instrument and equipment requirements and simple operation.

Drawings

FIG. 1 is a hydrogen spectrum of Compound 1.

FIG. 2 is a hydrogen spectrum of Compound 2.

FIG. 3 is a hydrogen spectrum of Compound 3.

FIG. 4 is a hydrogen spectrum of Compound 4.

FIG. 5 is a hydrogen spectrum of Compound 5.

FIG. 6 is a hydrogen spectrum of Compound 6.

FIG. 7 is a hydrogen spectrum of Compound 7.

FIG. 8 is a hydrogen spectrum of compound 8.

FIG. 9 is a hydrogen spectrum of Compound 9.

FIG. 10 is a hydrogen spectrum of compound 10.

FIG. 11 is a hydrogen spectrum of Compound 11.

FIG. 12 is a hydrogen spectrum of compound 12.

FIG. 13 is a hydrogen spectrum of Compound 13.

FIG. 14 is a hydrogen spectrum of compound 14.

FIG. 15 is a hydrogen spectrum of compound 15.

FIG. 16 is a graph showing the effect of nanoparticle compositions on delivery of Erythropoietin (EPO) mRNA expressed in mice.

Detailed Description

The following detailed description of the invention and the advantages achieved by the embodiments are intended to help the reader to better understand the nature and features of the invention, and are not intended to limit the scope of the invention.

Example 1

The synthetic route for compound 1 is as follows:

step 1:

compound 1-1 (5.0 g) was dissolved in DCM (200 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g) and n-decanol (6.79 g) were weighed out in order, and the reaction system was added in portions and stirred at room temperature for 16h. A small amount of the reaction solution was diluted with 1-1 standard control plates (PE/EA=10/1, phosphomolybdic acid and bromocresol green), and new spots of reduced polarity were observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (80. 80 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 40 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give the colorless oily compound 1-2 (6.0 g,54.5% yield).

Step 2:

to a solution of compound 1-2 (6.0 g) in methylene chloride (50 ml) was added trifluoroacetic acid (15 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compounds 1-2, with one point of increased polarity formation. The reaction solution was dried by spinning, and excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (100 ml), extracted with ethyl acetate (100 ml ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with an appropriate amount of silica gel and DCM (80 g normal phase column, PE/EA, 0-0% 5min,0-50% 20min,50-50% min, flow rate 50 ml/min) to give compounds 1-3 as a white solid (4.5 g,93% yield).

Step 3:

compounds 1 to 3 (300 mg) were dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (115 mg) and triphosgene (86 mg) were added in this order, followed by stirring in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 1-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (2-hydroxyethyl) -4-methylpiperazine (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 1 (200 mg,47% yield) as a pale yellow oil. The hydrogen spectrum of compound 1 is shown in FIG. 1.

1H NMR (400 MHz, Chloroform-d) δ 4.23 (t, J = 6.0 Hz, 2H), 4.16 – 4.03 (m, 8H), 2.75 – 2.30 (m, 10H), 2.28 (s, 3H), 1.67 – 1.57 (m, 8H), 1.31 – 1.24 (m, 24H), 0.88 (t, J = 8.0 Hz, 6H).

Example 2

The synthetic route for compound 2 is as follows:

step 1:

compounds 1 to 3 (300 mg) were dissolved in methylene chloride (5 ml), stirred in an ice bath, and super-dry pyridine (115 mg) and triphosgene (86 mg) were added in this order, followed by stirring in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 1-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, and the resulting solid was dissolved in methylene chloride (5 ml), and then added dropwise to a mixed solution of the compound 1- (3-hydroxypropyl) -4-methylpiperazine (500 mg) and pyridine (10 ml), followed by completion of the addition, the reaction mixture was cooled to 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 2 (214 mg,47% yield) as a pale yellow oil. The hydrogen spectrum of compound 2 is shown in fig. 2.

¹ H NMR (400 MHz, Chloroform-d) δ 4.15 – 4.10 (m, 6H), 4.06 (s, 2H), 2.68 – 2.30 (m, 8H), 2.28 (s, 3H), 1.83 – 1.76 (m, 2H), 1.64 – 1.52 (m, 12H), 1.36 – 1.13 (m, 24H), 0.88 (t,J= 6.8 Hz, 6H).

Example 3

The synthetic route for compound 3 is as follows:

step 1:

compound 1-1 (5.0 g) was dissolved in DCM (200 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g) and 6-undecanol (7.39 g) were weighed in order and added to the reaction system in portions, followed by stirring at room temperature for 16h. A small amount of the reaction solution was diluted with 1-1 standard control plates (PE/EA=10/1, phosphomolybdic acid and bromocresol green), and new spots of reduced polarity were observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (80 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 40 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 3-1 (8.0 g,68.9% yield) as colorless oily liquid compound.

Step 2:

to a solution of compound 3-1 (8.0 g) in methylene chloride (50 ml) was added trifluoroacetic acid (15 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 3-1 and the formation of a more polar spot. The reaction solution was dried by spinning, and excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (100 ml), extracted with ethyl acetate (100 ml ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then stirred with an appropriate amount of silica gel and DCM, and purified (80 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% min, flow rate 50 ml/min) to give 3-2 as a colorless oily compound (5.8 g,89% yield).

