Guanidyl derivative compound, composition containing guanidyl derivative compound and application of guanidyl derivative compound

Abstract

The invention belongs to the technical field of pharmaceutical and biological materials, and particularly relates to a guanidyl derivative compound, a composition containing the guanidyl derivative compound and application of the guanidyl derivative compound. The compound disclosed by the invention has a general formula as shown in a formula I. The invention also provides a lipid carrier, lipid nanoparticles and a pharmaceutical composition containing the lipid carrier, and application of the lipid carrier, the lipid nanoparticles and the pharmaceutical composition. The invention has the following technical effects: the invention provides a guanidyl derivative compound, a composition containing the guanidyl derivative compound and application of the guanidyl derivative compound. The novel guanidyl derivative compound can be used for preparing a lipid nanoparticle delivery carrier. Compared with lipid nanoparticles containing other ionizable lipids, the lipid nanoparticles or pharmaceutical compositions prepared from the guanidyl derivative compound have the advantages of high encapsulation efficiency, high transfection efficiency, weak hepatic tropism, good stability, good cytocompatibility and the like.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and biomaterials technology, specifically relating to guanidine-derived compounds, compositions containing them, and their applications. Background Technology

[0002] Nucleic acid therapy is a hot research topic in disease treatment, and clinically it is mainly used for genetic diseases, cancer, and infectious diseases. Nucleic acids are negatively charged hydrophilic macromolecules that face a series of physiological barriers in the body. Safe and efficient nucleic acid delivery systems are key to their effectiveness.

[0003] Nucleic acid therapy vectors are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors, such as adenovirus, retrovirus, adeno-associated virus, and herpes simplex virus, while exhibiting high transfection efficiency, also have drawbacks such as limited loading capacity, complex production processes, broad targeting, cytotoxicity, immunogenicity, and tumorigenicity, limiting their widespread application. Non-viral vectors, such as lipid nanoparticles, cationic polymers, and cationic liposomes, possess advantages such as high safety, low immunogenicity, low toxicity, high payload capacity, and ease of synthesis, and have attracted considerable attention in recent years. An ideal nucleic acid delivery system needs to assist nucleic acids in evading nuclease degradation, crossing cell membrane barriers, and endosome escape.

[0004] Lipid nanoparticles are commonly used nucleic acid delivery carriers, but current lipid nanoparticles prepared from ionizable lipids, such as ALC-0315 and SM-102, still have some drawbacks: ① Lipid nanoparticles have high hepatic tropism, and after local administration, they are prone to off-target transfection into the liver, causing hepatotoxicity. ② Low endosome escape rate of nucleic acids: After LNPs enter cells, only 1%–2% of the nucleic acids are released into the cytoplasm for protein translation, while the rest remain in intracellular vesicles and are degraded in lysosomes as the endosomes mature. ③ Low thermal stability of lipid nanoparticles: Currently commercially available lipid nanoparticle formulations require storage and transportation under ultra-low temperature conditions. For example, the mRNA-1273 vaccine can be stored at -20℃ to -15℃ for 6 months and at 2℃ to 8℃ for 30 days; the BNT162b2 COVID-19 vaccine can be stored at -80℃ to -60℃ for 6 months and at 2℃ to 8℃ for 5 days. This not only affects the maintenance of mRNA effectiveness but also means an increase in application costs, greatly limiting the development and application of LNPs.

[0005] LNPs can be administered via intravenous, intramuscular, intradermal, subcutaneous, intralymphatic, and intratumoral injections. Currently marketed mRNA LNP vaccines are all administered via intramuscular injection. The rich vascular network in muscle helps mRNA vaccines recruit immune cells from the bloodstream to the injection site, allowing for efficient activation of both innate and adaptive immunity with a relatively small amount of antigen. However, the hepatic tendency of intramuscularly injected mRNA vaccines can lead to adverse reactions such as liver damage, posing a significant challenge to in vivo delivery of mRNA vaccines. Therefore, developing safe and efficient novel delivery vectors is crucial for promoting the widespread application of gene therapy. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the types of lipid nanoparticle delivery carriers in the prior art are relatively limited. To this end, the present invention provides a class of guanidine-derived compounds, compositions containing the same, and applications thereof. This class of novel guanidine-derived compounds can be used to prepare lipid nanoparticle delivery carriers and has one or more of the following advantages: high encapsulation efficiency, high transfection efficiency and weak hepatic tropism, good stability, and good cell compatibility.

[0007] The technical solution of this invention to solve the technical problem is as follows:

[0008] In a first aspect of the invention, a class of guanidine derivatives is provided, specifically 1,3-isophthaloylguanidine derivatives or pharmaceutically acceptable salts thereof.

[0009] The compounds of the present invention have the general formula shown in Formula I:

[0010]

[0011] In Equation I, R1 is -(CH2). n - where n is an integer from 1 to 10, and (CH2) n Zero, one, or more methylene units in the R group are each independently controlled by R. 1a Substitute;

[0012] The R 1a Independently selected from R 1b -NH-, -O-, -S- or

[0013] The R mentioned 1b The structure is shown in Equation II:

[0014]

[0015] In Equation II, m, x, and z are each independently 0 or integers from 1 to 10; R 1b It is 1,3-isophthaloylguanidine, and its structure is shown in Formula III:

[0016]

[0017] In Formula I, R2 is CH or N. In some embodiments, R2 is N and R1 is -(CH2)n-; in some embodiments, R2 is CH and R1 has one or more methylene units, each independently coated with R. 1a Alternative (CH2) n .

[0018] In Formula I, R3 and R4 are each independently selected from R5, R6, and C. 5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl, C 5-30 One or more methylene units in the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0019] The R5 structure is -Rx-CH(Ry)(Rz);

[0020] Rx in R5 is -(CH2) k The k is selected from an integer from 1 to 10; the Rx terminal is connected to R2;

[0021] Ry and Rz in R5 are each independently selected from C 5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl, C 5-30 One or more methylene units in the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0022] The R6 structure is -Ra-N(Rb)(Rc);

[0023] In R6, Ra is -(CH2). y The y is selected from integers from 1 to 10; the Ra terminal is connected to R2;

[0024] In R6, Rb and Rc are each independently selected from C. 5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl, C 5-30One or more methylene units in the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0025] In a preferred embodiment of the present invention, R1 and R2 are selected from any one of the following groups:

[0026] 1) R1 is -(CH2) n - and R2 is N:

[0027] 2) R1 has the following structure: -(CH2) a -NH-(CH2) b -OP(=O)(OH)-O-(CH2) c -; the structure described (CH2) a The end connects to the parent core, (CH2) c Terminal R2 is connected; R2 is CH;

[0028] 3) R1 has the structure shown in Equation IV:

[0029]

[0030] (CH2) in Formula IV d The end connects to the parent core, (CH2) f Terminal R2 is connected; R2 is CH; R 1b’ As defined above.

[0031] The numbers n, z, a, b, c, d, e, and f are each independently selected from integers 1 to 5. (The sum of a, b, and c ≤ 9, and the sum of d, e, and f ≤ 9.)

[0032] In a further preferred embodiment of the present invention, R1 and R2 are selected from any one of the following groups:

[0033] 1) R1 is -CH2- and R2 is N;

[0034] 2) R1 has the following structure: -(CH2)-NH-(CH2)2-OP(=O)(OH)-O-(CH2)-, and R2 is CH;

[0035] 3) R1 has the structure shown in Equation V, and R2 is CH;

[0036]

[0037] In Equation V, R 1b’ As defined above.

[0038] In a preferred embodiment of the present invention, R3 and R4 are each independently selected from R5, R6, and C.5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl or C 5-30 The alkynyl group can be straight-chain or branched, and the C... 5-30 Alkyl, C 5-30 alkenyl, C 5-30 One or both methylene units of the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0039] The R5 structure is -Rx-CH(Ry)(Rz);

[0040] Rx in R5 is -(CH2) k The k is selected from an integer from 1 to 5; the Rx terminal is connected to R2;

[0041] Ry and Rz in R5 are each independently selected from C 5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl or C 5-30 The alkynyl group can be straight-chain or branched, and the C... 5-30 Alkyl, C 5-30 alkenyl, C 5-30 One or both methylene units of the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0042] The R6 structure is -Ra-N(Rb)(Rc);

[0043] In R6, Ra is -(CH2). y The y is selected from integers from 1 to 5; the Ra terminal is connected to R2;

[0044] In R6, Rb and Rc are each independently selected from C. 5-30 Alkyl, C 5-30 alkenyl or C 5-30 alkynyl group, the C 5-30 Alkyl, C 5-30 alkenyl or C 5-30 The alkynyl group can be straight-chain or branched, and the C... 5-30 Alkyl, C 5-30 alkenyl, C 5-30 One or both methylene units of the alkynyl group are each independently replaced by one or more of the following groups: -O-, -C(O)-,

[0045] In a further preferred embodiment of the present invention, R3 and R4 are each independently selected from R5, R6, and -(CH2). t1 -OC(O)-C 10-25 Alkyl group, -(CH2) t1 -C(O)-C 10-25 Alkyl group, -(CH2) t1 -OC(O)-C 10-25 Alkenyl or -(CH2) t1 -C(O)-C 10-25 Alkenyl; the t1 is independently selected from 0, 1, 2, 3, 4 or 5.

[0046] When R3 and R4 are each independently R5, their Ry and Rz are each independently selected from -(CH2). t1 -OC(O)-C 10-25 Alkyl group, -(CH2) t1 -C(O)-C 10-25 Alkyl group, -(CH2) t1 -OC(O)-C 10-25 Alkenyl or -(CH2) t1 -C(O)-C 10-25 Alkenyl; the t1 is independently selected from 0, 1, 2, 3, 4 or 5.

