Tertiary amine lipid compounds and their applications

Abstract

Provided are a tertiary amine lipid compound having a structure shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, and use thereof. The compound can efficiently and selectively destroy cell membranes or lysosomal membranes, thereby killing tumor cells.

Description

Technical Field

[0001] The invention relates to the technical field of materials and medicines, and in particular to a tertiary amine lipid compound and application thereof. Background Art

[0002] Malignant tumors have become a major threat to human life and health. Although current anti-tumor therapies such as chemotherapy, radiotherapy, and immunotherapy can inhibit tumor growth, prolong patient survival, and even completely cure tumors to a certain extent, they all face the problems of low specificity and large toxic side effects. For example, chemotherapy and radiotherapy can cause damage to normal tissues, and immune checkpoint therapy can cause immune-related toxic side effects.

[0003] Some cationic polymers can kill tumor cells and pathogens such as bacteria by destroying cell membranes or organelle membranes. They have the advantages of broad-spectrum killing effects and are not easy to develop drug resistance. They have broad application prospects in the treatment of diseases such as tumors. For example, LTX-315 cationic peptides are used in tumor treatment by intratumoral injection, which can cause a large number of tumor cells to necrotize. It has achieved good results in melanoma, head and neck cancer, lymphoma, and breast cancer in combination with drugs such as ipilimumab and pembrolizumab. Related research is in phase I clinical trials (https: / / clinicaltrials.gov / ). The study of LL-37 peptides for the treatment of melanoma by intratumoral injection is also in phase I clinical research (https: / / clinicaltrials.gov / ). However, since this type of drug can not only efficiently destroy tumor cells and pathogens, but also has strong cytotoxicity to normal tissue cells, and peptide drugs are easily degraded by proteases, most drugs have stopped at clinical research, and most of the drugs currently used in clinical practice are local injection treatments.

[0004] Therefore, there is an urgent need to develop drugs that have no toxic side effects on normal tissues and cells and can specifically kill tumor cells for tumor treatment. Summary of the invention

[0005] Based on this, the present invention provides a class of tertiary amine lipid compounds. The tertiary amine lipid compounds are hydrophobic and electrically neutral at normal physiological pH, and can self-assemble with amphiphilic auxiliary lipids such as DSPE-PEG into lipid nanoparticles with PEG as the shell, which have low cytotoxicity. Under slightly acidic pH conditions, they can be protonated to form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, which has a strong interaction with the cell membrane or organelle membrane, thereby producing strong cytotoxicity and specifically killing tumor cells.

[0006] The present invention includes the following technical solutions.

[0007] Application of a tertiary amine lipid compound having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material:

[0008]

[0009] Among them, each R 1 Each of the following is independently selected from: an alkyl group, an unsaturated chain hydrocarbon group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, an aryl group substituted with an alkyl group, and a cycloalkyl group substituted with an alkyl group;

[0010] Each R 2 Independently selected from: Or not;

[0011] R 3 and R 4 are independently selected from: H, R 9 Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group, -R 2 -R 1 , or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms R 8 substituted or unsubstituted 3-20 membered heterocyclic group; and when R 3 When connected directly to N, R 3 Not H; When R 4 When connected directly to N, R 4 Not for H;

[0012] Each R 5 are independently selected from: alkylene, cycloalkyl, aryl;

[0013] Each R 6 are independently selected from: alkyl, hydroxy substituted alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl substituted alkyl,

[0014] Each R 7 are independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group, aryl group substituted alkyl group, or R 6 , R 7 Together with the nitrogen atom to which it is attached, it forms R 8 a substituted or unsubstituted 5-8 membered heterocyclic group;

[0015] Each R 8are independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl substituted heterocyclic, alkenyl, alkynyl, aryl, aryl substituted alkyl, cycloalkyl substituted alkyl,

[0016] R 9 Selected from: hydroxyl, aryl, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic and heteroaryl groups;

[0017] Each X is independently O or NH;

[0018] Each q and each q' is independently 0, 1 or 2;

[0019] a is 0 or 1;

[0020] b is 1, 2, 3, 4 or 5;

[0021] c is 1, 2, 3, or 4;

[0022] d is an integer between 2 and 12.

[0023] The tertiary amine lipid compound may have a structure as shown in the following formula (II):

[0024]

[0025] The tertiary amine lipid compound may also have a structure as shown in the following formula (III):

[0026]

[0027] R 4 Selected from: R 9 Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group,

[0028] The tertiary amine lipid compound may also have a structure as shown in the following formula (IV):

[0029]

[0030] Where n is 1, 2, 3, 4 or 5;

[0031] R 3 and R 4 Each independently selected from: C 1 -C 10 Alkyl, hydroxy substituted C 1 -C 10 Alkyl, C 1 -C 10 Unsaturated chain hydrocarbon group, C3 -C 8 Cycloalkyl, or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms a 3-8 membered heterocyclic group.

[0032] The tertiary amine lipid compound may also have a structure as shown in the following formula (V):

[0033]

[0034] n 1 、n 2 、n 3 、n 4 Each is independently selected from: 0, 1, 2, 3 or 4.

[0035] On the other hand, the present invention also provides a lipid nanoparticle, which is formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic molecules in an aqueous medium, wherein the amphiphilic molecules are: amphiphilic polyethylene glycol lipid molecules, or a combination of amphiphilic polyethylene glycol lipid molecules and phospholipids.

[0036] On the other hand, the present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0037] dissolving the tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution;

[0038] dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution;

[0039] Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

[0040] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

[0041] On the other hand, the present invention also provides the use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, or the lipid nanoparticle, including the following technical solutions.

[0042] The use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating tumors.

[0043] The lipid nanoparticles are used in preparing drugs for preventing and / or treating tumors.

[0044] The tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt is used in combination with an anti-tumor drug in the preparation of a drug for preventing and / or treating tumors.

[0045] The lipid nanoparticles are used in combination with anti-tumor drugs in the preparation of drugs for preventing and / or treating tumors.

[0046] On the other hand, the present invention also provides a drug for preventing and / or treating tumors, which is prepared from active ingredients and pharmaceutically acceptable excipients and / or carriers, wherein the active ingredients include the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and / or the lipid nanoparticles.

[0047] On the other hand, the present invention also provides a combined drug for preventing and / or treating tumors, the active ingredients of which include:

[0048] Component 1: the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and / or the lipid nanoparticle; and,

[0049] Component 2: anti-tumor drugs other than component 1;

[0050] The component 1 and the component 2 are respectively independent administration units, or the component 1 and the component 2 together form a combined administration unit.

[0051] Based on the above technical solution, the present invention has the following beneficial effects:

[0052] The invention provides an acidity-activatable anti-tumor lipid tertiary amine small molecule compound, which consists of a tertiary amine head with convertible hydrophilic and hydrophobic properties and a tail containing a hydrophobic group. At normal physiological pH, the tertiary amine in the lipid material is hydrophobic and electrically neutral, and the tertiary amine head is in a hydrophobic conformation. Moreover, the lipid material can self-assemble with the assistance of auxiliary lipids such as DSPE-PEG to form acid-activatable anti-tumor lipid nanoparticles with PEG as the shell, and the interaction with the cell membrane is weak, so that it has the advantage of low toxicity to normal tissues when circulating in the body, and the PEG shell can improve the biocompatibility of the lipid material and prolong its blood circulation time; under the slightly acidic pH conditions of tumor tissue (a large number of studies have shown that tumor cells usually use glycolysis to produce more lactic acid and carbon dioxide, and because of the blockage of lymphatic reflux in solid tumor tissue, metabolic waste cannot be discharged normally, resulting in the peripheral environment pH of solid tumors being lower than the pH of normal body fluids, usually between 6.4 and 7.0), the tertiary amine part of the small molecule compound lipid material will be protonated, so that the nanoparticles form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, so that it has a very strong interaction with the cell membrane or organelle membrane, so that it can kill tumor cells efficiently and selectively. The tertiary amine small molecule compound and / or the acidity-activatable antitumor lipid nanoparticles of the present invention can be used to prepare antitumor drugs, and have the advantages of good therapeutic effect, high selectivity and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the hydrogen NMR spectrum of n-decyl acrylate (C10).

[0054] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of 9-octadecene acrylate (C181).

[0055] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of cis-9,12-octadecadienyl acrylate (C182).

[0056] Figure 4 This is the H NMR spectrum of phenyl butyl acrylate (Cp4).

[0057] Figure 5 This is a heat map of the hemolysis rate of the lipid nanoparticles prepared in Example 1 after incubation with 2% sheep blood at 400 μg / mL for 4 hours.

[0058] Figure 6 This is a graph showing the relationship between cell activity and pH after lipid nanoparticles with different carbon chain lengths prepared in Example 2 were co-incubated with mouse colorectal cancer MC38 cells at pH 7.4 and 6.8 for 24 hours.

[0059] Figure 7This is a graph showing the relationship between cell activity and pH after lipid nanoparticles with different carbon chain lengths prepared in Example 2 were co-incubated with mouse colorectal cancer MC38 cells at pH 7.4 and 6.8 for 24 hours.

[0060] Figure 8 This is the hydrogen nuclear magnetic resonance spectrum of the C8A45 lipid compound prepared in Example 1.

[0061] Fig. 9 This is the hydrogen nuclear magnetic resonance spectrum of the C8A49 lipid compound prepared in Example 1.

[0062] Fig.10 This is the hydrogen nuclear magnetic resonance spectrum of the C12A27 lipid compound prepared in Example 1.

