CN118791393A - A lipid nanoparticle based on asymmetric hydrophobic tail ionizable lipid and its preparation method and application - Google Patents

Abstract

The present invention provides a lipid nanoparticle based on an asymmetric hydrophobic tail ionizable lipid and a preparation method and application thereof. The blank lipid nanoparticle prepared by the present invention has strong designability, biodegradability and high in vivo and in vitro delivery efficiency. It is used as a drug carrier for siRNA delivery, and is superior to the currently marketed products at the in vivo and in vitro levels.

Description

A lipid nanoparticle based on an asymmetric hydrophobic tail ionizable lipid and its preparation method and application

Technical Field

The invention relates to a lipid nanoparticle based on an asymmetric hydrophobic tail ionizable lipid and a preparation method and application thereof, belonging to the technical field of drug delivery.

Background Art

With the discovery of RNA interference (RNAi) and the confirmation that small interfering RNA (siRNA) can transiently induce the degradation of specific messenger RNA (mRNA), people have made a lot of efforts in the past two decades to use gene silencing for disease treatment. Nucleic acid drugs such as siRNA and mRNA have become ideal drug candidates because they can be quickly chemically synthesized and act directly on target genes in a sequence-dependent manner. In principle, siRNA can target all genes including splice variants and mutants, which shows that siRNA is suitable for targeting non-drugifiable proteins. Therefore, siRNA has great application prospects for a variety of diseases.

Since siRNA is a medium-sized molecule with multivalent anions and high hydrophilicity, it can hardly be taken up by cells. In addition, siRNA is easily degraded by nucleases in the blood, resulting in the inability of siRNA to accumulate in target tissues. Therefore, the establishment of a suitable delivery carrier is crucial for the development of siRNA drugs. Lipid nanoparticles (LNPs) have become the most promising carriers for clinical application among non-viral vectors due to their excellent biodegradability, low toxicity, structural flexibility, biocompatibility, and ease of large-scale preparation. LNPs can protect siRNA from degradation, ensure its stability in the circulation, reduce immune activation, localize to target tissues, and promote intracellular delivery. The LNP delivery system is usually about 50 to 200 nm in diameter and is generally composed of ionizable cationic lipids, cholesterol, phospholipids, and polyethylene glycol (PEG)-lipids. It can effectively deliver siRNA to the cytoplasm, participate in the RNAi mechanism, and subsequently inhibit specific mRNA translation.

Ionizable cationic lipids play a key role in LNP-based siRNA delivery. First, under acidic conditions, lipids are positively charged and can load negatively charged nucleic acid drugs into nanoparticles. Second, the optimal acid dissociation constant (pKa) of LNPs containing ionizable lipids is 6.0-6.9, and the total LNP surface charge is close to neutral at physiological pH, which is less toxic during in vivo delivery. Third, ionizable lipids show positive charges in the endosomal acidic environment (pH = 5-6) in order to interact with endogenous anionic lipids to release siRNA into the cytoplasm. Finally, in order to effectively destabilize the endosomal membrane and release nucleic acids into the cell membrane, lipids must exhibit a physical shape that promotes the formation of a hexagonal (HII) lipid phase. Therefore, rational lipid design is combined with an iterative screening process to determine the optimal combination of alkyl chains, linking functional groups, and ionizable head groups. Although a series of ionizable cationic lipids have been reported, there is still a need for ionizable lipid compounds with more efficient and stable delivery performance. Efficient ionizable lipids provide RNA-LNPs with better transfection effects while maintaining low toxicity. Therefore, the research goal of this project is to design and synthesize new ionizable cationic lipids to efficiently encapsulate and deliver siRNA for disease treatment.

Summary of the invention

In order to improve the above technical problems, the present invention provides a compound represented by formula (I):

in,

L ₁ is selected from A ₁ , A ₂ , A ₃ are the same or different, independently absent or selected from the following groups which are unsubstituted or optionally substituted with one, two or more _Ra : C _1-6 alkylene, C _1-6 alkyleneoxy, C _2-6 alkenylene, C _3-8 cycloalkylene, 3-8 membered heterocyclylene, C _6-10 arylene, 5-10 membered heteroarylene, NH; each _Ra is the same or different and independently selected from C _1-10 alkylene; p and q are the same or different and independently selected from 0, 1, 2, 3, 4, 5 or 6;

J is absent or selected from H,

When J is absent, B ₂ -J is selected from -OC(=O)-, -C(=O)O-; when J is H, B ₂ -J is selected from -NH-, -NHC(=O)-, -C(=O)NH-; when J is When B ₂ is N;

B ₁ is selected from N,

U ₁ , U ₂ , G ₁ , G ₂ , E ₁ , E ₂ , J ₁ , J ₂ , K ₁ , K ₂ , Q ₁ , Q ₂ are independently selected from -OC(═O)-, -C(═O)O-, -C(═O)NH-, -NHC(═O)-, -C(═O)S-, the following groups which are unsubstituted or optionally substituted with one, two or more R _b : C _1-6 alkylene, C _1-6 alkyleneoxy, C _2-6 alkenylene; each R _b is the same or different and are independently selected from OH, C _1-10 alkylene;

U ₁ , U ₂ are different from J ₁ , J ₂ ; and/or,

G ₁ , G ₂ are different from K ₁ , K ₂ ; and/or,

E ₁ , E ₂ are different from Q ₁ , Q ₂ ;

n1 and n2 are the same or different and are independently selected from a number of 2-25, such as 3, 5, 7, 10, 12 and 13.

m1 and m2 are the same or different and are independently selected from a number of 1-25, for example 2, 4, 7, 9, 15, 18 and 22.

According to an embodiment of the present invention, A ₁ , A ₂ , and A ₃ are the same or different, independently absent or selected from the following groups which are unsubstituted or optionally substituted by one, two or more _Ra : methylene, ethylene, propylene, butylene, methyleneoxy, ethyleneoxy, propyleneoxy, O, NH, piperazinylene, and phenylene.

According to an embodiment of the present invention, each _Ra is the same or different and is independently selected from _C1-6 alkyl; for example, methyl, ethyl.

According to an embodiment of the present invention, L ₁ is selected from L-1, L-2, L-3, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11 or L-12;

According to an embodiment of the present invention, U ₁ and U ₂ are the same or different and are independently selected from -CH ₂ -, -(CH ₂ ) _U -, -CH(OH)-, -CH ₂ O-, -OC(═O)-, -C(═O)O-, -C(═O)NH-, -NHC(═O)-, -C(═O)S-; U is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

According to an embodiment of the present invention, J ₁ and J ₂ are the same or different and are independently selected from -CH ₂ -, -(CH ₂ ) _U -, -CH(OH)-, -CH ₂ O-, -OC(═O)-, -C(═O)O-, -C(═O)NH-, -NHC(═O)-, -C(═O)S-, -CH═CH-, -CH ₂ -CH═CH-; U is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.

According to an embodiment of the present invention, _G1 and _G2 are the same or different and are independently selected from _-CH2- , -CH(OH)-, _-CH2O- , -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-, -CH=CH-, _-CH2 -CH=CH-.

According to an embodiment of the present invention, _E1 and _E2 are the same or different and are independently selected from _-CH2- , -CH(OH)-, _-CH2O- , -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-, -CH=CH-, _-CH2 -CH=CH-.

According to an embodiment of the present invention, _K1 and _K2 are the same or different and are independently selected from _-CH2- , -CH(OH)-, _-CH2O- , -OC(=O)-, -C(=O)O-, -C(=O)NH-, -NHC(=O)-, -C(=O)S-, -CH=CH-, _-CH2- CH=CH-, -(CH=CH) _2- , -( _CH2 -CH=CH) _2- .

According to an embodiment of the present invention, Q ₁ and Q ₂ are the same or different and are independently selected from -CH ₂ -, -CH(OH)-, -CH ₂ O-, -OC(═O)-, -C(═O)O-, -C(═O)NH-, -NHC(═O)-, -C(═O)S-, -CH═CH-, -CH ₂ -CH═CH-, -(CH═CH) ₂ -, -(CH ₂ -CH═CH) ₂ -.

According to an embodiment of the present invention, The same or different, independently selected from -C ₈ H ₁₇ ,

According to an embodiment of the present invention, Different, independently selected from -C ₆ H ₁₃ , -C ₇ H ₁₅ , -C ₈ H ₁₇ , -C ₉ H ₁₉ , -C ₁₀ H ₂₁ , -C ₁₁ H ₂₃ , -C ₁₂ H ₂₅ , -C ₁₃ H ₂₇ , -C ₁₄ H ₂₉ , -C ₁₅ H ₃₁ , -C ₁₆ H ₃₃ , -C ₁₇ H ₃₅ , -(CH ₂ ) ₆ -CH＝CH-(CH ₂ ) ₇ CH ₃ , -(CH ₂ ) ₆ -CH＝CH-CH ₂ -CH＝CH-(CH ₂ ) ₄ CH ₃ , -(CH ₂ ) ₆ -CH＝CH-CH ₂ -CH＝CH-CH ₂ -CH＝CH-(CH ₂ ) ₂ CH ₃ ,

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the compounds of the following structures:

wherein L ₁ , B ₁ , B ₂ , U ₁ , U ₂ , G ₁ , G ₂ , E ₁ , E ₂ , J ₁ , K ₁ , Q ₁ , n1 , n2 , and m1 independently have the same meanings as described above.

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the compounds of the following structures:

wherein J, L ₁ , U ₁ , U ₂ , G ₁ , G ₂ , E ₁ , E ₂ , J ₁ , K ₁ , Q ₁ , n1 , n2 , and m1 independently have the same meanings as described above.

According to an embodiment of the present invention, the compound represented by formula (I) is selected from the compounds of the following structures:

The present invention provides a blank lipid nanoparticle, comprising a compound represented by formula (I), a neutral phospholipid, a steroid lipid and a polyethylene glycol lipid (PEG);

According to an embodiment of the present invention, the neutral phospholipid is selected from at least one of DSPC, DPPC, DPPE, DMPE, POPC, DOPG, DOPE, POPE and DEPC, preferably distearoylphosphatidylcholine (DSPC);

According to an embodiment of the present invention, the steroid lipid is selected from avenasterol, sitostanol, sitosterol, stigmasterol, stigmasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, hydroxycholesterol, lanosterol, dihydrocholesterol, dihydroergosterol, luminosterol, alginasterol, cholic acid, glycocholic acid, taurocholic acid, dehydrocholesterol, streptosterol, dihydroergocalciferol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol and deoxycholic acid, preferably cholesterol (CHO).