[0047] When R3 and R4 are each independently R6, their Rb and Rc are each independently selected from -(CH2). t1 -OC(O)-C 10-25 Alkyl group, -(CH2) t1 -C(O)-C 10-25 Alkyl group, -(CH2) t1 -OC(O)-C 10-25 Alkenyl or -(CH2) t1 -C(O)-C 10-25 Alkenyl; the t1 is independently selected from 0, 1, 2, 3, 4 or 5.

[0048] In a further preferred embodiment of the invention, R3 and R4 are each independently R5, R6, or any one of the following groups:

[0049] 1)-(CH2) t1 -OC(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0050] 2)-(CH2) t1-C(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0051] 3)-(CH2) t1 -C(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0052] Its structural formula is

[0053] 4)-(CH2) t1 -OC(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0054] Its structural formula is

[0055] The t1 is independently selected from 0, 1, 2, 3, 4, or 5, and the R 1-A and R 1-B Each independently is C 4-10 Straight-chain alkyl groups.

[0056] When R3 and R4 are each independently R5, their Ry and Rz are each independently selected from any of the following groups:

[0057] 1)-(CH2) t1 -OC(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0058] 2)-(CH2) t1 -C(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0059] 3)-(CH2) t1 -C(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0060] Its structural formula is

[0061] 4)-(CH2) t1 -OC(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0062] Its structural formula is

[0063] The t1 is independently selected from 0, 1, 2, 3, 4, or 5, and the R 1-A and R 1-B Each independently is C 4-10 Straight-chain alkyl groups.

[0064] When R3 and R4 are each independently R6, their Rb and Rc are each independently selected from any of the following groups:

[0065] 1)-(CH2) t1 -OC(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0066] 2)-(CH2) t1 -C(O)-C 10-25 alkenyl, the C 10-25 The alkenyl group is C 10-25 Straight-chain alkenyl; the C 10-25 The number of alkene bonds in a straight-chain alkenyl group can be 1, 2, or 3;

[0067] 3)-(CH2) t1 -C(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0068] Its structural formula is

[0069] 4)-(CH2) t1 -OC(O)-C 10-25 Alkyl, the C 10-25 Alkyl group is C 10-25 Branched alkyl groups;

[0070] Its structural formula is

[0071] The t1 is independently selected from 0, 1, 2, 3, 4, or 5, and the R 1-A and R 1-BEach independently is C 4-10 Straight-chain alkyl groups.

[0072] In certain specific embodiments of the present invention, Rb, Rc, Ry, and Rz are each independently selected from any one of the following groups:

[0073]

[0074]

[0075] In certain embodiments of the present invention, R3 and R4 are each independently selected from any one of the following groups:

[0076]

[0077]

[0078] In certain specific embodiments of the present invention, the 1,3-isophthaloylguanidine derivative compound is selected from any one of the following compounds: BGG1-link1-DOPE, BGG2-link1-DOPE, BGG-link2-OA3, BGG-link2-LA3, BGG-link3-LA3, BGG-link3-HA3, BGG-link4-LA2, BGG-link4-HA2, BGG-link5-LA2, and BGG-link5-HA2;

[0079] The specific structural formulas of each compound are shown in the table below:

[0080]

[0081]

[0082]

[0083] In the most preferred embodiment of the present invention, the 1,3-isophthaloylguanidine derivative compound is BGG-link2-LA3:

[0084]

[0085] The mechanism of action of the compounds in this invention is as follows: 1,3-Isobenzoylguanidine possesses a rigid planar structure, allowing the meta-biguanidin group on the benzene ring to align with the shape of nucleic acid molecules, resulting in high binding energy and effective nucleic acid encapsulation. Furthermore, it interacts with phospholipid molecules in biological membranes, promoting nucleic acid delivery. Based on this, this invention designs 1,3-m-benzoylguanidine as a nitrogen-containing head group in the ionizable lipid molecular backbone, creating a novel ionizable lipid. LNPs prepared from this type of ionizable lipid molecule can achieve efficient expression after intramuscular injection, reducing off-target accumulation in the liver and improving biosafety.

[0086] In a second aspect of the invention, the use of 1,3-isophthaloylguanidine derivatives or pharmaceutically acceptable salts thereof as described in the first aspect of the invention is provided in the preparation of lipid carriers.

[0087] In a third aspect of the invention, a lipid carrier comprising a 1,3-isophthaloylguanidine derivative compound or a pharmaceutically acceptable salt thereof as described in the first aspect of the invention is provided.

[0088] The lipid carrier comprises ionizable lipids, neutral lipids, structured lipids, and PEGylated lipids, wherein the ionizable lipids are selected from the 1,3-isophthaloylguanidine derivatives of this invention. The neutral lipids, structured lipids, and PEGylated lipids can be selected from commonly used components in the art.

[0089] The ionizable lipids are the core functional components of lipid nanoparticles, possessing the ability to encapsulate negatively charged nucleic acid molecules, facilitate cellular uptake, endosome / lysosome escape, and cross the nuclear membrane. The 1,3-isophthaloylguanidine derivative compounds of this invention can serve as a novel type of ionizable lipid, replacing existing ionizable lipids such as ALC-0315 and SM-102 in the preparation of lipid nanoparticles, thereby overcoming the shortcomings of existing ionizable lipids. Lipid nanoparticles or pharmaceutical compositions prepared from the 1,3-isophthaloylguanidine derivative compounds of this invention have the following advantages: high encapsulation efficiency, high transfection efficiency with weak hepatic tropism, good stability, and good cell compatibility.

[0090] The neutral lipids help promote the fusion of intracellular endosome membranes, thereby enhancing the release of nucleic acid molecules. The neutral lipids are selected from one or more of DSPC, DOPE, DPPC, DMPC, DOPC, POPC, DMPE, POPE, or DPPE, preferably DOPE.

[0091] The structural lipids stabilize the bilayer membrane structure of the LNP and include, but are not limited to, one or more of sterols, non-sterols, and their derivatives. The structural lipids are selected from at least one of the following: animal sterols, plant or fungal sterols, for example, the sterols are selected from cholesterol, β-sitosterol, ergosterol, campesterol, brassicosterol, and stigmasterol. Cholesterol is preferred.

[0092] The PEGylated lipids primarily function to improve the stability of LNPs, prolong their circulation time in the blood, and prevent particle aggregation. The PEGylated lipids are selected from at least one of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, such as PEG-modified phosphatidylethanolamine or PEG-modified dialkylamine, or DMG-PEG2000, DSPE-PEG2000, or ALC-0159. ALC-0159 is preferred.

[0093] The ionizable lipids, neutral lipids, structural lipids, and PEGylated lipids in the lipid carrier have a molar ratio of 40-80, 5-25, 20-60, and 0.1-2.5, respectively. Preferably, the molar ratio is 45-60:10-20:30-40:0.5-2.

[0094] In a preferred embodiment of the present invention, the lipid carrier component comprises: BGG-lipid (i.e., the compound described in the first aspect), DOPE, cholesterol, and ALC-0159, wherein the molar ratio of BGG-lipid, DOPE, cholesterol, and ALC-0159 is 47.6:14.3:36.7:1.4.

[0095] In another preferred embodiment of the present invention, the lipid carrier component includes: BGG-link2-LA3, DOPE, cholesterol, and ALC-0159, and the molar ratios of each component are shown in the table below:

[0096] BGG-link2-LA3 DOPE Chol ALC-0519 54.7 10 34.8 0.5

[0097] In a fourth aspect of the invention, the use of the compounds as described in the first aspect or pharmaceutically acceptable salts thereof, or lipid carriers as described in the third aspect, in the preparation of lipid nanoparticles is provided.

[0098] In a fifth aspect of the invention, a lipid nanoparticle carrying a nucleic acid drug is provided, comprising a compound as described in the first aspect or a pharmaceutically acceptable salt thereof, or a lipid carrier as described in the third aspect, and the nucleic acid drug.

[0099] The nucleic acid drug may be selected from one or more of RNA or DNA. The RNA may be selected from one or more of the following: mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, etc. The DNA may be selected from one or more of the following: cDNA, plasmid DNA, viral DNA, PCR product, oligonucleotide, etc. The RNA may be modified RNA. The DNA may be modified DNA.

[0100] In the lipid nanoparticles, the compound as described in the first aspect or its pharmaceutically acceptable salt or the lipid carrier as described in the third aspect is typically in a weight ratio of 5-45:1 to the nucleic acid drug, preferably 10-20:1.

[0101] In one preferred embodiment of the present invention, the lipid nanoparticles carrying nucleic acid drugs include a lipid carrier and mRNA. The lipid carrier component includes: BGG-lipid (i.e., the compound described in the first aspect), DOPE, cholesterol, and ALC-0159, wherein the molar ratio of BGG-lipid, DOPE, cholesterol, and ALC-0159 is 47.6:14.3:36.7:1.4; and the weight ratio of BGG-lipid to mRNA is 14:1.

[0102] In another preferred embodiment of the present invention, the lipid nanoparticles carrying nucleic acid drugs include a lipid carrier and mRNA, wherein the lipid carrier components include: BGG-link2-LA3, DOPE, cholesterol, and ALC-0159, and the molar ratios of each component are shown in the table below:

[0103] BGG-link2-LA3 DOPE Chol ALC-0519 54.7 10 34.8 0.5

[0104] The weight ratio of BGG-link2-LA3 to mRNA is 15:1.

[0105] In a preferred embodiment of the present invention, lipid nanoparticles encapsulating nucleic acid drugs of the present invention are prepared using a microfluidic method, the method comprising the following steps:

[0106] (1) The lipid compound prepared in this invention is dissolved in ethanol in a certain proportion with several other lipid substances to prepare a mixed lipid solution as the alcohol phase;

[0107] (2) Nucleic acid was added to citrate buffer to prepare nucleic acid-citrate buffer, which was used as the aqueous phase;

[0108] (3) Lipid nanoparticles were prepared by microfluidics. The total flow rate of the microfluidics was 4 ml / min and the flow rate ratio was alcohol phase:water phase = 1:3. The initial material was diluted by ultrafiltration and then the ultrafiltration was repeated three times.