Step 3:

compound 3-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (115 mg) and triphosgene (86 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 3-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (2-hydroxyethyl) -4-methylpiperazine (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH，0-0 % 5min，0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give pale yellow oily liquid compound 3 (162 mg,39% yield). The hydrogen spectrum of compound 3 is shown in FIG. 3.

¹ H NMR (400 MHz, Chloroform-d) δ 4.96 – 4.86 (m, 2H), 4.21 (t, J = 6.4 Hz, 2H), 4.12 (s, 2H), 4.04 (s, 2H), 2.71 – 2.32 (m, 10H), 2.28 (s, 3H), 1.61 – 1.51 (m, 10H), 1.30 – 1.24 (m, 22H), 0.90 – 0.85 (m, 12H).

Example 4

The synthetic route for compound 4 is as follows:

step 1:

compound 1-1 (1.5 g) was dissolved in DCM (50 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.70 g), 4-dimethylaminopyridine (DMAP, 79 mg), triethylamine (1.95 g) and pentadecan-8-ol (2.94 g) were weighed in order, and the reaction was added in portions and stirred at room temperature for 16h. A small amount of the reaction solution was diluted with 1-1 standard control plates (PE/EA=10/1, phosphomolybdic acid and bromocresol green), and new spots of reduced polarity were observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 4-1 (2.5 g,59.4% yield) as colorless oily compound.

Step 2:

to a solution of compound 4-1 (2.5 g) in methylene chloride (20 ml) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 4-1 and the formation of a more polar spot. The reaction solution was dried by spinning, and excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (50 ml), extracted with ethyl acetate (50 ml ×2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the concentrated crude product was stirred with an appropriate amount of silica gel and DCM, and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-50% 20min,50-50% 5min, flow rate 30 ml/min) to give 4-2 (1.8 g,85% yield) as a colorless oily liquid compound.

Step 3:

compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (58 mg) and triphosgene (43 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 4-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, the resulting solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of the compound 1- (3-hydroxypropyl) -4-methylpiperazine (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction mixture was added dropwise to 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give pale yellow oily compound 1 (130 mg,51% yield). The hydrogen spectrum of compound 4 is shown in fig. 4.

¹ H NMR (400 MHz, Chloroform-d) δ 4.95 – 4.86 (m, 2H), 4.14 (t,J= 8.0 Hz 2H), 4.11 – 4.03 (m, 4H), 2.72 – 2.31 (m, 10H), 2.30 (s, 3H), 1.84 – 1.77 (m, 2H), 1.56 – 1.48 (m, 8H), 1.30 – 1.21 (m, 40H), 0.90 – 0.85 (t,J= 6.8 Hz, 12H).

Example 5

The synthetic route for compound 5 is as follows:

step 1:

compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (58 mg) and triphosgene (43 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 4-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a very high degree of reaction was observedNew points of reduced sex. The reaction solution was evaporated under reduced pressure, the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of the compound 1- (2-hydroxyethyl) -4-methylpiperazine (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction solution was added dropwise to 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give pale yellow oily compound 5 (130 mg,50% yield). The hydrogen spectrum of compound 5 is shown in fig. 5.

1H NMR (400 MHz, Chloroform-d) δ 4.79 (s, 2H), 4.08 (s, 2H), 3.87 (s, 4H), 2.79 (s, 2H), 2.54 (s, 4H), 2.49 (s, 2H), 2.44 (s, 2H), 2.27 (s, 3H), 1.68 (d, J = 12.4 Hz, 4H), 1.56 (d, J = 12.4 Hz, 4H), 1.37 (d, J = 0.6 Hz, 8H), 1.34 (d, J = 1.2 Hz, 8H), 1.31 - 1.29 (m, 24H), 0.90 (s, 12H).

Example 6

The synthetic route for compound 6 is as follows:

step 1:

compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (58 mg) and triphosgene (43 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 1-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, the resulting solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of 3- (1-pyrrolidinyl) -1-propanol (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction mixture was added dropwise to a solution of 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, adding appropriate amount of silica gel and dichloromethane, mixing, purifying (10 g normal phase column, DCM: DCM-MeOH/NH ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow 20 ml/min) to give compound 6 (140 mg,54.7% yield) as a pale yellow oil. The hydrogen spectrum of compound 6 is shown in FIG. 6.

1H NMR (400 MHz, Chloroform-d) δ 4.79 (s, 2H), 4.14 (s, 2H), 3.87 (s, 4H), 2.67 (s, 2H), 2.56 (s, 4H), 1.90 (s, 2H), 1.75 (s, 4H), 1.69 (d, J = 12.4 Hz, 4H), 1.56(d, J = 12.4 Hz, 4H), 1.37 (d, J = 0.6 Hz, 8H), 1.33 (d, J = 1.2 Hz, 8H), 1.31 (s, 8H), 1.28 (d, J = 4.6 Hz, 16H), 0.90 (s, 12H).