According to an embodiment of the present invention, the PEG lipid is selected from at least one of DSPE-PEG, DMG-PEG, MG-PEG, DAG-PEG, DSG-PEG, DPPE-PEG and DMA-PEG, preferably 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000).

According to an embodiment of the present invention, the molar fraction of the compound represented by formula (I) is 15-65%, for example, 35-50%, such as 40%, 43%, 47%, 49%;

According to an embodiment of the present invention, the molar fraction of the PEG lipid is 0.1-6%, for example 0.5-3.5%, such as 0.8%, 1%, 1.5%, 1.7%, 2.7%, 3.4%;

According to an embodiment of the present invention, the molar fraction of the neutral phospholipids is 5-25%, for example, 10-15%, such as 11%, 13%, 14%, 15%;

According to an embodiment of the present invention, the molar fraction of the steroidal lipids is 10-50%, for example 30-45%, such as 32%, 37%, 39%, 40%, 41%, 43%, 45%.

According to one embodiment of the present invention, the molar ratio of the compound represented by formula (I), neutral phospholipid, steroid lipid and PEG lipid is 50:10:38.5:1.5.

The blank lipid nanoparticles of the present invention can be prepared by conventional lipid nanoparticle preparation methods in the art, such as high-pressure emulsification method, ethanol injection method, ultrasonic dispersion method, microfluidics method, etc.

The present invention also provides application of the blank lipid nanoparticles as drug carriers.

The present invention also provides a drug-loaded lipid nanoparticle composition, wherein the drug-loaded lipid nanoparticle comprises the blank lipid nanoparticle, a pharmaceutically acceptable excipient and a drug.

The pharmaceutically acceptable excipients described in the present invention include adjuvants, diluents, and the like.

According to an embodiment of the present invention, the mass ratio of the blank lipid nanoparticles to the drug is (5-30):1, for example (10-20):1, preferably 10:1, 15:1, 20:1, 25:1, 30:1, 40:1.

According to an embodiment of the present invention, the drug may be a small molecule compound, a nucleic acid, an oligopeptide, etc. Preferably, the drug is a nucleic acid.

Furthermore, the nucleic acid molecule can be selected from at least one of siRNA, mRNA, ASO, plasmid and saRNA.

Furthermore, the nucleic acid is used to prevent and/or treat cancer, inflammation, fibrotic diseases, autoimmune diseases, infections, psychiatric disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic diseases, oral diseases, musculoskeletal diseases, neonatal screening, nutritional diseases, parasitic diseases, skin diseases, etc.

The present invention also provides application of the drug-loaded lipid nanoparticle composition in delivering siRNA to cells or organs.

The present invention also provides a method for preparing the drug-loaded lipid nanoparticle composition, comprising the following steps: loading the drug onto the blank lipid nanoparticles to obtain the drug-loaded lipid nanoparticle composition.

According to an embodiment of the present invention, the preparation method comprises the following steps:

(1) dissolving the drug in a sodium citrate aqueous solution to obtain a drug solution;

(2) mixing the blank lipid nanoparticle solution with sodium citrate buffer to obtain a liposome solution;

(3) adding the drug solution to the liposome solution and heating them at an appropriate temperature to obtain the drug-loaded lipid nanoparticle composition.

According to an embodiment of the present invention, the heating temperature in step (3) is 25°C-100°C.

According to an embodiment of the present invention, the heating time in step (3) is 10 min to 60 min.

Preferably, the heating temperature is 50° C. and the heating time is 30 min.

According to an embodiment of the present invention, the volume ratio of the drug solution to the liposome solution in step (3) is 1:(0.5-5), for example 1:(0.8-3), preferably 1:1.

Beneficial Effects

1. The present invention designs and synthesizes a novel asymmetric ionizable lipid represented by formula (I);

2. The blank lipid nanoparticles prepared based on the asymmetric ionizable lipids of the present invention can be used as drug carriers. The lipid nanoparticles have uniform particle size, high drug loading and low toxicity, can be ionized in endosomes, and have excellent endosomal escape effects. Compared with the marketed ionizable lipid MC3 or the commercial transfection reagent Lipo2000, it has higher in vivo and in vitro transfection efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Flow cytometry of Cy5-siNC@LNP cell uptake ability;

Figure 2: Cy5-siNC@LNP cell uptake fluorescence intensity bar graph;

Figure 3: Bar graph of cytotoxicity test of lipid nanoparticles prepared by compounds;

Figure 4: ED ₅₀ determination of H18a-LNP and O14-LNP.

Definition and explanation of terms

Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and combined with each other. The definitions of groups and compound structures after such combinations and combinations should be understood to be within the scope of the specification and/or claims of this application.

Unless otherwise specified, the numerical ranges described in this specification and claims are equivalent to recording at least each specific integer value therein. For example, the numerical range "0-10" is equivalent to recording each integer value in the numerical range "0-10", namely 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

The term "C _1-10 alkyl" is understood to mean straight-chain and branched monovalent alkyl groups having 1 to 10 carbon atoms, and "C _1-6 alkyl" means straight-chain and branched alkyl groups having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or the like or isomers thereof.

The term "C _1-6 alkyloxy" is to be understood as "C _1-6 alkyl-O-", and C _1-10 alkyl is as defined above.

The term " _C2-6 alkenyl" is understood to mean a linear or branched monovalent hydrocarbon radical containing one or more double bonds and having 2 to 6 carbon atoms, for example, 2, 3, 4, 5 or 6 carbon atoms, 2 or 3 carbon atoms (i.e., _C2-3 alkenyl). It is understood that in the case where the alkenyl contains more than one double bond, the double bonds may be separated from each other or conjugated. The alkenyl is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)- Pent-1-enyl, (Z)-pent-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl , 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut-2-enyl, (Z)-1-methyl but-2-enyl, (E)-3-methylbut-1-enyl, (Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl, (E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.

The term "C _3-8 cycloalkyl" is understood to mean a saturated monovalent monocyclic or bicyclic (e.g. fused, bridged, spiro) hydrocarbon ring having 3 to 8 carbon atoms, for example 3, 4, 5, 6, 7 or 8 carbon atoms. The C _3-8 cycloalkyl may be a monocyclic hydrocarbon group, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, or a bicyclic hydrocarbon group such as bicyclo[2.1.1]hexyl.

The term " _C6-10 aryl" is to be understood as preferably meaning a monovalent aromatic or partially aromatic mono- or bicyclic ring having 6, 7, 8, 9 or 10 carbon atoms, in particular a ring having 6 carbon atoms (" _C6 aryl"), for example phenyl; or biphenyl, or a ring having 9 carbon atoms (" _C9 aryl"), for example indanyl or indenyl, or a ring having 10 carbon atoms (" _C10 aryl"), for example tetrahydronaphthyl, dihydronaphthyl or naphthyl.

The term "5-10 membered heteroaryl" is to be understood as including monovalent monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contain 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S and which, in addition, in each case may be benzo-fused. "Heteroaryl" also refers to radicals in which a heteroaromatic ring is fused to one or more aryl, alicyclic or heterocyclyl rings, wherein the radical or point of attachment is on the heteroaromatic ring.

The term "3-8 membered heterocyclyl" refers to a saturated or unsaturated non-aromatic ring or ring system, for example, a 4-, 5-, 6- or 7-membered monocyclic ring, a 7- or 8-membered bicyclic (such as fused, bridged, spiro) ring system, and contains at least one, for example 1, 2, 3, 4, 5 or more heteroatoms selected from O, S and N, wherein N and S may also be optionally oxidized to various oxidation states to form nitrogen oxides, -S(O)- or -S(O) ₂ -. The heterocyclyl may include fused or bridged rings as well as spiro rings. In particular, the heterocyclic group may include, but is not limited to: a 4-membered ring, such as azetidinyl, oxetanyl; a 5-membered ring, such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring, such as diazepanyl.

The term "sub-group" refers to a divalent organic group which is connected to the main body via two single bonds, such as "sub-group C _1-6 alkyl" refers to a sub-group formed by eliminating two single bonds from "C _1-6 alkane".

DETAILED DESCRIPTION

The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example 1: Synthesis of Compound C12-D3-A8

D3B (1.50 g, 8.6 mmol) and C12 (3.49 g, 18.9 mmol) were added to 15 mL of ethanol and stirred at room temperature for 36 h. After the reaction was completed by TLC, the mixture was washed with saturated NaCl for three times and separated by column chromatography to obtain white block solid C12-D3B (2.95 g) with a yield of 63.1%.

Under ice bath condition, TFA (5 mL) was slowly added to a dichloromethane solution of C12-D3B (1.00 g, 2.3 mmol), and the reaction was stirred for 1 h. After the reaction was completed by TLC, the solvent was removed by distillation under reduced pressure, and the product was washed with saturated NaHCO ₃ for 3 times. The organic phase was extracted with DCM and distilled under reduced pressure to obtain a light yellow transparent liquid C12-D3 (0.61 g) with a yield of 80.2%.

C12-D3 (300 mg, 0.7 mmol) and Na ₂ CO ₃ (574 mg, 5.4 mmol) were dissolved in DMF, reacted at room temperature for 2 h, A8 (327 mg, 1.7 mmol) was added, and then refluxed for 18 h. After the reaction was completed by TLC, the mixture was washed with saturated NaCl for 3 times and separated by column chromatography to obtain light yellow oily liquid C12-D3-A8 (360 mg) with a yield of 79.5%.

Compound C12-D3-A8 is a light yellow oily liquid. MS mass spectrum (MALDI) m/z: C ₄₃ H ₉₀ N ₂ O ₂ (M ⁺ ), theoretical calculated value: 667.700; measured value: 667.577 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ3.57(s,2H),2.93(dd,J＝10.6,5.2Hz,4H),2.59(t,J＝14.5Hz,2H),2.51–2.42(m,2H),2.30(d,J＝11.9Hz,2H),2.11(d,J＝12.6Hz,2H),1.82–1.73(m,2H ), 1.67 (d, J = 9.4Hz, 4H), 1.46–1.36 (m, 6H), 1.35–1.11 (m, 50H), 0.84 (t, J = 7.0Hz, 12H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ66.84,59.45,50.99,50.71,50.02,34.19,30.90,30.64,28.76,28.69,28.61,28.60,28.33,28.03,28.00,25.76,24.78,21.75,21.68,21.56, 13.10, 13.03.

