pH-responsive tertiary amine-based lipid compounds and uses thereof

Abstract

The application provides a tertiary amine lipid compound with a structure shown in formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, and an application thereof. The tertiary amine lipid compound is hydrophobic and electrically neutral at normal physiological pH, can be self-assembled into pH-responsive membrane-breaking material nanoparticles with a PEG shell layer together with materials such as DSPE-PEG, has weak interaction with membranes and is low in cytotoxicity; under a slightly acidic pH condition, part of the tertiary amine can be protonated to form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, has strong interaction with cell membranes and organelle membranes and strong membrane-breaking activity, and thus can efficiently and selectively destroy cell membranes or lysosome membranes, so as to kill tumor cells.

Description

Technical Field

[0001] This invention relates to the fields of materials and pharmaceutical technology, specifically to a pH-responsive tertiary amine lipid compound and its applications. Background Technology

[0002] Malignant tumors have become a major killer threatening 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 problems such as 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 materials can kill tumor cells and pathogens such as bacteria by disrupting cell membranes or organelle membranes, exhibiting advantages such as broad-spectrum killing effects and low likelihood of drug resistance, making them promising candidates for the treatment of tumors and other diseases. For example, the LTX-315 cationic peptide, administered intratumorally, can induce massive necrosis of tumor cells. Combined with drugs such as ipilimumab and pembrolizumab, it has shown promising results in melanoma, head and neck cancer, lymphoma, and breast cancer, and related research is currently in Phase I clinical trials (https: / / clinicaltrials.gov / ). Research on the intratumoral injection of the LL-37 peptide for melanoma treatment is also in Phase I clinical trials (https: / / clinicaltrials.gov / ). However, because these drugs not only efficiently destroy tumor cells and pathogens but also exhibit strong cytotoxicity to normal tissue cells, and because peptide drugs are easily degraded by proteases, most have stopped at the clinical research stage. Currently, most drugs used clinically are administered via local injection.

[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 pH-responsive tertiary amine lipid compounds. These tertiary amine lipids are prepared by ring-opening reactions of primary or secondary amines with epoxides, or by substituting the hydroxyl hydrogen with an alkyl group to obtain corresponding derivatives. The present invention discovers that partial modification of the hydroxyl group can reduce its cytotoxicity at pH 7.4, thereby improving its killing selectivity at normal physiological pH and tumor tissue pH. These tertiary amine lipid compounds can exhibit hydrophobic and electrically neutral behavior at normal physiological pH, and can self-assemble with amphiphilic auxiliary lipids such as DSPE-PEG to form lipid nanoparticles with a PEG shell, exhibiting low cytotoxicity. Under slightly acidic pH conditions, they can be protonated to form an amphiphilic structure composed of hydrophobic and cationic domains, exhibiting strong interactions with cell membranes or organelle membranes, thereby producing strong cytotoxicity and specifically killing tumor cells.

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

[0007] Application of tertiary amine lipid compounds having the structure shown in formula (I) or their stereoisomers or pharmaceutically acceptable salts in the preparation of pH-responsive membrane-breaking materials:

[0008]

[0009] Each R1 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, alkenyl, alkynyl, aryl, aryl-substituted alkyl, and cycloalkyl-substituted alkyl;

[0010] Each R2 is independently selected from: H, R1; where R is selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0011] R3 and R4 are each independently selected from: H, R9-substituted or unsubstituted alkyl groups, unsaturated chain hydrocarbon groups, cycloalkyl groups, heterocyclic groups, R8-substituted heterocyclic groups, alkenyl groups, alkynyl groups, aryl groups, etc. Alternatively, R3, R4, and the nitrogen atom attached to them can form an R8-substituted or unsubstituted 3-20 membered heterocyclic group; and when R3 is directly attached to N, R3 is not H; when R4 is directly attached to N, R4 is not H.

[0012] Each R5 is independently selected from: alkylene, cycloalkyl, and aryl;

[0013] Each R6 is independently selected from: alkyl, hydroxylated alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl-substituted alkyl,

[0014] Each R7 is independently selected from: alkyl, hydroxyl-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R8-substituted heterocyclic, alkenyl, alkynyl, aryl, aryl-substituted alkyl, Alternatively, R6, R7, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-8 membered heterocyclic groups;

[0015] Each R8 group is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl-substituted heterocyclic, alkenyl, alkynyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl.

[0016] R9 is selected from: hydroxyl, aryl, cycloalkyl, heterocyclic, R8-substituted heterocyclic, and heteroaryl;

[0017] Each q and each q' is independently 0, 1, or 2;

[0018] a can be 1, 2, 3, 4 or 5.

[0019] The tertiary amine lipid compound may have the structure shown in formula (II):

[0020]

[0021] The tertiary amine lipid compound may also have the structure shown in formula (III):

[0022]

[0023] R4 is selected from: R9-substituted or unsubstituted alkyl groups, unsaturated chain hydrocarbon groups, cycloalkyl groups, heterocyclic groups, R8-substituted heterocyclic groups, alkenyl groups, alkynyl groups, aryl groups, etc.

[0024] The tertiary amine lipid compound may also have the structure shown in formula (IV):

[0025]

[0026] Where n is 1, 2, 3, 4 or 5;

[0027] R3 and R4 are independently selected from: C1-C 10 Alkyl and hydroxyl substituted C1-C 10 Alkyl, C1-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, Alternatively, R3, R4, and the nitrogen atom attached to them can form 3-8 membered heterocyclic groups.