Example 7

The synthetic route for compound 7 is as follows:

step 1:

compound 1-1 (5.0 g) was dissolved in DCM (200 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 12.3 g), 4-dimethylaminopyridine (DMAP, 262 mg), triethylamine (6.51 g) and 1-tridecanol (8.59 g) were sequentially weighed into the reaction system, and stirred at room temperature for 16h. A small amount of the reaction solution was diluted with 1-1 standard control plates (PE/EA=10/1, phosphomolybdic acid and bromocresol green), and new spots of reduced polarity were observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (80 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 40 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 7-2 (8.5 g,66.3% yield) as colorless oily liquid compound.

Step 2:

to a solution of compound 7-2 (8.5 g) in methylene chloride (100 ml) was added trifluoroacetic acid (30 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 7-2 and the formation of a more polar spot. The reaction solution was dried by spinning, and excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 ml), extracted with ethyl acetate (100 ml ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with an appropriate amount of silica gel and DCM (120 g normal phase column, PE/EA, 0-0% 5min,0-50% 20min,50-50% min, flow rate 60 ml/min) to give compound 7-3 as a white solid (6.3 g,89% yield).

Step 3:

compound 7-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (96 mg) and triphosgene (72 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 7-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, the resulting solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of 3- (1-pyrrolidinyl) -1-propanol (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction mixture was added dropwise to a solution of 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow 20 ml/min) to give compound 7 (180 mg,45.7% yield) as a yellow oil. The hydrogen spectrum of compound 7 is shown in fig. 7.

1H NMR (400 MHz, Chloroform-d) δ 4.14 (s, 2H), 4.12 (s, 4H), 3.88 (s, 4H), 2.67 (s, 2H), 2.56 (s, 4H), 1.90 (s, 2H), 1.75 (s, 4H), 1.66 (s, 4H), 1.38 (s, 4H), 1.33 (d, J = 6.2 Hz, 8H), 1.30 – 1.23 (m, 28H), 0.90 (s, 6H).

Example 8

The synthetic route for compound 8 is as follows:

step 1:

compound 8-1 (2.0 g) was dissolved in DCM (30 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.28 g), 4-dimethylaminopyridine (DMAP, 140 mg), triethylamine (1.73 g) and (8 z,11 z) -heptadeca-8, 11-dien-1-ol (3.17 g) were sequentially weighed into the reaction system, and stirred at room temperature for 16h. A small amount of the reaction solution was diluted and used as a control point plate (PE/EA=10/1, potassium permanganate) for 8-1 standard samples, and a new point with reduced polarity was observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 8-2 (3.0 g,64% yield) as colorless oily liquid compound.

Step 2:

to a solution of compound 8-2 (3.0 g) in methylene chloride (30 ml) was added trifluoroacetic acid (10 m). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 8-2 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 ml), extracted with ethyl acetate (100 ml ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (120 g normal phase column, PE/EA, 0-0% 5min,0-50% 20min,50-50% min, flow rate 60 ml/min) to give 8-3 as a pale yellow oil (1.9 g,84% yield).

Step 3:

bromoacetic acid (2.0 g) was dissolved in DCM (40 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 4.14 g), 4-dimethylaminopyridine (DMAP, 176 mg), triethylamine (2.18 g) and pentadecan-8-ol (3.62 g) were weighed out in order, and the reaction was added in portions and stirred at room temperature for 16h. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 8-5 (3.6 g,72% yield) as colorless oily compound.

Step 4:

dissolving 8-3 (1.5. 1.5 g) in acetonitrile (10 ml), stirring at room temperature, sequentially weighing potassium carbonate (1.34. 1.34 g) and 8-5 (1.86. 1.86 g), adding into the reaction system, and reacting at 70 ^o C stirring for 3h. Reaction solution is passed throughThe mixture was filtered, the filter cake was washed with ethyl acetate, and the organic phase was stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-30% 20min,30-30% 10min, flow rate 30 ml/min), monitored on a spot-on plate, and the pure product fraction was evaporated to give 8-4 (800 mg,28.6% yield) as colorless oily liquid compound.

Step 5:

compound 8-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (83 mg) and triphosgene (62 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with 8-4 standard control plates (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, the resulting solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of 3- (1-pyrrolidinyl) -1-propanol (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction mixture was added dropwise to a solution of 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with appropriate amounts of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 8 (160 mg,42% yield) as a yellow oil. The hydrogen spectrum of compound 8 is shown in FIG. 8.

1H NMR (400 MHz, Chloroform-d) δ 5.35 (d, J = 16.0 Hz, 4H), 4.79 (s, 1H), 4.13 (d, J = 9.8 Hz, 4H), 3.88 (d, J = 4.4 Hz, 4H), 2.80 – 2.64 (m, 4H), 2.56 (s, 4H), 2.08 – 2.00 (dt, J = 9.0, 1.0 Hz, 4H), 1.90 (s, 2H), 1.75 (s, 4H), 1.72 – 1.63 (m, 4H), 1.55 (d, J = 12.6 Hz, 2H), 1.38 – 1.25 (m, 34H), 0.90 (s, 9H).