Example 2: Synthesis of Compound O12-D3-A8

TEA (1.45 g, 14.4 mmol) was slowly added dropwise to a solution of D3B (1.00 g, 5.7 mmol) in IPA (15 mL). After reacting for 1 h, O12 (3.45 g, 14.4 mmol) was added, and the reaction temperature was raised to 90°C and refluxed for 5 h. After the reaction was completed by TLC detection, the mixture was washed with saturated NaCl for 3 times, and the organic phase was extracted with DCM. Column chromatography was used to obtain a colorless, transparent, viscous liquid O12-D3B (0.75 g) with a yield of 20.1%.

Under ice bath conditions, TFA (5 mL) was slowly added to a dichloromethane solution of O12-D3B (1.00 g, 1.5 mmol), and the reaction was stirred for 1 h. After the reaction was completed as detected by TLC, the solvent was removed by distillation under reduced pressure, the mixture was washed with saturated NaHCO3 three times, and the organic phase was extracted with EA. Light yellow transparent liquid O12-D3 (1.18 g) was obtained by distillation under reduced pressure with a yield of 100%.

O12-D3 (300 mg, 0.5 mmol) and DIPEA (312 mg, 2.4 mmol) were dissolved in DMF, and the mixture was reacted at room temperature for 2 h. A8 (234 mg, 1.0 mmol) was added, and the mixture was reacted at room temperature for 18 h. After the reaction was completed by TLC, the mixture was washed with saturated NaCl for 3 times and separated by column chromatography to obtain light yellow oily liquid O12-D3-A8 (80.6 mg) with a yield of 20.8%.

Compound O12-D3-A8 is a light yellow oily liquid, MS mass spectrum (MALDI) m/z: C ₄₉ H ₉₈ N ₂ O ₄ (M ⁺ ), theoretical calculated value: 779.333; measured value: 779.575 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ7.97(t,J＝5.3Hz,1H),7.53(s,1H),6.81(s,1H),3.64–3.45(m,1H),3.26(q,J＝6.4Hz,2H),3.09–2.80(m,2H),2.42(dt,J＝39.5,7.3Hz,6H),1.71–1. 53(m,2H),1.45–1.36(m,4H),1.25(m,20H),0.85(t,J＝7.0Hz,6H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ173.78,134.13,54.26,52.95,51.11,37.23,30.83,28.55,28.31,26.58,25.56,21.64,13.09.

Example 3: Synthesis of Compound O12-D3-C12

Compound O12-D3-C12 was synthesized from compound C12-D3 (1.00 g, 2.2 mmol) in a similar manner to compound O12-D3B to obtain O12-D3-C12 as a light yellow oily liquid (0.60 g, 28.8%).

Compound O12-D3-C12 is a light yellow oily liquid, MS mass spectrum (MALDI) m/z: C ₅₇ H ₁₁₄ N ₂ O ₆ (M ⁺ ), theoretical calculated value: 923.868; measured value: 923.572 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ4.04(t,J＝6.8Hz,4H),3.76(s,2H),2.79(t,J＝7.0Hz,2H),2.74(t,J＝7.1Hz,4H),2.66(d,J＝11.6Hz,2H),2.58(dd,J＝21.4,7.4Hz,2H),2.47(t,J＝6.3Hz, 2H),2.42(d,J＝7.1Hz,4H),1.77–1.65(m,2H),1.62–1.55(m,4H),1.41(d,J＝9.4Hz,4H),1.30–1.21(m,68H),0.91–0.82(m,12H); ¹³ CNMR is: (150MHz, CDCl ₃ )δ171.65,67.72,63.76,61.53,53.30,49.89,47.79,34.05,34.00,30.96,30.92,28.76,28.72,28.67,28.64,28.62,28.36,28.31,27.63,24.94 ,24.61,21.69,13.11.

Example 4: Synthesis of Compound A8-D3-H8

A8-D3 (400 mg, 1.3 mmol), H8 (579 mg, 4.0 mmol), HATU (1.0 g, 2.7 mmol), and DIPEA (692 mg, 5.37 mmol) were dissolved in DCM (15 mL) and reacted at room temperature for 18 h. After the reaction was completed by TLC, the solvent was removed by distillation under reduced pressure, the mixture was washed with saturated NaCl for three times, and the organic phase was extracted with DCM. A8-D3-H8 (200 mg) was obtained as a light yellow transparent viscous liquid by column chromatography with a yield of 42.6%.

Compound A8-D3-H8 is a light yellow transparent viscous liquid, MS mass spectrum (ESI) m/z: C ₂₇ H ₅₆ N ₂ O (M ⁺ ), theoretical calculated value: 425.4393; measured value: 425.4465 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.99(t,J＝5.8Hz,1H),3.36–3.27(m,2H),3.10(d,J＝5.5Hz,2H),3.03(d,J＝8.3Hz,4H),2.31–2.25(m,2H),2.02(s,2H),1.68(p,J＝7.6Hz,4H),1.65– 1.55(m,2H),1.39–1.22(m,30H),0.88(t,J＝7.0Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ178.68,52.84,50.95,39.17,37.00,35.10,34.05,31.67,31.66,29.15,29.08,29.02,29.01,28.95,28.88,26.58,25.96,24.80,23.69,23.42 ,22.61,22.59,14.07,14.04.

Example 5: Synthesis of Compound C12-D3-H8

Compound C12-D3-H8 was synthesized from compound C12-D3 (500 mg, 1.1 mmol) in a similar manner to compound A8-D3-H8 to obtain C12-D3-H8 as a light yellow oil (130 mg, 32.5%).

Compound C12-D3-H8 is a light yellow oil, MS mass spectrum (ESI) m/z: C ₃₅ H ₇₂ N ₂ O ₃ (M ⁺ ), theoretical calculated value: 569.5543; measured value: 569.5616 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.69(s,1H),3.84(d,J＝7.5Hz,2H),3.28(q,J＝6.2Hz,2H),2.98(t,J＝7.1Hz,2H),2.78(d,J＝8.5Hz,4H),2.16–2.06(m,2H),1.83(p,J＝6.6Hz,2H),1.6 0–1.51(m,2H),1.45–1.28(m,6H),1.27–1.13(m,40H),0.89–0.71(m,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ173.27,66.83,61.23,52.80,35.67,35.65,34.21,33.99,30.91,30.73,28.65,28.62,28.34,28.09,24.82,24.48,21.68,21.63,13.11.

Example 6: Synthesis of Compound O12-D3-H8

Compound O12-D3-H8 was synthesized from compound O12-D3 (555 mg, 1.0 mmol) in a similar manner to compound A8-D3-H8 to obtain O12-D3-H8 as a light yellow oil (132 mg, 44.1%).

Compound O12-D3-H8 is a light yellow oil (MS mass spectrum (ESI) m/z: C ₄₁ H ₈₀ N ₂ O ₅ (M ⁺ ), theoretical calculated value: 681.61; measured value: 681.62 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.60(s,1H),4.05(t,J＝6.8Hz,4H),3.25(q,J＝5.9Hz,2H),2.78(s,4H),2.49(d,J＝22.3Hz,3H),2.24-2.11(m,2H),1.61(dt,J＝14.2,6.4Hz,8H),1.37-1.17(m,44H),0.87(t,J＝7.0Hz,9H); ¹³ C NMR is: (150 MHz, CDCl ₃ )δ 176.08, 172.54, 171.59, 63.92, 50.02, 48.09, 37.60, 35.78, 30.91, 30.72, 28.69, 28.65, 28.63, 28.59, 28.53, 28.34, 28.28, 28.06, 27.60, 24.92, 24.88, 21.68, 21.62.

Example 7: Synthesis of Compound A8-E3-H8

Under room temperature, E3 (1 g, 13.3 mmol) was dissolved in acetonitrile (50 mL), and Na ₂ CO ₃ (11.3 g, 106 mmol) was added. After reacting for 2 h, A8 (6.4 g, 33.1 mmol) was added, and then refluxed for 18 h. After the reaction was completed by TLC detection, it was distilled under reduced pressure, washed with saturated NaCl for 3 times, and the organic phase was extracted with DCM. After column chromatography, a light yellow liquid 2A8-E3 (3.64 g) was obtained with a yield of 91.4%.

H8 (722 mg, 5.0 mmol), A8-E3 (500 mg, 1.7 mmol), DMAP (224 mg, 1.8 mmol), EDCl (351 mg, 1.8 mmol), and DIPEA (237 mg, 1.8 mmol) were dissolved in DCM (20 mL) and refluxed at 50 °C for 28 h. After the reaction was completed as detected by TLC, the mixture was distilled under reduced pressure and washed with saturated NaHCO ₃ for three times. The organic phase was extracted with DCM and light yellow viscous liquid A8-E3-H8 (570 mg) was obtained after column chromatography with a yield of 80.2%.

Compound A8-E3-H8 is a light yellow oil, MS mass spectrum (ESI) m/z: C ₂₇ H ₅₅ NO ₂ (M ⁺ ), theoretical calculated value: 426.42; measured value: 426.43 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ ) δ4.03 (t, J = 6.5 Hz, 2H), 2.46-2.38 (m, 2H), 2.36-2.26 (m, 4H), 2.22 (t, J = 7.6 Hz, 2H), 1.68 (p, J = 6.6 Hz, 2H), 1.55 (p, J = 7.6 Hz, 2H), 1.34 (p, J = 7.2 Hz, 4H), 1.27-1.11 (m, 28H), 0.81 (t, J = 7.0 Hz, 9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ 172.87, 61.75, 53.18, 49.45, 33.39, 30.88, 30.68, 28.60, 28.34, 28.15, 27.96, 26.57, 26.07, 25.42, 24.03, 21.67, 21.61, 13.09.

Example 8: Synthesis of Compound C12-E3-H8

E3 (1 g, 13.3 mmol) and C12 (5.4 g, 29.3 mmol) were dissolved in EtOH (15 mL) and reacted at 50°C overnight. After the reaction was completed by TLC, it was evaporated under reduced pressure and column chromatography was performed to obtain a white solid C12-E3 (570 mg) with a yield of 84.7%.

Compound C12-E3-H8 was synthesized from compound C12-E3 (100 mg, 0.2 mmol) in a similar manner to compound A8-E3-H8 to obtain C12-E3-H8 as a light yellow oil (53.4 mg, 41.6%).

