WO2024187626A1 - Nano delivery system formed from amino acid lipids and use thereof - Google Patents

Abstract

A nano delivery system formed from amino acid lipids and the use thereof. Ionizable lipids for forming said amino acid lipid nano delivery system are amino acid lipids represented by general formula (I) or general formula (II) or salts thereof, img-content="drawing" img-, wherein R1 represents an amino acid residue in which a carboxyl group lacks a hydroxyl group, the structural formula thereof being NH2-CHR-CO-, R being the R group of an amino acid, and R2, R3 and R4 being independently linear hydrocarbon groups having 5-40 carbon atoms. The nano delivery system can enrich existing nucleic acid drug delivery systems and effectively reduce the immunogenicity and biotoxicity, thereby providing safer and more effective nucleic acid drugs for users.

Description

A nano delivery system formed by amino acid lipids and its application Technical Field

The invention belongs to the technical field of nucleic acid drug delivery systems, and in particular relates to a nano delivery system formed by amino acid lipids and applications thereof.

Background Art

With the development of mRNA vaccines, RNA interference (RNAi), RNAi therapy, RNA drugs, antisense therapy and gene therapy, the demand for introducing RNA into cells has increased. However, RNA is highly unstable. Generally speaking, RNA alone is stable in plasma for no more than a few hours. For RNA drugs to exert their therapeutic effects, an effective delivery system is required.

Liposome nanoparticles (LNP) are one of the most widely used delivery systems for nucleic acid drugs. LNP has a high nucleic acid encapsulation rate and can effectively transfect cells. It has strong tissue penetration and is more conducive to drug delivery. These advantages make LNP an excellent nucleic acid delivery system. The widely used LNP components at this stage mainly include the following four categories: liposomes, neutral phospholipid auxiliary lipids, cholesterol or its derivatives, and polyethylene glycol lipids. Liposomes are the key component. They are amphiphilic chemical molecules composed of a polar hydrophilic head, a hydrophobic tail, and a connecting group between the two.

The first generation of liposomes developed at the earliest was cationic liposomes, i.e., the hydrophilic head is positively charged, usually quaternary ammonium liposomes. Cationic liposomes and negatively charged nucleic acids can spontaneously self-assemble into stable nanoparticles through electrostatic interactions to deliver nucleic acids into cells. However, cationic liposomes have greater cytotoxicity and are gradually being replaced by ionizable liposomes, i.e., second-generation liposomes.

Ionizable liposomes are neutral liposomes, that is, the hydrophilic head is usually an amine group substituted by an alkyl group. Under acidic media (low pH), the amine groups in the ionizable liposomes combine with hydrogen ions to form positively charged compounds, which cover RNA molecules through ion interactions to form nanoparticles, and help RNA molecules be released from cell inclusion bodies/lysosomes into the cytoplasm by interfering with the stability and permeability of the inclusion body/lysosome membrane. Due to the improvement of effectiveness and toxicity characteristics, LNPs formed by ionizable liposomes have become the current mainstream nucleic acid delivery system.

The LNP delivery system that encapsulates RNA drugs and is stable in serum is also called SNALP (Serum-stable nucleic acid lipid particles, serum-stable nucleic acid lipid particles, US8058069). SNALP has helped multiple mRNA vaccines (Pfizer/BioNTech, Moderna) and the first siRNA drug (Patisiran, 2018, Alnylam) to obtain marketing approval. Figure 1 shows the structure of the liposome delivery system used in Patisiran. Typical liposomes used in the market are composed of a trisubstituted amine and two lipids containing linear or branched ester bonds (e.g. SM102, ALC0315).

One of the most important physicochemical properties of SNALP is the apparent dissociation constant (apparent pKa). The apparent pKa is an experimentally determined property of the nanoparticle. At this pH, the equivalents of dissociated and non-dissociated groups are equal. The most effective SNALP nanoparticles in RNA delivery have apparent pKa values between 6 and 7 (Cheng et al., Trends in Pharmacological Sciences, 42:448, 2021).

But SNALP also has disadvantages. The ionizable lipids in SNALP are synthetic chemicals with lower biocompatibility than natural lipids. Side effects such as inflammation and exacerbated inflammatory responses have been reported (Muzykantov et al., Journal of Controlled Release, 344:50, 2022).

Therefore, how to reduce the side effects of ionizable lipids and provide a safer nucleic acid drug delivery system while ensuring the encapsulation effect of nucleic acid molecules and the in vivo delivery function of nucleic acid molecules is one of the key issues in nucleic acid delivery technology.

Summary of the invention

The object of the present invention is to provide a new amino acid lipid or its salt, the drug delivery system prepared by the same has high encapsulation efficiency for nucleic acid molecules, can effectively deliver nucleic acid molecules and nucleic acid molecules can be successfully expressed in cells, and compared with the mainstream delivery system, the amino acid lipid or its salt can reduce immunogenicity and biotoxicity. Another object of the present invention is to provide a drug delivery system with low immunogenicity, low biotoxicity, and can effectively deliver nucleic acid molecules and ensure the successful expression of nucleic acid molecules. Another object of the present invention is to provide nucleic acid drugs with low immunogenicity and low biotoxicity.

To achieve the above object, the technical solution adopted by the present invention is:

An amino acid lipid or a salt thereof represented by the general formula (I) or the general formula (II),

in,

R ₁ represents an amino acid residue lacking a hydroxyl group on the carboxyl group, and its structural formula is NH ₂ -CHR-CO-, where R is the R group of the amino acid;

R ₂ , R ₃ and R ₄ are each independently a straight-chain hydrocarbon group having 5 to 40 carbon atoms.

In the present invention, the amino acids are not limited to L-type or D-type.

Preferably, the amino acid is glycine, methionine, serine, phenylalanine, alanine, threonine, tyrosine, hydroxyproline, glutamic acid, lysine, cysteine, proline, valine, leucine, isoleucine, tryptophan, glutamine, aspartic acid, asparagine, arginine or histidine.

More preferably, the amino acid is glycine, methionine, serine, phenylalanine, alanine, threonine, tyrosine, hydroxyproline, glutamic acid or lysine.