Example 9

The synthetic route for compound 9 is as follows:

step 1:

compound 8-1 (3.0 g) was dissolved in DCM (30 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 4.92 g), 4-dimethylaminopyridine (DMAP, 210 mg), triethylamine (2.60 g) and undecanol (3.25 g) were sequentially weighed into the reaction system and stirred at room temperature for 16h. A small dilution of the reaction was used to control the spot plate with 8-1 standard (PE/EA=20/1, phosphomolybdic acid) and a new spot with polarity approaching was observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (40 g normal phase column, PE/EA, 0-0% 10min,0-6% 30min,6-6% 20min, flow rate 30 ml/min), followed by spot-plate monitoring, and the pure product fraction evaporated to give compound 9-1 (3.2 g,57% yield) as a white solid.

Step 2:

to a solution of compound 9-1 (3.2 g) in methylene chloride (30 mL) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 9-1 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 mL), extracted with ethyl acetate (100 mL ×2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (120 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 15min, flow rate 30 mL/min) to give 9-2 as a colorless oil (2.0 g,90% yield).

Step 3:

bromoacetic acid (2.0 g) was dissolved in DCM (40 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 4.14 g), 4-dimethylaminopyridine (DMAP, 176 mg), triethylamine (2.18 g) and pentadecan-7-ol (3.62 g) were weighed out in order, and the reaction was added in portions and stirred at room temperature for 16h. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 9-4 (3.6 g,70% yield) as colorless oily compound.

Step 4:

9-2 (2.0 g) was dissolved in acetonitrile (20 ml), stirred at room temperature, and stirred at room temperatureWeighing potassium carbonate (2.41 g) and 9-4 (3.35 g) in batches, adding into the reaction system, and reacting at 70 ^o C stirring for 3h. The reaction solution was filtered, the filter cake was washed with ethyl acetate, and an appropriate amount of silica gel was added to the mixture and the mixture was stirred and purified (40 g normal phase column, PE/EA, 0-0% 5min,0-30% 20min,30-30% 20min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction was evaporated to give 9-3 (900 mg,20.7% yield) as colorless oily liquid compound.

Step 5:

compound 9-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (96 mg) and triphosgene (72 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 9-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (3-hydroxypropyl) -4-methylpiperazine (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with appropriate amounts of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow 20 ml/min) to give compound 9 (180 mg,43.9% yield) as a yellow oil. The hydrogen spectrum of compound 9 is shown in fig. 9.

1H NMR (400 MHz, Chloroform-d) δ 4.79 (s, 1H), 4.14 – 4.11 (m, 4H), 3.87 (d, J = 4.4 Hz, 4H), 2.66 (d, J = 1.6 Hz, 2H), 2.50 – 2.44 (m, 8H), 2.27(s, 3H), 2.01 – 1.83 (m, 2H), 1.73 – 1.63 (m, 4H), 1.55 (d, J = 12.4 Hz, 2H), 1.40 – 1.36 (m, 6H), 1.34 (d, J = 1.0 Hz, 2H), 1.34 – 1.30 (m, 10H), 1.30 – 1.25 (m, 18H), 0.90 (s, 9H).

Example 10

Synthesis of Compound 10

Step 1:

compound 3-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (115 mg) and triphosgene (86 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 3-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound N- (2-hydroxyethyl) hexamethylenediamine (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction solution was cooled to 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give pale yellow oily compound 10 (170 mg,41% yield). The hydrogen spectrum of compound 10 is shown in fig. 10.

¹ H NMR (400 MHz, Chloroform-d) δ 4.95 – 4.86 (m, 2H), 4.27 – 4.22 (m, 2H), 4.09 (d,J= 12.0 Hz, 4H), 2.78 – 2.70 (m, 2H), 2.65 – 2.50 (m, 4H), 1.80 – 1.30 (m, 16), 1.28 – 1.10(m, 24H), 0.87 (t,J= 4.0 Hz, 12H).

Example 11

The synthetic route for compound 11 is as follows:

step 1:

compound 11-1 (3.0 g) was dissolved in DCM (30 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.40 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g) and 5-undecanol (2.80 g) were weighed in order and added to the reaction system in portions, followed by stirring at room temperature for 16h. A small amount of the reaction solution was diluted into a control plate (PE/EA=10/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and added with an appropriate amount of silica gel for sample stirring and purification (40 g normal phase column, PE/EA, 0-0% 10min,0-6% 30min,6-6% 20min, flow rate 30 ml/min), followed by spot-plate monitoring, and the pure product fraction evaporated to give 11-2 (4.0 g,76% yield) as a white solid.

Step 2:

to a solution of compound 11-2 (4.0 g) in methylene chloride (30 ml) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 11-2 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 ml), extracted with ethyl acetate (100 ml ×2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (25 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 15min, flow rate 30 ml/min) to give 11-3 as a colorless oil (2.5 g,87% yield).

