Cationic lipid compounds, lipid nanoparticles constructed therefrom, pharmaceutical compositions and uses thereof

Abstract

The application discloses a cationic lipid compound, a lipid nanoparticle constructed by the cationic lipid compound, a pharmaceutical composition and purposes, the lipid nanoparticle constructed by the cationic lipid compound (formula I) has small particle size and low polymorphous index, has extremely high encapsulation efficiency on a drug molecule, can accurately deliver the mRNA drug loaded therein to the liver under the premise of keeping high expression intensity, is hardly expressed in other organs, such as lungs, hearts and kidneys, is slightly expressed in the spleen, and has important clinical application significance in cancer treatment.

Description

Technical Field

[0001] This invention relates to a cationic lipid compound and lipid nanoparticles thereof, pharmaceutical compositions and uses thereof, and more particularly to a cationic lipid compound with liver-targeting properties and lipid nanoparticles thereof, pharmaceutical compositions and uses thereof. Background Technology

[0002] Liver cancer, a common malignant tumor, has become one of the most pressing medical challenges worldwide due to its high malignancy and mortality rate. Traditional treatments such as surgical resection, chemotherapy, and radiotherapy have offered hope to patients to some extent, but they also have many limitations, such as significant side effects, high recurrence rates, and limited treatment scope. With the rapid development of medical technology, messenger RNA (mRNA) therapy, as an emerging treatment method, has brought new hope to liver cancer patients and plays an extremely important role in the comprehensive treatment of liver cancer.

[0003] Messenger RNA (mRNA) therapy, as a disruptive technology with broad application prospects, relies heavily on efficient and safe delivery systems for its success. Lipid nanoparticles (LNPs), currently the most clinically mature non-viral delivery system, are typically composed of cationic or ionizable lipids, phospholipids, steroids, and polyethylene glycol (PEG)-modified lipids. Among these, cationic or ionizable lipids are the core of the LNP delivery system, playing a crucial role in nucleic acid encapsulation, cellular uptake, and intracellular release, and even in targeting.

[0004] The efficient delivery of nucleic acid drugs remains a challenge, primarily because different types of nucleic acid drugs have varying requirements for delivery systems, and existing delivery systems based on traditional commercial lipids still have limitations in efficiency and applicability. For example, the commercially available MC3 lipid is mainly optimized for liver-targeted delivery of small nucleic acid drugs, while SM-102 and ALC-0315 are designed specifically for low-dose mRNA vaccines (such as COVID-19 vaccines), typically achieving high delivery efficiency in these applications. However, in emerging therapeutic scenarios such as mRNA-based gene editing therapies, not only does the required nucleic acid dose increase significantly, but higher demands are also placed on the targeting precision of the delivery system. Traditional lipid nanoparticles often fail to deliver efficiently in such high-dose, high-precision scenarios, limiting their therapeutic efficacy. Summary of the Invention

[0005] Objectives of the Invention: The first objective of this invention is to provide a cationic lipid compound with high liver-targeting properties. The second objective of this invention is to provide lipid nanoparticles constructed from the cationic lipid compound and other lipid components. The third objective of this invention is to provide a pharmaceutical composition constructed from the lipid nanoparticles that can efficiently deliver nucleic acid molecules to the liver. The fourth objective of this invention is to provide the pharmaceutical uses of the cationic lipid compound, the lipid nanoparticles constructed from it, and the pharmaceutical composition.

[0006] Technical solution: The cationic lipid compound of the present invention, or its pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer, wherein the compound has the structure of Formula I:

[0007] ,

[0008] in,

[0009] X1, X2, and X3 are each independently selected from -C-, -O-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -S-, -S(O)-, -C(O)S-, -SC(O)-, -OC(S)-, -C(S)O-, -SC(O)O-, -OC(O)S-, -SS-, -S(O)2-;

[0010] R1 and R2 are each independently selected from C 2~16 Straight-chain or branched alkyl, C 2~16 Straight-chain or branched alkenyl groups, C 2~16 A straight-chain or branched alkynyl group, or one or more of the methylene units therein, is independently replaced by one or more R' or -NR”-;

[0011] R' is independently selected from H and C. 1~14 Straight-chain or branched alkyl groups, -L a -OR a -、-L a -SR a -、-L a -NR a '-;

[0012] R”, ​​L a R a and R a Each is independently selected from H and C 1~14 Straight-chain or branched alkyl groups;

[0013] R3 is independently selected from -OH, -NC2H6, or -NC4H 10 ;

[0014] n1 and n6 are each independently selected from 1 to 6, n2 and n3 are each independently selected from 1 to 10, and n4 and n5 are each independently selected from 1 to 5.