More preferably, the amino acid is glycine, methionine, serine or phenylalanine.

Preferably, R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkyl group having 5 to 40 carbon atoms.

More preferably, R ₂ , R ₃ , and R ₄ are each independently a linear alkyl group having 7 to 30 carbon atoms.

More preferably, R ₂ , R ₃ , and R ₄ are each independently a linear alkyl group having 7 to 20 carbon atoms.

Preferably, R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkenyl group having 5 to 40 carbon atoms and containing 1 to 3 double bonds.

More preferably, R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkenyl group having 7 to 30 carbon atoms and containing 1 to 3 double bonds.

More preferably, R ₂ , R ₃ and R ₄ are each independently a straight-chain alkenyl group having 7 to 20 carbon atoms and containing 1 to 3 double bonds.

Preferably, the amino acid lipid or its salt is a compound represented by general formula (I).

In the present invention, R ₂ , R ₃ , and R ₄ may be the same or different from each other.

Preferably, R ₂ , R ₃ , and R ₄ are the same.

and/or, R ₂ , R ₃ , R ₄ are independently:

According to some embodiments, the amino acid lipid is:

The present invention also provides a delivery system, which comprises one or more of the above-mentioned amino acid lipids or salts thereof.

Preferably, the delivery system further comprises one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids.

Further preferably, the helper lipid is selected from phospholipids and their derivatives.

Still further preferably, the auxiliary lipid is selected from one or more of PC, DPPC, DOPC, DSPC, DOPE, and DPPG.

Further preferably, the PEGylated lipid is selected from one or more of PEG-DMG, PEG-C-DMG, and PEG-DSPE.

Further preferably, the molar ratio of the amino acid lipid or its salt, the auxiliary lipid, the cholesterol and its derivatives and the PEGylated lipid is (40-99.5):(0-15):(0-50):(0.5-3).

The present invention also provides application of the delivery system in preparing nucleic acid drugs.

The present invention also provides a nucleic acid drug, which comprises the above-mentioned delivery system and a nucleic acid molecule.

Preferably, the nucleic acid molecule includes one or more of a messenger nucleic acid molecule (mRNA), a small interfering nucleic acid molecule (siRNA), a micronucleic acid molecule (miRNA), a small activating nucleic acid molecule (saRNA), an antisense oligonucleotide molecule (ASO) or an aptamer.

Preferably, the nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50 to 300 nm.

Further preferably, the nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50 to 300 nm, for example, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, 220 nm, 250 nm, 280 nm, 300 nm.

Preferably, the nucleic acid drug is obtained by mixing the nucleic acid molecule with the amino acid lipid or its salt, and selectively with one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids (PEG-Lipid) through microfluidics for self-assembly.

Preferably, the nitrogen to phosphorus molar (N/P) ratio of the amino acid lipid or its salt to the nucleic acid molecule is (1-50):1.

More preferably, the nitrogen-phosphorus molar ratio of the amino acid lipid or its salt to the nucleic acid molecule is (3-15):1, for example 3:1, 5:1, 10:1, 15:1.

Preferably, the nucleic acid drug further comprises a pharmaceutically acceptable additive.

More preferably, the additives include excipients and the like.

Preferably, the nucleic acid drug is a lyophilized powder or an injection.

More preferably, the nucleic acid drug is used in mammals, preferably humans.

More preferably, the nucleic acid drug is administered by intramuscular injection, intravenous injection, etc.

The present invention has at least the following beneficial effects:

The present invention adopts amino acid substitution as the hydrophilic head of the ionizable lipid, combined with the coordination of the hydrophobic carboxylic acid tail, and the obtained amino acid lipid or its salt has low cytotoxicity. The drug delivery system prepared by the present invention can achieve the encapsulation efficiency of nucleic acid molecules as that of mainstream nanoliposomes, and can also deliver nucleic acid molecules into the body and realize the effective expression of nucleic acid molecules in cells. Compared with mainstream nanoliposomes, while ensuring the delivery function, the self-toxicity is lower.

Furthermore, the amino acid lipid or its salt formed by connecting the amino acid hydrophilic head and the hydrophobic hydrocarbon chain through tris(hydroxymethyl)aminomethane can not only reduce the immunogenicity and biological toxicity of the delivery system, but also has a significantly better in vivo delivery effect without affecting the encapsulation efficiency of nucleic acid molecules.

The present invention can enrich the delivery system of existing nucleic acid drugs, provide more choices for users, and is conducive to the development and application of nucleic acid drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of the liposome delivery system used in Patisiran

Fig. 2 is a diagram showing the cytotoxicity test results of amino acid lipids;

Fig. 3 is a graph showing the results of luciferase antibody detection;

FIG4 is a graph showing the gene knockdown effect corresponding to apparent pKa.

DETAILED DESCRIPTION

In order to solve the shortcomings of the existing nucleic acid molecule delivery system, such as complex composition, high preparation cost, high biological toxicity, strong immunogenicity, and low endosomal escape rate, the inventors have proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution, implementation process and principle will be further explained below.

After intensive research and long-term exploration, the inventors discovered that the hydrophilic head amine groups in ionizable liposomes can be formed by amino acids. Compared with non-natural and highly toxic substituted amine groups, amino acid substitutions have good biocompatibility and non-immunogenicity, and reduce the toxicity of ionizable liposomes to cells without causing a decrease in encapsulation efficiency.

Specifically, the present invention provides an amino acid lipid or a salt thereof represented by general formula (I) or general formula (II),

Wherein, _R1 represents an amino acid residue lacking a hydroxyl group on the carboxyl group, and its structural formula is _NH2 -CHR-CO-, R is the R group of the amino acid; _R2 , _R3 , and _R4 are independently straight-chain hydrocarbon groups with 5 to 40 carbon atoms.

The amino acid lipids of the present invention can disrupt the endosomal membrane and safely release the nucleic acid molecules into the cytoplasm to achieve expression.

One of the purposes of the present invention is to provide an amino acid lipid with low immunogenicity and low biotoxicity and a nucleic acid drug delivery system thereof. In addition to considering the selection of amino acids from natural sources, in the selection of carboxylic acid lipids, not only the encapsulation and delivery efficiency of nucleic acids is considered, but also carboxylic acids from natural sources are selected as much as possible, such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, etc., which can further reduce side reactions during the delivery process and reduce immunogenicity and biotoxicity.

