Chiral lipid compounds and compositions thereof, preparation and use

Abstract

The present application provides a chiral lipid compound and a composition thereof, preparation and use, wherein the chiral lipid compound at least comprises a structure of general formula 1 and / or general formula 2 or a pharmaceutically acceptable salt, solvate, chelate, non-covalent compound or precursor compound thereof. The chiral lipid compound of the present application can be used as a new carrier of nucleic acid drugs, has the characteristics of high chiral purity, improved distribution of RNA drugs, high safety and high drug delivery efficiency.

Description

Technical Field

[0001] This invention specifically relates to a class of lipid compounds with chiral centers, their compositions, preparation, and uses. The lipid compounds and their compositions with chiral centers involved in this invention can be used for in vivo delivery of nucleic acid drugs or vaccines, as well as for in vivo delivery of gene-editing drugs, wherein nucleic acid drugs include mRNA, siRNA, ASO, etc. Background Technology

[0002] RNA-based drugs have garnered significant attention worldwide, particularly with the FDA's approval of the mRNA vaccines BNT162b2 (Pfizer / BioNTech) and mRNA-1273 (Moderna) for the prevention of COVID-19. Nucleic acid drugs encompass not only messenger RNA (mRNA) but also antisense oligonucleotides (ASO), small interfering RNA (siRNA), microRNA (miRNA), and other types of messenger RNA. As of the end of 2021, the FDA had approved more than 10 nucleic acid drugs, with several candidates in clinical or preclinical trials.

[0003] However, nucleic acid drugs also face challenges such as poor stability, weak targeting, and difficulty in crossing in vivo barriers. Delivery via nucleic acid drug carriers can improve the stability of nucleic acid drugs in vivo, increase delivery efficiency, and reduce immunogenicity. Currently, commonly used delivery carriers include viral vectors, lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, protein carriers, and exosomes. Most currently marketed mRNA or DNA drugs utilize lipid nanoparticles (LNPs) for delivery. The development of simpler, more practical, less toxic, more efficient, and more precise nucleic acid drug delivery carriers will become a hot topic in nucleic acid drug development.

[0004] Lipid nanoparticles (LNPs) are a formulation that uses lipids to form nanoparticles. In the past, researchers typically used lipid nanoparticles to directly encapsulate chemical drugs. In gene therapy and vaccine research, researchers have begun using lipid nanoparticles to encapsulate nucleic acids, such as mRNA, siRNA, and pDNA, known as nucleic acid lipid nanoparticles. LNPs consist of a mixture of ionizable lipids, helper lipids, sterol lipids, and PEGylated lipids. Ionizable lipids are the main driving force behind nucleic acid payload encapsulation and LNP efficacy, but they are also intertwined with LNP tolerability, immunogenicity, and cytotoxicity.

[0005] Therefore, the present invention provides a new technical solution. Summary of the Invention

[0006] This invention provides a novel class of chiral lipid compounds for delivering therapeutic or preventative medicaments, including pharmaceutically usable salts, solvates, chelates, non-covalent compounds, or prodrugs. The chiral lipid compounds of this invention, together with auxiliary lipids, steroid lipids, and PEGylated lipids, form lipid nanoparticles for the delivery of nucleic acid drugs.

[0007] Neutral assist phospholipids are generally saturated phospholipids, which can increase the phase transition temperature of cationic liposomes, support the formation of layered lipid bilayers and stabilize their structural arrangement; cholesterol has strong membrane fusion properties, promoting intracellular uptake of mRNA and cytoplasmic entry; PEGylated phospholipids are located on the surface of lipid nanoparticles, improving the hydrophilicity of lipid nanoparticles, preventing them from being rapidly cleared by the human immune system, preventing LNP particle aggregation, increasing in vivo stability and prolonging the in vivo circulation time of LNPs.

[0008] Lipid nanoparticles transporting nucleic acids in the human body form "protein crowns" through interactions such as electrostatic and hydrophobic interactions, as well as interactions with various proteins and other macromolecules. These "protein crowns" influence the distribution and metabolism of nucleic acid drugs. Lipids with different chiralities forming LNPs may interact with different proteins or affect the strength of these interactions, thereby increasing or decreasing the nucleic acid transport capacity. Furthermore, the lipids themselves or the protein crown composition may interact with extracellular or intracellular receptors, thus affecting the uptake or release of nucleic acid drugs. Additionally, based on knowledge of small molecule drugs, the chirality of drugs can also affect drug safety and metabolism. Chiral purity ionizable lipids forming LNPs can improve nucleic acid delivery efficiency compared to LNPs formed by racemic compounds. The focus of this invention is to provide a novel delivery carrier with chiral purity for RNA drugs, which can improve RNA drug distribution, has high safety, and high drug delivery efficiency.