[0063] Fig.11 This is the hydrogen nuclear magnetic resonance spectrum of the C12A33 lipid compound prepared in Example 1.

[0064] Fig.12 This is the hydrogen nuclear magnetic resonance spectrum of the C14A33 lipid compound prepared in Example 1.

[0065] Fig.13 This is the hydrogen nuclear magnetic resonance spectrum of the C16A34 lipid compound prepared in Example 1.

[0066] Fig.14 This is the hydrogen nuclear magnetic resonance spectrum of the C181A17 lipid compound prepared in Example 1.

[0067] Fig.15 This is the hydrogen nuclear magnetic resonance spectrum of the C181A23 lipid compound prepared in Example 1.

[0068] Fig.16 This is the hydrogen nuclear magnetic resonance spectrum of the C181A24 lipid compound prepared in Example 1.

[0069] Fig.17 This is the hydrogen nuclear magnetic resonance spectrum of the C181A28 lipid compound prepared in Example 1.

[0070] Fig.18 This is the hydrogen nuclear magnetic resonance spectrum of the C182A17 lipid compound prepared in Example 1.

[0071] Fig.19 This is the hydrogen nuclear magnetic resonance spectrum of the C182A23 lipid compound prepared in Example 1.

[0072] Fig. 20 This is the hydrogen nuclear magnetic resonance spectrum of the C182A24 lipid compound prepared in Example 1.

[0073] Fig.21This is the hydrogen nuclear magnetic resonance spectrum of the C182A28 lipid compound prepared in Example 1.

[0074] Fig. 22 This is the hydrogen nuclear magnetic resonance spectrum of the C8A16 lipid compound prepared in Example 1.

[0075] Fig.23 This is the hydrogen nuclear magnetic resonance spectrum of the C8A19 lipid compound prepared in Example 1.

[0076] Fig.24 The concentration-dependent cytotoxicity of some lipid nanoparticles at pH 7.4 and 6.8.

[0077] Fig.25 The concentration-dependent cytotoxicity of some lipid nanoparticles at pH 7.4 and 6.8.

[0078] Fig.26 The particle size and distribution of lipid nanoparticles in aqueous solution were detected by dynamic light scattering.

[0079] Fig. 27 MTD of tertiary amine lipid nanoparticles tested in ICR mice.

[0080] Fig.28 The protonation rate of tertiary amine lipid nanoparticles changes with pH and its pKa.

[0081] Fig.29 Figure 3 is the concentration-dependent cytotoxicity of lipid nanoparticles assembled with C12A27 lipid compounds and different components under pH 7.4 and 6.8 conditions; Figure a shows the cytotoxicity of lipid nanoparticles assembled with C12A27 and different PEG lipids in a 1:1 ratio to MC38 at pH 7.4 and pH 6.8; Figure b shows the cytotoxicity of lipid nanoparticles assembled with DSPE-PEG and C12A27 in different mass ratios to MC38 at pH 7.4 and pH 6.8; Figures c and d show the cytotoxicity of lipid nanoparticles assembled with DSPE-PEG, C12A27 and different auxiliary lipids in different mass ratios to MC38 at pH 7.4 and pH 6.8, respectively.

[0082] Fig.30The present invention is a study on the mechanism of cytotoxicity of C12A27 lipid nanoparticles to MC38 cells at pH 6.8; Figure a shows the cytotoxicity of C12A27 lipid nanoparticles to MC38 cells at different temperatures at pH 6.8 for 4 hours; Figure b shows the leakage of ATP in the cytoplasm after C12A27 lipid nanoparticles were incubated with MC38 cells at different temperatures for 4 hours; Figure c shows the cytotoxicity of C12A27 lipid nanoparticles to MC38 cells at pH 6.8 for 4 hours. GFP / mCherry The leakage of mCherry in the cytoplasm and the changes of GFP on the cell membrane after co-incubation of cells.

[0083] Fig.31 C8A16 lipid nanoparticles and C8A19 lipid nanoparticles were mixed with MC38 at pH 6.8. GFP / mCherry The leakage of mCherry in the cytoplasm and the changes of GFP on the cell membrane after co-incubation of cells.

[0084] Fig.32 Cell morphology and cell staining after C8A16 lipid nanoparticles, PI and Annexin-FITC were co-incubated with MC38 at pH 6.8.

[0085] Fig.33 C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, C12A27 lipid nanoparticles and C12A33 lipid nanoparticles were mixed with Panc02 at pH 6.0. GFP / mCherry The leakage of mCherry in the cytoplasm and the changes of GFP on the cell membrane after co-incubation of cells.

[0086] Fig.34 Schematic diagram and experimental results of the therapeutic experimental administration of C12A27, C14A27, and C16A27 lipid nanoparticles on the mouse pancreatic cancer model Panc02.

[0087] Fig.35 Schematic diagram and experimental results of the therapeutic experimental administration of C8A16 lipid nanoparticles, C8A19 lipid nanoparticles, C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, C12A27 lipid nanoparticles, C12A33 lipid nanoparticles and C182A28 lipid nanoparticles on the mouse colorectal cancer model MC38.

[0088] Fig.36 Schematic diagram and experimental results of the therapeutic experimental administration of C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, C12A27 lipid nanoparticles, and C12A33 lipid nanoparticles on the mouse breast cancer model E0771.

[0089] Fig.37 Schematic diagram and experimental results of the therapeutic experiment of C12A27 and C14A27 lipid nanoparticles combined with immune checkpoint antibody aPD-1 on the mouse colorectal cancer model MC38.

[0090] Fig.38 Schematic diagram and experimental results of the therapeutic experiment of C12A27 lipid nanoparticles combined with the chemotherapy drug liposome doxorubicin Doxil on the mouse breast cancer model EMT-6. DETAILED DESCRIPTION

[0091] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0092] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0093] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0094] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0095] In the compounds described in the present invention, when any variable (such as R 1 , R 2, etc.) occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations render the compound stable. Lines drawn from substituents into the ring system indicate that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are attached only to any suitable carbon atom of the adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable. The phrase "optionally substituted with one or more substituents" is considered equivalent to the phrase "optionally substituted with at least one substituent" and in such cases preferred embodiments will have 0-3 substituents.

[0096] The term "alkyl" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. 1 -C 6 "C" in "alkyl" 1 -C 6 The definition of " includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched chain arrangement. For example, "C 1 -C 6 The "alkyl group" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl and hexyl.

[0097] The term "unsaturated chain hydrocarbon group" refers to a branched or straight chain unsaturated aliphatic hydrocarbon group having a specific number of carbon atoms, i.e., a non-cyclic unsaturated chain hydrocarbon group, and the carbon chain contains one or more carbon-carbon double bonds, or a carbon-carbon triple bond, such as: CH 2 =CHCH 2 -,-(CH 2 ) 8 (CH=CH)CH 3 ,-(CH 2 ) 7 CH=CH 2 ,-(CH 2 ) 8 CH=CH 2 , cis-9,12-octadecadienyl, etc.

[0098] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, fused, spirocyclic, bridged or other cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) and the remaining ring atoms are carbon, for example: piperidinyl, pyrrolidinyl, etc.

[0099] The "acidity-activatable anti-tumor lipids" described in the present invention refer to lipids that can specifically activate anti-tumor activity to kill tumor cells under specific acidity conditions.

[0100] The "pH responsive membrane-breaking material" described in the present invention refers to a material whose membrane-breaking activity can be activated under specific acidity conditions.

[0101] The "tertiary amine lipid compound" or "lipid" or "small molecule compound" or "small molecule lipid compound" or "tertiary amine small molecule compound" described in the present invention all refer to compounds within the structural range shown by formula (I) to formula (V) of the present invention.

[0102] In one embodiment of the present invention, there is provided a use of a tertiary amine lipid compound having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material:

[0103]

[0104] Among them, each R 1 Each of the following is independently selected from: an alkyl group, an unsaturated chain hydrocarbon group, a cycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, an aryl group substituted with an alkyl group, and a cycloalkyl group substituted with an alkyl group;

[0105] Each R 2 Independently selected from: Or not;

[0106] R 3 and R 4 are independently selected from: H, R 9 Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group, -R 2 -R 1 , or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms R 8 substituted or unsubstituted 3-20 membered heterocyclic group; and when R 3 When connected directly to N, R 3 Not H; When R 4 When connected directly to N, R 4 Not for H;

[0107] Each R 5 are independently selected from: alkylene, cycloalkyl, aryl;

[0108] Each R 6are independently selected from: alkyl, hydroxy substituted alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl substituted alkyl,

[0109] Each R 7 are independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group, aryl group substituted alkyl group, or R 6 , R 7 Together with the nitrogen atom to which it is attached, it forms R 8 a substituted or unsubstituted 5-8 membered heterocyclic group;

[0110] Each R 8 are independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl substituted heterocyclic, alkenyl, alkynyl, aryl, aryl substituted alkyl, cycloalkyl substituted alkyl,

[0111] R 9 Selected from: hydroxyl, aryl, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic and heteroaryl groups;

[0112] Each X is independently O or NH;

[0113] Each q and each q' is independently 0, 1 or 2;

[0114] a is 0 or 1;

[0115] b is 1, 2, 3, 4 or 5;

[0116] c is 1, 2, 3, or 4;

[0117] d is an integer between 2 and 12.