In some preferred embodiments, R ₂ , R ₃ , and R ₄ are independently:

After selecting the hydrophilic amino acid head and the hydrophobic carboxylic acid tail, the inventors selected pentaerythritol (PEL) as the connecting group between the two. The chemical structure of pentaerythritol is:

Pentaerythritol is a polyhydroxy compound that can be covalently linked to amino acids through ester bonds, and can also be covalently bound to carboxylic acid lipids through ester bonds. Pentaerythritol has good safety. At the same time, because pentaerythritol is covalently bound to amino acids and hydrophobic lipids through ester bonds, it is easily hydrolyzed into small molecular compounds after entering the cells and is promptly cleared from the body, effectively reducing immunogenicity and biological toxicity.

Based on this strategy, the inventors synthesized several amino acid liposomes (APL) with pentaerythritol as the linking group and prepared the corresponding liposome nanoparticles (APLNP). Physical and chemical and biological experiments found that APLNP can effectively encapsulate mRNA and siRNA.

The present invention further attempts to use Tris instead of pentaerythritol as a linking group in the amino acid liposome. The chemical structural formula of Tris is:

Similar to pentaerythritol, Tris is also a polyhydroxyl multifunctional compound, in which the three hydroxyl groups can be covalently linked to carboxylic acid lipids through ester bonds, while the amino groups can be covalently bound to amino acids through amide bonds. Compared with pentaerythritol, Tris not only has similar chemical structural characteristics to pentaerythritol and can be used as a linking group for amino acids and hydrophobic chains, but Tris itself also has low immunogenicity and low biological toxicity.

Tris, also known as ammonium tromethamine in medicine, is an alkaline buffer that has a good buffering effect on metabolic acidosis and enzyme activity reactions. It is suitable for diseases such as metabolic acidemia and respiratory acidemia, especially for hyperuricemia, and can effectively promote the dissolution of uric acid. The above applications of Tris in the biological or medical fields are sufficient to prove that Tris is biosafe.

Experiments have found that a reasonable combination of certain amino acids, Tris and lipids can achieve good delivery effects, and the effect of delivering fluorescent mRNA can reach or exceed the effect of mainstream LNP. The knockdown effect of the target gene after delivering siRNA can also reach or exceed the effect of mainstream LNP. In the control of biological experiments related to pentaerythritol analogs (APLNP), it was found that the amino acid lipid nanoparticle delivery system (Amino Acid-Tris-Lipid Nano Particles, ATLNP) prepared with Tris as the connecting group showed superior delivery efficiency than APLNP, whether it was delivering mRNA or siRNA.

The inventors believe that the amino group in Tris and the amide bond formed with amino acids play an important role in the nucleic acid delivery of ATLNP. The amino acid lipid or its salt (Amino Acid-Tris-Lipid, ATL) formed by connecting the hydrophilic head of amino acid with the hydrophobic hydrocarbon chain through tris (hydroxymethyl) aminomethane (Tris) has a much smaller charge than cationic lipids at physiological pH values, and mainly binds to nucleic acid molecules through hydrogen bonds to form lipid nanoparticles containing nucleic acid molecules. In ATL, which is a key component in ATLNP, the amide bond formed by Tris and amino acids has a strong hydrogen bond forming ability, thereby strengthening the binding ability of ATLNP with nucleic acids, which is greatly beneficial to the delivery of nucleic acid drugs. In APLNP, due to the absence of amide bonds, the hydrogen bond binding ability with nucleic acids is reduced. In the amino acid lipid ATL, which is the main component of ATLNP, amino acids and Tris are bound by amide bonds. The amide bonds can further enhance the hydrogen bond interactions between amino acid lipids and nucleic acid molecules, thereby further improving the delivery efficiency of nucleic acid molecules.

Therefore, the present invention provides a variety of amino acid lipids with high encapsulation efficiency, low toxicity and the ability to successfully deliver nucleic acid molecules into cells and express them, among which the amino acid lipid represented by general formula (I) is preferred, which has a better in vivo delivery effect on mRNA and siRNA.

The amino acid lipid nanoparticle delivery system (Amino Acid-Tris-Lipid Nano Particles, ATLNP) prepared using the ionizable liposome-amino acid lipid or its salt (Amino Acid-Tris-Lipid, ATL) of the present invention can be used for the delivery of nucleic acid molecules. It has low immunogenicity and low biological toxicity, and can effectively deliver nucleic acid molecules and achieve expression in vivo.

The technical scheme and technical effects of the present invention are further described below in conjunction with specific embodiments and comparative examples.

Unless otherwise specified, the raw materials and the like used in the following examples were commercially available.

Unless otherwise specified, the room temperature (RT) referred to in the following examples refers to 20-35° C., and the ratio of the eluents involved in the following examples is a volume ratio.

In the following examples and comparative examples, "C10" and "C ₁₀ " in the abbreviations are equivalent, and both represent a chain lipid containing 10 carbon atoms.

Unless otherwise specified, the experimental methods or test methods involved in the following embodiments adopt conventional methods in the art.

Synthesis Example

Example 1. Met-Tris-3MOA

Under _N2 protection, myristic acid (MOA, 298 mg, 1.31 mmol), N-tert-butyloxycarbonyl-tris(hydroxymethyl)aminomethane (Boc-Tris, 88.5 mg, 0.40 mmol), DMAP (50 mg, 0.40 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (307 mg, 1.60 mmol) was added. After the addition was completed, the mixture was stirred at room temperature for 18 h under _N2 protection. The reaction mixture was diluted with ethyl acetate (EA) (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, respectively, and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and purified by a silica gel column with an eluent (petroleum ether/ethyl acetate, PE/EA) = 30:1 to obtain a colorless oily liquid Boc-Tris-3MOA (259 mg, 0.31 mmol) with a yield of 77%.

Under _N2 protection, Boc-Tris-3MOA (250 mg, 0.30 mmol) was dissolved in 4 mL of dichloromethane (DCM), followed by the addition of trifluoroacetic acid (TFA, 1.0 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3MOA (216 mg, 0.29 mmol) with a yield of 97%.