Step 3:

4-Bromobutyric acid (2.0 g) was dissolved in DCM (40 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.44 g), 4-dimethylaminopyridine (DMAP, 147 mg), triethylamine (1.82 g) and 5-undecanol (2.27 g) were weighed in order and added to the reaction system in portions, followed by stirring at room temperature for 16h. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 11-5 (3.0 g,78% yield) as colorless oily compound.

Step 4:

dissolving 11-3 (2.0 g) in acetonitrile (20 ml), stirring at room temperature, sequentially weighing potassium carbonate (2.15 g) and 11-5 (2.75 g), adding into the reaction system, and reacting at 70 ^o C stirring for 3h. Filtering the reaction solution, washing the filter cake with ethyl acetate, adding appropriate amount of silica gel, stirring, purifying (40 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 20min, flow rate 30 ml/min), monitoring with spot plate, and evaporating the fraction of pure product to obtain colorless oily liquid compound11-4 (950 mg,24.6% yield).

Step 5:

compound 11-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (96 mg) and triphosgene (72 mg) were added in this order, followed by stirring in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 11-4 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction mixture was evaporated under reduced pressure, the resulting solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound N- (2-hydroxyethyl) -pyrrolidine (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction mixture was cooled to 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with appropriate amounts of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give 11 (160 mg,41.5% yield) as a yellow oily liquid. The hydrogen spectrum of compound 11 is shown in FIG. 11.

1H NMR (400 MHz, Chloroform-d) δ 4.77 (s, 2H), 4.10 (d, J = 0.8 Hz, 2H), 3.27 (d, J = 0.8 Hz, 4H), 2.85 – 2.70 (m, 6H), 2.40 – 2.29 (m, 4H), 2.28 – 2.16 (m, 4H), 1.76 (d, J = 4.0 Hz, 4H), 1.72 – 1.63 (dd, J = 12.4, 4.0 Hz, 4H), 1.61 – 1.51 (dd, J = 12.4, 8.4 Hz, 4H), 1.42 – 1.32 (m, 16H), 1.30(d, J = 8.4 Hz, 8H), 0.91 (d, J = 10.6 Hz, 12H).

Example 12

The synthetic route for compound 12 is as follows:

step 1:

compound 12-1 (3.00 g) was dissolved in DCM (30 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 4.24 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g) and pentadecan-7-ol (3.71 g) were weighed in sequence and added to the reaction system in portions and stirred at room temperature for 16h. A small amount of the reaction solution was diluted into a control plate (PE/EA=10/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (40 g normal phase column, PE/EA, 0-0% 10min,0-6% 30min,6-6% 20min, flow rate 30 ml/min), followed by spot-plate monitoring, and the pure product fraction evaporated to give compound 12-2 as a white solid (4.3 g,70.4% yield).

Step 2:

to a solution of compound 12-2 (4.3 g) in methylene chloride (30 ml) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 12-2 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 mL), extracted with ethyl acetate (100 mL ×2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (25 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 15min, flow rate 30 mL/min) to give 12-3 as a colorless oil (2.9 g,89% yield).

Step 3:

4-Bromobutyric acid (2.0 g) was dissolved in DCM (40 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.44 g), 4-dimethylaminopyridine (DMAP, 147 mg), triethylamine (1.82 g) and pentadecan-7-ol (3.01 g) were weighed in sequence and added to the reaction system in portions, followed by stirring at room temperature for 16h. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (25 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 12-5 (3.3 g,66.6% yield) as colorless oily liquid compound.

Step 4:

12-3 (2.0 g) is dissolved in acetonitrile (20 ml), stirred at room temperature, and then potassium carbonate (1.76 g) and 12-5 (2.65 g) are sequentially weighed and added into the reaction system in batches, and the reaction is carried out at 70 ^o C stirring for 3h. Filtering the reaction solution, washing a filter cake by ethyl acetate, adding a proper amount of silica gel to stir and purify(40 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 20min, flow rate 30 ml/min), spot-on monitoring, and evaporation of the pure product fraction gave compound 12-4 as a colourless oil (800 mg,22.6% yield).

Step 5:

compound 12-4 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (85 mg) and triphosgene (64 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 12-4 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (3-hydroxypropyl) -4-methylpiperazine (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with appropriate amounts of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 12 (180 mg,52.0% yield) as a yellow oil. The hydrogen spectrum of compound 12 is shown in fig. 12.

1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 2H), 4.15 (d, J = 0.8 Hz, 2H), 3.26 (d, J = 0.8 Hz, 4H), 2.65 (d, J = 1.2 Hz, 2H), 2.50 (s, 2H), 2.47 (s, 2H), 2.45 (s, 2H), 2.44 (s, 2H), 2.38 – 2.29 (m, 4H), 2.28 – 2.16 (m, 7H), 1.96 – 1.84 (m, 2H), 1.73 – 1.51 (m, 8H), 1.41 – 1.36 (m, 8H), 1.34 (d, J = 1.0 Hz, 4H), 1.31 (d, J = 6.8 Hz, 12H), 1.30 – 1.25 (dd, J = 4.9, 1.0 Hz, 16H), 0.90(s, 12H).