[0118] In some preferred embodiments, the tertiary amine lipid compound has a structure shown in the following formula (II):

[0119]

[0120] In some preferred embodiments, the tertiary amine lipid compound has a structure shown in the following formula (III):

[0121]

[0122] R 4 Selected from: R 9 Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R 8 Substituted heterocyclic group, alkenyl group, alkynyl group, aryl group,

[0123] In some preferred embodiments, R 4 Selected from: C 1 -C 22 Alkyl, R 9 Substituted C 1 -C 8 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 3 -C 22 Cycloalkyl, 3-22 membered heterocyclic group, R 8 Substituted 3-22 membered heterocyclic group, C 2 -C 22 Alkenyl, C 2 -C 22 Alkynyl, C 6 -C 22 Aryl,

[0124] In some preferred embodiments, R 4 Selected from: C 1 -C 10 Alkyl, R 9 Substituted C 1 -C 8 Alkyl, C 2 -C 10 Unsaturated chain hydrocarbon group, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, R 8 Substituted 3-8 membered heterocyclic group, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, phenyl, naphthyl, anthracenyl,

[0125] In some preferred embodiments, each R 5 Each independently selected from: C 1 -C 22 Alkylene, C 3 -C 8 Cycloalkyl, C 6 -C 14 Aryl.

[0126] In some preferred embodiments, each R 5 Each independently selected from: C 1 -C 10 Alkylene, C 3 -C 8 Cycloalkyl, phenyl, naphthyl, anthracenyl.

[0127] In some preferred embodiments, each R 5 Each independently selected from: C 1 -C 6 Alkylene, C 3 -C 8 Cycloalkyl.

[0128] In some preferred embodiments, each R 5 are independently selected from: methylene, ethylene, propylene, butylene,

[0129] In some preferred embodiments, each R 6 Each independently selected from: C 1 -C 22 Alkyl, hydroxy substituted C 1 -C 8 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 2 -C 22 Alkenyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 8 alkyl,

[0130] Each R 7 Each independently selected from: C 1 -C 22 Alkyl, hydroxy substituted C 1 -C 8 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 3 -C 22 Cycloalkyl, 3-22 membered heterocyclic group, R 8 Substituted 3-22 membered heterocyclic group, C 2 -C 22 Alkenyl, C 2 -C 22 Alkynyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 8 alkyl, or R 6 , R 7 Together with the nitrogen atom to which it is attached, it forms R 8 a substituted or unsubstituted 5-8 membered heterocyclic group;

[0131] Each R 8 Each independently selected from: C 1 -C 22 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 3 -C 22 Cycloalkyl, 3-22 membered heterocyclic group, C 1 -C 8 alkylalkyl substituted 3-22 membered heterocyclic group, C 2 -C 22 Alkenyl, C 2 -C 22 Alkynyl, C 6 -C 14 Aryl, C 6 -C 14 Aryl substituted C 1 -C 8 Alkyl, C 3 -C 22 Cycloalkyl substituted C 1 -C 8 alkyl,

[0132] In some preferred embodiments, each R 6 Each independently selected from: C 1 -C 10 Alkyl, hydroxy substituted C 1 -C 8 Alkyl, C 2 -C 10 Unsaturated chain hydrocarbon group, C 2 -C 10 Alkenyl, phenyl, naphthyl, anthracenyl, phenyl substituted C 1 -C 8 Alkyl, naphthyl substituted C 1 -C 8 Alkyl, anthracenyl substituted C 1 -C 8 alkyl,

[0133] Each R 7 Each independently selected from: C 1 -C 10 Alkyl, hydroxy substituted C 1 -C 8 Alkyl, C 2 -C 10 Unsaturated chain hydrocarbon group, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, R 8 Substituted 3-8 membered heterocyclic group, C 2 -C10 Alkenyl, C 2 -C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl substituted C 1 -C 8 Alkyl, naphthyl substituted C 1 -C 8 Alkyl, anthracenyl substituted C 1 -C 8 alkyl, or R 6 , R 7 Together with the nitrogen atom to which it is attached, it forms R 8 a substituted or unsubstituted 5-8 membered heterocyclic group;

[0134] Each R 8 Each independently selected from: C 1 -C 10 Alkyl, C 2 -C 10 Unsaturated chain hydrocarbon group, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, C 1 -C 6 alkylalkyl substituted 3-8 membered heterocyclic group, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl substituted C 1 -C 8 Alkyl, naphthyl substituted C 1 -C 8 Alkyl, anthracenyl substituted C 1 -C 8 Alkyl, C 3 -C 8 Cycloalkyl substituted C 1 -C 8 alkyl,

[0135] In some preferred embodiments, each R 6 Each independently selected from: C 1 -C 6 Alkyl; Hydroxyl substituted C 1 -C 6 alkyl,

[0136] Each R 7 Each independently selected from: C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 alkyl, or R6 , R 7 Together with the nitrogen atom to which it is attached, it forms R 8 a substituted or unsubstituted 5-8 membered heterocyclic group;

[0137] Each R 8 Each independently selected from: C 1 -C 6 Alkyl, 5-8 membered heterocyclic group, C 1 -C 6 Alkyl substituted 5-8 heterocyclic group, phenyl, phenyl substituted C 1 -C 3 alkyl,

[0138] In some preferred embodiments, R 9 Selected from: hydroxyl, C 6 -C 14 Aryl, C 3 -C 22 Cycloalkyl, 3-22 membered heterocyclic group, R 8 Substituted 3-22 membered heterocyclyl, 5-10 membered heteroaryl.

[0139] In some preferred embodiments, R 9 Selected from: hydroxyl, phenyl, naphthyl, anthracenyl, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, R 8 Substituted 3-8 membered heterocyclyl, 5-8 membered heteroaryl.

[0140] In some preferred embodiments, R 4 Selected from: C 1 -C 8 Alkyl, R 9 Substituted C 1 -C 8 Alkyl, C 3 -C 8 Cycloalkyl, phenyl substituted C 1 -C 3 Alkyl, naphthyl substituted C 1 -C 3 Alkyl, anthracenyl substituted C 1 -C 3 Alkyl, 3-8 membered heterocyclic group, R 8 Substituted 5-8 membered heterocyclic group, Substituted cyclohexyl;

[0141] R 5 Selected from: C 1 -C 4 Alkylene;

[0142] Each R6 Each independently selected from: C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, -CH 2 -R 2 -R 1 ;

[0143] Each R 7 Each independently selected from: C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, -CH 2 -R 2 -R 1 ;

[0144] Each R 8 Each independently selected from: C 1 -C 6 Alkyl, phenyl, phenyl-substituted C 1 -C 3 alkyl,

[0145] R 9 Selected from: hydroxyl, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, R 8 Substituted 3-8 membered heterocyclyl, 5-8 membered heteroaryl.

[0146] In some preferred embodiments, R 4 Selected from: C 4 -C 6 Alkyl, butyl substituted with dimethylamino, butyl substituted with diethylamino, n-propyl substituted with piperidinyl, R 9 Substituted C 1 -C 3 Alkyl, C 4 -C 8 Cycloalkyl, benzyl, 5-8 membered heterocyclic group, R 8 Substituted 5-6 membered heterocyclic group, Substituted cyclohexyl;

[0147] Each R 5 Each independently selected from: C 1 -C 3 Alkylene;

[0148] R 6 Selected from: C 3 -C 4 alkyl;

[0149] Each R7 Each independently selected from: C 3 -C 4 alkyl;

[0150] Each R 8 Each independently selected from: C 1 -C 6 Alkyl, benzyl,

[0151] R 9 Selected from:

[0152] In some preferred embodiments, the tertiary amine lipid compound has a structure as shown in the following formula (IV):

[0153]

[0154] Where n is 1, 2, 3, 4 or 5;

[0155] R 3 and R 4 Each independently selected from: C 1 -C 10 Alkyl, hydroxy substituted C 1 -C 10 Alkyl, C 1 -C 10 Unsaturated chain hydrocarbon group, C 3 -C 8 Cycloalkyl, or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms a 3-8 membered heterocyclic group.

[0156] In some preferred embodiments, R 3 and R 4 Each independently selected from: C 1 -C 6 Alkyl, hydroxy substituted C 1 -C 6 Alkyl, C 1 -C 6 Unsaturated chain hydrocarbon group, or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms a 5-8 membered heterocyclic group.

[0157] In some preferred embodiments, the tertiary amine lipid compound has a structure as shown in the following formula (V):

[0158]

[0159] n 1 、n 2 、n 3、n 4 Each is independently selected from: 0, 1, 2, 3 or 4.

[0160] In some preferred embodiments, each R 1 Each independently selected from: C 1 -C 22 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 3 -C 22 Cycloalkyl, C 2 -C 22 Alkenyl, C 2 -C 22 Alkynyl, C 6 -C 22 Aryl, C 6 -C 14 Aryl substituted C 1 -C 8 Alkyl, C 3 -C 22 Cycloalkyl substituted C 1 -C 8 alkyl.

[0161] In some preferred embodiments, each R 1 Each independently selected from: C 8 -C 20 Alkyl, C 8 -C 20 Unsaturated chain hydrocarbon group, C 5 -C 20 Cycloalkyl, C 8 -C 20 Alkenyl, C 8 -C 20 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl substituted C 1 -C 6 Alkyl, naphthyl substituted C 1 -C 6 Alkyl, anthracenyl substituted C 1 -C 6 Alkyl, C 5 -C 20 Cycloalkyl substituted C 1 -C 6 alkyl.

[0162] In some preferred embodiments, each R 1are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecylmethyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl , cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, docosene, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, docosene, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenemethyl, anthraceneethyl, anthracenepropyl, anthracenebutyl, anthracenehexyl, anthraceneoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

[0163] In some preferred embodiments, each R 1 Each of the following is independently selected from the group consisting of n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0164] In some preferred embodiments, each R 2 Independently selected from:

[0165] Or none, where c is 1 or 2; d is an integer between 2 and 6.