[0009] On the one hand, the present invention provides a lipid compound, said compound comprising at least one of the following structurally or pharmaceutically acceptable salts, solvates, chelates, non-covalent compounds or precursor compounds; General Formula 1: Where n1-n4 are each independently chosen as an integer between 0 and 15, and L1, L2, and L4 can be independently chosen. -CH2R1, -CH2OR1, -NHC(=O)R1, -NR2C(=O)R1, -C(=O)OR1,-C(=O)NR1R2, -OC(=O)OR1, -NHC(=O)OR1, -OC(=O)NR1R2, -NHC(=O)NR1R2; where R1 and R2 are independently selected from H, straight-chain or branched alkanes, respectively; Where C* represents a CH group, and there is a chiral center at C*, and its configuration is S configuration; L3 can be independently selected from the following structures: -NR3R4, -N(CH2)m, -N((CH2)aCH2Q)((CH2)bCH3), Among them, R3 and R4 can be independently selected as straight-chain or branched alkanes, or straight-chain or branched alkane alcohols. Where X can be independently chosen from N or CH; m can be independently selected as an integer between 3 and 8; a and b can each be independently chosen as integers between 1 and 5, and Q can be independently chosen as -OH or CN; General Formula 2: Where n1-n4 are each independently chosen as an integer between 0 and 15, and L1, L2, and L4 can be independently chosen. -CH2R1, -CH2OR1, -NHC(=O)R1, -NR2C(=O)R1, -C(=O)OR1, -C(=O)NR1R2, -OC(=O)OR1, -NHC(=O)OR1, -OC(=O)NR1R2, -NHC(=O)NR1R2; where R1 and R2 can be independently selected from H, straight-chain or branched alkanes; Where C* represents a CH group, and there is a chiral center at C*, with an R configuration; L3 can be independently selected from the following structures: -NR3R4, -N(CH2)m, -N((CH2)aCH2Q)((CH2)bCH3), Among them, R3 and R4 can be independently selected as straight-chain or branched alkanes, or straight-chain or branched alkane alcohols. Where X can be independently chosen from N or CH; m can be independently selected as an integer between 3 and 8; a and b can each be independently chosen as integers between 1 and 5, and Q can be independently chosen as -OH or CN.

[0010] Preferably, n1-n4 in Formula 1 and / or Formula 2 are integers between 3 and 10.

[0011] Preferably, in formula 1 and / or formula 2, L1, L2, and L4 are each independently preferably -CH2R1. -CH2OR1, -C(=O)OR1, -C(=O)NR1R2, where R1 and R2 can be independently selected from H, straight-chain or branched alkanes.

[0012] Preferably, in formula 1 and / or formula 2, L3 can be independently selected from the following structures: -NR3R4, -N(CH2)m,-N((CH2)aCH2Q)((CH2)bCH3), Among them, R3 and R4 can be independently selected as straight-chain or branched alkanes; Where X can be independently chosen from N or CH; m can be independently selected as an integer between 4 and 7.

[0013] In a preferred embodiment of the present invention, the lipid compound is an ionizable lipid or a cationic lipid.

[0014] On the one hand, the present invention provides a method for preparing lipid compounds according to the above-mentioned technical solution, using chiral amino acid derivatives as raw materials, and preparing lipid compounds by amide condensation, deprotection, alkylation and amide condensation.

[0015] The specific steps of its preparation method are as follows: a) A chiral amino acid derivative and a primary amine undergo a condensation reaction using tetramethylchlorourea hexafluorophosphate (TCFH) as the condensation reagent, followed by post-treatment to obtain product I; b) Product I was protected with diethylamine to remove Fmoc (Fmoc is 9-fluorenylmethoxycarbonyl), and then post-treated to obtain product II; c) Product II and the brominated product were alkylated using acetonitrile as solvent and diisopropylethylamine (DIEA) as base to give product III; d) Product III and the acid undergo a condensation reaction with the condensing agent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and the final product is obtained by passing through a silica gel column.

[0016] On the one hand, the present invention also provides a lipid composition comprising steroids, structured lipids, polymers and lipid compounds as described in the above technical solutions.

[0017] In a preferred embodiment of the present invention, the lipid compound is an ionizable lipid or a cationic lipid.

[0018] As a preferred embodiment of the present invention, the steroid includes at least the following structure: .

[0019] As a preferred embodiment of the present invention, the structural lipids include, but are not limited to, one or more of distearylphosphatidylcholine (DSPC), dilinoleylphosphatidylethanolamine (DOPE), and dioleoyllecithin (DOPC).

[0020] As a preferred embodiment of the present invention, the polymer lipids include, but are not limited to, polysarcosine, polybetaine methacrylate (DSPE-PCB20), dimyristic-sn-glycerol-polyethylene glycol 2K (DMG-PEG2k), distearate phosphatidylethanolamine-polyethylene glycol 2K (DSPE-PEG2k), or C14-polyethylene glycol 2K (C14-PEG2k).

[0021] In a preferred embodiment of the invention, the lipid composition includes a carrier for delivering a therapeutic or preventative agent, or the therapeutic or preventative agent itself. Preferably, the therapeutic or preventative agent is a nucleic acid drug, a nucleic acid-like drug, or a small molecule chemical drug. More preferably, nucleic acid drugs and nucleic acid-like drugs include nucleic acid aptamers. Nucleic acid aptamers include circular RNA, ASO, siRNA, sgRNA, miRNA, tRNA, or mRNA.