Compound C12-E3-H8 is a light yellow oil, MS mass spectrum (ESI) m/z: C ₃₅ H ₇₁ NO ₄ (M ⁺ ), theoretical calculated value: 570.54; measured value: 570.54 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ4.14–4.06(m,2H),3.68–3.55(m,2H),2.70–2.62(m,2H),2.56(dd,J＝13.2,3.4Hz,2H),2.40(t,J＝5.4Hz,1H),2.29(t,J＝8.0Hz,2H),1.80(p,J＝6.4Hz, 2H), 1.61 (p, J＝7.5Hz, 2H), 1.48–1.35 (m, 6H), 1.33–1.20 (m, 40H), 0.87 (t, J＝7.0Hz, 9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ172.88,68.53,66.79,61.63,61.32,60.08,51.68,50.71,34.06,33.86,33.29,30.90,30.66,28.76,28.61,28.60,28.12,27.93,25.35,24.64 ,23.95,21.68,21.59,13.10.

Example 9: Synthesis of Compound O12-E3-H8

TEA was slowly added dropwise to the IPA solution of E3, stirred at room temperature for 1 h, then O12 was added, and the reaction temperature was raised to 90°C and refluxed for 5 h. After the reaction was completed by TLC detection, it was distilled under reduced pressure, washed with saturated NaHCO ₃ three times, and the organic phase was extracted with EA. After column chromatography, a light yellow viscous liquid O12-E3 (9.9 g) was obtained with a yield of 66.8%.

Compound O12-E3-H8 was synthesized from compound O12-E3 (500 mg, 0.2 mmol) in a similar manner to compound A8-E3-H8 to obtain O12-E3-H8 as a light yellow oil (300 mg, 48.8%).

Compound O12-E3-H8 is a light yellow oil, MS mass spectrum (ESI) m/z: C ₄₁ H ₇₉ NO ₆ (M ⁺ ), theoretical calculated value: 682.59; measured value: 682.57 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ ) δ4.14-3.96 (m, 6H), 2.74 (t, J=7.0 Hz, 4H), 2.47 (t, J=6.8 Hz, 2H), 2.40 (t, J=7.0 Hz, 4H), 2.27 (t, J=7.6 Hz, 2H), 1.73 (p, J=6.5 Hz, 2H), 1.59 (p, J=6.8 Hz, 6H), 1.34-1.12 (m, 44H), 0.86 (t, J=7.0 Hz, 9H); ¹³ C NMR is: (150 MHz, CDCl ₃ )δ 172.83, 171.63, 63.63, 61.28, 52.41, 49.14, 48.28, 33.34, 31.71, 30.91, 30.67, 28.66, 28.64, 28.29, 27.94, 27.63, 25.57, 24.94, 24.00, 21.68, 21.60, 13.10.

Example 9: Synthesis of Compounds O12-D3n Series

The synthesis method of O12-D3n series refers to O12-D3-H8.

Mass spectrum of compound O12-D3-H18a: HRMS (ESI) m/z theoretical calculated value: C ₅₁ H ₉₈ N ₂ O ₅ (M ⁺ ) 818.748, measured value: 819.850 [M+H] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.54(t,J＝5.7Hz,1H),5.36–5.30(m,2H),4.05(t,J＝6.9Hz,4H),3.24(q,J＝6.0Hz,2H),2.75(t,J＝7.0Hz,4H),2.45(dt,J＝21.6,6.7Hz,6H),2.18–2.1 4(m,2H),2.00(q,J＝7.0Hz,4H),1.61(dt,J＝14.8,6.7Hz,8H),1.30–1.24(m,56H),0.87(t,J＝7.1Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ173.70,173.12,130.31,130.13,65.20,51.35,49.55,37.85,37.15,32.27,30.12,30.05,30.01,29.99,29.95,29.90,29.88,29.76,29.70,29 .67,29.64,29.60,29.57,28.97,27.57,26.29,26.24,23.04,14.46.

Mass spectrum of compound O12-D3-H18b: HRMS (ESI) m/z theoretical calculated value: C ₅₁ H ₉₆ N ₂ O ₅ (M ⁺ ) 816.732, measured value: 817.800 [M+H] ⁺ , measured value: 839.850 [M+Na] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.53(t,J＝5.8Hz,1H),5.38–5.29(m,4H),4.05(t,J＝6.8Hz,4H),3.24(q,J＝6.0Hz,2H),2.75(dt,J＝12.8,6.9Hz,6H),2.45(dt,J＝21.6,6.5Hz,6H),2. 16(t,J＝7.7Hz,2H),2.04(q,J＝7.2Hz,4H),1.62(dp,J＝14.0,6.5Hz,8H),1.37–1.25(m,50H),0.88(q,J＝7.1,6.6Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ172.71,130.14,130.00,127.93,127.84,64.75,50.90,49.13,37.42,36.72,32.40,31.84,31.44,29.58,29.55,29.52,29.47,29.32,29.27,2 9.21,29.13,28.54,27.14,27.12,25.92,25.86,25.80,25.54,22.60,22.49,14.03,13.98.

Mass spectrum of compound O12-D3-H18c: HRMS (ESI) m/z theoretical calculated value: C ₅₁ H ₉₄ N ₂ O ₅ (M ⁺ ) 814.716, found 815.850 [M+H] ⁺ , found 837.800 [M+Na] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.53(t,J＝5.9Hz,1H),5.59–5.13(m,6H),4.06(t,J＝6.8Hz,4H),3.26(q,J＝6.1Hz,2H),2.83–2.75(m,8H),2.47(s,6H),2.17(t,J＝7.7Hz,2H),2.05( dq,J＝14.2,7.3Hz,4H),1.82–1.49(m,10H),1.42–1.25(m,J＝30.3Hz,44H),0.97(t,J＝7.5Hz,3H),0.87(t,J＝6.9Hz,6H); ¹³ CNMR is: (150MHz, CDCl ₃ )δ172.98,172.91,132.31,130.64,128.64,128.05,127.47,65.24,51.43,49.57,38.95,37.07,31.87,30.48,30.01,29.99,29.95,29.89,29.74 ,29.70,29.64,29.56,28.95,27.58,27.54,26.28,26.19,25.96,25.87,23.03,20.90,14.62,14.46.

Embodiment 11:

The synthesis method of O18-D3 series refers to O12-D3-H8.

Mass spectrum of compound O18-D3-H7: HRMS (MALDI) m/z theoretical calculated value: C ₅₂ H ₁₀₂ N ₂ O ₅ (M ⁺ ) 835.782, measured value: 835.766 [M+H] ⁺ , measured value: 857.740 [M+Na] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.56(t,J＝5.8Hz,1H),4.06(t,J＝6.8Hz,4H),3.26(q,J＝6.1Hz,2H),2.80(s,4H),2.48(s,6H),2.17(t,J＝7.7Hz,2H),1.62(dt,J＝14.5,6.9Hz,8H),1. 32-1.25(m,66H),0.87(t,J＝7.0Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ172.81,172.71,65.12,51.28,49.33,37.45,36.87,32.06,31.72,29.84,29.80,29.75,29.69,29.50,29.43,29.21,28.74,26.07,25.95,22.8 2,22.68,14.25,14.18.

Mass spectrum of compound O18-D3-H8: HRMS (ESI) m/z theoretical calculated value: C ₅₃ H ₁₀₄ N ₂ O ₅ (M ⁺ ) 849.403, test value: 849.800 [M+H] ⁺ , test value: 871.850 [M+Na] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.65(t,J＝5.7Hz,1H),4.02(t,J＝6.8Hz,4H),3.21(q,J＝6.0Hz,2H),2.73(t,J＝6.9Hz,4H),2.44(dt,J＝27.7,6.5Hz,6H),2.18–2.11(m,2H),1.64–1.5 6(m,8H),1.30–1.18(m,68H),0.86–0.82(m,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ172.87,171.93,64.10,50.28,48.41,36.75,36.00,31.53,31.20,31.01,28.98,28.94,28.88,28.82,28.64,28.57,28.33,27.89,25.21,25.1 6,24.27,21.96,21.89,13.36,13.32.

Mass spectrum of compound O18-D3-H9: HRMS (ESI) m/z theoretical calculated value: C ₅₄ H ₁₀₆ N ₂ O ₅ (M ⁺ ) 863.430, measured value: 863.750 [M+H] ⁺ , measured value: 885.900 [M+Na] ⁺ ; ¹ H NMR: (600 MHz, CDCl ₃ )δ6.63(t,J＝5.8Hz,1H),4.04(t,J＝6.8Hz,4H),3.25(q,J＝6.0Hz,2H),2.81(t,J＝7.2Hz,4H),2.50(dt,J＝44.6,5.6Hz,6H),2.20–2.12(m,2H),1.72–1.6 5(m,2H),1.59(q,J＝7.0Hz,6H),1.32–1.21(m,70H),0.86(t,J＝7.0Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δ174.19,172.82,65.28,51.43,49.49,37.55,37.00,32.24,32.16,32.14,30.02,29.98,29.92,29.86,29.72,29.67,29.60,29.52,28.91,26.2 4,26.17,26.11,22.99,22.96,14.41,14.39.

Example 12: Synthesis experiment of O12-D3 series compounds

The synthesis method of O12-D3 series can refer to O12-D3-H8.

Mass spectrum of compound O12-D3-H9: MS (MALDI) theoretical calculated value: C ₄₂ H ₈₂ N ₂ O ₅ [M+H] ⁺ 695.626, measured value: 695.557, [M+Na] ⁺ 717.542, [M+K] ⁺ 733.529; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.57(t,J＝5.7Hz,1H),4.05(t,J＝6.8Hz,4H),3.25(q,J＝6.0Hz,2H),2.77(d,J＝7.1Hz,4H),2.50(s,2H),2.45(t,J＝6.9Hz,4H),2.20–2.14(m,2H),1.67 (t,J＝6.2Hz,2H),1.63–1.59(m,6H),1.32–1.24(m,46H),0.89–0.86(m,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm 173.97,173.02,65.25,51.40,49.50,38.96,37.12,32.26,32.20,32.15,30.00,29.98,29.95,29.89,29.77,29.70,29.63,29.58,29.55,29.48 ,29.46,28.96,26.28,26.23,25.18,23.03,22.98,14.45.