[0166] In some preferred embodiments, R 3 and R 4 are independently selected from: H, R 9 Substituted or unsubstituted C 1 -C 22 Alkyl, C 2 -C 22 Unsaturated chain hydrocarbon group, C 3 -C22 Cycloalkyl, 3-22 membered heterocyclic group, R 8 Substituted 3-22 membered heterocyclic group, C 2 -C 22 Alkenyl, C 2 -C 22 Alkynyl, C 6 -C 22 Aryl, C 6 -C 14 Aryl substituted C 1 -C 8 Alkyl, C 3 -C 22 Cycloalkyl substituted C 1 -C 8 Alkyl, -R 2 -R 1 , or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms R 8 A substituted or unsubstituted 3-10 membered heterocyclic ring.

[0167] In some preferred embodiments, R 3 and R 4 are independently selected from: H, R 9 Substituted or unsubstituted C 1 -C 10 Alkyl, C 2 -C 10 Unsaturated chain hydrocarbon group, C 3 -C 8 Cycloalkyl, 3-8 membered heterocyclic group, R 8 Substituted 3-8 membered heterocyclic group, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl substituted C 1 -C 6 Alkyl, naphthyl substituted C 1 -C 6 Alkyl, anthracenyl substituted C 1 -C 6 Alkyl, C 3 -C 8 Cycloalkyl substituted C 1 -C 6 Alkyl, -R 2 -R 1 , or R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms R 8 A substituted or unsubstituted 5-10 membered heterocyclic group.

[0168] In some preferred embodiments, R 3 , R 4 Together with the nitrogen atom to which it is attached, it forms the following group:

[0169]

[0170] In some preferred embodiments, the tertiary amine lipid compound is selected from the following compounds:

[0171]

[0172]

[0173]

[0174]

[0175] Among them, each R 1 Each of the following is independently: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0176] In some preferred embodiments, the tertiary amine lipid compound is selected from the following compounds:

[0177]

[0178]

[0179]

[0180] In one embodiment of the present invention, a lipid nanoparticle is also provided, which is formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic molecules in an aqueous medium, wherein the amphiphilic molecules are: amphiphilic polyethylene glycol lipid molecules, or a combination of amphiphilic polyethylene glycol lipid molecules and phospholipids.

[0181] Wherein, the amphiphilic polyethylene glycol lipid molecule can be selected from but not limited to: polyethylene glycol-dipalmitoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-(polyethylene glycol)-N,N-tetracosylacetamide, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyethylene glycol-polylactic acid-glycolate; preferably, the molecular weight of the polyethylene glycol in the amphiphilic polyethylene glycol lipid molecule is 500g / mol to 20000g / mol.

[0182] The phospholipids can be selected from, but are not limited to, cholesterol, soybean lecithin, hydrogenated soybean lecithin, soybean lecithin, egg yolk lecithin, highly purified egg yolk lecithin, dilauroyl lecithin, dieucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidyl ethyl Alcoholamine, palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl lysolecithin, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium-chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dioctadecenyloxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester.

[0183] In some preferred embodiments, the lipid nanoparticles are formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, amphiphilic polyethylene glycol lipid molecules and 1,2-palmitoylphosphatidylglycerol in an aqueous medium; or are formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic polyethylene glycol lipid molecules in an aqueous medium; preferably, the amphiphilic polyethylene glycol lipid molecule is distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0184] In some preferred embodiments, the mass ratio of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8, preferably 1:0.5-4, more preferably 1:0.5-2, more preferably 1:0.8-1.2; and most preferably 1:1.

[0185] In some preferred embodiments, the mass ratio of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt to the 1,2-palmitoylphosphatidylglycerol is 1:0.5-1.5, preferably 1:0.8-1.2.

[0186] In one embodiment of the present invention, a method for preparing the lipid nanoparticles is also provided, comprising the following steps:

[0187] dissolving the tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution;

[0188] dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution;

[0189] Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

[0190] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

[0191] In some preferred embodiments, the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran.

[0192] In some preferred embodiments, the concentration of the tertiary amine lipid compound is 10 mg / mL-100 mg / mL, more preferably 40 mg / mL-60 mg / mL; the concentration of the amphiphilic molecule solution is 20 mg / mL-200 mg / mL, more preferably 50 mg / mL-100 mg / mL.

[0193] In some preferred embodiments, the volume ratio of the mixed solution to the aqueous medium is 1:1-20, more preferably 1:2-10.

[0194] In one embodiment of the present invention, there is also provided the use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating tumors.

[0195] In one embodiment of the present invention, there is also provided the use of the lipid nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0196] In one embodiment of the present invention, there is also provided the use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in combination with an anti-tumor drug in the preparation of a drug for preventing and / or treating tumors.

[0197] In one embodiment of the present invention, there is also provided the use of the lipid nanoparticles combined with anti-tumor drugs in the preparation of drugs for preventing and / or treating tumors.

[0198] In some preferred embodiments, the anti-tumor drug is an immune checkpoint inhibitor and / or a chemotherapy drug.

[0199] In some preferred embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0200] In some preferred embodiments, the chemotherapy drug is doxorubicin.

[0201] In some preferred embodiments, the tumor is pancreatic cancer, melanoma, colorectal cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer, nasopharyngeal carcinoma, or brain cancer.

[0202] In one embodiment of the present invention, a drug for preventing and / or treating tumors is also provided, which is prepared from active ingredients and pharmaceutically acceptable excipients and / or carriers, wherein the active ingredients include the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and / or the lipid nanoparticles.

[0203] In one embodiment of the present invention, a combined drug for preventing and / or treating tumors is also provided, wherein the active ingredients include:

[0204] Component 1: the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and / or the lipid nanoparticle; and,

[0205] Component 2: anti-tumor drugs other than component 1;

[0206] The component 1 and the component 2 are respectively independent administration units, or the component 1 and the component 2 together form a combined administration unit.

[0207] In some preferred embodiments, component 2 is an immune checkpoint inhibitor and / or a chemotherapeutic drug.

[0208] In some preferred embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor.

[0209] In some preferred embodiments, the chemotherapy drug is doxorubicin.

[0210] The compounds of formula (I) to (V) of the present invention can be used in combination with other known anti-tumor drugs. When administered in combination, the compounds of formula (I) to (V) and the known drugs can be independent administration units, or together form a combined administration unit; the compounds of formula (I) to (V) can be administered simultaneously or separately with other known anti-tumor drugs. When the compounds of formula (I) to (V) are taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compounds of formula (I) to (V). Drug combination also includes taking the compounds of formula (I) to (V) and one or more other known drugs in overlapping time periods. When the compounds of formula (I) to (V) are used in combination with one or more other known drugs, the dosage of the compounds of formula (I) to (V) or the known drugs can be the same as the dosage of the single drug, or it can be lower than the dosage when they are taken alone.

[0211] The drugs or active ingredients that can be used in combination with the compounds of formula (I) to (V) include but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / Threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, etc.

[0212] In some embodiments, the drugs or active ingredients that can be used in combination with the compounds of formula (I) to (IV) include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosin, 5-azacytidine, azathioprine, bacillus Calmette-Guérin or tice bacillus Calmette-Guérin, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezosumab injection, capecitabine, carboplatin, castel, c Efesone, cimoleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, clodrophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin liposome, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin 2, depomet, deslorelin, delazosen, diethylstilbestrol, diflucan, docetaxel, doxorubicin, dronabinol, chin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-β-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphoric acid, vanbifu, etoposide, fa Drazolam, tamoxifen preparations, filgrastim, finasteride, filasti, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine nucleoside monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestan, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisiron hydrochloride, histrelin, andmeixin, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentan isobetumomab, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-nl, interferon α-n3, dry Interferon beta, interferon gamma-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levomizole, levofolinate calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, liposomal doxorubicin citrate, nedaplatin, pegfilgrastim, oprelleukin, neupogen, nilutamide, tamoxifen,NSC-631570, recombinant human interleukin 1-β, octreotide, ondansetron hydrochloride, dehydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate preparation, pegaspargase, PEGASYS, pentostatin, lysin, pilocarpine hydrochloride, pilocarpine, plicamycin, porfimer sodium, prednimustine, stiprenisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, libi, etidronate rhenium-186, rituximab, doxycycline-A, romotide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizoran, sobuzosine, sodium methylprednisolone, pafosic acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen Fen, tamsulosin, tasofenamin, tastolactone, taxotere, tesifazines, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid stimulating hormone, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, retinoic acid, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufodine, uridine, valrubicin, vesirinone, vinblastine, vincristine, vinamide, vinorelbine, virulizine, dextromethorphan, statins ester, zofran, paclitaxel protein stabilized preparation, acolbifene, interferon r-lb, affinitak, aminopterin, arzoxifene, a soprisnil, atamestane, atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, clenatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minocycline, MS- 209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratript, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium bromoglutamate, PN-401, QS-21, quasi-sea, R-154, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin αl, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100 and zoledronic acid or a combination thereof.

[0213] The drug for preventing and / or treating tumors of the present invention can be used in non-human mammals or humans.

[0214] The pharmaceutically acceptable excipients used in the drug for preventing and / or treating tumors of the present invention refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0215] "Compatibility" herein means that the components in the composition can be blended with the active ingredient of the present invention (tertiary amine lipid compounds represented by Formula I to Formula VII) and with each other without significantly reducing the efficacy of the active ingredient.