Under _N2 protection, Tris-3MOA (107.5 mg, 0.14 mmol), N-tert-butyloxycarbonyl-L-methionine (Boc-Met, 70 mg, 0.28 mmol), HBTU (115 mg, 0.30 mmol), HOBt (41 mg, 0.30 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (108 mg, 0.83 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent PE/EA = 10:1 to obtain a colorless oily liquid Boc-Met-Tris-3MOA (123 mg, 0.12 mmol) with a yield of 86%.

Under _N2 protection, Boc-Met-Tris-3MOA (123 mg, 0.12 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain colorless oily liquid Met-Tris-3MOA (97 mg, 0.11 mmol), the yield was 92%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.65 (s, 1H), 5.32 (s, 6H), 4.40 (s, 6H), 3.46 (d, J = 12.6Hz, 1H), 2.59 (d, J ＝3.9Hz,2H),2.32(d,J＝15.2Hz,6H),2.10(s,3H),2.01(s,12H),1.77(d,J＝28.4Hz,2H),1.59(s,6H ),1.30(s,36H),0.90(d,J=14.0Hz,9H).

Example 2. Gly-Tris-3MOA

Under _N2 protection, Tris-3MOA (168 mg, 0.22 mmol), N-tert-butyloxycarbonyl-glycine (Boc-Gly, 77 mg, 0.44 mmol), HBTU (167 mg, 0.44 mmol), HOBt (59 mg, 0.44 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (170 mg, 1.32 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3MOA (159 mg, 0.18 mmol) with a yield of 81%.

Under _N2 protection, Boc-Gly-Tris-3MOA (159 mg, 0.18 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, and a colorless oily liquid Gly-Tris-3MOA (117 mg, 0.15 mmol) was obtained, the yield was 83%, and the results of H NMR were as follows:

¹ H NMR(400MHz,Chloroform-d)δ7.62(s,1H),5.33(s,6H),4.44(s,6H),3.31(s,2H),2.34(s,6H),2.08–2.00 (m,12H),1.60(d,J＝6.8Hz,6H),1.31(d,J＝9.4Hz,36H),0.88(s,9H).

Example 3. Gly-Tris-3MA

Under _N2 protection, myristic acid (MA, 0.75 g, 3.3 mmol), Boc-Tris (0.22 g, 1.0 mmol), DMAP (0.12 g, 1.0 mmol) and DMF (10 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (0.85 g, 4.4 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (300 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid, which became a white solid Boc-Tris-3MA (0.78 g, 0.9 mmol) after standing, with a yield of 90%.

Under _N2 protection, Boc-Tris-3MA (0.78 g, 0.90 mmol) was dissolved in 8 mL of dichloromethane, followed by the addition of trifluoroacetic acid (2.0 mL). The reaction was allowed to react at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel. 200 mL of dichloromethane was added. methane, and adjust the organic layer to pH = 7-8 with saturated sodium bicarbonate solution, separate the organic layer, extract the aqueous layer twice with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, filter and concentrate to obtain white solid Tris-3MA (0.64 g, 0.85 mmol) with a yield of 94%.

Under _N2 protection, Tris-3MA (0.64 g, 0.85 mmol), Boc-Gly (0.30 g, 1.71 mmol), HBTU (0.65 g, 1.71 mmol), HOBt (0.23 g, 1.70 mmol) and DMF (10 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (0.81 g, 6.3 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (300 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3MA (0.66 g, 0.72 mmol) with a yield of 85%.

Under _N2 protection, Boc-Gly-Tris-3MA (0.66 g, 0.72 mmol) was dissolved in 8.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (2.0 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 200 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain a white solid Gly-Tris-3MA (0.48 g, 0.59 mmol), the yield was 82%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.62 (s, 1H), 4.44 (s, 6H), 3.32 (s, 2H), 2.32 (d, J = 15.1Hz, 6H), 1.61 (d, J =6.8Hz, 6H), 1.26 (s, 60H), 0.88 (d, J = 13.5Hz, 9H).

Example 4. Gly-Tris-3POA

Under _N2 protection, palmitoleic acid (POA, 251.7 mg, 0.99 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.7 mg, 0.3 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (230.3 mg, 1.2 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3POA (231 mg, 0.25 mmol), with a yield of 83%.

Under _N2 protection, Boc-Tris-3POA (231 mg, 0.25 mmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3POA (205 mg, 0.24 mmol) with a yield of 96%.

Under _N2 protection, Tris-3POA (205 mg, 0.24 mmol), Boc-Gly (87.6 mg, 0.5 mmol), HBTU (190 mg, 0.50 mmol), HOBt (68.2 mg, 0.50 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (195 mg, 1.5 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3POA (166 mg, 0.17 mmol) with a yield of 71%.

Under _N2 protection, Boc-Gly-Tris-3POA (166 mg, 0.17 mmol) was dissolved in 2.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was allowed to react at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel. 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained product was purified by silica gel column, and the eluent was DCM/ _CH3OH = 40:1 to obtain a colorless oily liquid. Gly-Tris-3POA (132 mg, 0.15 mmol), the yield is 88%, the results of H NMR are as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.64 (s, 1H), 5.33 (s, 6H), 4.44 (s, 6H), 3.28 (s, 2H), 2.32 (d, J = 15.2

Hz, 6H), 2.01 (d, J = 18.6Hz, 12H), 1.60 (d, J = 6.5Hz, 6H), 1.29 (d, J = 22.7Hz, 48H), 0.90 (s, 9H).

Example 5. Gly-Tris-3DOA

Under _N2 protection, cis-5-dodecenoic acid (DOA, 196.8 mg, 0.99 mmol), Boc-Tris (66.2 mg, 0.3 mmol), DMAP (36.8 mg, 0.3 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (230.2 mg, 1.2 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with saturated sodium bicarbonate and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column, eluent PE/EA = 30:1, to obtain colorless oily liquid Boc-Tris-3DOA (208 mg, 0.27 mmol), with a yield of 90%.