[0109] In a sixth aspect of the invention, a pharmaceutical composition capable of delivering nucleic acids is provided, comprising a compound as described in the first aspect, a lipid carrier as described in the third aspect, or lipid nanoparticles encapsulating a nucleic acid drug as described in the fifth aspect.

[0110] In the pharmaceutical composition described herein, lipid nanoparticles prepared using the compound as described in the first aspect or the lipid carrier as described in the third aspect are used as nucleic acid delivery carriers, and the nucleic acid is used as a preventive or therapeutic agent.

[0111] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, including but not limited to fillers, binders, disintegrants, diluents, lubricants, flavorings, or preservatives.

[0112] The pharmaceutical composition described herein can be administered via any of the following methods: intramuscular injection, intravenous injection, skin administration, oral mucosal administration, nasal mucosal administration, pulmonary administration, ocular administration, etc.

[0113] The dosage of the pharmaceutical composition described herein is a therapeutically effective amount. Specific dosing regimens can be determined based on factors such as the type and severity of the disease to be treated, the patient's age, weight, immune status, and route of administration.

[0114] In a seventh aspect of the invention, the use of the compounds described in the first aspect, the lipid carriers described in the second or third aspect, the lipid nanoparticles described in the fourth or fifth aspect, and the pharmaceutical compositions described in the sixth aspect in the preparation of gene vaccines or nucleic acid drugs for the prevention and / or treatment of tumor diseases, infectious diseases, genetic diseases, cardiovascular diseases, or autoimmune diseases is provided.

[0115] The present invention has the following technical effects:

[0116] This invention provides a class of 1,3-isophthaloylguanidine derivatives, which can be used as ionizable lipids to prepare lipid nanoparticles or pharmaceutical compositions. Compared with lipid nanoparticles containing other ionizable lipids, lipid nanoparticles or pharmaceutical compositions prepared from these 1,3-isophthaloylguanidine derivatives have the following advantages: high encapsulation efficiency, high transfection efficiency with weak hepatic tropism, good stability, and good cell compatibility.

[0117] Specifically:

[0118] As can be seen from Table 1 of Example 11 of the present invention, the prepared lipid nanoparticles BGG-LNP have the advantage of high encapsulation efficiency.

[0119] From Embodiment 12 of the present invention Figure 15 , Figure 16As shown in Table 2, the bioluminescent signal of BGG-LNP / mRNA injected intramuscularly was concentrated at the injection site, with no bioluminescent signal observed in the liver. In contrast, ALC-0315LNP / mRNA showed strong bioluminescent signals at both the injection site and in the liver. For intravenous injection, ALC-0315LNP / mRNA exhibited significantly higher transfection efficiency in the liver than BGG-LNP / mRNA, demonstrating stronger hepatic affinity. BGG-LNP / mRNA constructed using BGG-link2-LA3 showed the highest local transfection efficiency after intramuscular injection, but its bioluminescent intensity in the liver after intravenous injection was only 0.38% of that of ALC-0315LNP / mRNA. Therefore, BGG-LNP / mRNA exhibits a weak hepatic affinity.

[0120] As can be seen from Table 4 of Example 13 of the present invention, under storage conditions of 4°C, the particle size of BGG-link2-LA3 / mRNA remained stable, with none exceeding 200 nm and the PDI not exceeding 0.200. This indicates that BGG-link2-LA3 / mRNA preferably exhibits good storage stability at 4°C.

[0121] As can be seen from Table 5 of Example 14 of this invention, within the serum concentration range of 10% to 80%, the cell transfection efficiency of BGG-LNP / mRNA gradually decreases with increasing serum concentration. However, when the serum concentration is below 40%, the transfection efficiency of BGG-LNP / mRNA can still maintain a high level (greater than 10%). 8 RLU / mg protein).

[0122] As can be seen from Table 6 of Example 15 of the present invention: when the drug concentration is two times or less, the BGG-link2-LA3 / mRNA lipid carrier has high safety for HEK 293T cells; when the drug concentration is four times, the BGG-link2-LA3 / mRNA lipid carrier has no significant effect on the viability of HEK 293T cells.

[0123] From Embodiment 16 of the present invention Figure 17 As can be seen, HE section scanning results showed no significant changes in cell morphology or structure in any organ of the BGG-link2-LA3 / mRNA group. Cardiac cells were morphologically normal and spindle-shaped; liver cells were polygonal with clear boundaries; and the boundary between the cortex and medulla of the spleen was obvious. These results indicate that BGG-LNP / mRNA has good organ safety.

[0124] The following is a description of some of the terms used in this invention:

[0125] DSPC: Chemical name is 1,2-distearate-sn-glycerol-3-phosphorylcholine.

[0126] DOPE: Chemical name is 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine.

[0127] DPPC: Chemical name is 1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine.

[0128] DMPC: Chemical name is 1,2-dimyristoyl-sn-glycerol-3-phosphocholine.

[0129] DOPC: Chemical name is 1,2-dioleoyl-sn-glycerol-3-phosphorylcholine.

[0130] POPC: Chemical name is 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphorylcholine.

[0131] DMPE: Chemical name is 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine.

[0132] POPE: Its chemical name is 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine.

[0133] DPPE: Chemical name is 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine. Attached Figure Description

[0134] Figure 1 The compounds are 5-bromomethyl-1,3-isophthalic acid (Compound B) and 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (Compound C). 1 H-NMR and ESI-MS. Where a represents the ¹H-NMR and ESI-MS of Compound B. 1 H-NMR; b is Compound C 1 H-NMR; c is ESI-MS of Compound B; d is ESI-MS of Compound C.

[0135] Figure 2 For BGG1-link1-DOPE and BGG2-link1-DOPE 1 H-NMR and ESI-MS. Where 'a' represents Boc-BGG1-link1-DOPE (left) and Boc-BGG2-link1-DOPE (right). 1 H-NMR. b is ESI-MS of Boc-BGG1-link1-DOPE (top left), Boc-BGG2-link1-DOPE (top right), BGG1-link1-DOPE (bottom left), and BGG2-link1-DOPE (bottom right).

[0136] Figure 3 For Boc-BGG-link2 1 H-NMR and ESI-MS. Where 'a' represents Boc-BGG-link2. 1 H-NMR; b is ESI-MS of Boc-BGG-link2.

[0137] Figure 4 ESI-MS of Boc-BGG-link2-OA3 and BGG-link2-OA3. Where, a is the ESI-MS of Boc-BGG-link2-OA3; b is the ESI-MS of BGG-link2-OA3.

[0138] Figure 5 For Boc-BGG-link2-LA3 and BGG-link2-LA3 1 1H-NMR and ESI-MS. In the image, a represents the 1H-NMR of Boc-BGG-link2-LA3 (left) and BGG-link2-LA3 (right). b represents the ESI-MS of Boc-BGG-link2-LA3 (left) and BGG-link2-LA3 (right).

[0139] Figure 6 For Boc-BGG-link3 1 H-NMR and ESI-MS. Where α represents Boc-BGG-link3. 1 H-NMR, b is ESI-MS of Boc-BGG-link3.

[0140] Figure 7 ESI-MS of Boc-BGG-link3-LA3 and BGG-link3-LA3. Where, a is the ESI-MS of Boc-BGG-link3-LA3 and b is the ESI-MS of BGG-link3-LA3.

[0141] Figure 8 ESI-MS of Boc-BGG-link3-HA3 and BGG-link3-HA3. Where, a is the ESI-MS of Boc-BGG-link3-HA3 and b is the ESI-MS of BGG-link3-HA3.

[0142] Figure 9 For Boc-BGG-link4 1 H-NMR and ESI-MS. Where α represents Boc-BGG-link4. 1 H-NMR, b is ESI-MS of Boc-BGG-link4.

[0143] Figure 10 ESI-MS of Boc-BGG-link4-LA2 and BGG-link4-LA2. Where, a is the ESI-MS of Boc-BGG-link4-LA2 and b is the ESI-MS of BGG-link4-LA2.

[0144] Figure 11 ESI-MS of Boc-BGG-link4-HA2 and BGG-link4-HA2. Where, a is the ESI-MS of Boc-BGG-link4-HA2 and b is the ESI-MS of BGG-link4-HA2.

[0145] Figure 12 For Boc-BGG-link5 1 H-NMR and ESI-MS. Where α represents Boc-BGG-link5. 1 H-NMR, b is ESI-MS of Boc-BGG-link5.

[0146] Figure 13 ESI-MS of Boc-BGG-link5-LA2 and BGG-link5-LA2. Where, a is the ESI-MS of Boc-BGG-link5-LA2 and b is the ESI-MS of BGG-link5-LA2.

[0147] Figure 14 ESI-MS of Boc-BGG-link5-HA2 and BGG-link5-HA2. Where, a is the ESI-MS of Boc-BGG-link5-HA2 and b is the ESI-MS of BGG-link5-HA2.

[0148] Figure 15 The transfection efficiency of LNP intramuscular injection in each group was calculated.

[0149] Figure 16 The transfection efficiency of LNP intravenous injection in each group was calculated.

[0150] Figure 17 For histopathological sections. Detailed Implementation

[0151] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0152] The mRNA used in these embodiments of the invention is firefly luciferase mRNA, purchased from APE×Bio.

[0153] In the embodiments of this invention, all raw materials used for synthesis were purchased from TCI reagents.

