Cationic lipid compound, and preparation method therefor and use thereof

Abstract

A cationic lipid compound, and a preparation method therefor and use thereof are provided. The cationic lipid compound features a hydroxyl group at the head part, and its overall structure resembles a cone with a small head and a large tail. The LNPs prepared using the cationic lipid compounds with the aforementioned optimal structure usually exhibit enhanced biocompatibility and higher in vivo mRNA transfection efficiency, achieving unexpected technical effects. The synthesis route of the cationic lipid compounds is straightforward and practicable, with inexpensive and readily available raw materials, facilitating industrial production. Furthermore, the LNPs produced from the cationic lipid compounds possess a stable nanostructure that can be stored at low temperatures for a long time, thereby prolonging the shelf life of the pharmaceutical products while reducing the transportation requirements.

Claims

1. A cationic lipid compound selected from the group consisting of: ##STR00146## ##STR00147## ##STR00148## ##STR00149## or a stereoisomer thereof, tautomer thereof, or pharmaceutically acceptable salt thereof.

2. A cationic lipid compound selected from the group consisting of: ##STR00150## or a stereoisomer thereof, tautomer thereof, or pharmaceutically acceptable salt thereof.

3. A method for preparation of a composition, which comprises: using the cationic lipid compound of claim 1, a stereoisomer thereof, tautomer thereof, or pharmaceutically acceptable salt thereof as the cationic lipid compound to prepare the composition.

4. The method of claim 3, wherein the composition further comprises: a carrier, a loaded drug, a pharmaceutical adjuvant.

5. The method of claim 4, wherein the carrier is LNPs, the average size of the LNPs ranges from 30-200 nm, and the polydispersity index of the LNPs is 0.5.

6. The method of claim 5, wherein the carrier comprises one or more ionizable lipid compounds.

7. The method of claim 6, wherein the carrier further comprises: helper lipids, and the molar ratio of cationic lipid compounds to helper lipids ranges from 0.5:1-10:1; the helper lipid comprises one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, sterol and derivative thereof, ceramide, and charged lipid.

8. The method of claim 6 for the composition comprising the cationic lipid compounds, wherein the carrier further comprises structural lipids or polymer-conjugated lipids.

9. The method of claim 8, wherein the carrier further comprises: structural lipids, and the molar ratio of the cationic lipid compounds to structural lipids ranges from 0.5:1 to 5:1.

10. The method of claim 7, wherein the carrier further comprises: polymer-conjugated lipids, and the molar ratio of the cationic lipid compounds to polymer-conjugated lipids ranges from 20:1-250:1; the polymer-conjugated lipid is PEGylated lipid.

11. The method of claim 4, wherein the loaded drug comprises one or more of nucleic acid molecules, small molecule compounds, peptides, or proteins.

12. The method of claim 4, wherein the pharmaceutical adjuvant comprises one or more of diluents, stabilizers, preservatives, or lyoprotectants.

Description

DETAILED DESCRIPTION

(1) The present invention is introduced in detail with the drawings and examples.

(2) Cationic Lipid Compound

(3) The cationic lipid compound described in this invention is a compound of the following structure:

(4) ##STR00030## R.sub.1 is

(5) ##STR00031## R.sub.2 is

(6) ##STR00032## R.sub.3 is H,

(7) ##STR00033## R.sub.4 is

(8) ##STR00034## R.sub.5 is

(9) ##STR00035## R.sub.6 is

(10) ##STR00036## R.sub.7 is

(11) ##STR00037## R.sub.8 is

(12) ##STR00038## R.sub.9 is

(13) ##STR00039## R.sub.10 is

(14) ##STR00040## R.sub.11 is

(15) ##STR00041## M.sub.0 and M.sub.1 are:

(16) ##STR00042## M.sub.2 and M.sub.3 are:

(17) ##STR00043##

(18) The structure of compounds described in this invention resembles a cone with a small head and a large tail. The cationic lipid compounds with the aforementioned optimal structure and the hydroxyl group introduced in the head have achieved better biocompatibility and higher in vivo mRNA transfection efficiency.

Preparation of Cationic Lipid Compounds

(19) This invention provides a preparation method for the cationic lipid compound, which comprises the following steps: Synthesis of the first intermediate: the long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with olefin-ended long-chain alkyl alcohol with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator to afford the first intermediate; Synthesis of the second intermediate: the double bond is oxidized by meta-Chloroperbenzoic acid to form epoxy to obtain the second intermediate; Synthesis of cationic lipid compounds: the amine undergoes the ring-opening reaction with the epoxy under heating so as to form the cationic lipid compound by linking the hydrophilic head and hydrophobic tail of the cation.

(20) The method is preferably used for the preparation of compounds H-1, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H-11, H-12, H-13, H-14, H-15, H-16, H-18, H-19, H-20.

(21) This invention provides another method for the preparation of cationic lipid compound, which comprises the following steps: Synthesis of the first intermediate: long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with long-chain alkyl alcohol compound with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator to obtain the first intermediate; Synthesis of cationic lipid compounds: the first intermediate, long-chain alkyl carboxylic acid, is dissolved in dichloromethane and then esterified with triethanolamine with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator.

(22) The method is preferably used for the preparation of compound H-21 or H-23.

(23) This invention provides another method for the preparation of cationic lipid compound, which comprises the following steps: Synthesis of the first intermediate: long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with triethanolamine with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator; Synthesis of the second intermediate: long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with long-chain alkyl alcohol compound with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as activators to obtain the second intermediate; Synthesis of cationic lipid compounds: the second intermediate dissolved in dichloromethane is esterified with the first intermediate with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator.

(24) The method is preferably used for the preparation of compounds H-22 and H-24.

(25) This invention provides another method for the preparation of cationic lipid compound, which comprises the following steps: Synthesis of the first intermediate: under alkalines condition, using long-chain alkyl amine and carbon disulfide to form the corresponding isothiocyanate with the existence of 4-dimethylaminopyridine and di-tert-butyl dicarbonate ester as an catalyst; Synthesis of the second intermediate: isothiocyanate reacts with amine through affinity substitution in solvent to form the corresponding thiourea; Synthesis of the third intermediate: long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with long-chain alkyl alcohol compound with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator; Synthesis of cationic lipid compounds: the third intermediate dissolved in dichloromethane is esterified with the second intermediate with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator.

(26) The method is preferably used for the preparation of compounds H-25, H-26, H-27.

(27) This invention provides another method for the preparation of cationic lipid compound, which comprises the following steps: Synthesis of the first intermediate: amine is used to react with triphosgene to form isocyanate under alkaline conditions, then the corresponding first intermediate containing urea unit was formed by the reaction between the isocyanate reacts and amine; Synthesis of the second intermediate: long-chain alkyl carboxylic acid dissolved in dichloromethane is esterified with long-chain alkyl alcohol compound with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator; Synthesis of cationic lipid compounds: the second intermediate dissolved in dichloromethane is esterified with the first intermediate with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator.