Under _N2 protection, Boc-Tris-3DOA (208 mg, 0.27 mmol) was dissolved in 2 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3DOA (179 mg, 0.27 mmol) with a yield of 99%.

Under _N2 protection, Tris-3DOA (179 mg, 0.27 mmol), Boc-Gly (94.8 mg, 0.54 mmol), HBTU (205 mg, 0.54 mmol), HOBt (73.2 mg, 0.54 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (210 mg, 1.63 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3DOA (161 mg, 0.20 mmol) with a yield of 74%.

Under _N2 protection, Boc-Gly-Tris-3DOA (154 mg, 0.19 mmol) was dissolved in 2.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.5 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain a colorless oily liquid Gly-Tris-3DOA (97 mg, 0.13 mmol), the yield was 68%, and the results of H NMR were as follows:

¹ H NMR(400MHz,Chloroform-d)δ7.65(s,1H),5.31(s,6H),4.45(s,6H),3.29(s,2H),2.31(s,6H),2.09–1.99 (m,12H),1.66(d,J＝14.9Hz,6H),1.29(s,24H),0.89(d,J＝6.6Hz,9H).

Example 6. Gly-Tris-3OA

Under _N2 protection, oleic acid (OA, 282.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (230.5 mg, 1.2 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3OA (272 mg, 0.27 mmol), with a yield of 90%.

Under _N2 protection, Boc-Tris-3OA (272 mg, 0.27 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3OA (240 mg, 0.26 mmol) with a yield of 96%.

Under _N2 protection, Tris-3OA (240 mg, 0.26 mmol), Boc-Gly (93 mg, 0.53 mmol), HBTU (201 mg, 0.53 mmol), HOBt (72 mg, 0.53 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (205 mg, 1.58 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with saturated sodium bicarbonate and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column, eluent PE/EA = 10:1, to obtain colorless oily liquid Boc-Gly-Tris-3OA (189 mg, 0.18 mmol), with a yield of 69%.

Under _N2 protection, Boc-Gly-Tris-3OA (189 mg, 0.18 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain a colorless oily liquid Gly-Tris-3OA (139 mg, 0.14 mmol), the yield was 78%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.64(s,1H),5.34(s,6H),4.44(s,6H),3.34(s,2H),2.32(t,J＝7.5Hz,6H ),2.02(d,J＝12.1Hz,12H),1.56(s,6H),1.28(d,J＝12.7Hz,60H),0.86(s,9H).

Example 7. Gly-Tris-3Lin

Under _N2 protection, linoleic acid (Lin, 280.6 mg, 1.0 mmol), Boc-Tris (66.3 mg, 0.3 mmol), DMAP (36.9 mg, 0.3 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (230.5 mg, 1.2 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3Lin (241 mg, 0.24 mmol), with a yield of 80%.

Under _N2 protection, Boc-Tris-3Lin (241 mg, 0.24 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3Lin (213 mg, 0.23 mmol) with a yield of 96%.

Under _N2 protection, Tris-3Lin (213 mg, 0.23 mmol), Boc-Gly (82 mg, 0.47 mmol), HBTU (178 mg, 0.47 mmol), HOBt (63.5 mg, 0.47 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (182 mg, 1.40 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3Lin (166 mg, 0.16 mmol), with a yield of 69%.

Under _N2 protection, Boc-Gly-Tris-3Lin (136 mg, 0.13 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain a colorless oily liquid Gly-Tris-3Lin (101 mg, 0.11 mmol), the yield was 85%, and the results of hydrogen nuclear magnetic resonance spectrum are as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.65 (s, 1H), 5.39–5.30 (m, 12H), 4.44 (s, 6H), 3.28 (s, 2H), 2.77 (d, J＝13.0Hz ,6H),2.33(d,J＝7.5Hz,6H),2.06(s,12H),1.62(d,J＝7.0Hz,6H),1.31(tdd,J＝9.8,4.5,2.2Hz,42H) ,0.89(d,J＝13.7Hz,9H).

Example 8. Gly-Tris-3PA

Under _N2 protection, palmitic acid (PA, 260 mg, 1.01 mmol), Boc-Tris (68.2 mg, 0.31 mmol), DMAP (40.5 mg, 0.33 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (235 mg, 1.22 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3PA (235 mg, 0.25 mmol), with a yield of 81%.

Under _N2 protection, Boc-Tris-3PA (235 mg, 0.25 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain white solid Tris-3PA (212 mg, 0.25 mmol), with a yield of 98%.

Under _N2 protection, Tris-3PA (212 mg, 0.25 mmol), Boc-Gly (90 mg, 0.51 mmol), HBTU (193 mg, 0.51 mmol), HOBt (69 mg, 0.51 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (195 mg, 1.51 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Gly-Tris-3PA (193 mg, 0.19 mmol) with a yield of 76%.

Under _N2 protection, Boc-Gly-Tris-3PA (193 mg, 0.19 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (, 0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain a white solid Gly-Tris-3PA (151 mg, 0.17 mmol), the yield was 89%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.65 (s, 1H), 4.44 (s, 6H), 3.28 (s, 2H), 2.32 (d, J = 15.2Hz, 6H), 1.61 (d, J =14.2Hz, 6H), 1.25 (s, 72H), 0.88 (d, J = 13.7Hz, 9H).

Example 9. Met-Tris-3MA

Under _N2 protection, Tris-3MA (212 mg, 0.28 mmol), Boc-Met (140 mg, 0.56 mmol), HBTU (215 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (230 mg, 1.78 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Met-Tris-3MA (215 mg, 0.22 mmol) with a yield of 78%.

Under _N2 protection, Boc-Met-Tris-3MA (215 mg, 0.22 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 200 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent dichloromethane/ _CH3OH = 40:1, and white solid Met-Tris-3MA (179 mg, 0.20 mmol) was obtained, the yield was 91%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.67 (s, 1H), 4.43 (s, 6H), 3.45 (d, J = 12.6Hz, 1H), 2.58 (s, 2H), 2.32 (d, J ＝15.2Hz,6H),2.11(s,3H),1.72(s,1H),1.61(d,J＝14.2Hz,7H),1.26(s,60H),0.88(d,J＝13.7Hz,9H ).