Mass spectrum of compound O12-D3-H16: MS (MALDI) theoretical calculated value: C ₄₉ H ₉₆ N ₂ O ₅ [M+H] ⁺ 793.735, measured value: 793.578, [M+Na] ⁺ 815.870; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.56(t,J＝5.9Hz,1H),4.07(t,J＝6.8Hz,4H),3.28(s,2H),2.80(s,4H),2.51(s,4H),2.34(t,J＝7.5Hz,2H),2.18(t,J＝7.7Hz,2H),1.65–1.65(m,8H), 1.30–1.25(m,60H),0.88(t,J＝7.0Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm 178.52,172.19,65.03,51.04,48.98,31.83,29.63,29.59,29.57,29.55,29.52,29.48,29.46,29.33,29.27,29.20,28.48,25.82,25.74,24.75 ,14.01.

Mass spectrum of compound O12-D3-H17: MS (MALDI) theoretical calculated value: C ₅₀ H ₉₈ N ₂ O ₅ [M+H] ⁺ 807.751, measured value: 807.696, [M+Na] ⁺ 829.679; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.58(t,J＝5.7Hz,1H),4.05(t,J＝6.9Hz,4H),3.25(q,J＝6.0Hz,2H),2.76(t,J＝7.0Hz,4H),2.47(dt,J＝26.4,6.6Hz,6H),2.17(t,J＝7.7Hz,2H),1.66(t, J＝6.5Hz, 2H), 1.62 (q, J＝6.9Hz, 6H), 1.29–1.24 (m, 62H), 0.87 (t, J＝7.0Hz, 9H); ¹³ C NMR is: (150MHz, CDCl ₃ ) δppm 178.12,172.63,64.81,50.94,49.06,37.44,36.72,31.85,29.64,29.63,29.59,29.57,29.53,29.50,29.48,29.39,29.36,29.29,29.22,29.05 ,28.54,25.86,25.83,24.74,22.61,14.04.

Mass spectrum of compound O12-D3-H18: MS (ESI) theoretical calculated value: C ₅₁ H ₁₀₀ N ₂ O ₅ [M+H] ⁺ 821.767, measured value: 821.850; ¹ HNMR: (600 MHz, CDCl ₃ )δppm 1.7 5–1.69 (m, 2H), 1.61 (s, 6H), 1.32–1.25 (m, 64H), 0.86 (t, J = 7.4Hz, 9H); ¹³ C NMR is: (150MHz, CDCl ₃ ) δppm 174.21,172.29,64.99,50.99,48.97,37.11,36.59,34.07,31.84,29.64,29.61,29.58,29.53,29.47,29.39,29.34,29.28,29.21,29.05,28.50 ,25.83,25.78,25.51,24.73,22.61,14.02.

Example 13: Synthesis experiment of O14-D3 series compounds

The synthesis method of O14-D3 series refers to O12-D3-H8.

Mass spectrum of compound O14-D3-H9: MS (ESI) m/z theoretical calculated value: C ₄₆ H ₉₀ N ₂ O ₅ [M+H] ⁺ 752.692, measured value: 752.700, [M+Na] ⁺ 773.750; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.58(s,1H),4.06(t,J＝6.8Hz,4H),3.26(q,J＝6.1Hz,2H),2.76(s,4H),2.46(s,4H),2.34(t,J＝7.5Hz,2H),2.17(t,J＝7.7Hz,2H),1.64–1.58(m,8H), 1.33–1.26(m,54H),0.89–0.87(m,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm 65.27,51.39,49.47,37.16,32.28,32.21,32.16,31.86,31.79,30.05,30.02,29.96,29.91,29.78,29.72,29.65,29.56,29.46,28.97,26.29,2 6.25,25.11,23.04,22.99,14.47.

Mass spectrum of compound O14-D3-H13: MS (MALDI) theoretical calculated value: C ₅₀ H ₉₈ N ₂ O ₅ [M+H] ⁺ 807.751, measured value: 807.650; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.57 (t, J＝6.0 Hz, 1H), 4.07 (t, J＝6.8 Hz, 4H), 3.29 (q, J＝8.7 Hz, 2H), 2.89 (s, 6H), 2.53 (d, J＝9.9 Hz, 4H), 2.17 (t, J＝7.7 Hz, 2H), 1.74 (s, 2H), 1.64–1.58 (m, 6H), 1.33–1.23 (m, 62H), 0.87 (t, J＝7.0 Hz, 9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ ppm 172.77, 172.60, 65.51, 51.63, 49.56, 36.98, 32.27, 30.04, 30.01, 29.96, 29.90, 29.77, 29.71, 29.64, 28.92, 26.26, 26.15, 23.03, 14.45.

Mass spectrum of compound O14-D3-H14: MS (MALDI) theoretical calculated value: C ₅₁ H ₁₀₀ N ₂ O ₅ [M+H] ⁺ 821.767, measured value: 821.644; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.58 (t, J＝6.5 Hz, 1H), 4.25–3.99 (m, 4H), 3.46–3.38 (m, 4H), 3.17 (s, 2H), 2.97–2.71 (m, 4H), 2.22 (t, J＝7.8 Hz, 2H), 2.10–1.96 (m, 2H), 1.83–1.43 (m, 8H), 1.32–1.22 (m, 64H), 0.88 (t, J＝7.0 Hz, 9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ ppm 176.86, 170.72, 66.00, 51.83, 49.12, 35.99, 31.86, 29.66, 29.64, 29.61, 29.60, 29.57, 29.50, 29.49, 29.31, 29.30, 29.23, 28.37, 25.77, 25.45, 22.62, 14.04.

Mass spectrum of compound O14-D3-H15: MS (ESI) theoretical calculated value: C ₅₂ H ₁₀₂ N ₂ O ₅ [M+H] ⁺ 835.377, measured value: 835.850, [M+Na] ⁺ 857.900; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.60(t,J＝5.7Hz,1H),4.05(t,J＝6.9Hz,4H),3.26(q,J＝6.0Hz,2H),2.81(s,4H),2.51(dt,J＝41.2,6.9Hz,6H),2.17(t,J＝7.7Hz,2H),1.70–1.59(m,8H ), 1.29–1.24 (m, 66H), 0.86 (t, J = 7.1Hz, 9H); ¹³ C NMR is: (150MHz, CDCl ₃ ) δppm 173.71,172.46,64.89,51.02,49.07,37.24,36.66,33.74,31.85,29.62,29.59,29.53,29.48,29.41,29.35,29.28,29.22,29.08,28.53,25.85 ,25.80,25.71,22.61,14.03.

Example 14: Synthesis experiment of O16-D3 series compounds

The synthesis method of O16-D3 series refers to O12-D3-H8.

Mass spectrum of compound O16-D3-H9: MS (MALDI) theoretical calculated value: C ₅₀ H ₉₈ N ₂ O ₅ [M+H] ⁺ 807.324, measured value: 807.387; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 1 .61 (dq, J=30.0, 8.1Hz, 8H), 1.34-1.20 (m, 62H), 0.88 (d, J=7.0Hz, 9H); ¹³ C NMR is: (150MHz, CDCl ₃ ) δppm 166.21,166.13,66.39,49.54,38.96,34.15,32.27,32.18,30.05,30.02,29.98,29.89,29.71,29.64,29.58,29.53,29.48,28.80,26.19,25.89 ,25.20,23.03,22.99,14.44,14.43.

Mass spectrum of compound O16-D3-H10: MS (MALDI) caled for C ₅₁ H ₁₀₀ N ₂ O ₅ [M+H] ⁺ 821.320, test value: 821.500; ¹ H NMR is: ¹ H NMR (600 MHz, CDCl ₃ ) δ ppm 6.73(t,J＝6.5Hz,1H),4.11(t,J＝7.3Hz,4H),3.39(q,J＝6.6Hz,4H),3.14(t,J＝6.3Hz,2H),2.82(s,4H),2.20(t,J＝7.8Hz,2H),2.01(p,J＝6.1Hz,2H),1. 64–1.53(m,6H),1.31–1.24(m,66H),0.86(t,J＝7.0Hz,9H); ¹³ CNMR is: (150MHz, CDCl ₃ )δppm 170.73,165.65,65.84,51.82,49.08,38.47,35.87,31.80,31.76,29.59,29.58,29.54,29.45,29.38,29.24,29.20,28.32,25.72,25.39,22.55 ,13.97.

Mass spectrum of compound O16-D3-H11: MS (MALDI) caled for C ₅₂ H ₁₀₂ N ₂ O ₅ [M+H] ⁺ 835.377, measured value: 835.519; ¹ HNMR: (600MHz, CDCl ₃ )δppm 6.59(t,J＝6.5Hz,1H),4.17–4.08(m,4H),3.42(t,J＝6.3Hz,4H),3.19–3.15(m,2H),2.83(t,J＝15.7Hz,4H),2.29–2.16(m,2H),2.04(p,J＝5.9Hz,2H), 1.66–1.54(m,6H),1.43(dd,J＝17.9,6.7Hz,2H),1.37–1.25(m,66H),0.87(t,J＝7.2Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm 176.0,169.93,65.23,54.88,51.07,48.35,42.78,35.17,34.08,31.06,28.85,28.83,28.81,28.78,28.70,28.50,28.44,27.57,24.98,24.65, 23.93,21.82,13.25.

Mass spectrum of compound O16-D3-H12: MS (MALDI) caled for C ₅₃ H ₁₀₄ N ₂ O ₅ [M+H] ⁺ 849.403, test value: 849.405; ¹ H NMR: (600MHz, CDCl ₃ )δppm 6.55 (t, J＝5.8Hz, 1H), 4.05 (t, J＝6.9Hz, 4H), 3.25 (q, J＝6.0Hz, 2H), 2.77 (s, 4H), 2.58–2.34 (m, 6H), 2.19–2.14 (m, 2H), 1.63 (tq, J＝14.2, 6.9, 6.5Hz, 8H), 1.33–1.25 (m, 68H), 0.87 (t, J＝6.9Hz, 9H); ¹³ C NMR is: ¹³ C NMR (150 MHz, CDCl ₃ ) δ ppm 173.80, 173.04, 65.25, 51.45, 49.57, 37.15, 32.28, 30.05, 30.02, 29.97, 29.91, 29.79, 29.71, 29.65, 28.97, 26.29, 26.24, 23.04, 14.46.

Example 15: Synthesis experiment of O12-K6 series compounds

Preparation of intermediate K6-B: In a 250mL round-bottom flask equipped with a magnetic, add K6 (1.5g, 10.33mmol) and 20mL THF solution and mix and stir evenly, then mix B2 (1.13g, 5.18mmol) and 20mL THF solution and stir evenly; then use a constant pressure funnel to slowly add the THF solution of B2 to the THF solution of K6, react under ice bath conditions for 5h, and detect the reaction progress by TLC. After the reaction is completed, wash with saturated NaHCO ₃ solution and saturated NaCl solution and extract with DCM solution 3 times, column chromatography to obtain white solid K6-B (795mg, 3.24mmol), with a yield of 31.4%. The synthesis methods of other intermediates and target compounds refer to the synthesis of compound O12-D3-H8.