[0015] The term "pharmaceutically acceptable" in this invention means that the compound or composition is chemically and / or toxicologically compatible with other components constituting the preparation and / or with humans or mammals using it to prevent or treat diseases or conditions. The term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic acid or organic acid addition salt of the compounds of this invention.

[0016] It should be further understood that compounds of formula I or pharmaceutically acceptable salts thereof can be isolated as solvates, and therefore any such solvates are included within the scope of this invention. For example, compounds of formula I or pharmaceutically acceptable salts thereof may exist in unsolvated forms as well as in solvated forms formed with pharmaceutically acceptable solvents (such as water, ethanol, etc.).

[0017] The term "solvent" in this invention refers to a complex formed by combining a compound of Formula I or a pharmaceutically acceptable salt thereof with a solvent (e.g., ethanol or water). It should be understood that any solvate of a Formula I compound used in the treatment of a disease or condition, although it may provide different properties (including pharmacokinetic properties), will yield a Formula I compound once absorbed into the subject, such that the use of a Formula I compound separately encompasses the use of any solvate of a Formula I compound.

[0018] The term "hydrate" refers to the case where the solvent in the aforementioned term "solvent" is water.

[0019] Some compounds of this invention can exist in the form of one or more stereoisomers. Stereoisomers include geometric isomers, diastereomers, and enantiomers. Therefore, the compounds claimed in this invention also include racemic mixtures, single stereoisomers, and optically active mixtures. Those skilled in the art will understand that one stereoisomer may have better efficacy and / or fewer side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained using chiral source synthesis, chiral catalysis, chiral resolution, etc. Racemates can be chirally resolved by chromatographic or chemical resolution.

[0020] This invention also includes all suitable isotopic variants of the compounds of this invention. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but whose atomic mass differs from that of atoms commonly or predominantly found in nature.

[0021] Preferably, in the structure, X1 is selected from -O- or -C(O)O-.

[0022] Preferably, in the structure, X2 and X3 are each independently selected from -C(O)O- or -OC(O)-.

[0023] Preferably, in the structure, R1 and R2 are each independently selected from any of the following substituents:

[0024] .

[0025] Preferably, the cationic lipid compound, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, is selected from any of the following compounds:

[0026] .

[0027] Further preferably, the cationic lipid compound, or its pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer, is selected from any of the following compounds:

[0028] .

[0029] The compounds designed in this invention are cationic lipids, which are lipids that carry a positive charge at a selected pH value. Cationic liposomes readily bind to negatively charged nucleic acids, that is, they interact with the negatively charged phosphate groups present in nucleic acids through electrostatic forces to form lipid nanoparticles (LNPs). LNPs are one of the mainstream delivery carriers currently available.

[0030] The lipid nanoparticle (LNP) of the present invention is composed of the aforementioned cationic lipid compound and phospholipids, steroids and polyethylene glycol lipids, in a molar ratio of (20~70):(0~30):(10~80):(0~5).

[0031] Preferably, the cationic lipid compound has a molar percentage of 30% to 60% in the lipid nanoparticles, more preferably 35% to 45%.

[0032] Preferably, the phospholipid has a molar percentage of 5% to 12% in the lipid nanoparticles, and the phospholipid may be selected from at least one of distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), tetramethylbranched chain phosphatidylcholine (4ME-PC), and egg-derived phosphatidylcholine (Egg-PC).

[0033] Preferably, the steroid in the lipid nanoparticles has a molar percentage of 30% to 50%, and the steroid may be cholesterol.

[0034] Preferably, the polyethylene glycol lipid has a molar percentage of 2% to 3% in the lipid nanoparticles, and the polyethylene glycol lipid may be selected from at least one of distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecyl)acetamide (ALC-0159).

[0035] Preferably, in the lipid nanoparticles (LNPs) of the present invention, the N / P molar ratio is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.

[0036] Preferably, the lipid nanoparticles (LNPs) of the present invention include an internal aqueous phase that can dissolve hydrophilic cargo molecules, such as nucleic acids, in the LNPs.