Example 10. Met-Tris-3LA

Under _N2 protection, dodecanoic acid (LA, 220 mg, 1.1 mmol), Boc-Tris (70 mg, 0.31 mmol), DMAP (60 mg, 0.49 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (260 mg, 1.35 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3LA (239 mg, 0.31 mmol), with a yield of 99%.

Under _N2 protection, Boc-Tris-3LA (239 mg, 0.31 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3LA (197 mg, 0.29 mmol), with a yield of 93%.

Under _N2 protection, Tris-3LA (197 mg, 0.29 mmol), Boc-Met (159 mg, 0.60 mmol), HBTU (230 mg, 0.60 mmol), HOBt (819 mg, 0.60 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (238 mg, 1.83 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Met-Tris-3LA (210 mg, 0.23 mmol) with a yield of 79%.

Under _N2 protection, Boc-Met-Tris-3LA (210 mg, 0.23 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain white solid Met-Tris-3LA (157 mg, 0.20 mmol), the yield was 87%, and the results of hydrogen nuclear magnetic resonance spectrum are as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.66 (s, 1H), 4.46 (s, 6H), 3.45 (d, J = 12.6Hz, 1H), 2.60 (d, J = 11.2Hz, 2H), 2.34–2.29(m,6H),2.11(s,3H),1.76(d,J＝29.5Hz,1H),1.63(s,7H),1.26(s,48H),0.88(d,J＝13.7Hz ,9H).

Example 11. Met-Tris-3C10

Under _N2 protection, decanoic acid (C10, 228 mg, 1.32 mmol), Boc-Tris (89 mg, 0.40 mmol), DMAP (49 mg, 0.40 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (307 mg, 1.60 mmol) was added. After addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 30:1, to obtain a colorless oily liquid Boc-Tris-3C10 (156 mg, 0.23 mmol), with a yield of 58%.

Under _N2 protection, Boc-Tris-3C10 (156 mg, 0.23 mmol) was dissolved in 3 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution. The organic layer was separated, and the aqueous layer was extracted twice with dichloromethane. The organic layers were combined and dried with anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily liquid Tris-3C10 (138 mg, 0.23 mmol) with a yield of 99%.

Under _N2 protection, Tris-3C10 (138 mg, 0.23 mmol), Boc-Met (125 mg, 0.50 mmol), HBTU (190 mg, 0.50 mmol), HOBt (68 mg, 0.50 mmol) and DMF (3.5 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and DIPEA (195 mg, 1.51 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Met-Tris-3C10 (165 mg, 0.20 mmol) with a yield of 79%.

Under _N2 protection, Boc-Met-Tris-3C10 (165 mg, 0.20 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was tracked by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 100 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain white solid Met-Tris-3C10 (131 mg, 0.18 mmol), the yield was 90%, and the results of hydrogen nuclear magnetic resonance spectrum were as follows:

¹ H NMR (400MHz, Chloroform-d) δ7.67 (s, 1H), 4.43 (d, J = 24.6Hz, 6H), 3.45 (d, J = 11.5Hz, 1H), 2.58 (d, J = 31.9 Hz,2H),2.32(d,J＝15.2Hz,6H),2.10(s,3H),1.76(d,J＝27.6Hz,1H),1.63(s,7H),1.27(d,J＝7.3 Hz,36H),0.89(d,J＝6.6Hz,9H).

Example 12. Met-PEL-3MOA

Under _N2 protection, pentaerythritol (PEL, 5.0 g, 36.6 mmol), imidazole (2.67 g, 38.6 mmol) and anhydrous DMF (235 mL) were added to a 500 mL reaction bottle, and a solution of tert-butyldimethylsilyl chloride (TBDMS-Cl, 2.94 g, 19.6 mmol) dissolved in 15 mL anhydrous DMF was added dropwise under stirring. After addition, the mixture was stirred at room temperature for 24 h under _N2 protection. The mixture was concentrated under reduced pressure, diluted with ethyl acetate (300 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 1:1, to obtain a colorless oily liquid (solidified after standing at room temperature) PEL (TBS) (2.26 g, 9.02 mmol), with a yield of 46%.

Under _N2 protection, PEL (TBS) (65.8 mg, 0.26 mmol), myristic acid (MOA, 198 mg, 0.87 mmol), DMAP (32.5 mg, 0.26 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (203.2 mg, 0.26 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, with an eluent of PE/EA = 50:1, to obtain a colorless oily liquid PEL (TBS) -3MOA (210 mg, 0.24 mmol), with a yield of 92%.

Under _N2 protection, PEL(TBS)-3MOA (210 mg, 0.24 mmol) was dissolved in 5 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride in tetrahydrofuran solution (TBAF, 1 M, 1.5 mL) and acetic acid (AA, 0.5 mL). After 3 days of reaction at room temperature, the mixture was concentrated under reduced pressure, diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product purified by a silica gel column, eluent PE/EA = 10:1, to obtain a colorless oily liquid PEL-3MOA (144 mg, 0.19 mmol), with a yield of 79%.

Under _N2 protection, PEL-3MOA (144 mg, 0.19 mmol), Boc-Met (101 mg, 0.40 mmol), DMAP (24.5 mg, 0.20 mmol) and DMF (3 mL) were added to a 10 mL reaction bottle, stirred to obtain a clear solution, and EDCI.HCl (76.7 mg, 0.40 mmol) was added. After the addition, the mixture was stirred at room temperature for 18 h under _N2 protection. The mixture was diluted with ethyl acetate (150 mL), and the organic phase was washed once with a saturated sodium bicarbonate solution and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by a silica gel column, and the eluent was PE/EA = 10:1 to obtain a colorless oily liquid Boc-Met-PEL-3MOA (149 mg, 0.15 mmol), with a yield of 79%.