[0022] As a preferred embodiment of the present invention, the mass ratio of the carrier to the treatment or preventive agent is 1:1 to 100:1.

[0023] In a preferred embodiment of the present invention, the lipid composition is a nanoparticle formulation. Specifically, the average size of the nanoparticle formulation is 20 nm to 1000 nm, and the polydispersity index of the nanoparticle formulation is ≤0.5.

[0024] On one hand, the present invention provides a pharmaceutical composition, a lipid composition, and a nucleic acid composition to form a pharmaceutical composition. Preferably, the pharmaceutical composition further comprises one or more of a buffer, carbohydrate, mannitol, protein, polypeptide or amino acid, antioxidant, antibacterial agent, chelating agent, and adjuvant.

[0025] On the one hand, lipid compositions can serve as carriers for delivering therapeutic or preventative agents.

[0026] On the one hand, the drug compositions provided by the present invention can be administered via methods including but not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, ear administration, nasal administration, oral administration, etc.

[0027] As a preferred embodiment of the present invention, the target of administration of the pharmaceutical composition includes, but is not limited to, mammals, birds, non-human primates, or humans. Among them, mammals include cattle, horses, mules, donkeys, camels, pigs, sheep, dogs, foxes, rabbits, etc.; birds include chickens, ducks, geese, pigeons, quails, etc.; and non-human primates include monkeys, apes, humans, etc.

[0028] This document includes the following summary: SHA-001 SHA-002 SHA-003 SHA-004 SHA-005 SHA-006 SHA-007 SHA-008 SHA-009 SHA-010 SHA-011 SHA-012 SHA-013 SHA-014 SHA-015 SHA-016 SHA-017 SHA-018 SHA-019 SHB-001 SHB-002 SHB-003 SHB-004 SHB-005 SHB-006 SHB-007 SHB-008 SHB-009 SHB-010 SHB-011 SHB-012 SHB-013 SHB-014 SHB-015 SHB-016 SHB-017 SHB-018 SHB-019 Attached Figure Description Figure 1 This is a synthetic route diagram for chiral molecules; Figure 2 The proton NMR spectrum of compound II-3; Figure 3 The proton NMR spectrum of compound II-4; Figure 4 The proton NMR spectrum of compound SHA-001; Figure 5 Whole-body imaging of mice after drug administration; Figure 6 Whole-body imaging of mice after drug administration; Figure 7 Liver and spleen imaging in mice after drug administration. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the present invention will be described in further detail and completely below with reference to the embodiments. Where specific conditions are not specified in the embodiments, they can be obtained under conventional conditions. Where the detailed source of reagents or instruments is not specified in the embodiments, they can all be obtained through commercially available or conventional methods. Furthermore, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0030] Example 1: Synthesis of chiral lipid compounds Step 1: Synthesis of intermediate I-3: Succinic acid I-1 (10 g, 57.3 mmol), EDCI (5.9 g, 30.4 mmol), and DMAP (0.93 g, 7.8 mmol) were added to dichloromethane (150 mL), followed by DIPEA (7.89 g, 61.3 mmol). The reaction mixture was stirred at room temperature for 15 min, and then 9-heptadecyl alcohol I-2 (4.9 g, 19.1 mmol) was added. The reaction mixture was stirred at room temperature for 16 h.

[0031] The solvent was removed by rotary evaporation. The remaining oily substance was added to 100 mL of ethyl acetate and 100 mL of saturated sodium bicarbonate aqueous solution and separated. The organic phase was washed once with 50 mL of saturated sodium bicarbonate aqueous solution. Then it was washed once with 0.5 M (50 mL) hydrochloric acid aqueous solution and once with 50 mL of saturated saline solution. The solvent was then removed by evaporation to obtain 7.5 g of crude pale yellow oily substance.

[0032] The product-containing fraction was collected by column chromatography using PE / EA as the mobile phase and the solvent was evaporated to obtain 6 g of colorless oil (yield 76%), HPLC: 98%.

[0033] Step Two: Synthesis of intermediate II-3: (S)-N-Fmoc-ortholeucine (6.00 g, 16.98 mmol), octylamine (2.41 g, 18.67 mmol), and N-methylimidazole (4.18 g, 50.94 mmol) were dissolved in acetonitrile (50 mL) and dichloromethane (100 mL), and then TCFH (5.24 g, 18.67 mmol) was added. The reaction mixture was stirred at room temperature for 2 h.

[0034] The solvent in the reaction mixture was evaporated using a rotary evaporator. 100 mL of dichloromethane was added, followed by washing once with 100 mL of water. The organic phase was then washed once with 20 mL of saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated from the organic phase to obtain the crude product. This crude product was then subjected to silica gel column chromatography with DCM / MeOH (99:1–98:2) as elution, yielding 7.00 g of a white waxy solid (yield 88.7%).