[0037] Within the scope of this invention, the term "lipid nanoparticles" includes solid lipid nanoparticles composed of one or more lipids, structured lipid nanoparticles, liposomes or multilayer lipid vesicles, and micelles.

[0038] The pharmaceutical composition of the present invention is constructed from the aforementioned lipid nanoparticles loaded with a drug.

[0039] Furthermore, the drug is selected from one or more of the following: nucleic acids, small molecule compounds, and bioactive lipids.

[0040] Preferably, the nucleic acid is selected from at least one of siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), a nucleic acid associated with a regularly spaced short palindromic repeat sequence (CRISPR), single-stranded RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA).

[0041] Preferably, the nucleic acid is messenger RNA (mRNA), selected from at least one of self-replicating mRNA, non-self-replicating mRNA, and circular mRNA.

[0042] Preferably, the small molecule compound is a hydrophilic drug.

[0043] In some implementations, when LNP is administered to mammals, at least about 50%, 60%, 70%, 75%, 80%, 85%, or 90% of the therapeutic agent is delivered to the liver.

[0044] In some implementations, when LNP is administered to mammals, no more than about 45%, 30%, 25%, 20%, 15%, 10%, or 6% of the therapeutic agent is delivered to the spleen (or a combination of other non-hepatic organs).

[0045] In some embodiments, the average size of the LNP described in this invention is less than about 100 nanometers, less than about 90 nanometers, less than about 80 nanometers, or less than about 70 nanometers.

[0046] In some embodiments, the LNP of the present invention has a polydispersity index (PDI) between about 0.05 and 0.20 or between about 0.05 and 0.15.

[0047] In some embodiments, the LNP described in this invention has a packaging efficiency (EE%) of 91% to 99%.

[0048] The method for preparing lipid nanoparticles according to the present invention includes mixing a first organic liquid phase in an organic solvent (such as ethanol) miscible with water, the liquid phase comprising the cationic lipid composition, phospholipids, steroids and polyethylene glycol lipids disclosed in the present invention; and a second aqueous phase comprising the therapeutic agent disclosed in the present invention to form LNPs.

[0049] In some implementations, the method is performed in a microfluidic mixing device / chip. Automated microfluidic devices or microfluidic chips offer a rapid and efficient method for preparing LNPs. These devices enable rapid mixing in a highly controllable and reproducible manner, resulting in uniform LNPs and high encapsulation efficiency. In these devices, single streams of an ethanol-lipid mixture and an aqueous nucleic acid solution are rapidly mixed. The resulting mixture forms the tested LNP and is collected in a collection tube. The LNP can be fine-tuned by changing parameters such as the flow rate ratio and the total flow rate.

[0050] In some implementations, the method is performed in a T-type or Y-type mixer. These mixers can be assembled using inexpensive materials commonly found in laboratories. The T-type or Y-type connector can be fitted with two inlets, each connected to a syringe containing a lipid mixture or nucleic acid solution, and one outlet leading the LNP to a collection tube. An inlet flow rate can optionally be controlled using a syringe pump.

[0051] In some implementations, the method employs an ethanol injection method. The ethanol-lipid mixture and the nucleic acid aqueous solution are mixed using a magnetic stirring plate. While continuously stirring, the ethanol-lipid mixture is injected into the acidic nucleic acid aqueous solution, and stirring continues for 30 minutes. Alternatively, a simpler method is manual stirring. The ethanol-lipid mixture is transferred into the acidic nucleic acid aqueous solution and rapidly stirred for 15 seconds using a pipette. The mixture is then allowed to stand for 10 minutes.

[0052] In some implementations, the method further includes conditions or devices such as extrusion, high-pressure microjets to reduce particle size and produce a uniform particle size distribution.

[0053] In some embodiments, the method further includes dialyzing the LNP in a storage buffer using an appropriate molecular weight cutoff (MWCO) tube. This step removes unencapsulated cargo, excess lipid components, and ethanol from the final preparation. Dialysis also adjusts the pH of the LNP from the acidic preparation buffer to the neutral storage solution.

[0054] In some embodiments, the method further includes filtration to remove bacteria from the LNP solution. In some embodiments, the filtration step removes bacteria or other contaminants by using a 0.22 µm filter.

[0055] In some embodiments, the method further includes stepwise purification of LNPs using a tangential flow filtration (TFF) system.