[0216] The pharmaceutically acceptable excipients used in the drug for preventing and / or treating tumors of the present invention include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, solubilizers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents and fragrances.

[0217] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as silymarin, silymarin, etc.), and glycerol. ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0218] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0219] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0220] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:

[0221] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0222] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic;

[0223] (c) humectants, for example, glycerin;

[0224] (d) disintegrating agents, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0225] (e) a buffering solvent, for example, paraffin;

[0226] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0227] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0228] (h) adsorbents, for example, kaolin; and

[0229] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0230] The solid dosage forms can also be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0231] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

[0232] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0233] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0234] The following are specific embodiments.

[0235] Example 1: Preparation of tertiary amine lipid compounds obtained by reaction of acrylates with amine molecules

[0236] In this example, tertiary amine lipid compounds containing different types of hydrophobic groups and tertiary amine structures were prepared. These lipids were synthesized by Michael addition reaction of acrylates containing different hydrophobic carbon chains (C8, C10, C10i, C12, C14, C16, C18, C181, C182, Cp2, Cp4) and amine molecules containing primary and / or secondary amines (A1 to A51, a total of 51). C8, C10, C10i, C12, C14, C16, C18, C181, C182, Cp2, Cp4 are n-octyl acrylate (C8), n-octyl acrylate (C10 ... Decyl acrylate (C10), isodecyl acrylate (C10i), n-dodecyl acrylate (C12), n-tetradecyl acrylate (C14), n-hexadecyl acrylate (C16), n-octadecyl acrylate (C18), 9-octadecene acrylate (C181), cis-9,12-octadecadienyl acrylate (C182), 2-phenylethyl acrylate (Cp2), phenylbutyl acrylate (Cp4); A1 to A51 are:

[0237]

[0238] That is, in this example, a series of tertiary amine lipid compounds represented by formula (VIII) were synthesized by Michael addition reaction of acrylate and primary amine-containing molecules. The reaction formula is as follows:

[0239]

[0240] Among them, R 1 is: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0241] Among them, R 3 Selected from the following groups:

[0242]

[0243]

[0244] A series of tertiary amine lipid compounds were synthesized by Michael addition reaction of acrylate and secondary amine-containing molecules. The exemplary reaction formula is as follows:

[0245]

[0246] Among them, R 1 is: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0247] A series of tertiary amine lipid compounds were synthesized by Michael addition reaction of acrylate and molecules containing both primary and secondary amines. The exemplary reaction formula is as follows:

[0248]

[0249] Among them, R 1 is: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0250] The acrylate n-decyl ester, 9-octadecene acrylate, cis-9,12-octadecadienyl acrylate and phenyl butyl acrylate required for the Michael addition reaction were synthesized in the laboratory, and the NMR was shown in Figure 1-Figure 4 The remaining acrylates and molecules containing primary amines and / or secondary amines were purchased directly and used without purification.

[0251] The specific steps are as follows:

[0252] 100 mg (0.20 mmol-0.60 mmol) of acrylate (respectively C8, C10i, C10, C12, C14, C16, C18, C181, C182, Cp2, Cp4) was placed in a reaction container, and A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A47, A48, A49, A50, A51, A52, A53, A54, A55, A56, A57, A58, A59, A50, A51, A52, A53, A54, A55 2. A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A47, A48, A49, A50, A51, mix well, and react in a sealed container at 90°C for 3 days. After the reaction, collect the product and use it directly.

[0253] The tertiary amine lipid compound prepared in this example is referred to as C n A n , where C n Refers to the acrylate in the raw material, An Refers to the amine molecules in the raw materials, such as C 8 A 1 It refers to R 1 The tertiary amine lipid compound is prepared by the above-mentioned reaction of n-octyl acrylate (C8) and amine molecule (A1).

[0254] Example 2 Preparation of lipid nanoparticles

[0255] The tertiary amine lipid compound synthesized in Example 1 has a hydrophobic carbon chain portion and a hydrophobic tertiary amine portion at pH 7.4, and can self-assemble in water to form nanoparticles with the assistance of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG).

[0256] The specific operation is as follows: dissolve the tertiary amine lipid compound in ethanol to obtain a lipid solution with a concentration of 50 mg / mL; take DSPE-PEG 2000 Dissolve in ethanol to obtain DSPE-PEG with a concentration of 50 mg / mL 2000 Solution; take 50 μL lipid solution and 50 μL DSPE-PEG 2000 The solutions were mixed in a 1.5 mL centrifuge tube to obtain a mixed solution; 20 μL of the mixed solution was taken into another 1.5 mL centrifuge tube, 180 μL of deionized water or 1×PBS was added with a pipette, and the mixture was quickly added to the mixed solution, and the mixture was repeatedly blown with a pipette to obtain lipid nanoparticles; or 3 mL of pure water or 1×PBS was taken into a 10 mL round-bottom flask, 1 mL of the mixed solution was taken with a pipette and slowly added dropwise to the water under stirring, and lipid nanoparticles were obtained after stirring for 10 minutes; the mixture was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da, and the ethanol was removed by dialyzing in pure water.

[0257] Example 3 Hemolytic activity of lipid nanoparticles

[0258] In this example, the hemolytic activity of the series of lipid nanoparticles prepared in Example 2 was detected, and lipid materials with a concentration of 400 μg / mL and a hemolytic activity of less than 5% after incubation for 4 hours were screened out.

[0259] The specific experimental steps are as follows: wash sheep blood with 1×PBS twice until the supernatant is clear, transparent and colorless after centrifugation, and then dilute the sheep blood with 1×PBS to 4%; take 16 μL of lipid nanoparticles prepared with 1×PBS in Example 2 into a 184-well cell culture plate, add 34 μL of 1×PBS, and then add 50 μL of 4% sheep blood, and incubate in a 37°C incubator for 4 hours; centrifuge at 3000 rpm for 10 minutes, then aspirate 50 μL of supernatant into another 384-well plate, and detect the absorbance at 576 nm with a multifunctional microplate reader; in addition, set up a PBS control group without lipid nanoparticles (50 μL 1×PBS+50 μL 4% sheep blood), a nanoparticle control group containing only lipid nanoparticles, and a positive control group containing 0.2% triton X (50 μL 0.2%c+50 μL 4% sheep blood).

[0260] Test results such as Figure 5 As shown, it can be seen that the hemolytic activity of most lipid nanoparticles is low (<5%), and the hemolytic activity of a small number of lipid nanoparticles is high (≥5%), indicating that the hemolytic toxicity of most lipid nanoparticles is low. Lipid nanoparticles with a hemolysis rate of less than 5% were selected for the next experiment.

[0261] Example 4 Cytotoxicity of lipid nanoparticles at characteristic pH of normal tissues and tumor tissues

[0262] In this example, the CCK8 method (Cell Counting Kit-8) was used to evaluate the killing effect of the drug on tumor cells under pH 7.4 and pH 6.8 conditions, and the cytotoxicity of the series of lipid nanoparticles prepared in Example 2 at the characteristic pH of normal tissues and tumor tissues was studied.

[0263] The specific experimental steps are as follows: 6 mol / L HCl solution was used to adjust the DMEM medium to pH = 6.8 and 7.4 for standby use, and 25 mM HEPES was added to maintain pH stability. Mouse colorectal cancer cell line MC38 (purchased from ATCC) was cultured in DMEM medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, the lipid nanoparticles (100 μg / mL) prepared in Example 2 were incubated with cells (concentration of 1×10 6 The cells were incubated in a 96-well plate (cells / mL), and the original culture medium was discarded after culturing at 37°C and in a carbon dioxide incubator for 24 hours. CCK8 (5 μL CCK-8 stock solution + 95 μL DMEM culture medium) solution was added, and the cells were placed in a 37°C, carbon dioxide incubator for further culturing for 2 hours. The OD 450nm absorbance was tested using an ELISA instrument and the cell survival rate was calculated. The cell survival rate was calculated as follows: OD 背景The blank wells contain no cells and only CCK-8. The control group contained cells and CCK-8 but was not treated with drugs.

[0264] Test results such as Figure 6 and Figure 7 As shown: the cytotoxicity of the lipid nanoparticles prepared by the present invention to tumor cells is basically located below the curve y=x in the figure, indicating that the cytotoxicity of these lipid nanoparticles at pH=7.4 is lower than that at pH=6.8, indicating that the lipid nanoparticles of the present invention have pH-responsive cytotoxicity.

[0265] Example 5 Column Chromatography Purification of Preferred Tertiary Amine Lipid Compounds

[0266] According to the cytotoxicity results of Example 4, lipid nanoparticles whose cytotoxicity at pH 6.8 was 50% higher than that at pH 7.4 were screened out, and the corresponding tertiary amine lipid compounds were purified and characterized by column chromatography.

[0267] Silica gel was used as column filler and dichloromethane:methanol = 2.5% to 10% was used as eluent for gradient elution to purify the above-selected tertiary amine lipid compound. Its NMR characterization was as follows: Figure 8 to Figure 23 As shown, analysis showed that relatively pure target products C8A16, C8A19, C8A45, C8A49, C12A27, C12A33, C14A33, C16A34, C181A17, C181A23, C181A24, C181A28, C182A17, C182A23, C182A24, and C182A28 were obtained.

[0268] The purified tertiary amine lipid compound was prepared into lipid nanoparticles according to the method described in Example 2. The particle size and distribution of lipid nanoparticles in aqueous solution were detected by dynamic light scattering. The results showed that the average particle size of the assembled lipid nanoparticles was about 200 nm ( Fig.26 ).