[0035] The proton NMR spectrum of intermediate II-3 is as follows: Figure 2 As shown, 1 HNMR (400 MHz, CDCl3) δ 7.76 (d, J =8.0,2H), 7.58 (d, J = 8.0, 2H), 7.40 (t, J = 8.0, 2H), 7.33-7.29 (m, 2H), 5.96 (br,0.65H), 5.36 (br, 0.68H), 4.44-4.39 (m, 2H), 4.21 (t, J = 8.0, 1H), 4.13-4.07(m, 1H), 3.28-3.19 (m, 2H), 1.83 (br, 1H), 1.71 (s, 2H), 1.50-1.46 (m, 2H), 1.32-1.24 (m, 15H), 0.92-0.85 (m, 6H). Step 3: Synthesis of intermediate II-4: II-3 (7.00 g, 15.06 mmol) was dissolved in dichloromethane (80 mL), and then diethylamine (54 mL) was added. The reaction solution was stirred at room temperature for 2 h. The solvent was then evaporated from the reaction solution using a rotary evaporator to obtain the crude product. Then, silica gel column chromatography was used to elute with DCM / MeOH (98:2-94:6) to give 1.63 g of colorless oil (yield 44.9%).

[0036] The proton NMR spectrum of intermediate II-4 is as follows: Figure 3 As shown, 1 HNMR (400 MHz, CDCl3) δ 7.33-7.30(m,1H), 3.44-3.41 (m, 1H), 3.26-3.20 (m,2H), 2.23 (s,3H), 1.88-1.80 (m, 1H),1.58-1.46 (m, 3H), 1.39-1.25 (m, 15H), 0.92-0.85 (m, 6H). HPLC: 92.7%.

[0037] Step Four: Synthesis of intermediate II-6: II-4 (0.80 g, 3.30 mmol), 3-bromo-N,N-dimethyl-1-propanamine hydrobromide (1.14 g, 4.62 mmol), and DIPEA (1.7 mL) were dissolved in DMF (5 mL), and the reaction mixture was stirred at 60 °C for 15 h.

[0038] The solvent in the reaction solution was directly evaporated under reduced pressure using a rotary evaporator to obtain the crude product; then, silica gel column chromatography was performed, eluting with DCM / MeOH (98:2-90:10) + 0.5% ammonia, to give 170 mg of a yellow oil (yield 15.7%). HPLC: 95.1%.

[0039] Step 5: Synthesis of SHA-001: II-6 (170 mg, 0.52 mmol / L), I-3 (257 mg, 0.62 mmol / L), HATU (276 mg, 0.73 mmol / L), and DIPEA (0.2 mL) were dissolved in DMF (2 mL), and the reaction mixture was stirred at room temperature for 15 h.

[0040] Add 50 mL of ethyl acetate to the reaction solution, then wash three times with 50 mL of water, and wash the organic phase once with 10 mL of saturated brine. Dry the phase with anhydrous sodium sulfate and evaporate the solvent using a rotary evaporator to obtain the crude product. Then, use silica gel column chromatography to elute with DCM / MeOH (98:2-94:6) + 0.5% ammonia to give 280 mg of a pale yellow oil (yield 53%).

[0041] The proton NMR spectrum of compound SHA-001 is shown below. Figure 4 As shown, 1 HNMR (400 MHz, CDCl3) δ6.66(br, 1H), 4.89-4.82 (m, 1H), 4.70 (t, J = 8.0,1H), 3.40-3.23 (m, 2H), 3.19-3.14(m, 2H), 2.45-2.33 (m, 2H), 2.29-2.23 (m, 3H), 2.22 (s, 6H), 2.00-1.91 (m,1H), 1.80-1.70 (m, 2H), 1.68-1.59 (m, 6H), 1.52-1.42 (m, 6H), 1.37-1.32 (m, 6H), 1.29-1.15 (m, 38H), 0.93-0.86 (m, 12H). HPLC: 94.4%, M+H + =722.8.

[0042] 1. Synthesis of SHA-002: Synthesis of raw materials: Following the synthetic route of intermediate I-3 in step one of the examples, intermediate I-4 was obtained by using 7-tridecyl alcohol. Then, (S)-N-Fmoc-octylglycine was substituted for (S)-N-Fmoc-ortholeucine, following steps two to five of the examples, to obtain the final product SHA-002, a pale yellow oil. Its MS [ESI] was measured: MS+H + =722.8.

[0043] 2. Synthesis of SHA-003: Synthesis of intermediate III-3: Synthesis of intermediate III-3: Boc-6-aminohexanoic acid III-1 (5 g, 20.2 mmol), EDCI (5.9 g, 30.4 mmol), and DMAP (0.24 g, 2 mmol) were added to dichloromethane (100 mL), followed by DIPEA (7.81 g, 60.6 mmol). The reaction mixture was stirred at room temperature for 15 min, and then 9-heptadecyl alcohol I-2 (5.2 g, 20.2 mmol) was added. The reaction mixture was stirred at room temperature for 6 h.