Under _N2 protection, Boc-Met-PEL-3MOA (149 mg, 0.15 mmol) was dissolved in 3.0 mL of dichloromethane, followed by the addition of trifluoroacetic acid (0.75 mL). The reaction was carried out at room temperature, and the reaction was followed by LC-MS. The reaction was stopped after 2 h. The reaction solution was concentrated under reduced pressure and transferred to a separatory funnel, 120 mL of dichloromethane was added, and the organic layer was adjusted to pH = 7-8 with a saturated sodium bicarbonate solution, the organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained product was purified by silica gel column, eluent DCM/ _CH3OH = 40:1, to obtain colorless oily liquid Met-PEL-3MOA (105 mg, 0.12 mmol), the yield was 80%, and the results of H NMR were as follows:

¹ H NMR (400MHz, Chloroform-d) δ5.34(d,J＝11.8Hz,6H),4.14(d,J＝15.3Hz,8H),3.62(d,J＝15.5Hz,1H),2.62( d,J＝14.4Hz,2H),2.31(d,J＝15.1Hz,6H),2.10(s,3H),2.02(s,12H),1.78(d,J＝26.5Hz,2H),1.59( d, J＝6.8Hz, 6H), 1.32 (d, J＝17.4Hz, 36H), 0.90 (d, J＝14.0Hz, 9H).

Cytotoxicity testing of amino acid lipids

The CTG method was used to investigate the toxicity of the compounds obtained in Examples 1 to 7 to 293T cells. 2.5×10 ⁵ cell/mL of 293T cells were placed in a 24-well plate, and then a certain concentration of the test compound was added and incubated for 24 hours. After transferring to a 96-well plate and continuing to culture for 24 hours, 100 μL of CTG and complete culture medium were added. After incubation at room temperature for 10 minutes, the absorbance at a wavelength of 540 nm was detected using an ELISA instrument to calculate the cell survival rate. The results are shown in Figure 2. The results show that all the amino acid lipids tested have low cytotoxicity (MC3 is an ionizable lipid used in the Onpattro preparation). DLin-MC3-DMA).

Preparation of lipid nanoparticles

The amino acid lipids obtained in the above examples 1 to 7, 10 to 12 and the ionizable lipid SM102 used in Moderna Covid-19 Vaccine (Spikevax) were mixed with DSPC, cholesterol, and PEG-DMG at a molar ratio of 50/10/38.5/1.5 and dissolved in anhydrous ethanol to obtain a lipid solution (a). According to the N/P of amino acid lipids and mRNA = 5:1, Fluc mRNA was dissolved in a citric acid buffer to obtain a Fluc mRNA solution (b). The lipid solution (a) and the mRNA solution (b) were mixed by microfluidics at a flow rate ratio of 1:3 to obtain a product (c). The product (c) was dialyzed with a PBS buffer at pH = 7.2 to 7.4 for 24 hours and then ultrafiltered and concentrated at 4°C to obtain amino acid lipid nanoparticles ATLNP 1 to 7, 10, 11, APLNP1 and SM102-LNP (mRNA) encapsulating Fluc mRNA. The encapsulation efficiency of all LNP samples was greater than 90%. The Fluc mRNA used in this example was provided by Trilink. The particle size potential detection results of ATLNP and APLNP loaded with Fluc mRNA are shown in Table 1.

Table 1

The amino acid lipids synthesized in Examples 1 to 7, 9, and 12 and the ionizable lipid SM102 used in the Moderna Covid-19 Vaccine (Spikevax) preparation were mixed with DSPC, cholesterol, and PEG-C-DMG at a molar ratio of 50/10/38.5/1.5 and dissolved in anhydrous ethanol to obtain a lipid solution (a). According to the N/P of amino acid lipids and siRNA = 5:1, siRNA was dissolved in a citric acid buffer to obtain a siRNA solution (b). The lipid solution (a) and the siRNA solution (b) were mixed by microfluidics at a flow rate ratio of 1:3 to obtain a product (c). The product (c) was dialyzed with a PBS buffer at pH = 7.2-7.4 for 24 hours and then ultrafiltered and concentrated at 4°C to obtain amino acid lipid nanoparticles ATLNP 12 to 18, 20, APLNP 2 and SM102-LNP (siRNA) loaded with siRNA, and the sample encapsulation efficiency was greater than 90%. The siRNA sequence used in this example is hmTF-25-2 (US20210324384A1). The particle size potential detection results of ATLNP and APLNP loaded with siRNA are shown in Table 2.

Table 2

Cell and animal experiments

Nanoparticles ATLNP 1-7, ATLNP 10, ATLNP 11, APLNP 1 were taken, 20 μg each, and injected intramuscularly into the inner thigh of BABL/C mice, and the experiment was carried out in parallel three times. The mice were observed for in vivo imaging at two time points of 24h and 72h, and the fluorescence intensity was measured. The results are shown in Table 3. As can be seen from Table 3, all mice injected intramuscularly with ATLNP 1-7, ATLNP 10, and ATLNP 11 samples showed fluorescence after 24h, indicating that the ATLNP loaded with mRNA can effectively deliver Fluc mRNA into cells and express fluorescent protein in mice. The fluorescence intensity of mice injected intramuscularly with APLNP1 samples was significantly lower than that of the corresponding ATLNP (ATLNP 1 vs APLNP 1) after 24h, indicating that the efficiency of APLNP samples with pentaerythritol as the linker group in delivering Fluc mRNA into cells is lower than that of the corresponding ATLNP samples with Tris as the linker group.

Table 3

ATLNP 1-7 and SM102-LNP (mRNA) were taken, and the ATLNP sample containing 20 μg Fluc mRNA was injected intramuscularly into BABL/C mice, and the experiment was carried out in parallel three times. Two weeks after the first administration, the same dose was intramuscularly injected once for reinforcement, and then one week after the second administration, 100-200 μL of blood samples were collected from the mouse orbital venous plexus. The blood sample was placed in a 4°C refrigerator overnight to separate the serum, and luciferase was coated on the ELISA plate. After overnight at room temperature, the diluted mouse serum was added to the ELISA plate (using the luciferase antibody as a positive control and a mixture of phosphate and Tween 20 as a negative control), and the plate was washed after sufficient reaction, and then the IgG-HRP antibody was added. After sufficient reaction, the plate was washed, and then the TMB reaction solution was added. After sufficient reaction, the stop solution was added to stop the reaction. The absorbance value was read at a wavelength of 450nm on the ELISA instrument, and the luciferase antibody content was detected. The results are shown in Figure 3. The results showed that the immunogenicity of ATLNP 1 to 7 was lower than or equivalent to that of SM102-LNP, among which the antibody levels of ATLNP 1, 2, and 3 were significantly lower than that of SM102-LNP (mRNA), indicating that ATLNP1, 2, and 3 had lower immunogenicity.