(28) The method is preferably used for the preparation of compounds H-28, H-29.

(29) This invention provides another method for the preparation of cationic lipid compounds, characterized in that it comprises the following steps: Synthesis of the first intermediate: esterification occurs between linoleic alcohol and bromoacyl chloride under alkaline condition to obtain brominated long-chain alkane; Synthesis of the second intermediate: the double bond is oxidized by meta-Chloroperbenzoic acid to form epoxy; Synthesis of the third intermediate: the intermediate was formed by the nucleophilic substitution reaction between the brominated long-chain alkane of the first intermediate under alkaline conditions; Synthesis of cationic lipid compounds: under heating condition, the compound was formed by ring opening reaction between the epoxy of the second intermediate and the amine of the third intermediate.

(30) The method is preferably used for the preparation of compound H-17.

(31) This invention provides another method for the preparation of cationic lipid compound which comprises the following steps: Synthesis of the first intermediate: long-chain alkyl carboxylic acid compound dissolved in dichloromethane is esterified with dihydric alcohol with the existence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as an activator; Synthesis of the second intermediate: the first intermediate long-chain alkyl alcohol is oxidized to long-chain alkyl aldehyde using Dess-Martin periodinane; Synthesis of cationic lipid compounds: obtained by reduction amination reaction between amine and aldehyde group of the second intermediate.

(32) The method is preferably used for the preparation of compound H-2.

(33) This invention provides another method for the preparation of cationic lipid compound which comprises the following steps: The synthesis of the first intermediate: the double bond is oxidized by meta-Chloroperbenzoic acid to form epoxy to obtain the first intermediate; Synthesis of the second intermediate: long-chain epoxy alkyl carboxylic acid dissolved in dichloromethane is esterified with terminal long-chain alkyl alcohol under the activation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to obtain the second intermediate; Synthesis of cationic lipid compounds: cationic lipid compound was formed by the ring opening reaction between amine and epoxy under heating condition to link the hydrophilic head and hydrophobic tail of the cation.

(34) The method is preferably used for the preparation of compounds H-30, H-31.

(35) Pharmaceutical Compositions Comprising the Cationic Lipid Compounds

(36) As an application, the above compounds can be used to prepare a combination for pharmaceutical use, including: carriers, loaded drug reagents, and drug adjuvants, wherein: The carriers comprise one or more ionizable lipid compounds, helper lipids, structural lipids, or polymer-conjugated lipids. As an embodiment, the carriers are LNPs with an average size ranging from 30-200 nm and the polydispersity index of the nanoparticles is 0.5. It should be noted that any nanoparticles prepared using the lipid compounds described in this invention fall within the scope of this patent and are disclosed by this invention. For example, aside from LNPs, they may also be one or more doped nanoparticles formed by lipid compounds and polymers containing carbamate bonds, such as PLGA-PEG, PLA-PEG, PCL, etc. The examples are not exhaustive.

(37) The helper lipids include: phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), sterols and their derivatives, ceramides, and combinations of one or more charged lipids. Preferred phosphatidylcholines include: DSPC, DPPC, DMPC, DOPC, POPC; DOPE is a preferred phosphatidylethanolamine; cholesterol is a preferred sterol; as an embodiment, charged lipids such as DOTAP, DOTMA, or 18PA can be used. The examples are not exhaustive, any combination of lipid compounds using the structure described in present invention falls within protection scope and is disclosed by the present invention. The examples are not exhaustive, and the selection of helper lipids is unrestricted. As long as the lipid compounds utilize the structure described in present invention, they fall within the protection scope and disclosed by this invention.

(38) The structural lipids include one or more cholesterols, nonsterols, sitosterols, ergosterols, campesterols, stigmasterols, brassisterols, tomatines, tomatines, ursolic acids, -tocopherols, or corticosteroids. The examples are not exhaustive, and the selection of structural lipids is not restricted. Any lipid compounds with the structure listed in this invention fall within the scope of protection of this invention and are disclosed by this invention.

(39) The polymer-conjugated lipids are pegylated lipids; as an embodiment, the pegylated lipids include one or more PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, or PEG-modified dialkylglycerols. The examples are not exhaustive, and the selection of polymer-conjugated lipids is not limited. Any lipid compounds with the structure listed in the present invention fall within the scope of protection described in present invention and are inspired by this invention.

(40) The loaded drugs include one or more nucleic acid molecules, small molecule compounds, peptides, or proteins. The examples are not exhaustive, as any lipid compounds with the structure listed in the present invention, regardless of the selected drug, they fall within the protective scope described in present invention and are inspired by this invention.

(41) The pharmaceutical adjuvants include one or more diluents, stabilizers, preservatives, or lyoprotectants. The examples are not exhaustive, as any lipid compounds with the structure listed in the present invention, regardless of the selected pharmaceutical adjuvants, they fall within the protective scope described in present invention and are disclosed by this invention.

(42) The cationic lipid compounds were prepared using the preparation methods described in Examples 1-7.

Example 1

(43) ##STR00044##

(44) Synthesis of compound c: 2-Hexidodecanoic acid (compound b, 9.22 g, 36 mmol) was dissolved in 100 mL dichloromethane (DCM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 9.2 g, 36 mmol), 4-dimethylaminopyridine (DMAP, 1.46 g, 12 mmol) and N,N-diisopropylethylamine (DIPEA, 7.74 g, 60 mmol) were added to the solution and stirred for 10 minutes. 5-Hexen-1-ol (compound a, 3 g, 30 mmol) was added and the solution was stirred overnight at room temperature. After the reaction, monitored by TLC, was completed, dichloromethane was removed by rotary evaporation, then 200 mL of ethyl acetate was added. The residue was washed with equal volume of saturated sodium chloride solution for three times, the organic phase was dried by anhydrous sodium sulfate for 30 minutes. After the ethyl acetate was removed by rotary evaporation, and the residue was]purified by column (silica gel column, the eluent was PE: EA=3:1 (volume ratio)) to obtain 8.2 g of colorless liquid, yield: 81%.