[0044] The solvent was removed by rotary evaporation. The remaining oily substance was added to 80 mL of ethyl acetate and 80 mL of saturated sodium bicarbonate aqueous solution, and the mixture was separated. The organic phase was washed once with 50 mL of saturated sodium bicarbonate aqueous solution, and then once with 50 mL of saturated brine. The solvent was removed by evaporation to obtain 11 g of crude, pale yellow oily substance.

[0045] The product-containing fraction was collected by column chromatography using PE / EA as the mobile phase and the solvent was evaporated to obtain a colorless oily substance III-28.2 g (yield 84%), HPLC: 98%.

[0046] The product III-2 (8g) obtained in the previous step was dissolved in dichloromethane (120ml), and trifluoroacetic acid (TFA) (12ml) was added under ice bath. The reaction was stirred at room temperature for 3h, and the reaction solution was directly evaporated to dryness. Then, dichloromethane and saturated sodium bicarbonate aqueous solution were added for extraction. The organic phase was washed once with 80 mL of saturated brine and evaporated to dryness to obtain oily product III-3 (5g), which was directly used in the next step.

[0047] Referring to Example 1, in step two, n-octylamine was replaced with III-3 to obtain SHA-003 oily substance, and its MS [ESI] was detected: MS+H + =962.9.

[0048] 3. Synthesis of SHA-004: Referring to the synthesis of SHA-003, 7-tridecyl alcohol was used instead of 9-heptadecyl alcohol in the synthesis of intermediate III-3 to obtain SHA-004 as an oil. Its MS [ESI] was analyzed: MS+H + =906.8.

[0049] 4. Synthesis of SHA-005: Referring to the synthesis of SHA-004, intermediate I-3 was used instead of I-4 to obtain the oily SHA-005. Its MS[ESI] was analyzed: MS+H + =962.8.

[0050] 5. Synthesis of SHA-006: Referring to the synthesis of SHA-003, 7-tridecyl alcohol was replaced with 9-heptadecyl alcohol in the synthesis of intermediate I-3 to obtain SHA-006 as an oil. Its MS [ESI] was analyzed: MS+H + =1018.9.

[0051] 6. Synthesis of SHA-007: Referring to the synthesis of SHA-006, Boc-6-aminohexanoic acid was substituted for Boc-5-aminovaleric acid in the synthesis of intermediate III-3 to obtain SHA-007 oil. Its MS [ESI] was analyzed: MS+H + =1004.9.

[0052] 7. Synthesis of SHA-008: Referring to the synthesis of SHA-002, in step four of Example 1, N,N-dimethylaminobromoethane hydrobromide was used instead of 3-bromo-N,N-dimethyl-1-propylaminohydrobromide, and intermediate I-3 was used instead of intermediate I-4 to obtain SHA-008 oily precipitate. Its MS [ESI] was analyzed: MS + H + =764.8.

[0053] 8. Synthesis of SHA-009 Synthesis of intermediate IV-1: Referring to the synthesis of SHA-008, in step five of the example, IV-1 was used instead of I-3, and in step two, n-octylamine (II-2) was replaced with III-3, resulting in an oily SHA-008 compound. Its MS [ESI] was analyzed: MS+H + =892.9.

[0054] 9. Synthesis of SHA-010 Referring to the synthesis of SHA-008, in step three of Example 1, 4-bromo-N,N-dimethyl-1-butylamine hydrobromide was used instead of N,N-dimethylaminobromoethane hydrobromide to obtain SHA-010 oily precipitate. Its MS [ESI] was analyzed: MS + H + =792.8.

[0055] 10. Synthesis of SHA-011 Referring to the synthesis of SHA-008, in step three of Example, 1-(3-bromopropyl)-1H-imidazolium hydrobromide was used instead of N,N-dimethylaminobromoethane hydrobromide to obtain SHA-011 oily substance, and its MS[ESI] was measured to be MS+H+=801.9.

[0056] 11. Synthesis of SHA-012 Referring to the synthesis of SHA-005, in step three of Example 1, 4-bromo-N,N-dimethyl-1-butylamine hydrobromide was used instead of 3-bromo-N,N-dimethyl-1-propylamine hydrobromide to obtain SHA-012, a yellow oily substance, whose MS[ESI]:MS+H+=976.9.

[0057] 12. Synthesis of SHA-013 Referring to the synthesis of SHA-002, in step three of Example 1, 1-(3-bromopropyl)pyrrolidine hydrobromide was used instead of 3-bromo-N,N-dimethyl-1-propanamine hydrobromide to obtain SHA-013, a pale yellow oily substance, whose MS[ESI]:MS+H+=748.7.

[0058] 13. Synthesis of SHA-014 Referring to the synthesis of SHA-008, in step three of Example 1, 1-(3-bromopropyl)pyrrolidine hydrobromide was used instead of N,N-dimethylaminobromoethane hydrobromide to obtain SHA-014, a pale yellow oily substance, whose MS[ESI]:MS+H+=804.8 was measured.

[0059] 14. Synthesis of SHA-015 Referring to the synthesis of SHA-014, in step two of the example, (S)-N-Fmoc-octylglycine was replaced with (S)-2-(Fmoc-amino)heptanoic acid to obtain SHA-015, a pale yellow oily substance, whose MS[ESI]:MS+H+=762.8.