[0269] The lipid nanoparticles prepared in this example were tested for concentration-dependent cytotoxicity at pH values ​​characteristic of normal tissues and tumor tissues according to the method described in Example 4: the lipid nanoparticles were incubated with MC38 cells at different concentrations at pH = 6.8 and pH = 7.4 for 24 h, and their cytotoxicity was detected using the MTT method. The results are shown in Table 4. Fig.24 and Fig.25As shown, consistent with the results of Example 4, the lipid nanoparticles prepared with the purified tertiary amine lipid compounds have pH-responsive tumor cell killing ability, that is, the cytotoxicity is lower under the condition of pH = 7.4, and the cytotoxicity is higher under the condition of pH = 6.8, indicating that this type of lipid nanoparticles does have pH-activated cytotoxicity and is dose-dependent.

[0270] Example 6 Protonation of tertiary amine lipid nanoparticles at different pH

[0271] The preferred tertiary amine lipid compound of Example 4 was prepared into lipid nanoparticles according to the method of Example 2, and then the lipid nanoparticles were mixed with buffer solutions of different pH values ​​and TNS solutions, 100 μL of the solution was added to a black flat-bottom 96-well plate, and then the fluorescence intensity was measured using a full-wavelength microplate spectrophotometer, and the protonation degree and pKa were calculated by simulating the change curve. The specific method is as follows:

[0272] Buffer preparation: First, use ultrapure water to prepare a buffer solution containing 20mM HEPES, 20mM MES, 20mM ammonium acetate, and 260mM NaCl. Then, take four 25mL portions of 2× buffer solution, add 2mL and 1mL of 1M HCl solution (No. 1 and No. 2) and 2mL and 1mL of 1M NaOH solution (No. 3 and No. 4), respectively, and finally fill to 50mL with ultrapure water. Finally, use No. 1 and No. 4 solutions, No. 2 and No. 3 solutions to prepare buffer solutions with pH values ​​of 6.5-10 and 2.5-6.5 by mutual titration;

[0273] Measurement method: Take 980 μL of buffer solutions of different pH values ​​and add them to 1.5 mL EP tubes, then add 10 μL of 2.5 mg / mL lipid nanoparticles and 100 μM TNS aqueous solution in sequence. After mixing evenly, take 100 μL of the solution and add it to a black flat-bottom 96-well plate. Use a full-wavelength microplate spectrophotometer to measure the fluorescence intensity at excitation and emission wavelengths of 321 nm and 445 nm, respectively.

[0274] Calculation method: The measured data was imported into Origin and analyzed using the Boltzmann function in the S-type function simulation. The point with the maximum fluorescence intensity in the curve was defined as a protonation rate of 100%, and the point with the minimum fluorescence intensity in the curve was defined as a protonation rate of 0%. The data was then normalized to obtain a simulation curve graph. The pH value corresponding to a protonation rate of 50% in the graph is the pKa of the lipid nanoparticle.

[0275] Test results such as Fig. 27As shown: The results show that the carbon chain length and the type of tertiary amine significantly affect the protonation rate and pKa of lipids at different pH; at the same time, except for C8A45, the pka of the remaining lipid nanoparticles are between 6.0 and 7.0, indicating that these lipid nanoparticles can be protonated within this pH range, thereby activating the anti-tumor activity mediated by cell membrane disruption.

[0276] Example 7 Maximum Tolerated Dose of Lipid Nanoparticles in ICR Mice

[0277] In this example, the toxicity of the lipid nanoparticles of the present invention was studied by testing the maximum tolerated dose (MTD) in mice. The preferred lipid nanoparticles of Example 4 were injected into ICR mice via the tail vein. Each material was administered starting from 100 mg / kg body weight until the death of experimental mice occurred. The maximum dose without death of mice after continuous administration of 5 mice was recorded as the maximum tolerated dose of mice, and a graph was plotted, as shown in FIG. Fig.28 As shown: the MTD of C8A45, C8A49, C12A33 and C14A33 are all greater than 200 mg / kg, while the MTD of C16A34, C181A17, C181A23, C181A24, C181A28, C182A17, C182A23 and C182A24 are less than 100 mg / kg. In addition, the MTD of C12A27 and C182A28 are 120 and 140 mg / kg, respectively.

[0278] Example 8 Effects of lipid nanoparticle components on their cytotoxicity at characteristic pH of normal and tumor tissues

[0279] (1) Effect of the type of polyethylene glycol lipid on the cytotoxicity of lipid nanoparticles

[0280] Taking C12A27 as an example, three clinically approved polyethylene glycol lipids were selected to study the effects of PEG lipid types and their ratios to C12A27 on the cytotoxicity of lipid nanoparticles at different pH. C12A27 was respectively mixed with DSPE-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG), and 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159) according to the component dosage and experimental procedures described in Example 2 to prepare lipid nanoparticles, and the cytotoxicity of lipid nanoparticles at the characteristic pH of normal tissues and tumor tissues was studied according to the procedures described in Example 5.

[0281] The results are as follows Fig.28As shown in a: the three PEG lipids have no effect on the cytotoxicity of the obtained lipid nanoparticles at pH 6.8, but have an effect on their cytotoxicity at pH 7.4; specifically, the lipid nanoparticles containing DSPE-PEG have the least cytotoxicity at pH 7.4, followed by DMG-PEG, and ALC-0159 has the greatest toxicity.

[0282] (2) Effect of the ratio of DSPE-PEG to C12A27 on the cytotoxicity of the obtained lipid nanoparticles

[0283] DSPE-PEG and C12A27 were mixed at mass ratios of 0.2:1, 0.5:1, 1:1, 2:1, 4:1, and 8:1, and lipid nanoparticles were prepared according to the experimental steps in Example 2, and then the cytotoxicity of the lipid nanoparticles was detected referring to the method described in Example 4.

[0284] The results are as follows Fig.29 As shown in b: when the mass ratio of DSPE-PEG and C12A27 is ≤4:1, there is no significant effect on the cytotoxicity of lipid nanoparticles at pH 6.8; when the mass ratio of DSPE-PEG and C12A27 is 8:1, the cytotoxicity of lipid nanoparticles at pH 6.8 is significantly reduced; when the mass ratio of DSPE-PEG and C12A27 is 0.2:1, the cytotoxicity of lipid nanoparticles at pH 7.4 increases; when the mass ratio of DSPE-PEG and C12A27 is ≥0.5:1, there is no significant effect on the cytotoxicity of lipid nanoparticles at pH 7.4.

[0285] (3) Effects of other auxiliary lipid types on the cytotoxicity of lipid nanoparticles

[0286] Cholesterol (Chol), dioleoylphosphatidylcholine (DOPC), DSPE and 1,2-palmitoylphosphatidylglycerol (DPPG) were used as other auxiliary lipids. According to the steps described in Example 2, the above auxiliary lipids were added to the tertiary amine lipid compound and DSPE-PEG to prepare lipid nanoparticles. The specific operation was as follows: the tertiary amine lipid compound C12A27 was dissolved in ethanol to obtain a lipid solution with a concentration of 50 mg / mL; DSPE-PEG was taken 2000 Dissolve in ethanol to obtain DSPE-PEG with a concentration of 50 mg / mL 2000 Solution; take other auxiliary lipids and dissolve them in ethanol to obtain an auxiliary lipid solution with a concentration of 50 mg / mL; take 50 μL of lipid solution, 50 μL of DSPE-PEG 2000The solution and 25 μL (or 50 μL) of auxiliary lipid solution are mixed in a 1.5 mL centrifuge tube to obtain a mixed solution; 20 μL of the mixed solution is taken into another 1.5 mL centrifuge tube, 180 μL of deionized water is added with a pipette, and it is quickly added to the above mixed solution, and the lipid nanoparticles are repeatedly blown with a pipette to obtain lipid nanoparticles; the lipid nanoparticles are transferred to a dialysis bag with a molecular weight cutoff of 14000 Da, and dialyzed in pure water to remove ethanol.

[0287] The cytotoxicity of the obtained lipid nanoparticles was then tested according to the method described in Example 4. Fig.29 As shown in c and d: the incorporation of different auxiliary lipids has no significant effect on the cytotoxicity of lipid nanoparticles at pH 6.8, but the addition of Chol, DOPC, and DSPE will increase the cytotoxicity of lipid nanoparticles at pH 7.4, and the addition of DPPG has no obvious effect on the cytotoxicity of lipid nanoparticles at pH 7.4.

[0288] Example 9 Mechanism of lipid nanoparticles killing tumor cells at the characteristic pH of tumor tissue

[0289] C12A27 lipid nanoparticles were co-incubated with MC38 tumor cells at pH 6.8 and 37°C or 4°C. The activity of the cells was detected by CCK8 at 4 h. It was found that low temperature did not inhibit the killing of tumor cells by C12A27 lipid nanoparticles ( Fig.30 Figure a), indicating that this killing process is not dependent on energy. It was also found that C12A27 can cause tumor cells to rapidly release ATP in the cytoplasm ( Fig.30 This suggests that C12A27 may kill tumor cells by destroying the tumor cell membrane.