Mass spectrum of compound O12-K6-H7: MS (MALDI) caled for C ₄₄ H ₈₇ N ₃ O ₅ [M+H] ⁺ , 738.179, test value: 738.438; ¹ H NMR is: ¹ H NMR (600MHz, CDCl ₃ )δppm 6.78 (t, J＝6.4Hz,1H), 4.02 (t, J＝6.8Hz,4H), 3.32 (q, J＝6.2Hz,2H), 3.15 (t, J＝7.3Hz,2H), 3.09 (t, J＝7.0Hz,2H), 2.82 (s,3H), 2.74 (t, J＝6.6Hz,4H), 2.59 (t, J＝6.0Hz,2H ),2.43(t,J＝6.6Hz,4H),2.23(t,J＝7.7Hz,2H),1.98(p,J＝6.7Hz,2H),1.90(p,J＝6.7Hz,2H),1.59(dq,J＝12.0,6.1,5.4Hz,6H),1.32-1.24(m,42H),0.8 5(q,J=4.0Hz,9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ ppm 172.67, 172.62, 64.89, 55.19, 53.55, 51.24, 48.25, 39.94, 35.89, 35.39, 31.79, 31.36, 31.30, 29.54, 29.52, 29.49, 29.43, 29.23, 29.20, 28.78, 28.46, 25.81, 25.48, 24.23, 22.55, 22.37, 21.21, 13.97, 13.89.

Mass spectrum of compound O12-K6-H8: MS (MALDI) caled for C ₄₅ H ₈₉ N ₃ O ₅ [M+H] ⁺ 752.205, found: 752.453; ¹ H NMR: (600MHz, CDCl ₃ )δppm 6.90(t, J＝6.2Hz,1H),4.03(t, J＝6.8Hz,4H),3.36(q, J＝6.1Hz,2H),3.08(dt, J＝17.1,7.2Hz,4H),2.78(s,3H),2.72(t, J＝6.6Hz,4H),2.56(t, J＝5.9Hz,2H),2.42(t ,J＝6.6Hz,4H),2.26–2.23(m,2H),2.03(q,J＝6.1Hz,2H),1.92(dt,J＝12.3,6.8Hz,2H),1.60(q,J＝6.5Hz,6H),1.34–1.25(m,J＝14.3Hz,44H),0.87(t,J =4.5Hz,9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ ppm 172.62, 64.88, 55.15, 53.76, 51.31, 48.45, 40.07, 36.21, 35.73, 31.85, 31.77, 31.64, 29.62, 29.57, 29.49, 29.29, 29.25, 29.19, 28.94, 28.54, 25.88, 25.64, 24.85, 24.19, 22.62, 22.54, 14.04, 13.99.

Mass spectrum of compound O12-K6-H9: MS (MALDI) caled for C ₄₆ H ₉₁ N ₃ O ₅ [M+H] ⁺ 766.232, test value: 766.450; ¹ H NMR is: ¹ H NMR (600 MHz, CDCl ₃ )δppm 6.75(t,J＝6.5Hz,1H),4.03(t,J＝6.8Hz,4H),3.34(q,J＝6.2Hz,2H),3.14(dt,J＝37.4,7.0Hz,4H),2.84(s,3H),2.76(t,J＝6.5Hz,4H),2.61(t,J＝6.1Hz,2 H),2.45(t,J＝6.6Hz,4H),2.25(t,J＝7.7Hz,2H),2.00(p,J＝6.6Hz,2H),1.92(p,J＝6.7Hz,2H),1.61(p,J＝6.5Hz,6H),1.34–1.25(m,46H),0.87(d,J＝7. 2Hz,9H); ^13C NMR is: (150 MHz, CDCl ₃ ) δ ppm 175.80, 171.66, 63.99, 54.23, 52.61, 50.32, 47.30, 39.06, 34.97, 34.44, 30.84, 30.75, 30.72, 30.28, 28.59, 28.57, 28.54, 28.48, 28.25, 28.22, 28.14, 28.10, 28.03, 27.50, 24.86, 24.58, 23.71, 23.24, 21.60, 21.56, 13.02, 13.00.

Mass spectrum of compound O12-K6-H16: MS (MALDI) caled for C ₅₃ H ₁₀₅ N ₃ O ₅ [M+H] ⁺ 864.809, measured value: 864.718, [M+Na] ⁺ 886.695; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.69(t,J＝6.4Hz,1H),4.04(t,J＝6.8Hz,4H),3.36(q,J＝6.2Hz,2H),3.15(dt,J＝38.0,7.0Hz,4H),2.84(s,3H),2.77(s,2H),2.64(s,2H),2.46(t,J＝6 .6Hz,4H),2.34(t,J＝7.5Hz,2H),2.26(t,J＝7.7Hz,2H),2.02(p,J＝6.5Hz,2H),1.94(p,J＝6.5Hz,2H),1.62(p,J＝6.9Hz,6H),1.29–1.25(m,60H),0.87(d,J ＝7.2Hz,9H); ¹³ C NMR is: (150 MHz, CDCl ₃ ) δ ppm 177.24, 173.07, 65.51, 54.18, 51.82, 48.80, 40.63, 36.47, 35.90, 34.11, 32.28, 30.08, 30.06, 30.03, 30.01, 29.98, 29.95, 29.92, 29.80, 29.74, 29.72, 29.68, 29.66, 29.60, 29.43, 28.94, 26.29, 26.04, 25.08, 24.67, 23.05, 14.47.

Example 16: Synthesis experiment of O12-P6 series compounds

The synthesis method of O12-P6 series refers to O12-D3-H8.

Mass spectrum of compound O12-P6-H7: MS (ESI) caled for C ₄₇ H ₉₂ N ₄ O ₅ [M+H] 793.257, test value: [M+2H] ²⁺ 397.650, [M+H] ⁺ 793.750; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.95(d,J＝24.4Hz,1H),4.03(t,J＝6.8Hz,4H),3.32(q,J＝5.8Hz,2H),2.74(t,J＝7.1Hz,4H),2.70–2.57(m,4H),2.60–2.51(m,4H),2.42(dt,J＝20.1,7.0Hz,10H),2.14(t,J＝7.7Hz,2H),1.72(p,J＝6.4Hz,2H),1.61(tt,J＝13.9,7.0Hz,8H),1.31–1.24(m,42H),0.86(t,J＝6.9Hz,9H)； ¹³ CNMR为：(150MHz,CDCl ₃ )δppm 173.40,172.78,64.74,56.89,56.03,52.67,51.51,49.33,38.84,37.11,32.75,32.02,31.70,29.80,29.76,29.74,29.71,29.65,29.45,29.40 ,29.15,28.75,26.05,25.98,25.03,22.79,22.64,14.22,14.16.

Mass spectrum of compound O12-P6-H8: MS (ESI) caled for C ₄₈ H ₉₄ N ₄ O ₅ [M+H] ⁺ 807.284, measured value: 807.800; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 6.89(s,1H),4.03(t,J＝6.8Hz,4H),3.32(q,J＝5.9Hz,2H),2.73(t,J＝7.1Hz,4H),2.72–2.61(m,4H),2.56(t,J＝6.7Hz,4H),2.42(dt,J＝20.9,7.1Hz,10H ),2.14(t,J＝7.6Hz,2H),1.73(t,J＝6.6Hz,2H),1.60(p,J＝7.0Hz,8H),1.36–1.24(m,44H),0.85(t,J＝6.6Hz,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm 173.67,172.99,64.96,58.65,56.96,56.21,51.71,49.53,38.93,37.31,32.95,32.23,32.04,30.61,30.01,29.98,29.95,29.92,29.87,29.67 ,29.66,29.61,29.38,28.97,26.27,26.23,25.26,24.40,23.00,22.94,14.43,14.39.

Mass spectrum of compound O12-P6-H9: MS (ESI) caled for C ₄₉ H ₉₆ N ₄ O ₅ [M+H] ⁺ 821.310, measured value: 821.800; ¹ H NMR: (600 MHz, CDCl ₃ )δppm 7.03–6.88(m,1H),4.01(t,J＝6.8Hz,4H),3.31(q,J＝5.9Hz,2H),2.72(t,J＝7.2Hz,4H),2.71–2.59(m,4H),2.53(t,J＝6.5Hz,4H),2.41(dt,J＝20.9,7. 0Hz,10H),2.12(t,J＝7.6Hz,2H),1.71(q,J＝6.4Hz,2H),1.59(tt,J＝14.0,7.1Hz,8H),1.30–1.22(m,46H),0.86–0.83(m,9H); ¹³ C NMR is: (150MHz, CDCl ₃ )δppm173.37,172.74,64.69,56.85,55.99,52.64,51.48,49.31,38.80,37.07,32.72,31.98,31.91,29.76,29.73,29.70,29.67,29.62,29.46,29 .43,29.36,29.26,28.72,26.02,25.99,25.02,24.24,22.75,22.71,14.18.

Example 17: Preparation of blank lipid nanoparticles and drug-loaded lipid nanoparticles based on ionizable lipids

1. Experimental methods

The compound prepared in Example 1-16 was used as an ionizable lipid to prepare blank lipid nanoparticles and drug-loaded lipid nanoparticles. The lipid nanoparticle raw material (ionizable lipid, DSPC, cholesterol, DMG-PEG 2000 in a molar ratio of 50:10:38.5:1.5) was prepared into blank lipid nanoparticles by ethanol injection method, and the mass ratio of blank lipid nanoparticles to siRNA was 15:1. The lipid nanoparticle raw material was dissolved in ethanol in proportion to obtain a lipid stock solution, and three times the volume of ethanol siRNA citrate buffer (0.05M, pH=4.0) was placed in a penicillin bottle, and the lipid stock solution was quickly injected into the siRNA citrate buffer under stirring, and the mixed solution was incubated in a 50°C water bath for 20 minutes for siRNA encapsulation, and then the mixed solution was dialyzed in not less than 1000 times the volume of 1×PBS for at least 2 hours to remove sodium citrate and unencapsulated siRNA to obtain siRNA@LNPs.