[0060] 15. Synthesis of SHA-016 Referring to the synthesis of SHA-014, in step two of the example, (S)-N-Fmoc-octylglycine was replaced with Fmoc-L-ortholeucine to obtain SHA-016, a pale yellow oily substance, whose MS[ESI]:MS+H+=748.9.

[0061] 16. Synthesis of SHA-017 Referring to the synthesis method of SHA-014, the starting material in step five was replaced with 3-methylpentanoic acid instead of I-3. Other steps were performed according to the synthesis method of SHA-014. The final product SHA-017 was obtained as a pale yellow oily substance, and its MS[ESI]:MS+H+=508.5 was measured.

[0062] 17. Synthesis of SHA-018 Referring to the synthesis method of SHA-014, the intermediate 1-(3-bromopropyl)pyrrolidine hydrobromide in step three of Example was replaced with 3-bromo-N,N-dimethyl-1-propanamine hydrobromide. Other steps were performed according to the synthesis method of SHA-014. The final product SHA-018 was a pale yellow oily substance, and its MS[ESI]:MS+H+=778.8 was detected.

[0063] 18. Synthesis of SHA-019 Referring to the synthesis of SHA-002, in step two of the embodiment, ( S Replacing )-N-Fmoc-octylglycine with (S)-2-(Fmoc-amino)octanoic acid yielded SHA-019, a pale yellow oily substance. Its MS [ESI] assay was performed using MS + H₂O. + =750.8.

[0064] The above describes the synthetic method for synthesizing chiral lipids using S-configured amino acid derivatives as starting materials. The synthetic methods for lipid compounds SHB-001 to SHB-019 are similar to those described above, using R-configured amino acid derivatives as starting materials. The purity of all compounds is greater than 90% (HPLC). Several pairs of compounds were randomly selected from all compounds for specific rotation testing to confirm chirality. The specific rotation test results are shown in Table 1 below.

[0065] Example 2: Specific curl test: Using a Shanghai Shenguang automatic polarimeter (model: WZZ-ZS), the instrument was first calibrated using standard colorimetric tubes (calibrating levorotatory and dextrorotatory polarimetry separately) to verify the accuracy of the equipment and ensure the reliability of the sample detection data. Then, ethanol solutions of the sample at different concentrations (2g / 100ml, 1g / 100ml, 0.5g / 100ml, 0.25g / 100ml) were prepared, and the instrument temperature was set to 22°C. o C. Test the optical rotation values ​​of samples with different concentrations. Each sample is tested in parallel three times. Then, the specific rotation is calculated according to the specific rotation calculation formula.

[0066] The formula for calculating specific curl is as follows: The specific rotations of the levorotatory and dextrorotatory lipid compounds are calculated and shown in Table 1 below: Table 1: Specific rotation of lipid compounds Chiral molecules possess an asymmetric structure, resulting in optical activity. Specific rotation is an important physical constant for chiral molecules, used to characterize their properties and playing a crucial role in the quality control of chiral molecules. The optical rotation of chiral molecules can be levorotatory or dextrorotatory, indicated by "-" and "+". If a molecule is racemic, its specific rotation is 0. From the data in Table 1, the specific rotation of the R configuration is positive, while that of the S configuration is negative, indicating that the specific rotation values ​​of the R and S configurations are symmetrical. Example 3: Preparation and testing of lipid nanoparticle assemblies 1) Preparation of lipid nanoparticle compositions To investigate safe and effective lipid nanoparticle compositions for delivering therapeutic or preventative agents to cells, a series of nanolipid compositions were prepared and tested. Specific components and their ratios within the lipid components of the lipid nanoparticle compositions were optimized. For better data analysis and comparison, a control, SM102 (an ionizable cationic lipid from the FDA-approved COVID-19 vaccine Spikevax), was introduced into the experiments.

[0067] Lipid nanoparticles were prepared using an optimized process. LNPs were prepared using the aforementioned ionizable lipids and other components, including DSPC, cholesterol, and PEG lipids. The specific process is as follows: Ionizable lipids:DSPC:cholesterol:PEG lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. mRNA was diluted with citrate buffer at pH 4. The total flow rate of the aqueous and ethanol phases was controlled within the range of 6-30 ml / min using a microfluidic device. The ethanol solution of lipids and the aqueous solution of mRNA were mixed at a volume ratio of 1:3. LNP-mRNA was prepared at a total lipid to mRNA mass ratio of approximately 10:1 to 30:1. The ethanol was then removed by dialysis and replaced with PBS (phosphate-buffered saline). Finally, the lipid nanoparticles were filtered through a sterile filter to obtain the final product, LNP-mRNA.

[0068] 2) Characterization of nanoparticle assemblies Determination of physicochemical properties of nanolipid particles: The particle size and polydispersity index (PDI) of the nanolipid particle composition were determined using a Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). The nanolipid particle size was determined in 1×PBS.