[0056] In some embodiments, the organic solvent in the first organic liquid phase is composed of ethanol, or substantially composed of ethanol.

[0057] In some embodiments, the organic solvent in the first organic liquid phase is composed of methanol, or substantially of methanol.

[0058] In some embodiments, the organic solvent in the first organic liquid phase is composed of other alcohols or other organic phases, such as tetrahydrofuran, dimethylformamide, chloroform, etc.

[0059] In some embodiments, the first organic liquid phase and the second aqueous phase are mixed in a microfluidic device, with optional volume ratios of 1:2, 1:3, 1:4, 1:5, and / or optional total flow rates of approximately 10 to 25 mL / min (e.g., approximately 15 mL / min).

[0060] In some implementations, the aqueous phase includes 10 mM, 25 mM, or 50 mM citrate buffer (pH 3.0, 4.0, 5.0, or 6.0).

[0061] In some implementations, stepwise purification is performed in a TFF system containing 3%–10% sucrose citrate buffer (pH=5.0) and Tris-HCl buffer (pH=7.2).

[0062] In some implementations, the method further includes measuring the granularity of the LNP (e.g., average granularity and / or granularity distribution).

[0063] In some embodiments, particle size is determined by dynamic light scattering (DLS) measurements, such as using the Zetasizer Nano ZS from Malvern Instruments Ltd.

[0064] In some implementations, the method also includes measuring the encapsulation efficiency (EE%) of the cargo (such as mRNA).

[0065] In some implementations, EE% is determined by the RIBOGREEN® assay. RIBOGREEN® is a dye that fluoresces when it binds to single-stranded mRNA, but cannot enter the LNP.

[0066] In some embodiments, the method further includes imaging the LNP sample using cryo-electron microscopy (Cryo-EM) to study its nanoprecipitate core structure. Cryo-electron microscopy images can reveal that mRNA is encapsulated within a substance resembling an LNP, exhibiting a multi-layered core structure.

[0067] The present invention also provides the use of the cationic lipid compound or a pharmaceutically acceptable salt, isotope variant, tautomer or stereoisomer thereof, the lipid nanoparticle or the pharmaceutical composition thereof in the preparation of nucleic acid drugs or gene editing therapies.

[0068] Preferably, the use is for systemic administration, such as intravenous injection / administration.

[0069] Within the scope of this invention, the term "systemic administration" has the meaning generally accepted in the art, referring to a method or technique for administering molecules, drugs, formulations, or compounds in a manner that results in the systemic absorption of the drug in the body or its accumulation in the bloodstream and subsequent distribution throughout the body. Systemic administration includes in vivo administration.

[0070] In some embodiments, systemic administration includes pulmonary administration (inhalation, nebulization, etc.), intravenous injection, subcutaneous injection, catheter injection, nasopharyngeal administration, or oral / gastrointestinal administration, all of which are commonly known in the art.

[0071] Other non-limiting examples of systemic administration methods of the present invention include oral administration, sublingual administration, parenteral administration (i.e., intravenous injection, intraperitoneal injection, subcutaneous injection, or intramuscular injection), local rectal administration, or other local administration that causes the administration composition to be absorbed or accumulated in the bloodstream and then distributed throughout the body.

[0072] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention designs a series of cationic lipid compounds, and the lipid nanoparticles constructed therefrom have small particle size and low polymorphism index, and have extremely high encapsulation efficiency for drug molecules. At the same time, it can accurately deliver the mRNA drugs loaded therein to the liver while maintaining high expression intensity, and hardly expresses them in other organs, such as the lungs, heart and kidneys, and express them in small amounts in the spleen. It has important clinical application significance in cancer treatment. Attached Figure Description

[0073] Figure 1 This is the NMR spectrum of the BK-13 lipid molecule in this invention;

[0074] Figure 2 The NMR spectrum of the BK-16 lipid molecule in this invention;

[0075] Figure 3 The NMR spectrum of the BK-18 lipid molecule in this invention;

[0076] Figure 4 This is the NMR spectrum of the BK-19 lipid molecule in this invention;

[0077] Figure 5 The images shown are fluorescence images of isolated mouse organs of BK-13:F0, BK-13:F1, BK-13:F2, BK-13:F3, and SM-102 in Table 1 of this invention.