[0290] C12A27 lipid nanoparticles were further combined with MC38 cells expressing GFP on the cell membrane and mCherry in the cytoplasm. GFP / mCherry Incubated in a medium with pH 6.8 and observed under a laser confocal microscope, the red fluorescence (mCherry) of the cells gradually weakened over time and almost completely disappeared at 3 hours. In addition, the cell membranes of a large number of cells were observed to swell and spit bubbles under the bright field ( Fig.30 Figure c in the figure shows that the lipid nanoparticles of the present invention destroy the cell membrane of tumor cells, causing cell membrane damage and cell content release, thereby achieving the effect of killing tumor cells. GFP / mCherry Similar phenomena were observed when cells were co-incubated in a medium with pH 6.8 and observed using a high-content imaging system ( Fig.31). C8A16 lipid nanoparticles were co-incubated with propidium iodide (PI) and Annexin-V-FITC at pH 6.8 with MC38 cells and observed using a high-content imaging system. After incubation of C8A16 lipid nanoparticles with MC38 for 2 hours, the cells died rapidly, with double positive staining of PI and Annexin-V-FITC ( Fig.32 ). In addition, the lipid nanoparticles of the present invention have an effect on Panc02 GFP / mCherry Cells also have a similar killing mechanism that causes membrane rupture and rapid release of cytoplasmic contents ( Fig.33 ).

[0291] Example 10 In vivo tumor inhibition experiment of lipid nanoparticles

[0292] The tumor inhibition effect of lipid nanoparticles was verified by in vivo experiments in C57 mouse pancreatic cancer subcutaneous tumor model (Panc-02 cells), C57 mouse colorectal cancer subcutaneous tumor model (MC38 cells), and C57 mouse breast cancer model (E0771 cells). The operation is as follows:

[0293] Female mice (C57BL / 6, 6-8 weeks old) were injected subcutaneously on the back with 1×10 7 Panc02 cell suspension (100 μL) was used to establish a subcutaneous pancreatic cancer tumor model. 1×10 7 To establish a subcutaneous colorectal cancer tumor model, 1×10 cells / mL MC38 cell suspension (100 μL) was injected into the second mammary pad of the right side of female mice (C57BL / 6, 6-8 weeks old). 7 E0771 cell suspension (100 μL) was added to establish an orthotopic breast cancer tumor model. 3 (Tumor volume = length × width × width / 2), administered via tail vein, the dosage is as follows, Panc02 model: C12A27 (20 mg / kg), C14A27 (50 mg / kg), C16A27 (67 mg / kg); MC38 model: C8A16 (100 mg / kg), C8A19 (100 mg / kg), C8A45 (100 mg / kg), C8A49 (100 mg / kg), C12A27 (30 mg / kg); E0771 model: C8A45 (80 mg / kg), C8A49 (80 mg / kg), C12A27 (30 mg / kg), C12A33 (80 mg / kg). A negative control group was set up, and only an equal volume of PBS solution was injected. The tumor size was measured using a vernier caliper and the weight of the mice was recorded.

[0294] In vivo tumor inhibition effect of Panc02 model Fig.34 As shown in the figure, according to the tumor growth curve, compared with the PBS group, the tumor volume of the three drug-treated groups was inhibited to a certain extent, among which the growth of the tumor volume of the C12A27 lipid nanoparticle-treated group was significantly slowed down. At the same time, after the mice underwent the entire treatment cycle, there was no difference in body weight between the experimental group and the PBS control group. In the MC38 tumor model ( Fig.35 ), C8A16 lipid nanoparticles, C8A19 lipid nanoparticles, C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, and C12A27 lipid nanoparticles all had a certain inhibitory effect on tumor growth, among which C8A49 lipid nanoparticles had the best therapeutic effect. At the same time, this treatment regimen had no significant effect on the weight of mice. Lipid nanoparticles also have a good therapeutic effect on mouse breast cancer E0771. Among them, C8A49 lipid nanoparticles and C12A27 lipid nanoparticles can cause tumor regression in all mice, while C8A45 lipid nanoparticles and C12A33 lipid nanoparticles can cause tumor regression in some mice ( Fig.36 ).

[0295] Example 11 In vivo tumor inhibition experiment of lipid nanoparticles combined with immune checkpoint inhibitors

[0296] The tumor inhibition effect of lipid nanoparticles combined with immune checkpoint antibody, anti-programmed cell death receptor 1 antibody (αPD-1), was verified by in vivo experiments in a C57 mouse colorectal cancer subcutaneous tumor model (MC38 cells). The operation is as follows:

[0297] Female mice (C57BL / 6, 6-8 weeks old) were injected subcutaneously on the back with 1×10 7 100 μL of MC38 cell suspension was added to establish a subcutaneous colorectal cancer tumor model. 3 (Tumor volume = length × width × width / 2), administered via tail vein, the dosage is as follows: C12A27 (20 mg / kg), C14A27 (50 mg / kg), αPD-1 (0.75 mg / kg). A negative control group was set up, and only an equal volume of PBS solution was injected. Tumor size was measured with a vernier caliper, and the weight of the mice was recorded.

[0298] From the mouse tumor growth curve ( Fig.37) It can be seen that the combined treatment of C12A27 and C14A27 lipid nanoparticles and αPD-1 can enhance the therapeutic efficacy of αPD-1, among which C14A27 combined with αPD-1 has the best tumor inhibition effect, and the tumors of 4 mice have completely disappeared. The tumors of 2 mice in the C12A27 combined group have completely disappeared. At the same time, this treatment regimen has no significant effect on the weight of mice. The above results show that the lipid nanoparticles of the present invention can enhance the efficacy of immune checkpoint antibodies used clinically, thereby increasing the response rate of tumor patients to immune checkpoint antibodies and improving the anti-tumor effect.

[0299] Example 12 In vivo tumor inhibition experiment of lipid nanoparticles combined with chemotherapeutic drugs

[0300] The tumor inhibition effect of lipid nanoparticles combined with the chemotherapy drug liposomal doxorubicin Doxil was verified by in vivo experiments in the Balb / c mouse breast cancer orthotopic model (EMT-6 cells). The operation is as follows:

[0301] Female mice (Balb / c, 6-8 weeks old) were injected with 3 × 10 6 100 μL of EMT-6 cell suspension was added to establish a breast cancer subcutaneous tumor model. 3 (Tumor volume = length × width × width / 2), administered via tail vein, the dosage is as follows: C12A27 (20 mg / kg), C14A27 (50 mg / kg), Doxil (2.5 mg / kg). A negative control group was set up, and only an equal volume of PBS solution was injected. Tumor size was measured with a vernier caliper, and the weight of the mice was recorded.

[0302] From the growth curve of mouse tumors ( Fig.38 ) It can be seen that the combination of C12A27 lipid material and Doxil can significantly enhance the anti-tumor efficacy, and the tumors of all mice in the combination group completely disappeared. The above results show that the lipid nanoparticles of the present invention can enhance the anti-tumor efficacy of chemotherapeutic drugs.

[0303] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0304] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A tertiary amine lipid compound or a pharmaceutically acceptable salt thereof, characterized in that: The tertiary amine lipid compound is selected from the following compounds:

2. A lipid nanoparticle, characterized in that: It is formed by self-assembly of a tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof, and an amphiphilic molecule in an aqueous medium, wherein the amphiphilic molecule is: an amphiphilic polyethylene glycol lipid molecule, or a combination of an amphiphilic polyethylene glycol lipid molecule and a phospholipid; The tertiary amine lipid compound has a tertiary amine lipid compound having a structure shown in formula (I) or a pharmaceutically acceptable salt thereof: Wherein, each R1 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon, C6-C 14 Aryl-substituted C1-C8 alkyl; Each R2 is independently selected from: R3 and R4 are independently selected from: H, R9 substituted or unsubstituted C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, -R2-R1, Or R3, R4 and the nitrogen atom connected thereto together form a 3-10 membered heterocyclic ring which is substituted or unsubstituted with R8; and when R3 is directly connected to N, R3 is not H; when R4 is directly connected to N, R4 is not H; Each R5 is independently selected from: C1-C 22 Alkylene, C3-C8 cycloalkyl, C6-C 14 Aryl; Each R6 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon, C2-C 22 Alkenyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, Each R7 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, C1-C8 alkyl substituted 3-22 membered heterocyclic group, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl, R9 is selected from: hydroxyl, C6-C 14 Aryl, C3-C 22 Cycloalkyl, 3-22 membered heterocyclyl, 3-22 membered heterocyclyl substituted by R8, 5-10 membered heteroaryl; Each q and each q' is independently 0, 1 or 2; a is 0 or 1; b is 1, 2, 3, 4 or 5.

3. The lipid nanoparticle according to claim 2, characterized in that The tertiary amine lipid compound has a structure shown in the following formula (II):

4. The lipid nanoparticle according to claim 3, characterized in that The tertiary amine lipid compound has a structure shown in the following formula (III): R4 is selected from: R9 substituted or unsubstituted C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, 5. The lipid nanoparticle according to claim 4, characterized in that R4 is selected from: C1-C 22 Alkyl, R9 substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, 6. The lipid nanoparticle according to claim 5, characterized in that R4 is selected from: C1-C 10 Alkyl, R9 substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, 7. The lipid nanoparticle according to any one of claims 2 to 6, characterized in that Each R5 is independently selected from: C1-C 10 Alkylene, C3-C8 cycloalkyl, phenyl, naphthyl, anthracenyl.

8. The lipid nanoparticle according to claim 7, characterized in that Each R5 is independently selected from: C1-C6 alkylene, C3-C8 cycloalkyl.