[0069] Determination of nanoparticle encapsulation efficiency: For nanolipid particle compositions containing LNP-mRNA, the encapsulation of mRNA by the nanoparticle composition can be evaluated using QUANT-IT™ RNA (Invitrogen Corporation Carlsbad, CA). The specific test method is as follows: Dilute the sample to approximately 5 μg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Transfer 50 μL of the diluted sample to a polystyrene 96-well plate and add 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution to each well. Incubate at 37°C for 15 minutes. Dilute the reagent 1:100 in TE buffer and add 100 μL of this solution to each well. The fluorescence intensity can be measured using a fluorescent plate reader (VICTOR® Nivo™ Multimode Plate Readers, PerkinElmer, GER) at an excitation wavelength of approximately 480 nm and an emission wavelength of approximately 520 nm. The percentage of free RNA was determined by subtracting the fluorescence value of the reagent blank from the fluorescence value of each sample, and by dividing the fluorescence intensity of the complete sample (without Triton X-100) by the fluorescence value of the destroyed sample (caused by the addition of Triton X-100). Specific data are shown in Table 2.

[0070] Table 2: Characteristics of nanoparticle assemblies prepared from different compounds Example 4: In vitro cell transfection experiment of lipid nanoparticles 293T cells were seeded at a density of 3 x 10^4 cells / well in 96-well cell culture plates and cultured for 24 h until adherence. Following the preparation method in Example 3, the compounds listed in Table 2 above were prepared into lipid nanoparticle formulations according to the method in Example 3 and added to the cells. After culturing for 24 h, chromogenic buffer was added, and the luminescence value of each well was detected using a microplate reader. Table 3 below shows that the lipid nanoparticle formulation can carry mRNA and transfect into 293T cells to express the target fluorescent protein. Commercially available SM102 was used as a positive control. A, B, and C represent the normalized results after comparing the luminescence flux of the compound and SM102-LNP, as shown in Table 3.

[0071] Note: A, B, and C represent the normalized results after comparing the luminous flux of the compound with that of SM102-LNP, with the following ratio ranges: A: ≥0.8 B: ≥0.1 and <0.8 C: <0.1 Table 3: Evaluation results of in vitro cell transfection with lipid nanoparticles Example 5: Fluorescent protein expression experiment of lipid nanoparticle formulation in mice Samples were prepared according to the method for preparing nanoparticle assemblies in Example 3, with SM102-LNP as a positive reference.

[0072] Female SPF-grade BALB / c mice (n=3) aged 6-8 weeks were housed in an SPF-grade animal facility at a temperature of 20-26℃ and humidity of 40-70%, with ample feed and water. The formulation was prepared using the method described in Example 3, with 100 μL (0.05 μg / μL) administered intramuscularly. The control group used SM102-LNP for in vivo efficacy comparison. Six hours after administration, 200 μL of D-fluorescein potassium salt at a concentration of 15 mg / ml was injected intraperitoneally. In vivo imaging was performed 5 minutes after D-fluorescein potassium salt injection. After in vivo imaging of all groups was completed, D-fluorescein potassium salt was injected again, and organ imaging was performed 5 minutes later. Commercially available SM102 was used as a control group. In whole-body imaging, as shown... Figure 5-7 As shown, SM102 muscle expression reached 6*10. 9 p / s, some lipids of this invention can reach 1*10 10 p / s; SM102 expression in the spleen reached 3*10 7 p / s, some lipids of this invention can reach 8*10 7 p / s, the results show that the LNPs prepared by compounds SHA-005, SHA-009, SHA-010, SHA-013, SHB-007, and SHB-018 in this invention achieve mRNA delivery effects reaching even the level of SM102. Furthermore, this experiment also observed the systemic and liver / spleen distribution of mRNA. We observed that different conformations of chiral compounds exhibited different systemic and liver / spleen distributions. For example, SHA-014 and SHB-014, SHA-005 and SHB-005 showed differences in liver expression, while SHA-018 and SHB-018, SHA-011 and SHB-011 showed differences in muscle expression.

[0073] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. The above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, and variations to the above embodiments within the scope of the present invention.