[0078] Figure 6 For the present invention Figure 5 A bar chart of the average radiation intensity of organs in medullomyeloid mice;

[0079] Figure 7 For the present invention Figure 5 A bar chart showing the percentage of organ distribution in medullomyeloid mice;

[0080] Figure 8 The images show fluorescence imaging of isolated mouse organs of BK-16:F1, BK-18:F1, BK-19:F1, BK-13:F1, and ALC-0315 in Table 1 of this invention.

[0081] Figure 9 For the present invention Figure 8 A bar chart of the average radiation intensity of organs in medullomyeloid mice;

[0082] Figure 10 For the present invention Figure 8 A bar chart showing the percentage of organ distribution in medullomyeloid mice;

[0083] Figure 11 The images shown are fluorescence images of isolated mouse organs of BK-13:F1, P-4, C14-4, and C12-200 in Table 1 of this invention.

[0084] Figure 12 For the present invention Figure 11 A bar chart of the average radiation intensity of organs in medullomyeloid mice;

[0085] Figure 13 For the present invention Figure 11 A bar chart showing the percentage distribution of organs in mice. Detailed Implementation

[0086] The technical solution of the present invention will be further described below with reference to specific embodiments. Without additional explanation, all reagents used are commercially available and are used directly without purification.

[0087] Example 1: Synthesis of BK-13

[0088] The synthesis route is shown below:

[0089] Step 1: Dissolve EDCI (12 mmol, 2.30 g) and DMAP (0.5 mmol, 0.61 g) in 30 mL of DCM, add compound 1a (10 mmol, 1.81 g) and compound 2a (11 mmol, 2.81 g), and stir at room temperature for 12 hours. After the reaction is complete, concentrate under vacuum. The crude product is purified by silica gel column chromatography to give compound 1b (2.64 g, pale yellow liquid), in 63% yield.

[0090]

[0091] Step 2: Compound 2b (5 mmol, 1.15 g), K₂CO₃ (15 mmol, 2.07 g), and KI (1 mmol, 0.17 g) were dissolved in 20 mL of acetonitrile. Compound 1b (5.25 mmol, 2.20 g) was added, and the mixture was heated at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered under reduced pressure, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 1c (1.68 g, pale yellow oil), with a yield of 59%.

[0092]

[0093] Step 3: Dissolve compound 1c (1.68 g) in 10 mL DCM, add 2 mL TFA, react at room temperature for 2 hours, concentrate under vacuum after the reaction is complete, and compound 1d can be obtained without further purification, with a yield of 94%.

[0094]

[0095] Step 4: Compound 1d (2.77 mmol, 1.30 g), K₂CO₃ (11.08 mmol, 1.53 g), and KI (0.55 mmol, 0.09 g) were dissolved in 15 mL of acetonitrile. Compound 1b (2.91 mmol, 1.22 g) was added, and the mixture was heated at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered under reduced pressure, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 1e (1.25 g, yellow oil), with a yield of 56%.

[0096]

[0097] Step 5: Compound 1e (1.60 mmol, 1.25 g) and NaH (1.60 mmol, 0.04 g) were dissolved in 5 mL of DMF. After stirring at room temperature for 45 minutes, compound 2e (1.92 mmol, 0.46 g) was added dropwise. The mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was poured into water and extracted with EA (10 mL × 3 times). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 1f (0.77 g, yellow oil), with a yield of 50%.

[0098]

[0099] Step Six: Compound 1f (0.82 mmol, 0.77 g) was dissolved in 5 mL of THF, and TBAF (0.90 mmol, 0.23 g) was added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the solution was concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 1 g (0.60 g, yellow oil) in 86% yield, namely compound BK-13. Its NMR spectra are as follows: Figure 1 As shown.

[0100]

[0101] Example 2: Synthesis of BK-16

[0102] The synthesis route is shown below:

[0103] Step 1: Compound 1A (20 mmol, 2.34 g) was dissolved in 20 mL of toluene. A mixture of 2A (16 mmol, 4.33 g) and toluene (20 mL) was added under controlled temperature. The mixture was heated at 70 °C for 9 hours. After the reaction was completed, the temperature was lowered and 43 mL of water and 5.53 g of diatomaceous earth were added sequentially with stirring. The mixture was filtered under pressure, and the aqueous phase was collected. The aqueous phase was extracted with DCM (30 mL × 5 times). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Compound 1B (grayish-white solid) was obtained without further purification, with a yield of 18%.