Example 13

The synthetic route for compound 13 is as follows:

step 1:

the compound 4- (tert-butoxycarbonylamino) butanoic acid (3.00 g) was dissolved in DCM (30 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 3.40 g), 4-dimethylaminopyridine (DMAP, 180 mg), triethylamine (2.24 g) and 1-nonanol (2.13 g) were weighed in sequence and added to the reaction system in portions, followed by stirring at room temperature for 16h. A small amount of the reaction solution was diluted into a control plate (PE/EA=10/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (40 g normal phase column, PE/EA, 0-0% 10min,0-6% 30min,6-6% 20min, flow rate 30 ml/min), followed by spot-plate monitoring, and the pure product fraction evaporated to give compound 13-1 as a white solid (3.4 g,69.9% yield).

Step 2:

to a solution of compound 13-1 (3.4 g) in methylene chloride (30 ml) was added trifluoroacetic acid (10 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 13-1 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (150 mL), extracted with ethyl acetate (100 mL ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (40 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 15min, flow rate 30 mL/min) to give 13-2 as a colorless oil (2.1 g,89% yield).

Step 3:

13-2 (2.0 g) is dissolved in acetonitrile (20 ml), stirred at room temperature, and then potassium carbonate (1.76 g) and 12-5 (2.65 g) are sequentially weighed and added into the reaction system in portions for reaction at 70 ^o C stirring for 3h. The reaction solution was filtered, the filter cake was washed with ethyl acetate, and an appropriate amount of silica gel was added to the mixture and the mixture was stirred and purified (40 g normal phase column, PE/EA, 0-0% 5min,0-40% 20min,40-40% 20min, flow rate 30 ml/min), followed by spot-on-plate monitoring, and the pure product fraction was evaporated to give 13-3 (800 mg,22.6% yield) as colorless oily compound.

Step 4:

compound 13-3 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (90 mg) and triphosgene (6) were added sequentially8 mg), stirred under ice bath for 0.5h. A small amount of the reaction solution was taken with a 13-3 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (2-hydroxyethyl) -4-methylpiperazine (500 mg) and pyridine (10 mL), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring at C for three hours, the solution was orange-red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with appropriate amounts of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 13 (200 mg,50.4% yield) as a yellow oil. The hydrogen spectrum of compound 13 is shown in FIG. 13.

1H NMR (400 MHz, Chloroform-d) δ 4.78 (s, 1H), 4.15 – 4.08 (m, 4H), 3.27 (d, J = 0.8 Hz, 4H), 2.79 (d, J = 1.6 Hz, 2H), 2.56 (s, 2H), 2.52 (s, 2H), 2.49 (s, 2H), 2.44 (s, 2H), 2.40 – 2.31 (m, 4H), 2.30 – 2.24 (m, 6H), 2.22 – 2.20 (m, 1H), 1.72 – 1.61 (m, 4H), 1.55 (d, J = 12.4 Hz, 2H), 1.39 (d, J = 2.4 Hz, 4H), 1.36 (d, J = 0.6 Hz, 2H), 1.34 (d, J = 1.0 Hz, 2H), 1.33 (d, J = 1.0 Hz, 4H), 1.31 (s, 6H), 1.30 – 1.27 (m, 14H), 0.90(d, J = 10.6 Hz, 9H).

Example 14

The synthetic route for compound 14 is as follows:

step 1:

compound 1-1 (1.0 g) was dissolved in DCM (20 ml), stirred at room temperature, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 2.47 g), 4-dimethylaminopyridine (DMAP, 524 mg), triethylamine (1.30 g) and (8 z,11 z) -heptadeca-8, 11-dien-1-ol (2.16 g) were weighed in order and added to the reaction system in portions, followed by stirring at room temperature for 16h. A small amount of the reaction solution was diluted with 1-1 standard control plates (PE/EA=10/1, phosphomolybdic acid and bromocresol green), and new spots of reduced polarity were observed. The reaction solution was quenched with water, separated, the organic phase evaporated under reduced pressure, and then stirred with an appropriate amount of silica gel and purified (80 g normal phase column, PE/EA, 0-0% 5min,0-10% 20min,10-10% 10min, flow rate 40 ml/min), followed by spot-on-plate monitoring, and the pure product fraction evaporated to give 14-1 (2.1 g,69.8% yield) as colorless oily compound.

Step 2:

to a solution of compound 14-1 (2.1 g) in methylene chloride (20 ml) was added trifluoroacetic acid (5 ml). The mixture was stirred at room temperature for 16 hours. TLC showed complete disappearance of compound 14-1 and the formation of a more polar spot. The reaction was dried by spinning, excess trifluoroacetic acid was quenched by addition of saturated aqueous sodium bicarbonate (100 ml), extracted with ethyl acetate (100 ml ×2), and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and then purified by stirring with appropriate amounts of silica gel and DCM (80 g normal phase column, PE/EA, 0-0% 5min,0-50% 20min,50-50% min, flow rate 50 ml/min) to give 14-2 as a colorless oil (2.1 g,88.9% yield).