[0154] Example 1: Synthesis of 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine

[0155] Synthetic route of 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine:

[0156]

[0157] Dimethyl 5-bromomethyl-1,3-isophthalic acid (8.0 g, 27.97 mmol) was added to 100 mL of glacial acetic acid, followed by the slow addition of 100 mL of 40% hydrobromic acid. The mixture was refluxed at 120 °C for 12 h. The reaction system was cooled to room temperature, and the mixture was added dropwise to 1000 mL of ice water with thorough stirring. The solid was collected by filtration under reduced pressure, and water was added to suspend it. After freeze-drying, a white solid of 5-bromomethyl-1,3-isophthalic acid (5.6 g, 21.71 mmol) was obtained, with a yield of 77.6%. 1 H-NMR (400MHz, DMSO-d6)δ=13.29(2H,s,-COOH),8.39(1H,s,Ph-H),8.24(2H,s,Ph-H),4.86(2H,s,-CH2Br)ppm.ESI-MS(m / z)259.18[MH] - .

[0158] 5-Bromomethyl-1,3-isophthalic acid (1.0 g, 3.86 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (5.9 g, 15.44 mmol) were dissolved in 20 mL of tetrahydrofuran. N,N-diisopropylethylamine (2.7 mL, 15.44 mmol) was added dropwise under ice bath conditions, and the reaction was continued under ice bath conditions for 30 min. 1-(tert-Butoxycarbonyl)guanidine (2.0 g, 12.56 mmol) was dissolved in 20 mL of a mixed solution of tetrahydrofuran and dichloromethane (1:1, v / v). This solution was added dropwise to the above reaction system under ice bath conditions for 5 min, and the reaction was continued at room temperature for 3.5 h. The organic solvent was removed by rotary evaporation, yielding an orange-yellow viscous liquid. The liquid was extracted with ethyl acetate, and the upper organic phase was collected. An appropriate amount of anhydrous magnesium sulfate solid was added, and the mixture was dried overnight. The crude product was purified by silica gel column chromatography (200 mesh) using petroleum ether:ethyl acetate = 4:1 + 2% triethylamine, v / v, to give a white solid 5-bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (478.5 mg, 0.89 mmol), with a yield of 23.0%. 1 H-NMR (400MHz, DMSO-d6)δ=11.02(2H,s,-NH-),9.68(2H,s,=NH),8.72(1H,s,Ph-H),8.63(2H,m,Ph -H),8.30(2H,s,-NH-),4.77(2H,s,-CH2Br),1.48(18H,s,Boc)ppm.ESI-MS(m / z)341.15[M-2Boc+H] + 440.95[M-Boc+H] + 540.93 [M+H] + . 1 H-NMR and ESI-MS are shown below Figure 1 .

[0159] Example 2: Synthesis of BGG1-link1-DOPE and BGG2-link1-DOPE

[0160] Synthetic routes of BGG1-link1-DOPE and BGG2-link1-DOPE:

[0161]

[0162] 5-Bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (100.0 mg, 0.19 mmol) and 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine (138 mg, 0.19 mmol) were dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was slowly added dropwise with stirring, and the mixture was refluxed at 40 °C. The reaction was monitored by thin-layer chromatography under a 254 nm UV lamp (developing solvent: cyclohexane:ethyl acetate = 2:1, v / v) until the starting material was completely reacted. The reaction system was purified by preparative thin-layer chromatography (developing solvent: chloroform:methanol:n-butanol:ammonia = 15:1:0.5:0.5%, v / v). The positions of the Boc-BGG1-link1-DOPE and Boc-BGG2-link1-DOPE silica gel bands were determined using Dittmer-Lester molybdenum blue reagent. The corresponding silica gel bands of Boc-BGG1-link1-DOPE and Boc-BGG2-link1-DOPE were scraped off, crushed, and eluted (eluent:dichloroform:methanol = 5:1, v / v). The organic solvent was removed by rotary evaporation to obtain yellow oily products Boc-BGG1-link1-DOPE and Boc-BGG2-link1-DOPE. 1 mL of 50% trifluoroacetic acid in dichloroform solution was added dropwise to each product under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 using saturated sodium carbonate solution. Extraction with ethyl acetate was performed, and the upper organic phase was collected. An appropriate amount of anhydrous magnesium sulfate solid was added and the mixture was dried overnight. The organic solvent was removed by rotary evaporation to obtain yellow solids BGG1-link1-DOPE (73.3 mg, 0.07 mmol) and BGG2-link1-DOPE (109.5 mg, 0.09 mmol), with yields of 38.4% and 45.6%, respectively. Boc-BGG1-link1-DOPE: 1H NMR(400MHz,DMSO-d6)δ=11.25(2H,s,-NH-),9.76(2H,s,-NH-),8.80(1H,s,Ph-H),8.60(2H,s,=NH),8.36(2H,s,Ph-H),5.76(1H,s,-OH),5.33(2H,d,J=5.2Hz,-OCH2CH2-),5.30(2H,d,J=4.8Hz,-OCH2CH2-),5.10-5.06(1H,m,-OCH2-),4.32-4.24(4H,m,-CH=CH-),3.96-3.92(2H,m,-CH=CH-),3.40-3.35(2H,m,-CH=CH-),3.81(2H,t,J=5.6Hz,-CH2N-),3.65-3.58(10H,m,-CH2CH2CH2-),3.17-3.10(12H,m,-CH2CH2CH2-),2.26-2.21(4H,m,-CH2CH2CH2-),1.99-1.94(10H,m,-CH2CH2CH2-),1.48(18H,s,-Boc),1.29-1.23(20H,m,-CH2CH2CH2-)ppm.ESI-MS(m / z)1204.75[M+H] + .BGG1-link1-DOPE:ESI-MS(m / z)1004.73[M+H] + 。Boc-BGG2-link1-DOPE: 1 HNMR(400MHz,DMSO-d6)δ=11.34(4H,s,-NH-),9.68(4H,s,-NH-),8.66(2H,s,Ph-H),8.58(4H,s,=NH),8.32(4H,s,Ph-H),5.76(1H,s,-OH),5.28(4H,s,-NCH2-),5.05(1H,m,-OCH2-),4.32-3.84(12H,m,-OCH2CH2-,-NCH2C-,-COCH2-),3.39(4H,t,J=6.0Hz,-COCH2-),2.19-2.16(4H,m,-CH=CH-),1.96-1.94(4H,m,-CH=CH-),1.48(36H,s,-Boc),1.33-1.18(50H,m,-CH2CH2CH2CH3)ppm.ESI-MS(m / z)633.22[M-4Boc+2H] 2+ ,1664.63[M+H] +. BGG2-link1-DOPE:ESI-MS(m / z)422.68[M+3H] 3+ 633.24[M+2H] 2+ 964.03[M-3Boc+H] + 1264.71 [M+H] +1 . 1 H-NMR and ESI-MS are shown below Figure 2 .

[0163] Example 3: Synthesis of BGG-link2-OA3 The synthetic route of BGG-link2-OA3 is as follows:

[0164]

[0165] 5-Bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (100.0 mg, 0.19 mmol) and 1-amino-2,3-propanediol (25 μL, 0.33 mmol) were dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was slowly added dropwise with stirring, and the reaction was refluxed at 40 °C. The reaction progress was monitored by thin-layer chromatography. The reaction system was purified by preparative thin-layer chromatography (developing solvent: chloroform:methanol:n-butanol:ammonia = 10:1:0.5:0.5%, v / v). The corresponding silica gel band of Boc-BGG-link2 was scraped off and eluted (eluting solvent:dichloroform:methanol = 5:1, v / v). The organic solvent was removed by rotary evaporation to give a yellow oily product Boc-BGG-link2 (72.3 mg, 0.13 mmol), with a yield of 69.1%. 1 H NMR(400MHz,DMSO-d6)δ=11.03(2H,s,-NH-),9.62(2H,s,-NH-),8.66(1H,d,J=1.7Hz,Ph-H),8.59 (2H,s,=NH),8.21(2H,d,J=1.7Hz,Ph-H),4.61(1H,s,-OH),4.31(1H,t,J=5.2Hz,-NH-),3.78(2H,s ,PhCH2-),3.57(1H,s,-OH),3.40-3.36(3H,m,-CHOH,-CH2OH),2.61(1H,dd,J=11.8,4.3Hz,-NHCH2 -),2.45(1H,dd,J=11.8,7.4Hz,-NHCH2-),1.48(18H,s,-Boc)ppm.ESI-MS(m / z)352.33[M-2Boc+H] + 452.28[M-Boc+H] +552.27 [M+H] + . 1 H-NMR and ESI-MS are shown below Figure 3 .

[0166] Boc-BGG-link2 (96.0 mg, 0.17 mmol) was dissolved in 2 mL of dichloromethane. Oleic acid (55 μL, 0.17 mmol), N,N-dicyclohexylcarbodiimide (86 mg, 0.42 mmol), and 4-dimethylaminopyridine (6 mg, 0.05 mmol) were added with stirring, and the reaction was carried out at room temperature for 6 h. 55 μL of oleic acid was added to the above reaction system, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the solution was reconstituted in cold dichloromethane. After filtering through a 0.22 μm filter, the solution was purified by preparative thin-layer chromatography (developing solvent: dichloromethane: methanol: ammonia = 40:1:0.5%, v / v). The corresponding silica gel band of Boc-BGG-link2-OA3 was scraped off and eluted (eluting solvent: dichloromethane: methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link2-OA3. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to give a yellow solid BGG-link2-OA3 (85.0 mg, 0.07 mmol), with a yield of 43.7%. ESI-MS: 573.29 [M + 2H] 2+ 1144.94 [M+H] + ESI-MS (see) Figure 4 .