[0104]

[0105] Step 2: Compound 1e was prepared using the same method as steps 1 to 4 in Example 1. Compound 1e (1.55 mmol, 1.25 g) was dissolved in 10 mL of DMF. NaH (24.8 mmol, 0.99 g) was added at 0 °C, and the mixture was stirred at room temperature for 0.5 hours. Then, 1B (7.75 mmol, 1.39 g) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was poured into water and extracted with EA (10 mL × 3 times). The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography to obtain compound 1h (0.38 g, yellow oil), with a yield of 27%, i.e., compound BK-16. Its NMR spectra are as follows: Figure 2 As shown.

[0106]

[0107] Example 3: Synthesis of BK-18

[0108] The synthesis route is shown below:

[0109] Step 1: Dissolve EDCI (12 mmol, 2.30 g) and DMAP (0.5 mmol, 0.61 g) in 30 mL of DCM, add compound 3a (11 mmol, 2.23 g) and compound 4a (10 mmol, 2.56 g), and stir at room temperature for 12 hours. After the reaction is complete, concentrate under vacuum. The crude product is purified by silica gel column chromatography to give compound 3b (2.17 g, colorless liquid), in 47% yield.

[0110]

[0111] Step 2: Dissolve EDCI (12 mmol, 2.30 g) and DMAP (0.5 mmol, 0.61 g) in 30 mL of DCM, add compound 3c (11 mmol, 1.95 g) and compound 4c (10 mmol, 1.72 g), and stir at room temperature for 12 hours. After the reaction is complete, concentrate under vacuum. The crude product is purified by silica gel column chromatography to give compound 3d (2.20 g, colorless liquid), in 63% yield.

[0112]

[0113] Step 3: Compound 3e (5 mmol, 1.15 g), K₂CO₃ (15 mmol, 2.07 g), and KI (1 mmol, 0.17 g) were dissolved in 20 mL of acetonitrile. Compound 3b (5.25 mmol, 2.54 g) was added, and the mixture was heated at 60 °C for 24 hours. After the reaction was complete, the mixture was filtered under reduced pressure, and the filtrate was concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 3f (1.62 g, pale yellow oil), with a yield of 53%.

[0114]

[0115] Step 4: Dissolve compound 3f (1.62 g) in 10 mL DCM, add 2 mL TFA, react at room temperature for 2 hours, concentrate under vacuum after the reaction is complete, and obtain compound 3 g without further purification, with a yield of 93%.

[0116]

[0117] Step 5: Dissolve 3 g of compound (2.46 mmol, 1.26 g), K₂CO₃ (9.84 mmol, 1.36 g), and KI (0.49 mmol, 0.08 g) in 15 mL of acetonitrile. Add compound 3d (2.58 mmol, 0.90 g) and heat at 60 °C for 24 hours. After the reaction is complete, filter under reduced pressure. Concentrate the filtrate under vacuum. Purify the crude product by silica gel column chromatography to give compound 3h (0.73 g, yellow oil), yield 38%.

[0118]

[0119] Step Six: Compound 3A (40 mmol, 2.92 g) was dissolved in 100 mL of EA, and 4A (10 mmol, 1.81 g) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was washed successively with water, sodium bicarbonate, and brine to separate the organic layer. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 3B (1.32 g, colorless liquid), with a yield of 76%.

[0120]

[0121] Step 7: Compound 3B (7.60 mmol, 1.32 g) was dissolved in 10 M hydrochloric acid (76 mmol), heated under reflux at 110 °C for 4 hours, cooled to room temperature and concentrated under vacuum, leaving an oily residue, which was then dissolved in distilled water and concentrated twice to remove hydrochloric acid, finally yielding a white solid as compound 3C (0.53 g), with a yield of 48%.

[0122]

[0123] Step 8: Dissolve compound 3C (0.98 mmol, 0.14 g), EDCI (2.80 mmol, 0.38 g), and DMAP (0.93 mmol, 0.11 g) in 10 mL of DCM, add compound 3h (0.93 mmol, 0.73 g), and stir at room temperature for 12-16 hours. After the reaction is complete, concentrate under vacuum. Purify the crude product by silica gel column chromatography to give compound 3i (0.14 g, colorless liquid), in 17% yield, i.e., compound BK-18. Its NMR spectra are as follows: Figure 3 As shown.