(45) Synthesis of compound d: compound c (8 g, 24 mmol) was dissolved in 100 mL of DCM, meta-Chloroperbenzoic acid (m-CPBA, 7.2 g, 36 mmol, mass fraction 85%) was added under ice bath and stirred for 15 minutes. After the ice bath was removed, the mixture was stirred overnight. TLC monitored that the reaction was completed, then excess amount of saturated sodium bisulfite solution (10 mL) was added to consume the remaining meta-Chloroperbenzoic acid, and dichloromethane was removed by rotary evaporation, then 200 mL of ethyl acetate was added. The mixture was washed with 200 mL of saturated sodium bicarbonate solution three times and 200 mL of saturated sodium chloride solution for one time, then the organic phase was dried by anhydrous sodium sulfate for 30 minutes. The ethyl acetate was removed by evaporation, separated and purified by column (silica gel column, the eluent was PE: EA=1:1 (volume ratio)) to obtain 6 g of colorless liquid, yield: 72%.

(46) Synthesis of compound H-15: diethylenetriamine (compound e, 0.6 g, 6 mmol) was dissolved in anhydrous methanol, compound d (6 g, 0.17 mmol) was added and stirred at room temperature for 10 minutes, then heated to reflux for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation, and the residue was purified by column (silica gel column, the eluent was DCM: MeOH=200:1 (volume ratio)) to obtain 3.9 g of colorless liquid, yield: 85%. MS m/z (ESI): 814.72 [M+H].sup.+

(47) The following compounds were prepared by the method of Example 1: H-1, H-3, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H-11, H-12, H-13, H-14, H-15, H-16, H-18, H-19, H-20.

(48) TABLE-US-00001 .sup.1H NMR (400 MHz, Chloroform-d) 4.05 (t, J = 6.5 Hz, 4H), 3.86-3.74 (m, 2H), 3.69-3.57 (m, 2H), 2.80- 2.52 (m, 5H), 2.47-2.40 (m, 1H), 2.28 (td, J = 8.9, 5.2 Hz, 2H), 1.74- 1.49 (m, 14H), 1.47-1.34 (m, 10H), 1.29-1.17 (m, 40H), 0.85 (t, J = 6.6 Hz, 12H). H-1 .sup.1H NMR (400 MHz, Chloroform-d) 4.05 (t, J = 6.3 Hz, 4H), 3.80-3.70 (m, 2H), 3.68-3.57 (m, 2H), 2.73- 2.47 (m, 5H), 2.38 (d, J = 12.5 Hz, 1H), 2.27 (t, J = 7.5 Hz, 4H), 1.78- 0.97 (m, 70H), 0.90-0.76 (m, 12H). H-3 .sup.1H NMR (400 MHz, Chloroform-d) 4.05 (t, J = 6.5 Hz, 4H), 3.81-3.53 (m, 8H), 3.18-2.55 (m, 8H), 2.27 (t, J = 7.6 Hz, 4H), 1.81-0.98 (m, 64H), 0.93-0.73 (m, 12H). H-4 .sup.1H NMR (400 MHz, Chloroform-d) 3.94 (d, J = 5.8 Hz, 4H), 3.76- 3.53 (m, 8H), 2.99-2.50 (m, 8H), 2.38-2.28 (m, 4H), 1.84-1.50 (m, 12H), 1.46-1.17 (m, 76H), 0.86 (t, J = 6.6 Hz, 12H). H-5 .sup.1H NMR (400 MHz, Chloroform-d) 4.85 (p, J = 6.4 Hz, 2H), 3.81-3.53 (m, 8H), 3.02-2.59 (m, 6H), 2.25 (t, J = 7.5 Hz, 4H), 1.64-1.19 (m, 56H), 0.86 (t, J = 6.5 Hz, 12H). H-6 0 .sup.1H NMR (400 MHz, Chloroform-d) 4.85 (p, J = 6.3 Hz, 2H), 3.99-3.86 (m, 2H), 3.71-3:64 (m, 2H), 2.99- 2.87 (m, 2H), 2.86-2.74 (m, 3H), 2.61 (d, J = 11.9 Hz, 1H), 2.26 (t, J = 7.5 Hz, 4H), 1.83-1.74 (m, 2H), 1.71-1.19 (m, 58H), 0.94-0.74 (m, 12H). H-7 .sup.1H NMR (400 MHz, Chloroform-d) 4.04 (t, J = 6.7 Hz, 4H), 3.96-3.86 (m, 2H), 3.71-3.64 (m, 2H), 2.97- 2.86 (m, 2H), 2.83-2.72 (m, 3H), 2.59 (d, J = 12.9 Hz, 1H), 2.29 (tt, J = 9.4, 5.3 Hz, 2H), 1.82-1.73 (m, 2H), 1.71-1.64 (m, 2H), 1.63-1.15 (m, 76H), 0.86 (t, J = 6.6 Hz, 12H). H-8 .sup.1H NMR (400 MHz, Chloroform-d) 4.04 (t, J = 6.7 Hz, 4H), 3.80-3.54 (m, 8H), 3.02-2.62 (m, 6H), 2.33- 2.24 (m, 2H), 1.70-1.09 (m, 76H), 0.86 (t, J = 6.6 Hz, 12H). H-9 .sup.1H NMR (400 MHz, Chloroform-d) 4.85 (p, J = 6.1 Hz, 2H), 3.82-3.63 (m, 4H), 2.94 (s, 1H), 2.81-2.53 (m, 5H), 2.26 (t, J = 7.5 Hz, 4H), 1.66- 1.13 (m, 56H), 0.94-0.77 (m, 12H). H-10 .sup.1H NMR (400 MHz, Chloroform-d) 4.04 (t, J = 6.7 Hz, 4H), 3.92-3.75 (m, 4H), 3.06 (s, 1H), 2.92-2.66 (m, 5H), 2.33-2.24 (m, 2H), 1.67-1.14 (m, 76H), 0.86 (t, J = 6.7 Hz, 12H). H-11 .sup.1H NMR (400 MHz, Chloroform-d) 4.03 (t, J = 6.7 Hz, 4H), 3.82-3.54 (m, 8H), 2.94-2.50 (m, 6H), 2.27 (t, J = 7.5 Hz, 4H), 1.84-0.98 (m, 82H), 0.93-0.74 (m, 12H). H-12 .sup.1H NMR (400 MHz, Chloroform-d) 4.03 (t, J = 6.8 Hz, 4H), 3.99-3.88 (m, 2H), 3.72-3.64 (m, 2H), 2.99- 2.77 (m, 5H), 2.63 (d, J = 12.9 Hz, 1H), 2.27 (t, J = 7.5 Hz, 4H), 1.89- 0.98 (m, 86H), 0.93-0.75 (m, 12H). H-13 .sup.1H NMR (400 MHz, Chloroform-d) 4.03 (t, J = 6.8 Hz, 4H), 3.86-3.68 (m, 4H), 2.99 (s, 1H), 2.85-2.60 (m, 5H), 2.27 (t, J = 7.5 Hz, 4H), 1.79- 0.92 (m, 82H), 0.90-0.75 (m, 12H). H-14 .sup.1H NMR (400 MHz, Chloroform-d) 4.04 (t, J = 6.5 Hz, 4H), 3.72 (q, J = 4.2 Hz, 2H), 3.68- 3.49 (m, 6H), 3.01-2.21 (m, 8H), 1.69-1.49 (m, 10H), 1.47-1.15 (m, 10H), 1.29- 1.32 (m, 40H), 0.85 (t, J = 6.6 Hz, 12H). H-15 .sup.1H NMR (400 MHz, Chloroform-d) 4.05 (t, J = 6.6 Hz, 4H), 3.92-3.71 (m, 4H), 3.03 (s, 1H), 2.89-2.63 (m, 5H), 2.29 (tt, J = 9.2, 5.3 Hz, 2H), 1.67-1.16 (m, 60H), 0.86 (t, J = 6.7 Hz, 12H). H-16 0 .sup.1H NMR (400 MHz, Chloroform-d) 4.84 (p, J = 6.1 Hz, 2H), 3.78-3.55 (m, 8H), 2.94-2.54 (m, 6H), 2.25 (t, J = 7.5 Hz, 4H), 1.71-1.11 (m, 80H), 0.86 (t, J = 6.6 Hz, 12H). H18 .sup.1H NMR (400 MHz, Chloroform-d) 4.84 (p, J = 6.2 Hz, 2H), 3.98-3.85 (m, 2H), 3.71-3.63 (m, 2H), 2.97- 2.74 (m, 5H), 2.60 (d, J = 12.9 Hz, 1H), 2.25 (t, J = 7.5 Hz, 4H), 1.83- 1.12 (m, 84H), 0.86 (t, J = 6.7 Hz, 12H). H-19 .sup.1H NMR (400 MHz, Chloroform-d) 4.84 (p, J = 6.3 Hz, 2H), 3.92-3.73 (m, 4H), 3.05 (s, 1H), 2.92-2.64 (m, 5H), 2.25 (t, J = 7.5 Hz, 4H), 1.69- 1.05 (m, 80H), 0.86 (t, J = 6.7 Hz, 12H). H-20