[0167] Example 4: Synthesis of BGG-link2-LA3

[0168] Synthetic route of BGG-link2-LA3:

[0169]

[0170] Boc-BGG-link2 (70.0 mg, 0.13 mmol) was dissolved in 2 mL of dichloromethane. Linoleic acid (40 μL, 0.13 mmol), N,N-dicyclohexylcarbodiimide (63 mg, 0.31 mmol), and 4-dimethylaminopyridine (5 mg, 0.04 mmol) were added with stirring. The mixture was reacted at room temperature for 6 h under N2 protection. 40 μL of linoleic acid was added to the reaction mixture, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the mixture was reconstituted in cold dichloromethane. After filtering through a 0.22 μm filter, the mixture was purified by preparative thin-layer chromatography (developing solvent: dichloromethane: methanol: ammonia = 60:1:0.5%, v / v). The corresponding silica gel band of Boc-BGG-link2-LA3 was scraped off and eluted (eluting solvent: dichloromethane: methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link2-LA3. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to obtain a yellow solid BGG-link2-LA3 (71.3 mg, 0.06 mmol), with a yield of 48.2%. Boc-BGG-link2-LA3: 1 H NMR (400MHz, DMSO-d6)δ=11.02(2H,s,-NH-),9.64(2H,s,-NH-),8.73(1H,s,Ph-H),8.56(2H,s,=NH), 8.01(2H,s,Ph-H),5.76(2H,s,Ph-CH2N-),5.33-5.25(10H,m,-NCH2CHCH2O-,-COCH2-),4.73-4.61(1H ,m,-NHCH2CHCH2-),2.73-2.70(6H,m,-CH=CH-),2.27-2.19(6H,m,-CH=CH-),2.00-1.99(16H,m,-CH2C H2CH2CH2-),1.42(18H,s,-Boc),1.28-1.23(60H,m,-CH2CH2CH2CH2-)ppm; ESI-MS(m / z)1338.48[M+H] + .BGG-link2-LA3: 1HNMR (400MHz, DMSO-d6)δ=8.67(2H,s,-NH-),8.61(2H,s,-NH-),7.93(1H,s,Ph-H),7.91(2H,s,Ph-H),5.33-5.25(10H,m,-NCH2CH CH2O-,-COCH2-),4.73-4.62(1H,m,-NHCH2CHCH2-),3.52(2H,s,Ph-CH2N-),2.73-2.71(6H,m,-CH=CH-),2.27-2.22(6H, m,-CH=CH-),2.00-1.99(16H,m,-CH2CH2CH2CH2-),1.28-1.17(60H,m,-CH2CH2CH2CH2-)ppm.ESI-MS(m / z)1138.85[M+H] + 1156.84[M+H2O+H] + 1174.88[M+2H2O+H] + 570.26 [M+2H] 2+ . 1 H-NMR and ESI-MS are shown below Figure 5 .

[0171] Example 5: Synthesis of BGG-link3-LA3

[0172] Synthetic route of BGG-link3-LA3:

[0173]

[0174] 5-Bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (100 mg, 0.19 mmol) and N-3-(aminopropyl)diethanolamine (25 μL, 0.33 mmol) were dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was slowly added dropwise with stirring. The reaction was refluxed at 40 °C, and the reaction progress was monitored by thin-layer chromatography. The reaction system was purified by preparative thin-layer chromatography (developing solvent: chloroform:methanol:n-butanol:ammonia = 10:1:0.5:1%, v / v). The corresponding silica gel band of Boc-BGG-link3 was scraped off and eluted (eluting solvent: dichloroform:methanol = 5:1, v / v). The organic solvent was removed by rotary evaporation to give a yellow oily product Boc-BGG-link3 (79.3 mg, 0.13 mmol), with a yield of 67.1%. 1HNMR(400MHz,DMSO-d6)δ=10.92(2H,s,-NH-),9.62(2H,s,-NH-),8.85(1H,s,Ph-H),8. 64(2H,s,=NH),8.29(2H,s,Ph-H),5.75(2H,s,-OH),4.14(2H,s,PhCH2-),3.53(4H,t,J= 5.6Hz,-CH2OH),3.37(2H,t,J=6.4Hz,-CH2N-),3.00(2H,t,J=6.0Hz,-NHCH2-),2.73-2 .64(6H,m,-NHCH2CH2-,-NCH2),1.49(18H,s,-Boc)ppm.ESI-MS(m / z)423.41[M-2Boc+H] + 523.40[M-Boc+H] + 623.44 [M+H] + . 1 H-NMR and ESI-MS are shown below Figure 6 .

[0175] Boc-BGG-link3 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and linoleic acid (15 μL, 0.05 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), and N,N-dicyclohexylcarbodiimide (24 mg, 0.12 mmol) were added. The mixture was reacted under N2 protection at room temperature for 6 h. 15 μL of linoleic acid was then added to the reaction mixture, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the mixture was reconstituted in cold dichloromethane. After filtering through a 0.22 μm filter, the mixture was purified by preparative thin-layer chromatography (developing solvent: dichloromethane:methanol = 40:1, v / v). The corresponding silica gel band of Boc-BGG-link3-LA3 was scraped off and eluted (eluting solvent: dichloromethane:methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link3-LA3. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to give a yellow solid BGG-link3-LA3 (22.9 mg, 0.02 mmol), with a yield of 39.4%. Boc-BGG-link3-LA3: ESI-MS (m / z) 605.80 [M-2Boc+2H] 2+ 1409.58 [M+H] + . BGG-link3-LA3:ESI-MS(m / z)605.83[M+2H] 2+,1210.10[M+H] + ESI-MS (see) Figure 7 .

[0176] Example 6: Synthesis of BGG-link3-HA3

[0177] Synthetic route of BGG-link3-HA3:

[0178]

[0179] Boc-BGG-link3 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and 2-hexyldecanoic acid (15 μL, 0.05 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), and N,N-dicyclohexylcarbodiimide (24 mg, 0.12 mmol) were added. The reaction was carried out at room temperature for 6 h. 15 μL of 2-hexyldecanoic acid was added to the above reaction system, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the solution was redissolved in cold dichloromethane. After filtering through a 0.22 μm filter membrane, the solution was purified by preparative thin-layer chromatography (developing solvent: dichloromethane:methanol = 20:1, v / v). The corresponding silica gel band of Boc-BGG-link3-HA3 was scraped off and eluted (eluting solvent: dichloromethane:methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link3-HA3. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to obtain a yellow solid BGG-link3-HA3 (23.3 mg, 0.02 mmol), with a yield of 40.9%. Boc-BGG-link3-HA3: ESI-MS (m / z) 569.76 [M-2Boc+2H] 2+ 681.83[M+Na+H] 2+ 1337.77 [M+H] + . BGG-link3-HA3:ESI-MS(m / z)569.78[M+2H] 2+ 1137.97 [M+H] + ESI-MS (see) Figure 8 .

[0180] Example 7: Synthesis of BGG-link4-LA2

[0181] Synthetic route of BGG-link4-LA2:

[0182]

[0183] 5-Bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (100 mg, 0.19 mmol) and 3,3'-iminobis-1-propanol (50 μL, 0.37 mmol) were dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was slowly added dropwise with stirring. The reaction was refluxed at 40 °C, and the reaction progress was monitored by thin-layer chromatography. The reaction system was purified by chromatographic separation using a 200-mesh silica gel column (eluent: chloroform:methanol:n-butanol:ammonia = 15:1:0.5:0.5%, v / v). The organic solvent was removed by rotary evaporation to give a yellow oily product, Boc-BGG-link4 (94.3 mg, 0.16 mmol), in 83.7% yield. 1 H NMR (400MHz, CDCl3) δ = 8.74 (1H, s, Ph-H), 8.63 (2H, s, = NH), 8.10 (2H, s, Ph-H), 3.70 (4H, t, J = 5.2Hz, -CH2OH) ,3.66(2H,s,PhCH2-),2.60(4H,t,J=6.0Hz,-NCH2-),1.75-1.73(4H,t,-NCH2CH2-),1.38(18H,s,-Boc)ppm. ESI-MS(m / z)394.34[M-2Boc+H] + 494.32[M-Boc+H] + 594.27 [M+H] + . 1 H-NMR and ESI-MS are shown below Figure 9 .

[0184] Boc-BGG-link4 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and linoleic acid (15 μL, 0.05 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), and N,N-dicyclohexylcarbodiimide (25 mg, 0.12 mmol) were added. The mixture was reacted under N2 protection at room temperature for 6 h. 15 μL of linoleic acid was then added to the reaction mixture, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the mixture was reconstituted in cold dichloromethane. After filtering through a 0.22 μm filter, the mixture was purified by preparative thin-layer chromatography (developing solvent: cyclohexane: ethyl acetate: triethylamine = 3:1:1.5%, v / v). The corresponding silica gel band of Boc-BGG-link4-LA2 was scraped off and eluted (eluting solvent: ethyl acetate). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link4-LA2. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to give a yellow solid BGG-link4-LA2 (27.0 mg, 0.03 mmol), with a yield of 58.9%. Boc-BGG-link4-LA2: ESI-MS (m / z) 460.16 [M-2Boc+2H] 2+ 1018.76[M-Boc+H] + 1118.61[M+H] + . BGG-link4-LA2:ESI-MS(m / z)460.14[M+2H] 2+ 918.74 [M+H] + ESI-MS (see) Figure 10 .