Step 3:

compound 14-2 (300 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (79 mg) and triphosgene (60 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 14-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, and the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then added dropwise to a mixed solution of the compound 1- (2-hydroxyethyl) -4-methylpiperazine (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction solution was added dropwise to a solution of 70 ^o Stirring was performed for three hours at C, the solution was orange-yellow, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new point of formation, and was purified by mixing with an appropriate amount of silica gel and dichloromethane (10 g normal phase column, DCM: DCM/MeOH/NH ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow 20 ml/min) to give compound 14 (160 mg,41.6% yield) as a pale yellow oil. The hydrogen spectrum of compound 14 is shown in fig. 14.

1H NMR (400 MHz, Chloroform-d) δ 5.37 (s, 4H), 5.33 (s, 4H), 4.12 (s, 4H), 4.08 (s, 2H), 3.88 (s, 4H), 2.79 (s, 2H), 2.78 – 2.66 (m, 4H), 2.54 (s, 4H), 2.49 (s, 2H), 2.44 (s, 2H), 2.27 (s, 3H), 2.07 – 2.01 (dt, J = 9.0 Hz, 1.0 Hz, 8H), 1.37 (s, 4H), 1.35 – 1.30 (m, 20H), 1.29 (s, 4H), 0.90 (s, 6H).

Example 15

The synthetic route for compound 15 is as follows:

step 1:

compound 4-2 (200 mg) was dissolved in ultra-dry dichloromethane (5 ml), stirred in an ice bath, and ultra-dry pyridine (58 mg) and triphosgene (43 mg) were sequentially added and stirred in an ice bath for 0.5h. A small amount of the reaction solution was taken with a 4-2 standard control plate (PE/EA=1/1, phosphomolybdic acid), and a new point of reduced polarity was observed. The reaction solution was evaporated under reduced pressure, the obtained solid was dissolved in ultra-dry dichloromethane (5 ml), and then the solution was added dropwise to a mixed solution of compound 1-piperidinopropanol (500 mg) and pyridine (10 ml), and after the completion of the addition, the reaction solution was cooled to 70 ^o Stirring was carried out for three hours at C, the solution was red, TLC (DCM/MeOH/NH) ₄ OH=10/1/0.1, phosphomolybdic acid) showed a new spot formation under pyridine, and was stirred with appropriate amount of silica gel and dichloromethane and purified (10 g normal phase column, DCM: DCM/MeOH/NH) ₄ OH, 0-0% 5min,0-70% 20min,70-70% 10min, flow rate 20 ml/min) to give compound 15 (164 mg,62.8% yield) as a yellow oil. The hydrogen spectrum of compound 15 is shown in fig. 15.

1H NMR (400 MHz, Chloroform-d) δ 4.79 (s, 2H), 4.14 (s, 2H), 3.87 (s, 4H), 2.66 (s, 2H), 2.47(s, 4H), 1.90 (s, 2H), 1.69 (d, J = 12.4 Hz, 4H), 1.60 – 1.52 (d, J = 12.4 Hz, 8H), 1.46 (s, 2H), 1.37 (d, J = 0.6 Hz, 8H), 1.35 – 1.25 (m, 32H), 0.90 (s, 12H).

Example 16

Preparation and character characterization of lipid nano-particles

Preparing an aqueous phase: mRNA (LUC-mRNA, corresponding nucleotide sequence to LUC-mRNA see SEQ ID NO:1 of patent application 202210286081.0) was diluted in citrate-sodium citrate buffer at a final concentration of 0.144 mg/mL.

Preparing an organic phase: mixing the above cationic lipid, DSPC, cholesterol and PEG _2k DMG was dissolved in ethanol at a total concentration of 10mg/mL (composition ratio of cationic lipid: DSPC: cholesterol: PEG) _2k -DMG=50:10:38.5:1.5）。

Adding 3ml of water phase buffer solution and 1ml of lipid organic phase into a 15ml centrifuge tube, respectively connecting the water phase buffer solution and the 1ml of lipid organic phase with two ends of a flow control A, B, mounting a chip into microfluidic equipment, setting a certain flow rate proportion, performing pre-experiment with pure water and pure ethanol, adding feed liquid when the pressure and the flow rate are stable, allowing the feed liquid to flow through the chip while observing the color of a sample at the outlet of the chip, discarding front and rear 3-5 milky white liquid drops (about 100 mu L), collecting a middle-end sample into an EP tube, then quickly transferring the sample into a dialysis bag, dialyzing for 12-24h in 20mM Tris-HCl buffer solution, and transferring to a refrigerator at 4 ℃ for preservation after the dialysis is finished.

The encapsulation efficiency of the sample is determined according to the operation instructions by using a Ribogreen kit, the fluorescence of the sample is determined at 485nm of excitation light and 535nm of emission light by using an enzyme-labeled instrument, and the encapsulation efficiency of the sample is calculated by the fluorescence value of the sample.