(49) ##STR00063##

Example 2

(50) Synthesis of compound c: succinic acid (compound b, 1.52 g, 8.71 mmol) was dissolved in 50 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 3.34 g, 17.4 mmol), 4-dimethylaminopyridine (DMAP, 0.3 g, 2.4 mmol) and N,N-diisopropylethylamine (DIPEA, 1.5 g, 11.6 mmol) were added to the solution and stirred for-10 minutes, then 6-undecyl alcohol (compound a, 1 g, 5.8 mmol) was added and stirred at room temperature for 12 hours. After the reaction, monitored by TLC, was completed, the dichloromethane was removed by rotary evaporation, then 100 mL of ethyl acetate was added. The mixture was washed with equal volume of saturated sodium chloride solution for three times, the organic phase was dried by anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=20:1 (volume ratio)) to obtain 1.2 g of colorless liquid, yield: 63%.

(51) Synthesis of compound H-21: compound c (0.88 g, 2.68 mmol) was dissolved in 20 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 0.64 g, 3.35 mmol), 4-dimethylaminopyridine (DMAP, 0.065 g, 0.54 mmol) and N, N-diisopropylethylamine (DIPEA, 0.35 g, 2.68 mmol) were added to the solution and stirred for 10 minutes. Triethanolamine (0.2 g, 1.34 mmol) was added and stirred at room temperature for 12 hours. After the reaction, monitored by TLC, was completed, the solvent was removed by rotary evaporation, then 50 mL of ethyl acetate was added. The mixture was washed with saturated sodium chloride solution of an equal volume for three times, then the organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:1 (volume ratio)) to obtain 0.4 g of colorless liquid, yield: 39%. MS m/z (ESI): 770.71 [M+H].sup.+

(52) The compounds H-21 and H-23 were prepared using the method of Example 2:

(53) TABLE-US-00002 .sup.1H NMR (400 MHz, Chloroform-d) 4.84 (p, J = 6.3 Hz, 2H), 4.15 (s, 4H), 3.55 (s, 2H), 2.85 (s, 4H), 2.76 (s, 2H), 2.28 (dt, J = 14.1, 7.5 Hz, 8H), 1.66-1.57 (m, 8H), 1.53-1.43 (m, 8H), 1.36-1.18 (m, 32H), 0.94-0.78 (m, 12H). H-21 .sup.1H NMR (400 MHz, Chloroform-d) 4.43- 3.82 (m, 8H), 3.55 (s, 2H), 3.08-2.58 (m, 6H), 2.38-2.20 (m, 8H), 1.67-1.58 (m, 8H), 1.36-1.20 (m, 58H), 0.93-0.81 (m, 12H). H-23

Example 3

(54) ##STR00066##

(55) Synthesis of compound b: Tetradecanoic acid (compound a, 1.57 g, 6.48 mmol) was dissolved in 70 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 1.86 g, 9.72 mmol), 4-dimethylaminopyridine (DMAP, 0.32 g, 2.6 mmol), and N,N-diisopropylethylamine (DIPEA, 1.67 g, 12.3 mmol) were added to the solution and stirred for 10 minutes, then triethanolamine (2.9 g, 19.44 mmol) was added and stirred at room temperature for 12 hours. After the reaction, monitored by TLC, was completed, the solvent was removed by rotary evaporation, and 120 mL of ethyl acetate was added. The mixture was washed with equal volume of saturated sodium chloride solution for three times. The organic phase was dried by anhydrous sodium sulfate for 30 minutes, after the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:5 (volume ratio)) to obtain 1.8 g of colorless liquid, yield: 74%.

(56) Synthesis of compound c: the compound is synthesized by referring to the method of compound c in Example 2.

(57) Synthesis of compound H-22: compound c (0.88 g, 2.68 mmol) was dissolved in 30 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 0.51 g, 2.68 mmol), 4-dimethylaminopyridine (DMAP, 0.13 g, 1.07 mmol) and N,N-diisopropylethylamine (DIPEA, 0.52 g, 4.02 mmol) were added to the solution and stirred for 10 minutes, then compound b (1 g, 2.68 mmol) was added and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation, then 70 mL of ethyl acetate was added. The mixture was washed with equal volume of saturated sodium chloride solution for three times, and the organic phase was dried by anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:1 (volume ratio)) to obtain 1.0 g of colorless liquid, yield: 55%. MS m/z (ESI): 684.57 [M+H].sup.+

(58) The compounds H-22 and H-24 were prepared by the method of Example 3.