[0185] Example 8: Synthesis of BGG-link4-HA2

[0186] Synthetic route of BGG-link4-HA2:

[0187]

[0188] Boc-BGG-link4 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and 2-hexyldecanoic acid (15 μL, 0.05 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), and N,N-dicyclohexylcarbodiimide (25 mg, 0.12 mmol) were added. The reaction was carried out at room temperature for 6 h. 15 μL of 2-hexyldecanoic acid was added to the above reaction system, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the solution was redissolved in cold dichloromethane. After filtering through a 0.22 μm filter membrane, the solution was purified by preparative thin-layer chromatography (developing solvent: cyclohexane:ethyl acetate = 4:1, v / v). The corresponding silica gel band of Boc-BGG-link4-HA2 was scraped off and eluted (eluting solvent: ethyl acetate). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link4-HA2. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to give a yellow solid BGG-link4-HA2 (20.7 mg, 0.02 mmol), with a yield of 47.5%. Boc-BGG-link4-HA2: ESI-MS (m / z) 970.79 [M-Boc+H] + 1070.64 [M+H] + . BGG-link4-HA2:ESI-MS(m / z)436.18[M+2H] 2+ 870.73 [M+H] + ESI-MS (see) Figure 11 .

[0189] Example 9: Synthesis of BGG-link5-LA2

[0190] Synthetic route of BGG-link5-LA2:

[0191]

[0192] 5-Bromomethyl-N,N'-di-tert-butoxycarbonyl-1,3-isophthaloylguanidine (100 mg, 0.19 mmol) and 4,4'-iminobis-1-butanol (45 μL, 0.28 mmol) were dissolved in 2 mL of N,N-dimethylformamide. N,N-diisopropylethylamine (322 μL, 1.85 mmol) was slowly added dropwise with stirring. The reaction was refluxed at 40 °C, and the reaction progress was monitored by thin-layer chromatography. The reaction system was purified by chromatographic separation using a 200-mesh silica gel column (eluent: chloroform:methanol:n-butanol:ammonia = 15:1:0.5:0.5%, v / v). The organic solvent was removed by rotary evaporation to give a yellow oily product, Boc-BGG-link5 (102.3 mg, 0.16 mmol), in 86.6% yield. 1 H NMR(400MHz, CDCl3)δ=8.79(1H,s,Ph-H),8.61(2H,s,=NH),8.14(2H,s,Ph-H),3.71(2H,s,PhCH2-),3.55(4H,t,J=5.2Hz,-CH2OH),2.45 (4H,t,J=6.0Hz,-NCH2-),1.64(4H,t,-NCH2CH2CH2-),1.55(4H,t,-NCH2CH2-),1.36(18H,s,-Boc)ppm.ESI-MS(m / z)422.31[M-2Boc+H] + 522.25[M-Boc+H] + 622.21[M+H] + . 1 H NMR and ESI-MS are shown in the figure. Figure 12 .

[0193] Boc-BGG-link5 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and linoleic acid (15 μL, 0.05 mmol), 4-dimethylaminopyridine (2 mg, 0.02 mmol), and N,N-dicyclohexylcarbodiimide (25 mg, 0.12 mmol) were added. The mixture was reacted under N2 protection at room temperature for 6 h. 15 μL of linoleic acid was added to the above reaction system, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the mixture was reconstituted in cold dichloromethane. After filtering through a 0.22 μm filter membrane, the mixture was purified by preparative thin-layer chromatography (developing solvent: dichloromethane: methanol = 30:1, v / v). The corresponding silica gel band of Boc-BGG-link5-LA2 was scraped off and eluted (eluting solvent: DCM: methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link5-LA2. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to give a yellow solid BGG-link5-LA2 (25.4 mg, 0.03 mmol), with a yield of 53.7%. Boc-BGG-link5-LA2: ESI-MS (m / z) 474.10 [M-2Boc+2H] + 1046.61[M-Boc+H] + 1146.47 [M+H] + .BGG-link5-LA2:ESI-MS(m / z)474.14[M+2H] 2+ 946.69 [M+H] + ESI-MS (see) Figure 13 .

[0194] Example 10: Synthesis of BGG-link5-HA2

[0195] Synthetic route of BGG-link5-HA2:

[0196]

[0197] Boc-BGG-link5 (30 mg, 0.05 mmol) was dissolved in 2 mL of dichloromethane, and 2-hexyldecanoic acid (15 μL, 0.05 mmol), DMAP (2 mg, 0.02 mmol), and DCC (24 mg, 0.12 mmol) were added. The reaction was carried out at room temperature for 6 h. 15 μL of 2-hexyldecanoic acid was added to the above reaction system, and the reaction was continued at room temperature for another 6 h. The organic solvent was removed by rotary evaporation, and the solution was redissolved in cold dichloromethane. After filtering through a 0.22 μm filter membrane, the solution was purified by preparative thin-layer chromatography (developing solvent: dichloromethane:methanol = 30:1, v / v). The corresponding silica gel band of Boc-BGG-link5-HA2 was scraped off and eluted (eluting solvent: dichloromethane:methanol = 10:1, v / v). The organic solvent was removed by rotary evaporation to obtain a yellow solid, Boc-BGG-link5-HA2. 1 mL of a 50% trifluoroacetic acid solution in dichloromethane was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 6 h. The pH of the reaction system was adjusted to 8–9 with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The upper organic phase was collected, and an appropriate amount of anhydrous magnesium sulfate was added and dried overnight to obtain a yellow solid BGG-link5-HA2 (23.0 mg, 0.03 mmol), with a yield of 51.2%. Boc-BGG-link5-HA2: ESI-MS (m / z) 450.16 [M-2Boc+2H] + 998.68[M-Boc+H] + 1098.57 [M+H] + . BGG-link5-HA2:ESI-MS(m / z)450.15[M+2H] 2+ 898.72 [M+H] + ESI-MS (see) Figure 14 .

[0198] Example 11: Preparation and characterization of BGG-LNP / mRNA lipid nanoparticles

[0199] The synthesized BGG-lipids: BGG1-link1-DOPE and BGG2-link1-DOPE (Example 2), BGG-link2-OA3 (Example 3), BGG-link2-LA3 (Example 4), BGG-link3-LA3 (Example 5), BGG-link3-HA3 (Example 6), BGG-link4-LA2 (Example 7), BGG-link4-HA2 (Example 8), BGG-link5-LA2 (Example 9), and BGG-link5-HA2 (Example 10) were dissolved in anhydrous ethanol with DOPE, cholesterol, and ALC-0159 in a molar ratio of BGG-lipids:DOPE:Chol:ALC-0159 = 47.6:14.3:36.7:1.4 to prepare a mixed lipid solution with a total lipid concentration of 6240 nmol / ml. Positive control ALC-0315 was dissolved in anhydrous ethanol at a ratio of ALC-0315:DSPC:cholesterol:ALC-0159 = 46.3:9.4:42.7:1.6 to prepare a mixed lipid solution (i.e., the ethanol phase) with a total lipid concentration of 6240 nmol / ml. BGG-LNP / mRNA was prepared as a Fluci mRNA solution (luciferase mRNA) with a weight ratio of ionizable lipids (i.e., the compounds in the above examples) to mRNA of 14:1 using 50 mM citrate buffer at pH 3. Positive control Fluci mRNA solution (i.e., the aqueous phase) was prepared at a weight ratio of ALC-0315 to mRNA of 14:1. Lipid nanoparticles were prepared using microfluidics at a total flow rate of 4 ml / min and a flow rate ratio of ethanol phase:aqueous phase = 1:3, with a preparation volume of approximately 400 μl. The lipid nanoparticles were then ultrafiltered using a 100 kDa ultrafiltration tube. They were diluted 10-fold with 1×PBS (0.01 M) and ultrafiltered at 2300 g for 10 min. This process was repeated three times to obtain lipid nanoparticles with a buffer solution of 1×PBS. Thermo Fisher's Quant-iT... TM RiboGreen TM The LNP encapsulation efficiency was determined using the kit. Particle size and zeta potential were measured using a Malvern NanoZS particle size analyzer. The results are shown in Table 1.

[0200] Table 1

[0201] Size / nm PDI Zeta / mV EE / % BGG1-link1-DOPE 131.6 0.136 5.5 84.02 BGG2-link1-DOPE 126.7 0.136 -12.3 73.35 BGG-link2-OA3 123.0 0.172 5.5 65.67 BGG-link2-LA3 120.5 0.142 2.2 80.12 BGG-link3-LA3 81.8 0.121 3.4 86.52 BGG-link3-HA3 84.1 0.073 1.0 67.18 BGG-link4-LA2 86.5 0.105 8.4 74.72 BGG-link5-LA2 72.0 0.097 5.4 85.95 BGG-link5-HA2 98.8 0.074 -0.1 75.62

[0202] Example 12: In vivo transfection effect of BGG-LNP / mRNA lipid nanoparticles

[0203] Following the method described in Example 11, the prepared lipid nanoparticles were formulated into 50 μl solutions containing 1 μg mRNA using 1×PBS. These solutions were then administered via intramuscular injection or tail vein injection to the hind legs of ICR mice (BikKai, male, 20-25g, 3 mice per group). Six hours post-injection, each ICR mouse was intraperitoneally injected with 100 μl of 15 mg / ml D-Luciferin. Ten minutes later, the expression of luciferase in the mice was captured using a small animal in vivo imaging system. Results are shown below. Figure 15 , Figure 16 See Tables 2 and 3.