Claims

1. A chiral lipid compound, characterized in that, The compound comprises at least one general structural formula as follows: General Formula 1: Where n1-n4 are independently chosen as integers between 0 and 15, and L1, L2, and L4 are independently chosen as -CH2R1. -CH2OR1, -NHC(=O)R1, -NR2C(=O)R1, -C(=O)OR1, -C(=O)NR1R2, -OC(=O)OR1, -NHC(=O)OR1, -OC(=O)NR1R2, -NHC(=O)NR1R2; where R1 and R2 are independently selected from H, straight-chain or branched alkanes; Where C* represents a CH group, and there is a chiral center at C*, and its configuration is S configuration; L3 can independently choose from the following structures: -NR3R4, -N(CH2)m, -N((CH2)aCH2Q)((CH2)bCH3), Among them, R3 and R4 are independently selected as straight-chain or branched alkanes, or straight-chain or branched alkane alcohols, respectively. X can independently choose N or CH; m can be an integer between 3 and 8; a and b are each independently chosen as integers between 0 and 5, and Q is independently chosen as -OH or CN; General Formula 2: Where n1-n4 are independently selected as integers between 0 and 15, and L1, L2 and L4 are independently selected... -CH2R1, -CH2OR1, -NHC(=O)R1, -NR2C(=O)R1, -C(=O)OR1, -C(=O)NR1R2, -OC(=O)OR1, -NHC(=O)OR1, -OC(=O)NR1R2, -NHC(=O)NR1R2; where R1 and R2 are independently selected from H, straight-chain or branched alkanes; Where C* represents a CH group, and there is a chiral center at C*, with an R configuration; L3 can independently choose from the following structures: -NR3R4, -N(CH2)m, -N((CH2)aCH2Q)((CH2)bCH3), Among them, R3 and R4 are independently selected as straight-chain or branched alkanes, or straight-chain or branched alkane alcohols, respectively. X can independently choose N or CH; m can be an integer between 3 and 8; a and b each independently choose an integer between 0 and 5, and Q independently chooses OH and CN.

2. The lipid compound according to claim 1, characterized in that, In general formula 1 and / or general formula 2, n1-n4 are each independently chosen as an integer between 3 and 10.

3. The lipid compound according to claim 1, characterized in that, In general formula 1 and / or general formula 2, L1, L2 and L4 are independently -CH2R1, -CH2OR1, -C(=O)OR1 or -C(=O)NR1R2, respectively, wherein R1 and R2 are independently selected from H, straight-chain or branched alkanes.

4. The lipid compound according to claim 1, characterized in that, In general formula 1 and / or general formula 2, L3 is independently selected from the following structures: -NR3R4, -N(CH2)m, -N((CH2)aCH2Q)((CH2)bCH3), ; Among them, R3 and R4 can be independently selected as straight-chain or branched alkanes; Where X can be independently chosen from N or CH; m independently selects an integer between 4 and 7.

5. The lipid compound according to any one of claims 1-4, characterized in that, Lipid compounds also include their pharmaceutically available salts, solvates, chelates, non-covalent compounds, or precursor compounds.

6. The method for preparing lipid compounds according to any one of claims 1-4, using chiral amino acid derivatives as raw materials, prepares lipid compounds through amide condensation, deprotection, alkylation, and amide condensation; the specific steps are as follows: a) A chiral amino acid derivative and a primary amine undergo a condensation reaction using tetramethylchlorourea hexafluorophosphate as a condensing agent, followed by post-treatment to obtain product I; b) Product I was deprotected from Fmoc by diethylamine and then post-treated to obtain product II; c) Product II and the brominated product were alkylated using acetonitrile as solvent and diisopropylethylamine as base to give product III; d) Product III and the acid undergo a condensation reaction with the condensing reagent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and the final product is obtained by passing through a silica gel column.

7. A lipid composition, characterized in that, The lipid composition includes steroids, structured lipids, polymers, and lipid compounds as described in any one of claims 1-5, wherein the lipid compounds are ionizable lipids or cationic lipids.

8. The lipid composition according to claim 7, characterized in that, The steroids include at least the following structures: 。 9. The lipid composition according to claim 7, characterized in that, The structural lipids include one or more of distearylphosphatidylcholine, dilinoleylphosphatidylethanolamine, and dioleoyllecithin.

10. The lipid composition according to claim 7, characterized in that, The polymer lipids include polysarcosine, polybetaine methacrylate, dimyristicoyl-sn-glycerol-polyethylene glycol 2K, distearate phosphatidylethanolamine-polyethylene glycol 2K, or C14-polyethylene glycol 2K.

11. The lipid composition according to claim 7, characterized in that, The lipid composition includes a therapeutic or preventive agent and a carrier for delivering the therapeutic or preventive agent.

12. The lipid composition according to claim 11, characterized in that, The treatment or preventive agent is a nucleic acid drug, a nucleic acid-like drug, or a small molecule chemical drug. Nucleic acid-like drugs include nucleic acid aptamers, circular RNA, ASO, siRNA, sgRNA, miRNA, tRNA, or mRNA.

13. The lipid composition according to claim 11, characterized in that, The mass ratio of the carrier to the treatment or preventive agent is 1:1 to 100:

1.

14. The lipid composition according to any one of claims 7-13, characterized in that, The lipid composition is a nanoparticle formulation with an average size of 20 nm to 1000 nm and a polydispersity index of ≤0.

5.

15. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises nucleic acids and lipid compositions as described in any one of claims 7-14, and further comprises one or more of buffers, carbohydrates, mannitol, proteins, peptides or amino acids, antioxidants, antibacterial agents, chelating agents, and adjuvants.

16. The pharmaceutical composition according to claim 15, wherein the administration method includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intratumoral injection, ocular administration, ear administration, nasal administration, and oral administration.

17. The pharmaceutical composition according to claim 15 or 16, wherein the target of administration includes, but is not limited to, mammals, birds, non-human primates (monkeys, apes, or humans).

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