(59) TABLE-US-00003 .sup.1H NMR (400 MHz, Chloroform-d) 4.85 (t, J = 6.2 Hz, 1H), 4.16 (s, 4H), 3.56 (s, 2H), 3.04-2.66 (m, 6H), 2.39-2.17 (m, 6H), 1.65-1.56 (m, 6H), 1.51-1.44 (m, 4H), 1.36-1.21 (m, 38H), 0.93-0.80 (m, 9H). H-22 1H NMR (400 MHz, Chloroform-d) 5.44- 5.24 (m, 4H), 4.85 (p, J = 6.3 Hz, 1H), 4.15 (s, 4H), 3.55 (s, 2H), 3.00-2.62 (m, 10H), 2.28 (dt, J = 14.7, 7.6 Hz, 6H), 2.03 (q, J = 6.9 Hz, 4H), 1.65-1.56 (m, 6H), 1.52-1.44 (m, 4H), 1.38-1.18 (m, 28H), 0.92-0.81 (m, 9H). H-24

Example 4

(60) ##STR00069##

(61) Synthesis of compound a: oleylamine (2 g, 7.48 mmol) was dissolved in 50 mL of THF, triethylamine (1.13 g, 11.21 mmol) was added to the solution, and CS.sub.2 (0.74 g, 9.72 mmol) was added dropwise under ice bath, then the solution was stirred at room temperature for 12 hours. 4-Dimethylaminopyridine (DMAP, 0.27 g, 2.24 mmol) was added, and di-tert-butyl dicarbonate ((Boc).sub.2O, 2.12 g, 9.72 mmol) was added under ice bath condition and stirred at room temperature for 3 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added to the mixture and then washed with equal volume of saturated sodium chloride solution for three times, dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was separated and purified by column (silica gel column, the eluent was PE: EA=100:1 (volume ratio)) to obtain 1.78 g of light yellow liquid, yield: 77%.

(62) Synthesis of compound b: compound a (0.5 g, 1.62 mmol) was dissolved in 10 mL of DMF, compound d (0.24 g, 1.62 mmol) was added to the solution and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, most of the solvent was removed by rotary evaporation. 70 mL of ethyl acetate was added, and the mixture was then washed with equal volume of saturated sodium chloride solution for three times, then dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:1 (volume ratio)) to obtain 0.5 g of colorless liquid, yield: 68%.

(63) Synthesis of compound c: the compound is synthesized by referring to the method of compound c in Example 2.

(64) Synthesis of compound H-25: compound c (0.36 g, 1.09 mmol) was dissolved in 20 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 0.32 g, 1.64 mmol), 4-dimethylaminopyridine (DMAP, 0.05 g, 0.44 mmol), and N,N-diisopropylethylamine (DIPEA, 0.28 g, 2.81 mmol) were added to the solution and stirred for 10 minutes, then compound b (0.5 g, 1.09 mmol) was added and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. 50 mL of ethyl acetate was added, and the mixture was washed for three times with equal volume of saturated sodium chloride solution, then the organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=5:1 (volume ratio)) to obtain 0.5 g of colorless liquid, yield: 60%. MS m/z (ESI): 768.62 [M+H].sup.+.

(65) The compounds H-25, H-26, and H-27 were prepared by the method of Example 4.

(66) TABLE-US-00004 0 .sup.1H NMR (400 MHz, Chloroform-d) 7.02 (s, 1H), 5.46-5.24 (m, 2H), 4.85 (p, J = 6.2 Hz, 1H), 4.30 (t, J = 6.2 Hz, 2H), 3.97-3.75 (m, 6H), 3.57 (dq, J = 7.0, 4.0 Hz, 2H), 2.28 (dt, J = 14.9, 7.4 Hz, 4H), 2.05-1.91 (m, 4H), 1.83-1.70 (m, 2H), 1.65-1.56 (m, 6H), 1.52-1.45 (m, 4H), 1.41- 1.16 (m, 42H), 0.90-0.82 (m, 9H). H-25 .sup.1H NMR (400 MHz, Chloroform-d) 7.02 (s, 1H), 5.39-5.29 (m, 2H), 4.30 (t, J = 6.3 Hz, 2H), 3.94 (d, J = 5.8 Hz, 2H), 3.91-3.80 (m, 6H), 3.57 (t, J = 7.4 Hz, 2H), 2.29 (q, J = 7.7 Hz, 4H), 1.99 (q, J = 6.6 Hz, 4H), 1.72 (s, 6H), 1.64-1.56 (m, 7H), 1.38- 1.19 (m, 50H), 0.91-0.81 (m, 9H). H-26 .sup.1H NMR (400 MHz, Chloroform-d) 7.01 (s, 1H), 5.38-5.27 (m, 2H), 4.31 (t, J = 6.3 Hz, 2H), 3.96-3.80 (m, 8H), 3.62-3.53 (m, 2H), 2.29 (q, J = 8.0 Hz, 4H), 2.04-1.91 (m, 4H), 1.78-1.55 (m, 14H), 1.36-1.17 (m, 65H), 0.86 (t, J = 6.6 Hz, 9H). H-27

Example 5

(67) ##STR00073##

(68) Synthesis of compound b: oleyamine (1 g, 3.74 mmol) was dissolved in 50 mL of DCM, triethylamine (1.13 g, 11.21 mmol) was added to the solution, and triphosgene (0.44 g, 1.5 mmol) was added dropwise under ice bath condition, then stirred at room temperature for 3 hours. TLC monitored that the reaction was completed, the solvent was removed by rotary evaporation. 30 mL of DMF and compound a (1.11 g, 7.48 mmol) were added to the solution and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, most of the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium chloride solution for three times, then dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:1 (volume ratio)) to obtain 1.0 g of light yellow liquid, yield: 61%.

(69) Synthesis of compound c: the compound is synthesized by referring to the method of compound c in Example 2.

(70) Synthesis of compound H-28: compound c (0.5 g, 1.52 mmol) was dissolved in 20 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 0.44 g, 2.28 mmol), 4-dimethylaminopyridine (DMAP, 0.07 g, 0.61 mmol) and N,N-diisopropylethylamine (DIPEA, 0.39 g, 3.04 mmol) were added and stirred for 10 minutes, then compound b (0.67 g, 1.52 mmol) was added to the solution and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium chloride solution for three times. The organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=5:1 (volume ratio)) to obtain 0.4 g of colorless liquid, yield: 35%. MS m/z (ESI): 752.64 [M+H].sup.+.

(71) The compounds H-28 and H-29 were prepared by the method of Example 5.