After preparation, the fluid particle size and zeta potential of LNPs were determined using dynamic light scattering (DLS). The temperature of the dynamic light scattering instrument was set to 25°C and the scattering angle was fixed at 90°. 100 μL of the prepared LNPs, siRNA@LNPs or Fc-siRNA@LNPs solution was diluted to 3 mL with deionized water for particle size and zeta potential testing, and each sample was tested three times. The encapsulation efficiency of siRNA@LNPs was determined using the RiboGreen RNA assay kit according to the instructions. siRNA@LNPs and RNA standards were diluted into Tris-EDTA (TE) buffer, while siRNA@LNPs were diluted into TE buffer with 2% Triton X-100 and placed in a 96-well plate for 30 minutes. Afterwards, RiboGreen working solution was added to the corresponding wells, and the fluorescence intensity of the 96-well plate was read using a microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The concentration of siRNA was quantified using the siRNA standard curve. The encapsulation efficiency was calculated according to the following formula:

(Formula 2-1) Encapsulation efficiency (%) = [(total siRNA - free siRNA) / total siRNA] × 100%

2. Experimental results

The characterization data of lipid nanoparticles prepared with different ionizable lipids loaded with negative control siNC are shown in the table below, and the encapsulation efficiency is about 80-99%. The above results show that under the same preparation conditions, the particle size of siNC@LNP prepared with 34 ionizable lipids is uniform, and ionizable lipids have a high encapsulation effect on siRNA.

Example 18: Determination of apparent pKa of LNPs

1. Experimental methods

To determine the pKa of three imidazole-based ionizable lipid LNPs, LNPs were diluted to 100 μM using a buffer containing 130 mM NaCl, 10 mM ammonium acetate, 10 mM Hepes, and 10 mM MES, and then the diluted solution was divided into 19 equal parts, and the pH of each solution was adjusted in the range of 2.5-11 using NaOH or HCl, and the pH was increased by 0.5. Then TNS was dissolved in deionized water and added to the pH-adjusted LNPs solution at a final concentration of 1 μM. The fluorescence intensity of different pH solutions was measured by a microplate reader at room temperature using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. The pKa was defined as the pH value at half the maximum fluorescence intensity, and each sample was measured three times.

2. Experimental results

The pK _a values of 34 LNPs were measured by TNS method as shown in the following table. The results show that: compared with ester groups, lipids containing amides have higher pK _a values; the length of the hydrophobic tail and the degree of unsaturation have little effect on the pK _a value, but with the increase of the length of the hydrocarbon chain, the pK _a value gradually increases; with the increase of the number of unsaturated bonds in the hydrophobic tail chain, the pK _a value gradually decreases. Among them, the pK _a values of 14 lipids are in the range of 6.0 to 6.9, which have relatively ideal pK _a values for nucleic acid drug delivery.

Example 19: Determination of Cy5-siNC@LNP Cellular Uptake Ability

1. Experimental methods

Flow cytometry was used to analyze the cellular uptake of LNPs. HepG2 cells were seeded in 12-well plates at a density of 1×105 cells/well. After incubation at 5% CO ₂ and 37°C for 24 h, adherent cells were treated with Cy5-siNC@LNP, and cy5-siNC@MC3 and cy5-siNC@Lipo 2000 were used as positive controls. After 4 h, cells were washed 3 times with PBS, digested with trypsin (0.25%), resuspended with 350 μL 2% paraformaldehyde, and detected by flow cytometry using a flow cytometer. All samples were read ≥10,000 cells, and data were analyzed using FlowJo V10 software.

2. Experimental results

In order to explore the in vitro uptake level of this series of LNPs, the cellular uptake efficiency of LNPs was determined by flow cytometry. Lipo 2000, MC3-LNP and symmetrical four-tail lipids (the structure shown below) were used as controls.

Cy5-siRNA was encapsulated into lipid LNPs or Lipo 2000. This siRNA has no homology to any known mammalian gene and can be regarded as a negative control (NC) siRNA in this study. HepG2 cells were treated with 50nM Cy5-siRNA@LNPs or Cy5-siRNA@Lipo 2000 for 4 hours and then flow cytometry was performed. As shown in Figure 1, all 34 new LNPs showed higher cellular uptake efficiency than MC3-LNP and Lipo 2000. Compared with their symmetrical structures (2C12-D3 and 2O12-D3), except for O12-K6-H12, the remaining 33 LNPs with asymmetric hydrophobic tail lipids showed higher cellular uptake efficiency. The O12-K6 series showed the highest cellular uptake efficiency, among which the cellular uptake efficiency of O12-K6-H9 was 19.7 times and 35.5 times higher than that of MC3-LNP and Lipo 2000, respectively. In addition, the O18-D3, O12-D3n and O14-D3 series lipids all had high uptake efficiencies. The structure-activity relationship between the asymmetric hydrophobic tail lipid structure and the cellular uptake efficiency of LNPs is as follows: (1) Compared with the symmetric structure, LNPs containing asymmetric hydrophobic tail lipids have higher cellular uptake activity; (2) Asymmetric hydrophobic tail lipids containing both amide and ester functional groups have higher cellular uptake activity; (3) Compared with the head group and linker functional group, the length of the hydrophobic tail has less effect on the cellular uptake efficiency.

Example 20: In vitro knockdown activity test

1. Experimental methods

HepG2-Luc stably transfected with firefly luciferase was inoculated into a 96-well plate. The next day, A-LNPs, C-LNPs and O-LNPs were used to encapsulate siLuc targeting firefly luciferase, and MC3-LNPs were used as a positive control. Samples with a final concentration of 50nM of siLuc were added to the corresponding wells and incubated for 4h. The complete medium was replaced and the cells were cultured for another 20h. After that, the cells were collected using a cell lysis buffer, luciferin was added to the lysed cells, and the chemiluminescence intensity was immediately determined using a microplate reader.

2. Experimental results

HepG2 cells stably expressing the luciferase gene (HepG2-luc) were used to study the silencing effect of siLuc@LNPs loaded with the targeted luciferase gene. The results showed that the lipids with the best in vitro activity were O18-D3-H7, O12-D3-H18a, and O14-D3-H13, and the transfection efficiency of their LNPs was 84%, 83%, and 80%, respectively, which were about 1.7 and 2.7 times higher than the transfection efficiency of MC3-LNP and Lipo 2000, respectively, and about 1.8 and 2.2 times higher than the transfection efficiency of 2C12-D3 and 2O12-D3, respectively (Figure 2). The O18-D3 series and the O14-D3 series had the best transfection efficiency, and the transfection efficiency of the seven compounds in the two series was greater than 70%. Among the O12-D3n series of lipids, the transfection efficiencies of O12-D3-H18b LNP and O12-D3-H18c LNP were 52% and 22%, respectively. The transfection efficiency of O12-K6-H9 LNP, which had the highest cell uptake efficiency, was 31%. The structure-activity relationship between asymmetric hydrophobic tail lipid structure and cell transfection efficiency of LNPs is summarized as follows: (1) Compared with asymmetric four-tail lipids (4trail), lipids containing three hydrophobic tail chains have higher cell transfection efficiency; (2) Under the same structural conditions, O18-D3 series LNPs with more suitable _pKa values show higher transfection efficiency than O12-D3-H8 LNPs, although O12-D3-H8 LNPs have higher uptake efficiency; (3) Lipids containing amide and ester functional groups have higher transfection activity; (4) Compared with hydrophobic tails with multiple unsaturations, lipids containing one unsaturation in a single hydrophobic tail have higher transfection activity, and the transfection efficiency decreases significantly with the increase of unsaturation in the hydrophobic tail.

In summary, these results indicate that LNPs with high uptake efficiency usually have relatively higher transfection efficiency compared with cellular uptake efficiency, but the ratio is not absolutely proportional. The transfection efficiency in cells is determined by both cellular uptake efficiency and endosomal escape efficiency. Compared with the current benchmark lipid MC3-LNP and Lipo 2000 delivery systems for siRNA delivery, O18-D3-H7 LNP (O18-LNP), O12-D3-H18a LNP (H18a-LNP) and O14-D3-H13 LNP (O14-LNP) induced higher transfection efficiency, while O12-K6-H9 LNP (K6-LNP) had significantly higher cellular uptake efficiency than other lipids. Therefore, O18-LNP, H18a-LNP, O14-LNP and K6-LNP were selected as preferred LNPs for subsequent studies.

Example 21: LNPs cytotoxicity assay

1. Experimental methods

The CCK-8 method was used to detect the cytotoxicity of blank LNPs in HepG2, RAW 264.7, BASE-2B and HUVEC cells. HepG2, RAW 264.7 or BASE-2B cells in the logarithmic growth phase were collected, the concentration of the cell suspension was adjusted, and the cells were seeded in 96-well plates at a density of 8000 cells/well. After incubation at 5% CO ₂ and 37°C for 24 hours, the medium in the 96-well plate was replaced with a medium without dual antibodies, and ionizable cationic lipids were added at final concentrations of 10μM, 20μM, 40μM, 80μM, 160μM, and 320μM O18-LNPs, H18a-LNPs, O14-LNPs, K6-LNPs, and MC3-LNPs. After incubation at 37°C for 24 h, the culture medium was discarded, 90 μL PBS and 10 μL CCK-8 were added to each well, and the incubation was continued for 30 min. The absorbance of each well at 450 nm was detected using a microplate reader.

2. Experimental results

CCK-8 was used to determine the cell viability of O18-LNP, H18a-LNP, O14-LNP and K6-LNP in BASE 2B, RAW 264.7, HepG2 and HUVEC, and MC3-LNP was used as a control (Figure 3). The results showed that the five LNPs did not show significant toxicity at 320μM in BASE 2B (Figure 3A) and HUVEC (Figure 3C) cells. In RAW 264.7 (Figure 3B) and HepG2 (Figure 3D), O18-LNP and K6-LNP were more significantly toxic at 40μM and 320μM, respectively, compared with MC3-LNPs at this concentration. The toxicity of H18a-LNP and O14-LNP in the four cells was comparable to that of MC3-LNPs, indicating that H18a-LNP and O14-LNP have good in vitro biocompatibility. Therefore, H18a-LNP and O14-LNP were selected for in vivo activity testing.

Example 22: In vivo ED ₅₀ test

1. Experimental methods

75 male C57BL/6 mice were divided into 19 groups, 4 mice in each group, and injected with PBS, siALOX12@H18a-LNP, and siALOX12@MC3 (0.03-0.5 mg/kg) via the tail vein. The mice were killed 48 hours later, and the livers were collected to detect the ALOX12 mRNA content using qPCR.