[0204] Table 2

[0205]

[0206]

[0207] Table 3

[0208] BGG-lipid I.V. Liver Total Flux [p / s] BGG1-link1-DOPE 5.11E+06 BGG2-link1-DOPE 1.08E+06 BGG-link2-OA3 4.73E+06 BGG-link2-LA3 4.67E+07 BGG-link3-LA3 5.45E+07 BGG-link3-HA3 5.68E+07 BGG-link4-LA2 8.62E+06 BGG-link4-HA2 6.50E+07 BGG-link5-LA2 8.42E+06 BGG-link5-HA2 1.30E+07 ALC-0315 1.24E+10

[0209] like Figure 15 As shown in Table 2, for intramuscular injection, the total bioluminescence intensity at the injection site of 6 BGG-LNP / mRNAs exceeded 10. 7 p / s represent BGG-LNP / mRNAs constructed using BGG1-link1-DOPE, BGG-link2-LA3, BGG-link3-LA3, BGG-link4-LA2, BGG-link4-HA2, and BGG-link5-LA2, respectively. Among these, the BGG-LNP / mRNA constructed using BGG-link2-LA3 showed the highest intramuscular transfection efficiency. The bioluminescent signal of intramuscularly injected BGG-LNP / mRNA was concentrated at the injection site; no bioluminescent signal was observed in the liver, while ALC-0315LNP / mRNA showed strong bioluminescent signals at both the injection site and in the liver. Figure 16 As shown in Table 3, intravenous injection of ALC-0315LNP / mRNA significantly enhanced liver transfection efficiency compared to BGG-LNP / mRNA, demonstrating stronger hepatic affinity. While intramuscular injection of BGG-LNP / mRNA constructed using BGG-link2-LA3 showed the highest local transfection efficiency, intravenous injection resulted in only 0.38% of the bioluminescence intensity of ALC-0315LNP / mRNA in the liver. Therefore, BGG-LNP / mRNA exhibits a weaker hepatic affinity.

[0210] Example 13: Storage stability of BGG-link2-LA3 / mRNA lipid nanoparticles

[0211] Following the method described in Example 11 and the results described in Example 12, BGG-link2-LA3 / mRNA lipid nanoparticles were prepared using the formulation shown in Table 4. The BGG-link2-LA3 / mRNA lipid nanoparticles were placed at 4°C, and the changes in LNP particle size and PDI were examined on days 0, 2, 4, 6, 8, 10, 12, and 14, with the preparation day designated as day 0. The results are shown in Table 5.

[0212] Table 4

[0213]

[0214] In Table 4, the weight ratio is the weight ratio of BGG-link2-LA3 to mRNA.

[0215] Table 5

[0216]

[0217]

[0218] As shown in Table 5, under storage conditions of 4℃, the particle size of BGG-link2-LA3 / mRNA remained stable, all exceeding 200 nm, and the PDI did not exceed 0.200. This indicates that the preferred BGG-link2-LA3 / mRNA exhibits good storage stability at 4℃.

[0219] Example 14: Serum stability of BGG-link2-LA3 / mRNA lipid nanoparticles

[0220] The transfection efficiency of BGG-link2-LA3 / mRNA in HEK293t cells under different serum concentrations was compared in Example 13. HEK293t cells were counted and diluted to 8 × 10⁶ cells / mL in DMEM medium (complete medium) containing 10% FBS and 1% penicillin-streptomycin solution. 4Cell suspension was added to 96-well plates at a rate of 150 μL per well. After cell adhesion, the original culture medium was removed, and BGG-link2-LA3 / mRNA was added at a dose of 0.5 μg mRNA / well (50 μl lipid nanoparticles contain 0.5 μg mRNA). DMEM medium containing 10%, 20%, 40%, 60%, and 80% FBS was added to a final volume of 150 μL, respectively. Cells were cultured at 37°C and 5% CO2 for 12 h. The original culture medium was removed from each group, and each well was rinsed with 100 μL of 1×PBS, followed by 100 μL of cell lysis buffer. Cells were incubated on ice for 10 min, and the cells were thoroughly lysed by pipetting. The lysates were centrifuged at 15000g for 2 min at 4°C, and the supernatant was collected. The total protein concentration in the supernatant was determined using the BCA method. Add 20 μL of supernatant from each group to a black 96-well plate. Add 100 μL of firefly luciferase assay reagent to each well every 10 seconds, and measure the relative light unit (RLU) using a microplate reader. The final cell transfection efficiency of LNP in each group is expressed as total protein in milligrams (RLU / mg protein). The results are shown in Table 6.

[0221] Table 6

[0222] Serum concentration Total protein content (RLU / mg protein) 10％ 3.08E+08 20％ 2.28E+08 40％ 1.37E+08 60％ 7.70E+07 80％ 4.92E+07

[0223] Table 6 shows that within the serum concentration range of 10%–80%, the cell transfection efficiency of BGG-LNP / mRNA gradually decreased with increasing serum concentration. However, when the serum concentration was below 40%, the transfection efficiency of BGG-LNP / mRNA remained at a relatively high level (greater than 10%). 8 RLU / mg protein).

[0224] Example 15: Cell compatibility of BGG-link2-LA3 / mRNA lipid nanoparticles

[0225] The cytocompatibility of BGG-link2-LA3 / mRNA lipid nanoparticles in Example 13 was determined. HEK293T cells were plated and cultured as described in Example 14. The cytocompatibility of the LNP lipid carrier was examined using the CCK-8 assay. After cell adhesion, the original culture medium was removed, and complete culture medium containing BGG-link2-LA3 / mRNA at final concentrations (total lipid concentration) of 0.8 mM, 0.4 mM, 0.2 mM, 0.1 mM, and 0.05 mM was added, respectively. A negative control group with cells and complete culture medium, and a blank control group without cells and complete culture medium were also set up. After culturing at 37°C and 5% CO2 for 6 h, the medium was replaced with fresh complete culture medium, and culturing continued for 48 h. The original culture medium was removed, and 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well. The cells were incubated at 37°C and 5% CO2 for 1 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula. The results are shown in Table 7.

[0226]

[0227] Table 7

[0228] Total lipid concentration / mM Cell viability % 0.05 97 0.1 104 0.2 94 0.4 92 0.8 79

[0229] As shown in Table 7, when the total lipid concentration was below 0.4 mM (i.e., twice or less of the administered concentration), cell viability remained above 90%; when the total lipid concentration was 0.8 mM (i.e., four times the administered concentration), cell viability was 79.2%. These results indicate that the BGG-link2-LA3 / mRNA lipotransferase has high safety for HEK 293T cells at twice or less of the administered concentration; at four times the administered concentration, the BGG-link2-LA3 / mRNA lipotransferase had no significant effect on HEK 293T cell viability.

[0230] Example 16: Organ Safety of BGG-link2-LA3 / mRNA Lipid Nanoparticles

[0231] Following the method described in Example 13, BGG-link2-LA3 / mRNA lipid nanoparticles and the positive control ALC-0315 / mRNA lipid nanoparticles were prepared. ICR mice were intramuscularly injected with the same volume of BGG-LNP / mRNA, ALC-0315LNP / mRNA, or PBS containing 1 μg mRNA. Two days after administration, the mice were sacrificed, and the hearts were perfused with PBS. The hearts, livers, spleens, lungs, and kidneys were collected and fixed in 4% paraformaldehyde at room temperature for 24 h. Paraffin-embedded sections were prepared, stained with hematoxylin and eosin (HE), and scanned for observation. Results are shown in […]. Figure 17 .

[0232] from Figure 17As can be seen from the HE section scan results, no significant changes in cell morphology or structure were observed in any organ of the BGG-link2-LA3 / mRNA group. Cardiac cardiomyocytes showed normal morphology and a spindle-shaped distribution; liver cells were polygonal with clear boundaries; and the boundary between the spleen cortex and medulla was distinct. These results indicate that BGG-LNP / mRNA has good organ safety.

[0233] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A class of guanidine-derived compounds, characterized in that, The compound is a 1,3-isophthaloyl guanidine derivative compound or a pharmaceutically acceptable salt thereof, and the compound has a general formula as shown in formula I: In formula I, R1is -(CH2) n -, n is an integer from 1 to 10, and 0, 1, or more than one methylene unit in said (CH2) n is each independently replaced by R 1a ; said R 1a independently selected from R 1b -NH-, -O-, -S- or The R 1b The structure is shown in formula II: In formula II, m, x, z are each independently 0 or an integer from 1 to 10; R 1b’ is 1,3- isophthaloyl guanidine, and the structure is shown in formula III: In formula I, R2 is CH or N; In formula I, R3and R4are each independently selected from R5, R6, C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl, 1 or more methylene units of said C 5-30 alkyl, C 5-30 alkenyl, C 5-30 alkynyl are each independently replaced, one or more times, with one or more of the following: -O-, -C(O)-, said R5formula is -Rx-CH(Ry)(Rz); Rx in R5 is -(CH2) k , said k is selected from an integer number from 1 to 10; Rx is attached to R2 at the end; Ryand Rzin R5are each independently selected from C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl, 1 or more methylene units in said C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl are each independently replaced, one or more times, with one or more of the following: -O-, -C(O)-, R6 has a structure of -Ra-N(Rb)(Rc); Ra in R6 is -(CH2) y , said y is selected from an integer from 1 to 10; Ra is attached to R2; Rb and Rc in R6 are each independently selected from C 5-30 alkyl, C 5-30 alkenyl or C 5-30 alkynyl, 1 or more methylene units in said C 5-30 alkyl, C 5-30 alkenyl, C 5-30 alkynyl are each independently replaced, one or more times, with one or more of the following groups: -O-, -C(O)-, 2. The guanidine-based derivative compound according to claim 1, characterized by, R1 and R2 are selected from any one of the following groups: 1) R1is -(CH2) n - and R2is N: 2) R1has the structure: -(CH2) a -NH-(CH2) b -O-P(=O)(OH)-O-(CH2) c -; said (CH2) c terminal connection R2; R2is CH; 3) R1 has a structure as shown in formula IV; (CH2) f terminal connection R2; R2 is CH; R 1b’ as defined in claim 1 ; n, z, a, b, c, d, e, and f are each independently selected from an integer from 1 to 5; Preferably, R1 and R2 are selected from any one of the following groups: 1) R1 is -CH2-, and R2 is N; 2) R1 has a structure of -(CH2)-NH-(CH2)2-O-P(=O)(OH)-O-(CH2)-, and R2 is CH; 3) R1 has a structure as shown in formula V, and R2 is CH; In formula V, R 1b’ As defined in claim 1.