(72) TABLE-US-00005 .sup.1H NMR (400 MHz, Chloroform-d) 5.39- 5.27 (m, 2H), 4.85 (dt, J = 12.0, 6.1 Hz, 1H), 4.18 (t, J = 6.1 Hz, 2H), 3.77-3.72 (m, 2H), 3.49-3.40 (m, 4H), 3.18 (t, J = 7.3 Hz, 2H), 2.28 (dt, J = 14.7, 7.4 Hz, 4H), 2.06-1.81 (m, 8H), 1.66-1.55 (m, 4H), 1.53-1.44 (m, 6H), 1.35-1.19 (m, 38H), 0.92-0.76 (m, 9H). H-28 .sup.1H NMR (400 MHz, Chloroform-d) 5.40- 5.26 (m, 2H), 4.18 (t, J = 6.1 Hz, 2H), 3.95 (d, J = 6.0 Hz, 2H), 3.74 (s, 2H), 3.51- 3.38 (m, 4H), 3.18 (t, J = 7.2 Hz, 2H), 2.29 (q, J = 8.3, 7.9 Hz, 4H), 2.24-1.88 (m, 8H), 1.66-1.55 (m, 5H), 1.53-1.45 (m, 2H), 1.42-1.16 (m, 50H), 0.93-0.79 (m, 9H). H-29

Example 6

(73) ##STR00076##

(74) Synthesis of compound b: linoleic acid (1.25 g, 4.68 mmol) was dissolved in 50 mL of DCM, triethylamine (0.62 g, 6.09 mmol) was added and stirred in an ice bath for 10 minutes, then 6-bromohexanoyl chloride (compound a, 1 g, 4.68 mmol) was added dropwise, gradually returned to room temperature, then stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium bicarbonafe solution for three times, then washed with equal volume of saturated sodium chloride solution for three times, and dried by anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=20:1 (volume ratio)) to obtain 1.8 g of colorless liquid, yield: 87%.

(75) Synthesis of compound d: refer to the synthesis method of compound d in the synthesis of compound H-15.

(76) Synthesis of compound e: compound b (1.5 g, 3.38 mmol) dissolved in 50 mL of anhydrous ethanol, triethylamine (0.44 g, 4.4 mmol) and compound c (1.07 g, 10.25 mmol) were added to the solution, heated at 50 C. and stirred for 24 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium chloride for three times, then dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was DCM: MeOH=20:1 (volume ratio)) to obtain 1.1 g of colorless liquid, yield: 70%.

(77) Synthesis of compound H-17: compound e (0.4 g, 0.85 mmol) was dissolved in 10 mL of absolute ethyl alcohol, compound d (0.3 g, 0.85 mmol) was added and stirred for 10 minutes, then heated to reflux for 12 hours. TLC monitored that the reaction was completed, the solvent was removed by rotary evaporation, then the residue was separated and purified by column (silica gel column, the eluent was PE: EA=5:1 (volume ratio)) to obtain 0.4 g of colorless liquid, yield: 57%. MS m/z (ESI): 822.72 [M+H].sup.+

(78) The compound H-17 was prepared using the method of Example 6.

(79) TABLE-US-00006 .sup.1H NMR (400 MHz, Chloroform-d) 5.42-5.28 (m, 4H), 4.10-4.00 (m, 4H), 3.79-3.49 (m, 7H), 2.76 (t, J = 6.5 Hz, 2H), 2.65-2.44 (m, 3H), 2.37-2.24 (m, 4H), 2.03 (q, J = 6.9 Hz, 4H), 1.69-1.19 (m, 56H), 0.86 (q, J = 6.6 Hz, 9H). H-17

Example 7

(80) ##STR00078##

(81) Synthesis of compound c: compound b (1.6 g, 5.64 mmol) dissolved in 50 mL of dichloromethane, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC, 1.3 g, 6.77 mmol), 4-dimethylaminopyridine (DMAP, 0.28 g, 2.26 mmol) and N,N-diisopropylethylamine (DIPEA, 1.09 g, 8.46 mmol) were added and stirred for 10 minutes, then hexanediol (compound a, 1 g, 8.46 mmol) was added and stirred at room temperature for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. 100 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium chloride solution for three times. The organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=50:1 (volume ratio)) to obtain 1.5 g of colorless liquid, yield: 69%.

(82) Compound d (1.0 g, 2.6 mmol) was dissolved in 50 mL of DCM, NaHCO.sub.3 (1.75 g, 20.8 mmol) was added and stirred for 5 minutes, then compound Dess-Martin Periodinane (1.75 g, 4.16 mmol) was added and stirred at room temperature for 3 hours. TLC monitored that the reaction was completed, the solvent was removed by rotary evaporation. The petroleum ether was added, and the mixture was washed three times with equal volume of saturated sodium bicarbonate solution, then washed once with equal volume of saturated salt solution. The organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the solvent was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=10:1 (volume ratio)) to obtain 0.5 g of colorless liquid, yield: 50%.

(83) Synthesis of compound H-2: compound d (0.3 g, 0.78 mmol) was dissolved in 10 mL of DCM, compound e (0.08 g, 0.78 mmol) was added to the solution and stirred for 10 minutes, then sodium triacetoxyborohydride (0.22 g, 1.02 mmol) was added and stirred overnight at room temperature. TLC monitored that the reaction was completed, the solvent was removed by rotary evaporation. 50 mL of ethyl acetate was added, and the mixture was washed with equal volume of saturated sodium chloride solution for three times. The organic phase was dried with anhydrous sodium sulfate for 30 minutes. The solvent was removed by rotary evaporation, and the residue was purified by column (silica gel column, the eluent was PE: EA=5:1 (volume ratio)) to obtain 0.3 g of colorless liquid, yield: 46%. MS m/z (ESI): 838.79 [M+H]+

(84) The compound H-2 was prepared by the method of Example 7.

(85) TABLE-US-00007 .sup.1H NMR (400 MHz, Chloroform-d) 4.03 (t, J = 6.7 Hz, 4H), 3.75-3.55 (m, 6H), 2.75-2.38 (m, 6H), 2.27 (t, J = 7.5 Hz, 4H), 1.77-0.96 (m, 70H), 0.91-0.75 (m, 12H). H-2

Example 8

(86) ##STR00080##

(87) Synthesis of compound b: compound a (5 g, 29 mmol) was dissolved in 100 mL of DCM, meta-Chloroperbenzoic acid (m-CPBA, 8.9 g, 44 mmol, mass fraction 85%) was added to the solution under ice bath conditions and stirred for 15 minutes, then the ice bath was removed and the mixture was stirred overnight. TLC monitored that the reaction was completed, excess saturated sodium bisulfite solution (10 mL) was added to consume the unreacted meta-Chloroperbenzoic acid, and DCM was removed by rotary evaporation. 200 mL of ethyl acetate was added, washed with 200 mL of saturated sodium bicarbonate solution for three times, then washed with 200 mL of saturated sodium chloride solution for one time. The organic phase was dried with anhydrous sodium sulfate for 30 minutes, after the ethyl acetate was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=1:2 (volume ratio)) to obtain 4.5 g of colorless liquid, yield: 82%.