[0124]

[0125] Example 4: Synthesis of BK-19

[0126] The synthesis route is shown below:

[0127] Compound 3h was prepared using the same method as steps one through five in Example 3. Compound 3h (0.98 mmol, 0.11 g), EDCI (2.80 mmol, 0.38 g), and DMAP (0.93 mmol, 0.11 g) were dissolved in 10 mL of DCM. Compound 4h (0.93 mmol, 0.73 g) was added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, it was concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 3j (0.38 g, colorless liquid), with a yield of 47%, i.e., compound BK-19. Its NMR spectra are as follows: Figure 4 As shown.

[0128]

[0129] Examples 5-11: Preparation of mRNA@LNP

[0130] (1) In vitro transcription and aqueous phase preparation of mRNA: Luciferase mRNA was synthesized by in vitro transcription according to the literature method (J.Am.Chem.Soc. 2023, 145,4691-4706). After purification and verification, it was prepared into an aqueous phase using 50mM citrate buffer (pH=5.0).

[0131] (2) Preparation of LNP: The cationic lipid compound, DSPC (purchased from Jiangsu Southeast Nanomaterials), cholesterol (purchased from Jiangsu Southeast Nanomaterials) and DMG-PEG2000 (purchased from SINOPEG) were prepared into an ethanol phase in a molar ratio of (20~70):(0~30):(10~80):(0~5);

[0132] (3) LNP loaded with mRNA: Using a typical microfluidic device, the aforementioned aqueous phase and ethanol phase with a total flow rate of 15 mL / min were rapidly mixed at a volume ratio of 1:4 to obtain LNP loaded with mRNA (mRNA@LNP).

[0133] Using the methods described above, BK-13:F0, BK-13:F1, BK-13:F2, BK-13:F3, BK-16:F1, BK-18:F1, and BK-19:F1 were prepared according to the formulations shown in Table 1. The above mRNA@LNPs were purified by dialysis or a TFF system in citrate buffer (pH=5.0) and Tris-HCl buffer (pH=7.2) containing 8% sucrose. After purification, they were diluted to the target concentration and can be stored for short periods (up to 3 months) at 2–8°C and for long periods (up to 3 months) at -80°C.

[0134] Comparative Examples 1-5

[0135] mRNA@LNP was prepared using the same method as in Examples 5-11, wherein the cationic lipid compounds were selected from commercial compounds SM-102, ALC-0315, C12-200, C14-4, and P-4 (all purchased from SINOPEG), and their structures are shown below:

[0136] .

[0137] The specific formulations of mRNA@LNP for Comparative Examples 1-5 are shown in Table 1.

[0138] Table 1. LNP formulations of Examples 5-11 and Comparative Examples 1-5

[0139]

[0140] Note: Chol in the table refers to cholesterol; the comparative formula was prepared according to the literature (Nano Lett. 2022, 22, 533-542).

[0141] The mRNA@LNPs prepared in Examples 5-11 and Comparative Examples 1-2 were characterized by particle size analysis. Their nanoparticle size and polydispersity index (PDI) were determined using dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments Ltd.), and the concentration of loaded mRNA was determined using the Ribogreen® (Thermofisher) assay. Ethanol was removed from the mRNA@LNPs using a TFF purification process, and the mRNA was converted to a sucrose-containing Tris-HCl buffer suitable for intravenous injection. The prepared products were characterized by DLS and the Ribogreen® assay to determine their size and encapsulation efficiency (EE%) / concentration. The results are shown in Table 2.

[0142] Table 2. Morphological characterization results of mRNA@LNP in Examples 5-11 and Comparative Examples 1-2

[0143]