9. The lipid nanoparticle according to claim 8, characterized in that Each R5 is independently selected from: methylene, ethylene, propylene, butylene, 10. The lipid nanoparticle according to any one of claims 2 to 6, characterized in that Each R6 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon, C2-C 10 alkenyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, Each R7 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, C1-C6 alkyl substituted 3-8 membered heterocyclic group, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, C3-C8 cycloalkyl-substituted C1-C8 alkyl, 11. The lipid nanoparticle according to claim 10, characterized in that Each R6 is independently selected from: C1-C6 alkyl; C1-C6 alkyl substituted with hydroxyl; Each R7 is independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C6 alkyl, 5-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by C1-C6 alkyl, phenyl, C1-C3 alkyl substituted by phenyl, 12. The lipid nanoparticle according to claim 11, characterized in that R9 is selected from the group consisting of: hydroxy, phenyl, naphthyl, anthracenyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted by R8, and 5-8 membered heteroaryl.

13. The lipid nanoparticle according to claim 6, characterized in that R4 is selected from: C1-C8 alkyl, C1-C8 alkyl substituted by R9, C3-C8 cycloalkyl, C1-C3 alkyl substituted by phenyl, C1-C3 alkyl substituted by naphthyl, C1-C3 alkyl substituted by anthracenyl, 3-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by R8, Substituted cyclohexyl; R5 is selected from: C1-C4 alkylene; Each R6 is independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, -CH2-R2-R1; Each R7 is independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, -CH2-R2-R1; Each R8 is independently selected from: C1-C6 alkyl, phenyl, phenyl-substituted C1-C3 alkyl, R9 is selected from the group consisting of: hydroxy, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted by R8, and 5-8 membered heteroaryl.

14. The lipid nanoparticle according to claim 13, characterized in that R4 is selected from: C4-C6 alkyl, butyl substituted by dimethylamino, butyl substituted by diethylamino, n-propyl substituted by piperidinyl, C1-C3 alkyl substituted by R9, C4-C8 cycloalkyl, benzyl, 5-8 membered heterocyclic group, 5-6 membered heterocyclic group substituted by R8, Substituted cyclohexyl; Each R5 is independently selected from: C1-C3 alkylene; R6 is selected from: C3-C4 alkyl; Each R7 is independently selected from: C3-C4 alkyl; Each R8 is independently selected from: C1-C6 alkyl, benzyl, R9 is selected from:

15. The lipid nanoparticle according to claim 2, characterized in that The tertiary amine lipid compound has a structure as shown in the following formula (IV): Where n is 1, 2, 3, 4 or 5; R3 and R4 are independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C 10 Alkyl, C1-C 10 An unsaturated chain hydrocarbon group, a C3-C8 cycloalkyl group, or R3, R4 and the nitrogen atom to which they are attached together form a 3-8 membered heterocyclic group.

16. The lipid nanoparticle according to claim 15, characterized in that R3 and R4 are independently selected from: C1-C6 alkyl, C1-C6 alkyl substituted with hydroxyl, C1-C6 unsaturated chain hydrocarbon group, or R3, R4 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group.

17. The lipid nanoparticle according to claim 2, characterized in that The tertiary amine lipid compound has a structure as shown in the following formula (V): n1, n2, n3, n4 are independently selected from: 0, 1, 2, 3 or 4.

18. The lipid nanoparticle according to any one of claims 2 to 6 and claims 15 to 17, characterized in that: Each R1 is independently selected from: C8-C 20 Alkyl, C8-C 20 Unsaturated chain hydrocarbon group, phenyl-substituted C1-C6 alkyl group, naphthyl-substituted C1-C6 alkyl group, anthracenyl-substituted C1-C6 alkyl group.

19. The lipid nanoparticle according to any one of claims 2 to 6 and claims 15 to 17, characterized in that: Each R1 is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, dioctyl, Dodecyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, dodecenyl, linolenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenemethyl, anthraceneethyl, anthracenepropyl, anthracenebutyl, anthracenehexyl, anthraceneoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

20. The lipid nanoparticle according to claim 19, characterized in that Each R1 is independently selected from: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

21. The lipid nanoparticle according to any one of claims 2 to 6 and claims 15 to 17, characterized in that: R3 and R4 are independently selected from: H, R9 substituted or unsubstituted C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthracenyl-substituted C1-C6 alkyl, C3-C8 cycloalkyl-substituted C1-C6 alkyl, -R2-R1, Or R3, R4 and the nitrogen atom to which they are connected together form a 5-10 membered heterocyclic group which is substituted or unsubstituted with R8.

22. The lipid nanoparticle according to claim 21, characterized in that R3, R4 and the nitrogen atom connected thereto together form the following group:

23. The lipid nanoparticle according to claim 2, characterized in that The tertiary amine lipid compound is selected from the following compounds: Wherein, each R1 is independently: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

24. The lipid nanoparticle according to claim 23, characterized in that The tertiary amine lipid compound is selected from the following compounds:

25. The lipid nanoparticle according to claim 2, characterized in that The phospholipids are selected from cholesterol, soybean lecithin, hydrogenated soybean lecithin, soybean lecithin, egg yolk lecithin, highly purified egg yolk lecithin, dilauroyl lecithin, dieucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidylethanolamine, palm lysophosphatidylcholine, myristoyl lysophosphatidylcholine, stearoyl lysophosphatidylcholine, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium-chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dioctadecenyloxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester; and / or, The amphiphilic polyethylene glycol lipid molecule is selected from polyethylene glycol-dipalmitoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, 2-(polyethylene glycol)-N,N-tetracosyl acetamide, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyethylene glycol-polylactic acid-glycolate.

26. The lipid nanoparticle according to claim 25, characterized in that The molecular weight of the polyethylene glycol in the amphiphilic polyethylene glycol lipid molecule is 500 g / mol to 20000 g / mol.

27. The lipid nanoparticle according to claim 2, characterized in that It is formed by self-assembly of the tertiary amine lipid compound or its pharmaceutically acceptable salt, amphiphilic polyethylene glycol lipid molecules and 1,2-palmitoylphosphatidylglycerol in an aqueous medium; or it is formed by self-assembly of the tertiary amine lipid compound or its pharmaceutically acceptable salt and amphiphilic polyethylene glycol lipid molecules in an aqueous medium.

28. The lipid nanoparticle according to claim 27, characterized in that The amphiphilic polyethylene glycol lipid molecule is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

29. The lipid nanoparticle according to claim 27, characterized in that The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8; The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the 1,2-palmitoylphosphatidylglycerol is 1:0.5-1.

5.

30. The lipid nanoparticle according to claim 29, characterized in that The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the amphiphilic polyethylene glycol lipid molecule is 1:0.5-4.

31. The lipid nanoparticle according to claim 30, characterized in that The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the amphiphilic polyethylene glycol lipid molecule is 1:0.5-2.

32. The lipid nanoparticle according to claim 31, characterized in that The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the amphiphilic polyethylene glycol lipid molecule is 1:0.8-1.

2.

33. The lipid nanoparticle according to claim 29, characterized in that The mass ratio of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof to the 1,2-palmitoylphosphatidylglycerol is 1:0.8-1.

2.

34. A method for preparing lipid nanoparticles according to claim 2, characterized in that: The steps include: dissolving the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution; dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution; Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution; The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

35. The method for preparing lipid nanoparticles according to claim 34, characterized in that: The organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran.

36. The method for preparing lipid nanoparticles according to claim 34, characterized in that: The concentration of the tertiary amine lipid compound is 10 mg / mL-100 mg / mL; the concentration of the amphiphilic molecule solution is 20 mg / mL-200 mg / mL.

37. The method for preparing lipid nanoparticles according to claim 34, characterized in that: The volume ratio of the mixed solution to the aqueous medium is 1:1-20.

38. Use of the tertiary amine lipid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing and / or treating tumors.

39. Use of the lipid nanoparticles according to any one of claims 2 to 33 in the preparation of drugs for preventing and / or treating tumors.

40. Use of the tertiary amine lipid compound according to claim 1 or a pharmaceutically acceptable salt thereof in combination with an anti-tumor drug in the preparation of a drug for preventing and / or treating tumors.

41. Use of the lipid nanoparticles according to any one of claims 2 to 33 in combination with an anti-tumor drug in the preparation of a drug for preventing and / or treating tumors.

42. The use according to claim 40 or 41, characterized in that The anti-tumor drug is an immune checkpoint inhibitor and / or a chemotherapy drug.

43. The use according to claim 42, characterized in that The immune checkpoint inhibitor is a PD-1 inhibitor; and / or the chemotherapy drug is doxorubicin.

44. The use according to any one of claims 38 to 41, characterized in that The tumor is pancreatic cancer, melanoma, colorectal cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer, nasopharyngeal carcinoma, and brain cancer.

45. A drug for preventing and / or treating tumors, characterized in that: It is prepared from active ingredients and pharmaceutically acceptable excipients and / or carriers, wherein the active ingredients include the tertiary amine lipid compound according to claim 1 or a pharmaceutically acceptable salt thereof, and / or the lipid nanoparticles according to any one of claims 2-33.

46. ​​A combined drug for preventing and / or treating tumors, characterized in that: Its active ingredients include: Component 1: the tertiary amine lipid compound of claim 1 or a pharmaceutically acceptable salt thereof, and / or the lipid nanoparticle of any one of claims 2 to 33; and, Component 2: anti-tumor drugs other than component 1; The component 1 and the component 2 are respectively independent administration units, or the component 1 and the component 2 together form a combined administration unit.

47. The combined drug for preventing and / or treating tumors according to claim 46, characterized in that: The component 2 is an immune checkpoint inhibitor and / or a chemotherapy drug.

48. The combined drug for preventing and / or treating tumors according to claim 47, characterized in that: The immune checkpoint inhibitor is a PD-1 inhibitor; and / or the chemotherapy drug is doxorubicin.

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