(88) Synthesis of compound d: compound b (3.0 g, 16 mmol) was dissolved in 50 mL of dichloromethane (DCM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 4.01 g, 21 mmol), 4-dimethylaminopyridine (DMAP, 0.79 g, 6 mmol), and N,N-diisopropylethylamine (DIPEA, 3.12 g, 24 mmol) were added and stirred for 10 minutes, then hex-5-en-2-ol (compound c, 1.94 g, 19 mmol) was added and stirred overnight at room temperature. TLC monitored that the reaction was completed, then dichloromethane was removed by rotary evaporation. 100 mL of ethyl acetate was added, washed with equal volume of saturated sodium chloride solution for three times. The organic phase was dried with anhydrous sodium sulfate for 30 minutes. After the ethyl acetate was removed by rotary evaporation, the residue was purified by column (silica gel column, the eluent was PE: EA=20:1 (volume ratio)) to obtain 3.5 g of colorless liquid, yield: 81%.

(89) Synthesis of compound H-30: diethylenetriamine (compound e, 0.65 g, 6 mmol) was dissolved in anhydrous methanol, compound d (5 g, 0.19 mmol) was added to the solution and stirred at room temperature for 10 minutes, then heated to reflux for 12 hours. TLC monitored that the reaction was completed, then the solvent was removed by rotary evaporation. The residue was purified by column (silica gel column, the eluent was DCM: MeOH=100:1 (volume ratio)) resulted in 3.0 g of colorless liquid, yield: 75%. MS m/z (ESI): 642.47 [M+H].sup.+.

(90) The compounds H-30 and H-31 were prepared using the method of Example 8:

(91) ##STR00081##

(92) It should be noted that the aforementioned compounds are not exhaustive, and any cationic lipid compounds adopting the synthetic method described in this invention (with a hydroxyl group in the head part and having a small head part and a large tail part) fall within the scope of protection of this invention. The small head part and a large tail part mean that when the N atom is taken as a center, the molecular structure space occupied by the head part with hydroxyl group is smaller than that occupied by the tail part with alkane chain at the end.

Experimental Example 1: Preparation and Detection of the LNPs

(93) Compounds H-1 through H-29 were used to prepare mRNA-LNP for the following experiments: The cationic lipids, DSPC or DOPE (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), cholesterol (AVT (Shanghai) Pharmaceutical Tech Co., Ltd.), and PEG lipids were dissolved in ethanol (Lipid concentration 20 mg/mL) according to the designed prescription ratio (Lipid (cationic lipid compound)/DOPC/Cholesterol/DMG-PEG (conjugated lipid) a molar ratio of 40/10/50/1.7), then the solution was mixed thoroughly. The mass ratio of LNPs to mRNA ranges from 10:1 to 30:1. mRNA was diluted to 0.2 mg/mL using citrate or sodium acetate buffer (pH=3 or 5). The aforementioned lipid ethanol solution and mRNA solution were mixed thoroughly at a volume ratio ranging from 1:5 to 1:1. The yielded nanoparticles were purified by ultrafiltration and dialysis, followed by filtration and sterilization. The particle size and particle dispersion index (PDI) of mRNA-LNPs (mRNA loaded LNPs) were characterized by dynamic light scattering using Malvern Zetasizer Nano ZS in 173 reverse phase scattering detection mode. The particle dispersion index represents the degree of particle size uniformity and is an important index for particle size characterization. The encapsulation efficiency of mRNA was detected using the Ribogreen RNA quantification kit (Thermo Fisher), and the results are shown in Table 1.

(94) TABLE-US-00008 TABLE 1 PDI En- (part- cap- icle sul- dis- ation mRNA- per- effi- LNP Size sion ciency Sample Structure of the cationic lipid (nm) index) (%) Sample 1 85.25 0.112 96.1 Sample 2 91.23 0.134 92.2 Sample 3 93.23 0.126 93.4 Sample 4 92.14 0.136 89.1 Sample 5 82.26 0.118 86.2 Sample 6 80.51 0.125 94.7 Sample 7 78.32 0.141 90.8 Sample 8 98.45 0.131 91.3 Sample 9 0 105.18 0.137 96.5 Sample 10 87.55 0.125 94.2 Sample 11 79.89 0.127 91.1 Sample 12 85.58 0.135 92.3 Sample 13 91.63 0.126 93.6 Sample 14 97.18 0.133 91.3 Sample 15 85.34 0.119 96.2 Sample 16 83.66 0.128 94.8 Sample 17 69.36 0.155 88.2 Sample 18 79.15 0.158 93.6 Sample 19 00 81.48 0.161 91.8 Sample 20 01 99.12 0.164 93.2 Sample 21 02 81.87 0.147 81.1 Sample 22 03 102.46 0.161 85.8 Sample 23 04 84.31 0.164 84.3 Sample 24 05 81.56 0.181 92.7 Sample 25 06 77.38 0.172 78.2 Sample 26 07 65.12 0.157 76.6 Sample 27 08 70.43 0.163 73.9 Sample 28 09 110.25 0.192 70.1 Sample 29 0 67.12 0.187 71.7 Sample 30 87.52 0.156 93.3 Sample 31 97.43 0.171 85.2

Experimental Example 2: Animal Experiments for LNPs Using the Samples Prepared in Experimental Example 1

(95) Male ICR mice (6-8 weeks, Shanghai Jiesijie Experimental Animal Co., Ltd.) were housed under experimental conditions with a temperature of 222 C. and relative humidity of 45-75%, with a light/dark cycle of 12 hours. mRNA encoding luciferase was used as the reporter gene. Luciferase catalyzes fluorescein to produce biofluorescence, and the transfection efficiency of LNPs was determined by measuring the biofluorescence intensity per unit time. Taking luciferase mRNA (purchased from ApexBio Technology) as an Example, the mRNA-LNP samples 1-29 obtained above were compared with an existing commercially available compound MC3

(96) ##STR00113##
The LNPs used as a positive control were formulated based on the well-known optimal PEG-lipid composition for MC3, with the molar ratio of Lipid (cationic lipid)/DSPC/Cholesterol/DMG-PEG (conjugated lipid) set at 50/10/38.5/1.5. A dose of 150 g/kg mRNA was administered via intramuscular injection to two legs of one mouse in each group. At a specific time point, fluorescein (20 g/mL) was injected intraperitoneally into mice. After 5 minutes, the fluorescence intensity of each mouse were measured using a small animal in vivo imaging system. The final results were represented as the average fluorescence intensity. The experimental results of fluorescence intensity after intraperitoneal injection in mice are shown in Table 2.