Particle size and PDI detection and Zeta potential analysis were performed using standard detection methods on a Zetasizer nano instrument from malvern.

The particle size, PDI and encapsulation efficiency of the mRNA-loaded LNP prepared in this example are shown in table 1.

TABLE 1

Example 17

Delivery of Erythropoietin (EPO) mRNA in mice Using nanolipid particle compositions and efficacy assays

Female Balb/c mice of 6-8 weeks of age were injected with EPO-mRNA-lipid nanoparticles (nucleotide sequence corresponding to EPO-mRNA is shown in SEQ ID NO:2 of patent application 202210286081.0) via tail vein at 0.5mg/kg, 5 parallel mice were used for each group of formulations, and blood was collected at specific time points (6 h and 12 h) respectively. The mRNA used therein is characterized essentially by an ARCA cap structure with a polyA tail length of 100-120nt and a complete substitution of pseudouracil. The resulting blood was centrifuged at 5000g for 10min at 4℃to separate serum, which was analyzed by ELISA according to a commercially available kit, and the results of each assay are shown in Table 2 and FIG. 16.

TABLE 2

The cationic lipid compound control group SM102 of the present invention was purchased from mansion cinnabar (M2212000880009) and has the structure shown below:

control T13 was prepared according to CN114773217a (compound 35) and the structure is shown below:

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Claims

1. a cationic lipid compound of general formula (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof:

(I)

wherein,

ring a is methylpiperazinyl;

L ₁ a linear alkylene group of C1-C6;

G ₁ and G ₂ The same or a different one of the above, each independently of the other is- (C=O) O-, -O (c=o) -, -S (c=o) -,-O (c=s) -or- (c=o) S-;

R ₃ and R is ₄ The same or different, each independently is a C8-C22 branched acyclic alkyl group; the branched acyclic alkyl group is:

2. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R ₅ And R is ₆ Identical or different, each independently of the other is a C4-C8 straight-chain alkyl radical.

3. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L ₁ Is a C2-C4 linear alkylene group.

4. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L ₂ And L ₃ At least one of which is a C1-C6 alkylene group.

5. The cationic lipid compound of claim 4, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L ₂ And L ₃ And each independently is a C1-C6 alkylene group.

6. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein ring a is。

7. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein G ₁ And G ₂ Is- (c=o) O-.

8. The cationic lipid compound of claim 7, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein G ₁ And G ₂ Through which-O-is respectively with R ₃ And R is ₄ And (5) connection.

9. The cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R ₃ And R is ₄ Each independent is respectively:

。

10. the cationic lipid compound of claim 1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein the cationic lipid compound is selected from one or more of the following structures:

。

11. A liposome formulation comprising a cationic lipid compound of any one of claims 1-10, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, and a prophylactic or therapeutic nucleic acid.

12. The liposomal formulation of claim 11, wherein the molar ratio of the cationic lipid compound to the nucleic acid is 20:1 to 1:1.

13. the liposomal formulation of claim 11, wherein the liposomal formulation has an average particle size of 50nm to 200 nm.

14. The liposomal formulation of claim 11, wherein the liposomal formulation further comprises one or more additional lipid components including neutral lipids, steroids, and polymer conjugated lipids.

15. The liposomal formulation of claim 14, wherein the steroid is β -sitosterol, stigmasterol, ergosterol, cholesterol, or dihydrocholesterol.

16. The liposomal formulation of claim 15, wherein the steroid is cholesterol.

17. The liposome formulation of claim 14, wherein the molar ratio of steroid to cationic lipid compound is (0.5-1): 1.

18. the liposomal formulation of claim 14, wherein the polymer in the polymer-conjugated lipid is polyethylene glycol.

19. The liposomal formulation of claim 14 wherein the molar ratio of the cationic lipid compound to the polymer-conjugated lipid is from 100:1 to 20:1.

20. The liposomal formulation of claim 18 wherein the polyethylene glycol conjugated lipid is PEG2k-DSG, PEG2k-DMG, PEG2k-DPPE, PEG2k-DSPE, PEG2k-cer, PEG2k-DMG, or ALC-0159.

21. The liposomal formulation of claim 20, wherein the polyethylene glycol conjugated lipid is PEG2k-DMG.

22. The liposomal formulation of claim 14 wherein the neutral lipid is selected from one or more of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-3-phosphoethanolamine, sodium 1, 2-dipalmitoyl-sn-phosphoglyceride, sphingomyelin, ceramide, and sterols.

23. The liposomal formulation of claim 14 wherein the molar ratio of the cationic lipid compound to the neutral lipid is from 2:1 to 8:1.

24. The liposomal formulation of claim 11 wherein the nucleic acid is selected from mRNA, siRNA, miRNA or antisense oligonucleotides.

25. The liposomal formulation of claim 24, wherein the nucleic acid is selected from mRNA.

26. Use of a cationic lipid compound according to any one of claims 1-10 or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof or a liposome formulation according to any one of claims 11-25 for the manufacture of a medicament for inducing protein expression in a subject.