3. The guanidine-based derivative compound according to claim 1, characterized by, R3and R4are each independently selected from R5, R6, C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl, the C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl is straight-chained or branched, the C 5-30 alkyl, C 5-30 alkenyl, or C 5-30 alkynyl one or two methylene units are each independently replaced by one or more of the following: -O-, -C(O)-, R5 has a structure of -Rx-CH(Ry)(Rz); Rx in R5 is -(CH2) k , and k is an integer selected from 1-5; Rx is attached to R2; Ryand Rzin R5are each independently selected from C 5-30 alkyl, C 5-30 alkenyl or C 5-30 alkynyl, said C 5-30 alkyl, C 5-30 alkenyl or C 5-30 alkynyl is straight-chained or branched, said C 5-30 alkyl, C 5-30 alkenyl, C 5-30 one or two methylene units in the C R6 has a structure of -Ra-N(Rb)(Rc); Ra in R6 is -(CH2) y , said y is selected from an integer from 1 to 5; Ra is attached to R2; Rb and Rc in R6 are each independently selected from C 5-30 alkyl, C 5-30 alkenyl or C 5-30 alkynyl, said C 5-30 alkyl, C 5-30 alkenyl or C 5-30 alkynyl is straight-chained or branched, said C 5-30 alkyl, C 5-30 alkenyl, C 5-30 one or two methylene units in the C 4. The guanidine derivative compound of claim 1, wherein R3and R4are each independently selected from R5, R6, -(CH2) t1 -OC(O)-C 10-25 alkyl, -(CH2) t1 -C(O)-C 10-25 alkyl, -(CH2) t1 -OC(O)-C 10-25 alkenyl or -(CH2) t1 -C(O)-C 10-25 alkenyl; ti is independently selected from 0, 1, 2, 3, 4 or 5; when R3and R4are each independently R5, each of Ryand Rzis independently selected from -(CH2) t1 -OC(O)-C 10-25 alkyl, -(CH2) t1 -C(O)-C 10-25 alkyl, -(CH2) t1 -OC(O)-C 10-25 alkenyl or -(CH2) t1 -C(O)-C 10-25 alkenyl; said t1is independently selected from 0, 1, 2, 3, 4 or 5; when R3and R4are each independently R6, each Rb and Rc thereof is independently selected from -(CH2) t1 -OC(O)-C 10-25 alkyl, -(CH2) t1 -C(O)-C 10-25 alkyl, -(CH2) t1 -OC(O)-C 10-25 alkenyl or -(CH2) t1 -C(O)-C 10-25 alkenyl; said t1 is independently selected from 0, 1, 2, 3, 4 or 5; Preferably, R3 and R4 are each independently R5, R6, or any one of the following groups: 1) - (CH2) t1 -OC(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl bonds in said straight-chain alkenyl is 1, 2 or 3; 2) - (CH2) t1 -C(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl groups in straight-chain alkenyl is 1, 2 or 3; 3) - (CH2) t1 -C(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is 4) - (CH2) t1 -OC(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is said t1is independently selected from 0, 1, 2, 3, 4 or 5, said R 1-A and R 1-B each independently is C 4-10 a straight chain alkyl group; When R3 and R4 are each independently R5, Ry and Rz are each independently selected from any one of the following groups: 1) - (CH2) t1 -OC(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl bonds in straight-chain alkenyl is 1, 2 or 3; 2) - (CH2) t1 -C(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl groups in straight-chain alkenyl is 1, 2 or 3; 3) - (CH2) t1 -C(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is 4) - (CH2) t1 -OC(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is said t1is independently selected from 0, 1, 2, 3, 4 or 5, said R 1-A and R 1-B each independently is C 4-10 a straight-chain alkyl group; When R3 and R4 are each independently R6, Rb and Rc are each independently selected from any one of the following groups: 1) - (CH2) t1 -OC(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl bonds in said straight-chain alkenyl is 1, 2 or 3; 2) - (CH2) t1 -C(O)-C 10-25 alkenyl, said C 10-25 alkenyl is C 10-25 straight-chain alkenyl; said C 10-25 the number of alkenyl groups in straight-chain alkenyl is 1, 2 or 3; 3) - (CH2) t1 -C(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is 4) - (CH2) t1 -OC(O)-C 10-25 alkyl, said C 10-25 alkyl is C 10-25 branched alkyl; The structural formula is said t1is independently selected from 0, 1, 2, 3, 4 or 5, said R 1-A and R 1-B each independently is C 4-10 a straight chain alkyl.

5. The guanidine-based derivative compound according to claim 1, characterized by, Rb, Rc, Ry, and Rz are each independently selected from any one of the following groups:

6. The guanidine-based derivative compound according to claim 1, characterized by, R3 and R4 are each independently selected from any one of the following groups:

7. The guanidine-based derivative compound according to claim 1, characterized by, The compound is selected from any one of the following compounds: BGG1-link1-DOPE, BGG2-link1-DOPE, BGG-link2-OA3, BGG-link2-LA3, BGG-link3-LA3, BGG-link3-HA3, BGG-link4-LA2, BGG-link4-HA2, BGG-link5-LA2, and BGG-link5-HA2; The specific structural formula of each compound is shown in the following table:

8. Use of the guanidine derivative compound or a pharmaceutically acceptable salt thereof of any one of claims 1-7 in the preparation of a lipid carrier, a lipid nanoparticle, or a pharmaceutical composition.

9. A lipid carrier, characterized in that, The guanidine derivative compound or a pharmaceutically acceptable salt thereof of any one of claims 1-7 is contained; Preferably, the lipid carrier satisfies one or more of the following conditions: 1) further contains a neutral lipid selected from one or more of DSPC, DOPE, DPPC, DMPC, DOPC, POPC, DMPE, POPE, or DPPE; preferably DOPE; 2) further contains a structural lipid selected from at least one of an animal sterol, a plant or fungal sterol, for example, the sterol is selected from cholesterol, beta-sitosterol, ergosterol, campesterol, brassicasterol, and stigmasterol; preferably cholesterol; 3) further comprises a PEGylated lipid selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol and PEG-modified dialkylglycerol, for example PEG-modified phosphatidylethanolamine or PEG-modified dialkylamine, for example DMG-PEG2000, DSPE-PEG2000 or ALC-0159; preferably ALC-0159; 4) the lipid carrier comprises ionizable lipid, neutral lipid, structural lipid and PEGylated lipid, wherein the molar ratio of ionizable lipid, neutral lipid, structural lipid and PEGylated lipid is 40-80, 5-25, 20-60, 0.1-2.5, preferably the molar ratio is 45-60:10-20:30-40:0.5-2. 5) the lipid carrier component comprises BGG-lipid, i.e. the guanidyl derivative compound or pharmaceutically acceptable salt thereof as described in any one of claims 1-7, DOPE, Chol and ALC-0159, wherein the molar ratio of BGG-lipid, DOPE, Chol and ALC-0159 is 47.6:14.3:36.7:1.4; 6) the lipid carrier component comprises BGG-link2-LA3, DOPE, Chol and ALC-0159, and the molar ratio of each component is shown in the following table:

10. A lipid nanoparticle encapsulating a nucleic acid drug, characterized in that, which comprises the lipid carrier as described in claim 9, and a nucleic acid drug.

11. The lipid nanoparticle of claim 10, wherein, which satisfies one or more of the following conditions: 1) the nucleic acid drug can be selected from one or more of RNA and DNA, the RNA is selected from one or more of mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, and the DNA is selected from one or more of cDNA, plasmid DNA, viral DNA, PCR product, oligonucleotide; 2) in the nucleic acid drug-loaded lipid nanoparticle, the weight ratio of lipid carrier to nucleic acid drug is generally 5-45:1, preferably 10-20:1; 3) the nucleic acid drug-loaded lipid nanoparticle comprises lipid carrier and mRNA, the lipid carrier component comprises BGG-lipid, i.e. the guanidyl derivative compound or pharmaceutically acceptable salt thereof as described in any one of claims 1-7, DOPE, Chol and ALC-0159, wherein the molar ratio of BGG-lipid, DOPE, Chol and ALC-0159 is 47.6:14.3:36.7:1.4; and the weight ratio of BGG-lipid to mRNA is 14:1; 4) the nucleic acid drug-loaded lipid nanoparticle comprises lipid carrier and mRNA, the lipid carrier component comprises BGG-link2-LA3, DOPE, Chol and ALC-0159, and the molar ratio of each component is shown in the following table: the weight ratio of BGG-link2-LA3 to mRNA is 15:1; 5) the average particle size of the lipid nanoparticles is 50-200 nm; 6) the polydispersity index of the lipid nanoparticles is 0.05-0.3; 7) the encapsulation efficiency of the lipid nanoparticles is 55%-95%; 8) the nucleic acid drug-loaded lipid nanoparticles are prepared by a microfluidic method, and the method comprises the following steps: S1, dissolving the lipid compound prepared in the application and other several lipid substances in ethanol according to a certain proportion to prepare a mixed lipid solution as an alcohol phase; S2, adding nucleic acid into a citric acid buffer to prepare a nucleic acid-citric acid buffer as an aqueous phase; S3, preparing the lipid nanoparticles by using microfluidics, the total flow rate of the microfluidics is 4 ml / min-16 ml / min, the flow rate ratio is alcohol phase:aqueous phase=1:3, diluting the obtained initial product after ultrafiltration, and repeating the ultrafiltration three times.

12. A pharmaceutical composition capable of delivering a nucleic acid, characterized in that, which comprises the guanidyl derivative compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, the lipid carrier according to claim 9 or the nucleic acid drug-loaded lipid nanoparticles according to claim 10, and a pharmaceutically acceptable carrier.

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