(97) TABLE-US-00009 TABLE 2 mRNA- Average LNP intensity Sample Structure of the cationic lipid (p/s/cm.sup.2/sr) Comp- arative Sample MC3 4.2E+5 Sample 1 6.32E+7 Sample 2 7.13E+6 Sample 3 6.08E+6 Sample 4 8.12E+6 Sample 5 7.47E+6 Sample 6 0 1.27E+7 Sample 7 8.18E+7 Sample 8 7.2E+7 Sample 9 9.67E+7 Sample 10 8.59E+7 Sample 11 7.16E+7 Sample 12 1.26E+7 Sample 13 1.77E+7 Sample 14 2.71E+7 Sample 15 1.91E+8 Sample 16 0 8.18E+7 Sample 17 1.26E+7 Sample 18 1.11E+7 Sample 19 8.58E+7 Sample 20 1.69E+7 Sample 21 8.39E+6 Sample 22 5.13E+6 Sample 23 9.45E+6 Sample 24 6.5E+6 Sample 25 8.15E+7 Sample 26 0 1.2E+7 Sample 27 7.1E+6 Sample 28 5.76E+6 Sample 29 6.55E+6 Sample 30 7.17E+7 Sample 31 5.36E+7

(98) The results showed that compared to the existing commercially available cationic lipid compound MC3, the cationic lipid compounds described in the present invention achieved significantly better biocompatibility and higher in vivo mRNA transfection efficiency.

Experimental Example 3: Structural Stability Experiment of LNPs Using the Sample Prepared in Experimental Example 1

(99) Preparation and characterization of transmission electron microscopy samples (using sample 1 as an Example). 10 L prepared sample 1 was dripped onto a copper mesh and deposited for 10 minutes, then the extra sample was absorbed and dried. The sample was then stained with uranyl acetate for 5 minutes, followed by blotting with filter paper to remove excess stain. After drying overnight, the morphology was examined using transmission electron microscopy (TEM).

(100) As shown in the FIGURE, the LNPs described in the present invention can form stable nanostructure within a narrow size distribution range (small PDI), The particle size varies depending on the structure of the LNPs, ranging between 30 and 200 nm.

Experimental Example 4: Biocompatibility Verification Experiment Using the Samples Prepared in Experimental Example 1 and the Comparative Samples from Experimental Example 2

(101) Cell viability was detected using CCK-8 (cell counting kit-8) assay kit. Hep3B cells in exponential growth phase (100 L, cell density of 210.sup.4 cells/mL) were seeded into a 96-well plate and incubated in a cell culture incubator for 24 hours. After removal of the culture medium from each well, 100 L of fresh cell culture medium containing mRNA at 20 g/mL encapsulated in LNPs was added, and the cells were co-incubated for 4 hours. Subsequently, the cell supernatant was removed, fresh cell culture medium was added, and the cells were further incubated for 20 hours. Then, the supernatant was removed, and 100 L of fresh cell culture medium containing CCK-8 working solution (10 L/mL) was added and incubated for 2 hours. Blank wells were set up with cell culture medium containing the working solution of CCK-8. The absorbance at 450 nm of each well was measured using a multimode microplate reader (no bubbles should be present in the plate during measurement). Cells without LNPs treatment were used as the control group, and their cell viability was set as 100%.
Cell viability (%)=[A1-A0]/[A2-A0]100.

(102) A1 represents the absorbance of the treating group, A0 represents the absorbance of the blank group, and A2 represents the absorbance of the control group. The experimental results are shown in Table 3.

(103) TABLE-US-00010 TABLE 3 mRNA LNP Cell viability sample (%) Comparative 95% sample MC3 Sample 1 96% Sample 2 96% Sample 3 97% Sample 4 95% Sample 5 96% Sample 6 95% Sample 7 96% Sample 8 95% Sample 9 96% Sample 10 95% Sample 11 96% Sample 12 95% Sample 13 96% Sample 14 97% Sample 15 98% Sample 16 96% Sample 17 95% Sample 18 96% Sample 19 94% Sample 20 97% Sample 21 96% Sample 22 97% Sample 23 96% Sample 24 97% Sample 25 93% Sample 26 91% Sample 27 93% Sample 28 92% Sample 29 93% Sample 30 97% Sample 31 95%

(104) The experimental results showed that within the specified concentration range of LNPs, most cells had a viability no less than 95%, with no significant cytotoxicity observed.

Experimental Example 5: Low temperature Storage Effect Experiment of LNPs

(105) Taking sample 1 as an Example, the LNPs prepared according to the formulation were stored at a low temperature of 4 C. At different time points (0 day, 6 days, 10 days, 15 days, 30 days, 45 days, 60 days, 90 days), samples were taken to characterize the particle size and PDI of mRNA-LNPs(mRNA loaded LNPs) using Malvern Zetasizer Nano ZS. The encapsulation efficiency of mRNA was determined using the Ribogreen RNA quantification kit (Thermo Fisher). The results are shown in Table 4.

(106) TABLE-US-00011 TABLE 4 Storage Storage time Size Encapsulation condition (day) (nm) PDI efficiency (%) 4 C. 0 85.3 0.112 96.1 6 83.5 0.116 95.0 10 84.4 0.113 95.3 15 85.7 0.117 96.3 30 82.4 0.113 95.3 45 83.1 0.114 95.1

(107) According to Table 4, the LNPs formed by the lipid molecules described in the present invention can be stored at low temperatures for a long time, which facilitates the transportation and preservation of the product.

(108) In summary, the lipid compounds described in this invention feature hydroxyl group in the head structure, which confers hydrophilicity and capacities of fusing membrane. Meanwhile, the overall structure resembles a cone with a small head (nitrogen-containing moiety) and a large tail (long-chain alkane of hydrophobic moiety). The LNPs prepared using the cationic lipid compounds with the aforementioned optimal structure usually exhibit enhanced biocompatibility and higher in vivo mRNA transfection efficiency, achieving unexpected technical effects. Degradable ester bonds introduced into the hydrophobic tail part of the cationic lipid compounds described in this invention can be rapidly degraded by esterolytic enzymes in vivo. Compared with long-chain alkane in MC3, the introduction of ester bonds can alter the metabolic behavior of lipid molecules in vivo, thereby enhancing the biosafety of mRNA-LNPs. In terms of synthesis process, MC3 requires a five step reaction and involves highly dangerous Grignard reagents. Compared with MC3, the synthesis route described in this invention is simple and easy to implement, with inexpensive and readily available raw materials, which is conducive to industrial production. The LNPs prepared from the compounds described in this invention can be stored at low temperature for a long time, which facilitates the transportation and preservation of the product. Therefore, the novel cationic lipid compounds described in this invention have promising application prospects.

(109) The basic principles, main features, and advantages of the present invention are shown and described herein. Those skilled in the art should understand that the aforementioned embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the scope of protection of the present invention.