The samples ATLNP12-20 and APLNP 2 loaded with siRNA were transfected into 293T cells, and the SM102-LNP (siRNA) prescription was compared. After 24 hours, the total cell RNA was collected for reverse transcription and the mRNA expression level of the target gene was detected by QPCR technology. The knockdown effect of TGF-β1 siRNA on the expression level of the target gene TGF-β1 mRNA is shown in Table 4. The results show that ATLNP 13-18, 20 samples have good gene silencing efficiency on TGF-β1 at different concentrations. By comparing the knockdown effect of ATLNP 12 and APLNP 2 on TGF-β1 mRNA expression, it was found that the same amino acid head (Met) and carboxylic acid lipid tail (MOA), APLNP 21 (Met-PEL-3MOA) with pentaerythritol as the linker group had a significantly lower gene silencing efficiency at different concentrations than the corresponding ATLNP 12 (Met-Tris-3MOA) with Tris as the linker group.

Table 4

The apparent pKa (determination method see Hope et al., Angew. Chem. Int. Ed., 51:1, 2012) and the corresponding gene knockdown effect (KD) of ATLNP13, ATLNP15-18 and SM102-LNP (siRNA) loaded with siRNA were determined, and the results are shown in Figure 4. The pKa value of ATLNP nanoparticles is between 3-5, which indicates that ATLNP nanoparticles have a very low cationic charge at physiological pH (pH 7). There is no obvious correlation between the delivery performance of ATLNP and the apparent pKa value, indicating that ATLNP and SNALP have different delivery mechanisms for siRNA. Combined with the characteristics of the ATL chemical structure, it is inferred that hydrogen bonding is the main and most important interaction for RNA delivery in the ATLNP system.

Although the present invention describes certain embodiments of the amino acid lipids, the delivery systems containing the same, and the preparation methods, and many details have been described for the purpose of illustration, the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (15)

1. An amino acid lipid or a salt thereof represented by the general formula (I) or the general formula (II),
   

   in,

   R ₁ represents an amino acid residue lacking a hydroxyl group on the carboxyl group, and its structural formula is NH ₂ -CHR-CO-, where R is the R group of the amino acid;

   R ₂ , R ₃ and R ₄ are each independently a straight-chain hydrocarbon group having 5 to 40 carbon atoms.
2. The amino acid lipid or its salt according to claim 1, characterized in that the amino acid is glycine, methionine, serine, phenylalanine, alanine, threonine, tyrosine, hydroxyproline, glutamic acid, lysine, cysteine, proline, valine, leucine, isoleucine, tryptophan, glutamine, aspartic acid, asparagine, arginine or histidine.
3. The amino acid lipid or salt thereof according to claim 1, wherein R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkyl group having 5 to 40 carbon atoms or a straight-chain alkenyl group having 1 to 3 double bonds.
4. The amino acid lipid or salt thereof according to claim 3, wherein R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkyl group having 7 to 30 carbon atoms, or a straight-chain alkenyl group having 7 to 30 carbon atoms and containing 1 to 3 double bonds.
5. The amino acid lipid or salt thereof according to claim 4, characterized in that R ₂ , R ₃ , and R ₄ are each independently a straight-chain alkyl group having 7 to 20 carbon atoms, or a straight-chain alkenyl group having 7 to 20 carbon atoms and containing 1 to 3 double bonds.
6. The amino acid lipid or a salt thereof according to claim 1, wherein R ₂ , R ₃ , and R ₄ are the same.
7. The amino acid lipid or salt thereof according to claim 1, characterized in that R ₂ , R ₃ , and R ₄ are independently:
   
8. The amino acid lipid or its salt according to claim 1, characterized in that the amino acid lipid or its salt is a compound represented by general formula (I).
9. The amino acid lipid or its salt according to claim 1, characterized in that the amino acid lipid is:
   
   
10. A delivery system, characterized in that the delivery system comprises one or more of the amino acid lipids or salts thereof according to any one of claims 1 to 9.
11. The delivery system according to claim 10 is characterized in that the delivery system also includes one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids.
12. The delivery system according to claim 11, characterized in that the helper lipid is selected from phospholipids and their derivatives;

    And/or, the PEGylated lipid is selected from one or more of PEG-DMG, PEG-C-DMG, and PEG-DSPE;

    And/or, the molar ratio of the amino acid lipid or its salt, the auxiliary lipid, the cholesterol and its derivatives and the PEGylated lipid is (40-99.5):(0-15):(0-50):(0.5-3).
13. Use of the amino acid lipid or salt thereof according to any one of claims 1 to 9, or the delivery system according to any one of claims 10 to 7 in the preparation of a nucleic acid drug.
14. A nucleic acid drug, characterized in that it comprises the delivery system and nucleic acid molecule according to any one of claims 5 to 7.
15. The nucleic acid drug according to claim 14, characterized in that the nucleic acid molecule comprises one or more of a messenger nucleic acid molecule (mRNA), a small interfering nucleic acid molecule (siRNA), a micronucleic acid molecule (miRNA), a small activating nucleic acid molecule (saRNA), an antisense oligonucleotide molecule (ASO) or an aptamer;

    And/or, the nucleic acid drug is an amino acid lipid nanoparticle with a particle size of 50 to 300 nm;

    And/or, the nucleic acid molecule is self-assembled by mixing the nucleic acid molecule with the amino acid lipid or its salt, and selectively with one or more of auxiliary lipids, cholesterol and its derivatives, and PEGylated lipids through microfluidics to obtain the nucleic acid drug;

    and/or, the nitrogen-phosphorus molar ratio of the amino acid lipid or its salt to the nucleic acid molecule is (1-50):1;

    And/or, the nucleic acid drug further comprises a pharmaceutically acceptable additive;

    And/or, the nucleic acid drug is a lyophilized powder or an injection.