2. Experimental results

To evaluate the transfection activity of H18a-LNP and O14-LNP in vivo, the mouse arachidonic acid-12-lipoxygenase (ALOX12) gene silencing model was used. H18a-LNP and MC3-LNP containing siRNA targeting ALOX12 (siALOX) were administered to mice at a dose of 0.03-0.5 mg/kg via tail vein, resulting in potent and dose-dependent knockdown of ALOX12 protein. Compared with the siALOX12@MC3-LNP group (ED ₅₀ = 0.16 mg/kg), the siALOX12@H18a-LNP (ED ₅₀ = 0.11 mg/kg) and siALOX12@O14-LNP groups (ED ₅₀ = 0.08 mg/kg) decreased by 31% and 50%, respectively (Figure 4). The results showed that H18a-LNP and O14-LNP had higher in vivo transfection activity than MC3-LNP.

In summary, the present invention designed and synthesized asymmetric hydrophobic tail ionizable lipids with novel structures, wherein blank or drug-loaded lipid nanoparticles prepared using O12-D3-H18a and O14-D3-H13 had excellent in vitro and in vivo transfection activity, which was superior to the marketed positive control MC3, and had no significant toxicity to cells.

The above is an exemplary description of the implementation of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above implementation. Any modification, equivalent substitution, improvement, etc. made by those skilled in the art within the spirit and principle of the present invention should be included in the protection scope of the claims of this application.

Claims (10)

1. A compound of formula (I):

Wherein,

L ₁ is selected fromA ₁、A₂、A₃, identical or different, are, independently of one another, absent or selected from the following groups which are unsubstituted or optionally substituted by one, two or more R _a: c _1-6 -C _1-6 -alkyloxy, C _2-6 -alkenyl, C _3-8 -cycloalkyl, 3-8-membered heterocyclyl, C _6-10 -aryl, 5-10-membered heteroaryl, NH; each R _a, which are identical or different, are independently selected from the group consisting of C _1-10 alkyl; p and q are identical or different and are independently selected from 0, 1,2, 3, 4, 5 or 6;

j is absent or selected from H,

When J is absent, B ₂ -J is selected from-OC (=o) -, -C (=o) O-; when J is H, B ₂ -J is selected from-NH-, -NHC (=o) -, -C (=o) NH-; when J isWhen B ₂ is N;

B ₁ is selected from N,

U₁、U₂、G₁、G₂、E₁、E₂、J₁、J₂、K₁、K₂、Q₁、Q₂ Independently of each other, from-OC (=o) -, -C (=o) O-, -C (=o) NH-, -NHC (=o) -, -C (=o) S-, the following groups being unsubstituted or optionally substituted by one, two or more R _b: c _1-6 alkyl, C _1-6 alkyloxy, C _2-6 alkenyl; each R _b, which are identical or different, are independently selected from OH, C _1-10 alkyl;

U ₁、U₂ is different from J ₁、J₂; and/or the number of the groups of groups,

G ₁、G₂ is different from K ₁、K₂; and/or the number of the groups of groups,

E ₁、E₂ is different from Q ₁、Q₂;

n1, n2 are identical or different and are independently selected from the group consisting of numbers 2 to 25, such as 3, 5, 7, 10, 12 and 13;

m1, m2 are identical or different and are independently selected from the group consisting of numbers 1 to 25, such as 2, 4, 7, 9, 15, 18 and 22.

2. A compound of formula (I) according to claim 1, wherein a ₁、A₂、A₃, identical or different, are, independently of each other, absent or selected from the following groups which are unsubstituted or optionally substituted by one, two or more R _a: methylene, ethylene, propylene, butylene, methylene, ethylene, propylene, O, NH, piperazine, phenylene;

Preferably, each R _a, equal to or different from each other, is selected independently from C _1-6 alkyl; such as methyl and ethyl;

preferably, L ₁ is selected from the group consisting of L-1, L-2, L-3, L-4, L-5, L-6, L-7, L-8, L-9, L-10, L-11, or L-12;

Preferably, U ₁、U₂, which are the same or different, are independently selected from -CH₂-、-(CH₂)_U-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-;U from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

preferably, J ₁、J₂ are the same or different and are independently selected from -CH₂-、-(CH₂)_U-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-、-CH＝CH-、-CH₂-CH＝CH-;U from 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Preferably, G ₁、G₂, identical or different, are selected independently of one another from -CH₂-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-、-CH＝CH-、-CH₂-CH＝CH-;

Preferably E ₁、E₂ are identical or different and are selected independently of one another from -CH₂-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-、-CH＝CH-、-CH₂-CH＝CH-;

Preferably, K ₁、K₂, identical or different, are selected independently of one another from -CH₂-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-、-CH＝CH-、-CH₂-CH＝CH-、-(CH＝CH)₂-、-(CH₂-CH＝CH)₂-;

Preferably, Q ₁、Q₂ are identical or different and are selected independently of one another from -CH₂-、-CH(OH)-、-CH₂O-、-OC(＝O)-、-C(＝O)O-、-C(＝O)NH-、-NHC(＝O)-、-C(＝O)S-、-CH＝CH-、-CH₂-CH＝CH-、-(CH＝CH)₂-、-(CH₂-CH＝CH)₂-;

Preferably, the method comprises the steps of,Identical or different, independently of one another, from-C ₈H₁₇,

Preferably, the method comprises the steps of,Different from each other, independently selected from -C₆H₁₃、-C₇H₁₅、-C₈H₁₇、-C₉H₁₉、-C₁₀H₂₁、-C₁₁H₂₃、-C₁₂H₂₅、-C₁₃H₂₇、-C₁₄H₂₉、-C₁₅H₃₁、-C₁₆H₃₃、-C₁₇H₃₅、-(CH₂)₆-CH＝CH-(CH₂)₇CH₃、-(CH₂)₆-CH＝CH-CH₂-CH＝CH-(CH₂)₄CH₃、-(CH₂)₆-CH＝CH-CH₂-CH＝CH-CH₂-CH＝CH-(CH₂)₂CH₃、

3. The compound of formula (I) according to claim 1 or 2, wherein the compound of formula (I) is selected from the group consisting of compounds of the following structures:

wherein ,L₁、B₁、B₂、U₁、U₂、G₁、G₂、E₁、E₂、J₁、K₁、Q₁、n1、n2、m1, independently of one another, have the definition as claimed in claim 1 or 2;

preferably, the compound of formula (I) is selected from the group consisting of compounds of the following structures:

Wherein ,J、L₁、U₁、U₂、G₁、G₂、E₁、E₂、J₁、K₁、Q₁、n1、n2、m1, independently of one another, have the definition as claimed in claim 1 or 2.

4. A compound of formula (I) according to any one of claims 1 to 3, wherein the compound of formula (I) is selected from the group consisting of compounds of the following structures:

5. A blank lipid nanoparticle comprising a compound of formula (I) according to any one of claims 1 to 4, neutral phospholipid, steroid lipid and polyethylene glycol lipid (PEG);

Preferably, the neutral phospholipid is selected from at least one of DSPC, DPPC, DPPE, DMPE, POPC, DOPG, DOPE, POPE and DEPC, preferably distearoyl phosphatidylcholine (DSPC);

Preferably, the steroid lipid is selected from the group consisting of oat sterols, sitostanol, sitosterol, stigmastanol, stigmasterol, β -sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterols, hydroxycholesterol, lanosterol, dihydrocholesterols, photosterols, algal sterols, cholic acid, glycocholic acid, taurocholic acid, dehydrocholesterol, stigmasterol, dihydroergocalcitol, epicholesterol, ergosterol, fucosterol, hexahydro-sterols, and deoxycholic acid, preferably Cholesterol (CHO);

Preferably, the PEG lipid is selected from at least one of DSPE-PEG, DMG-PEG, MG-PEG, DAG-PEG, DSG-PEG, DPPE-PEG and DMA-PEG, preferably 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000);

Preferably, the compound of formula (I) comprises a mole fraction of 15-65%, for example 35-50%;

preferably, the PEG lipid comprises a mole fraction of 0.1-6%, for example 0.5-3.5%;

Preferably, the neutral phospholipids comprise a mole fraction of 5-25%, such as 10-15%;

Preferably, the mole fraction of steroid lipids is from 10 to 50%, for example from 30 to 45%;

Preferably, the molar ratio of the compound of formula (I), neutral phospholipid, steroid lipid, and PEG lipid is 50:10:38.5:1.5.

6. Use of the blank lipid nanoparticle of claim 5 as a drug carrier.

7. A drug-loaded lipid nanoparticle composition comprising the blank lipid nanoparticle of claim 5, a pharmaceutically acceptable adjuvant, and a drug;

Preferably, the mass ratio of the blank lipid nanoparticle to the drug is (5-30): 1, e.g., (10-20): 1;

preferably, the drug may be a small molecule compound, a nucleic acid, an oligopeptide, etc., preferably, the drug is a nucleic acid;

preferably, the nucleic acid molecule may be selected from at least one of siRNA, mRNA, ASO, a plasmid, and a saRNA;

preferably, the nucleic acid is used for preventing and/or treating cancer, inflammation, fibrosis disease, autoimmune disease, infection, psychotic disorder, hematopathy, chromosomal disease, genetic disease, connective tissue disease, digestive disease, otorhinolaryngologic disease, endocrine disease, ocular disease, reproductive disease, heart disease, kidney disease, lung disease, metabolic disorder, oral disease, musculoskeletal disease, neonatal screening, nutritional disease, parasitic disease, skin disease.

8. Use of the drug-loaded lipid nanoparticle composition of claim 7 for delivering siRNA to a cell or organ.

9. A method of preparing the drug-loaded lipid nanoparticle composition of claim 7, comprising the steps of: and loading the drug on the blank lipid nanoparticle to obtain the drug-loaded lipid nanoparticle composition.

10. The preparation method according to claim 9, characterized in that the preparation method comprises the steps of:

(1) Dissolving the medicine in a sodium citrate water solution to obtain a medicine solution;

(2) Mixing the solution of the blank lipid nanoparticle with a sodium citrate buffer solution to obtain a liposome solution;

(3) Adding the drug solution into the liposome solution, and heating at a proper temperature to obtain the drug-loaded lipid nanoparticle composition;

preferably, the volume ratio of the drug solution to the liposome solution in step (3) is 1 (0.5-5), such as1 (0.8-3), preferably 1:1.