[0144] The mRNA@LNP prepared in Examples 5-11 and Comparative Examples 1-5 were evaluated in vivo to detect their in vivo expression efficiency and organ expression distribution. BALB / c mice (female, 6-8 weeks old) were divided into n=3 groups. Mice were intravenously injected with luciferase mRNA@LNP (5 μg mRNA dispersed in Tris-HCl buffer containing 7.5% sucrose, pH=7.2). Six hours after injection, D-luciferin potassium (150 mg / kg, intraperitoneal injection) was injected into the mice. Ten minutes later, the average radiant intensity (corresponding to fluorescence expression intensity) of the bioluminescence produced by the interaction of luciferase mRNA carried by LNP with the substrate (D-luciferin potassium) in mice was detected using the IVIS Lumina small animal in vivo imaging system. After sampling, the mice were sacrificed, dissected, and the internal organs were isolated: heart, lungs, liver, kidneys, and spleen. IVIS Lumina imaging was used to test the average radiant intensity of the mouse organs and calculate the organ distribution percentage. The results are shown in Tables 3-4 and 4. Figures 5-13 As shown in the figure, Figures 5-7 , Figures 8-10 , Figures 11-13The BK-13:F1 used was prepared three times, and new mice were used in the experiments. Figures 5-7 From the same batch Figures 8-10 For the same batch, Figures 11-13 (Since these are from the same batch), differences in results are normal. The BK-13:F1 data in Tables 3 and 4 are the average values ​​of the three batches mentioned above. All animal experiments were approved by the Animal Care and Use Committee of Hangzhou Normal University and complied with the relevant regulations of the Chinese government regarding animal care and use.

[0145] Table 3. Mean radiation intensity of mouse organs after administration of drugs in Examples 5-11 and Comparative Examples 1-5.

[0146]

[0147] Table 4. Organ distribution percentages in Examples 5-11 and Comparative Examples 1-5

[0148]

[0149] As shown in Tables 2-4, when using the same cationic lipid compound BK-13 to prepare mRNA@LNP, BK-13:F1 prepared according to the molar ratio of BK-13:DSPC:Chol:DMG-PEG=35:15:48.5:1.5 exhibits a smaller PDI and a higher EE%, indicating that it has uniform particle size and better encapsulation efficiency for mRNA, resulting in higher mRNA loading efficiency. Subsequent analysis of in vivo expression efficiency and organ expression distribution further demonstrates that this formulation delivers LNPs more precisely to the liver. Therefore, this formulation is preferred for preparing BK-16:F1, BK-18:F1, and BK-19:F1.

[0150] Further comparison of the expression levels of mRNA@LNP prepared from BK-13, BK-16, BK-18, and BK-19 in different organs of mice revealed significant differences in expression. BK-13, BK-16, BK-18, and BK-19 were almost exclusively expressed in the liver, with a small amount expressed in the spleen, and virtually no expression in other organs such as the heart, lungs, and kidneys. This indicates that the delivery system prepared from the cationic lipid compounds of this invention can precisely target and deliver to the liver. Among them, BK-16 had the highest expression level in the liver, indicating the best delivery efficiency, followed by BK-13, but BK-13 had the highest expression percentage in the liver (better liver targeting).

[0151] Comparing the four cationic lipid compounds of the present invention with existing representative cationic lipids SM-102 and ALC-0315, it can be seen that BK-13, BK-16, BK-18, and BK-19 are superior to SM-102 and ALC-0315 in terms of delivery efficiency or expression intensity in the liver. Compared with existing piperazine-containing molecules P-4, C14-4, and C12-200 with similar structures, BK-13, BK-16, BK-18, and BK-19 also have advantages in delivery efficiency in the liver.

[0152] In summary, this invention designs a series of cationic lipid compounds, among which BK-13, BK-16, BK-18, and BK-19 can target mRNA delivery to the liver, are almost not expressed in other organs such as the lungs, heart, and kidneys, and are expressed in small amounts in the spleen, thereby improving delivery efficiency and liver targeting efficiency, which has important clinical significance.

Claims

1. A cationic lipid compound, or a pharmaceutically acceptable salt thereof, characterized in that, Selected from any of the following compounds: 。 2. A lipid nanoparticle, characterized in that, The compound is composed of the cationic lipid compound of claim 1 and phospholipids, steroids, and polyethylene glycol lipids, with a molar ratio of (20~70):(0~30):(10~80):(0~5).

3. A pharmaceutical composition, characterized in that, The drug is constructed from lipid nanoparticles loaded with the drug as described in claim 2.

4. The pharmaceutical composition according to claim 3, characterized in that, The drug is selected from one or more of the following: nucleic acids, small molecule compounds, and bioactive lipids.

5. The pharmaceutical composition according to claim 4, characterized in that, The nucleic acid in question is messenger RNA.

6. Use of a cationic lipid compound of claim 1 or a pharmaceutically acceptable salt thereof, a lipid nanoparticle of claim 2, or a pharmaceutical composition of any one of claims 3 to 5 in the preparation of a nucleic acid drug.

Images