[0028] On the other hand, the present invention also provides lipid nanoparticles formed by the self-assembly of the aforementioned 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.

[0029] On the other hand, the present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0030] The tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt is dissolved in an organic solvent to obtain a tertiary amine lipid compound solution;

[0031] The amphiphilic molecule is dissolved in an organic solvent to obtain an amphiphilic molecule solution;

[0032] The tertiary amine lipid compound solution and the amphiphilic molecule solution are mixed to obtain a mixed solution;

[0033] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing it with a pipette; or, the lipid nanoparticles are obtained by slowly adding the mixed solution dropwise to an aqueous medium under stirring and continuing to stir.

[0034] On the other hand, the present invention also provides the application of the aforementioned tertiary amine lipid compounds or their stereoisomers or their pharmaceutically acceptable salts, or the aforementioned lipid nanoparticles, including the following technical solutions.

[0035] The use of the aforementioned tertiary amine lipid compounds, or their stereoisomers, or their pharmaceutically acceptable salts, in the preparation of medicaments for the prevention and / or treatment of tumors.

[0036] The application of the lipid nanoparticles in the preparation of drugs for the prevention and / or treatment of tumors.

[0037] On the other hand, the present invention also provides a medicament for the prevention and / or treatment of tumors, prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt described in the present invention, and / or the lipid nanoparticles described therein.

[0038] Previous research by the inventors indicated that protonable tertiary amine-based membrane-breaking materials, due to their protonable tertiary amine structure, are hydrophobic and electrically neutral under normal physiological pH, exhibiting low membrane-breaking activity against cells and low cytotoxicity to normal tissue cells. However, under slightly acidic conditions, they can be protonated, changing from electrically neutral to electrically positive, activating strong membrane-breaking activity and efficiently killing tumor cells, thereby achieving selective killing of tumor cells. Building upon this, the inventors hypothesized that tertiary amine lipid compounds obtained by ring-opening primary or secondary amines with epoxides, due to their similar structures, might also have the same effect. However, studies have found that these tertiary amine lipid compounds exhibit poor selective killing effect on tumor cells under normal physiological and slightly acidic environments. This invention further reveals that partially modifying the hydroxyl groups of these tertiary amine lipid compounds can reduce their cytotoxicity under normal tissue pH. Partially modified tertiary amine lipid compounds exhibit higher selective cell-killing activity under tumor acidity than fully modified or unmodified tertiary amine lipid compounds.

[0039] Based on the above technical solution, the present invention has the following beneficial effects:

[0040] This invention provides an acidity-activated antitumor lipid tertiary amine small molecule compound, which consists of a tertiary amine head with hydrophilic-hydrophobic properties and a tail containing a hydrophobic group. Under normal physiological pH, the tertiary amine in this lipid material is hydrophobic and electrically neutral, and the tertiary amine head exhibits a hydrophobic conformation. Furthermore, this lipid material can self-assemble with the assistance of auxiliary lipids such as DSPE-PEG to form acid-activated antitumor lipid nanoparticles with a PEG shell. These nanoparticles exhibit weak interaction with cell membranes, thus exhibiting low toxicity to normal tissues during in vivo circulation. The PEG shell also enhances the biocompatibility of the lipid material and prolongs its blood circulation time. Under the slightly acidic pH conditions of tumor tissue (numerous studies have shown that tumor cells typically employ glycolysis, producing more lactic acid and carbon dioxide. Due to lymphatic obstruction in solid tumor tissues, metabolic waste cannot be properly eliminated, resulting in a lower pH in the peripheral environment of solid tumors compared to normal body fluids, typically between 6.4 and 7.0), the tertiary amine portion of this small molecule lipid material undergoes protonation, causing the nanoparticles to form an amphiphilic structure composed of hydrophobic and cationic domains. This results in a strong interaction with cell membranes or organelle membranes, enabling highly efficient and selective killing of tumor cells. The tertiary amine small molecule compounds and / or acidity-activated antitumor lipid nanoparticles of the present invention can be used to prepare antitumor drugs, which have the advantages of good therapeutic effect, high selectivity and low toxicity. Attached Figure Description

[0041] Figure 1The 1H NMR spectra of C4OA25, C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25 are shown.

[0042] Figure 2 The 1H NMR spectra of C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 are shown.

[0043] Figure 3 The 1H NMR spectra of C14OEtA25, C14OBuA25, and C14OHexA25 are shown.

[0044] Figure 4 The 1H NMR spectra of C12OEtA25, C12OBuA25, and C12OHexA25 are shown.

[0045] Figure 5 The 1H NMR spectra of C12OEt2A25, C12OBu2A25, and C12OHex2A25 are shown.

[0046] Figure 6 The 1H NMR spectra of C12OOctA25, C10OHexA25, C10OOctA25, C10ODecA25, and C10ODodA25 are shown.

[0047] Figure 7 The hydrogen nuclear magnetic resonance spectra of C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, and C8OPalA25 are shown.

[0048] Figure 8 The graph shows the relationship between cell viability and pH after co-incubating mouse pancreatic cancer Panc02 cells with lipid nanoparticles of C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25 prepared in Example 2 at pH 7.4 and 6.8 for 24 h.

[0049] Figure 9 The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles of C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0050] Figure 10The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles of C6OMyrA25, C6OPalA25, and C6OSetA25 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0051] Figure 11 The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles of C8ODecA25, C8ODodA25, C8OMyrA25, and C8OPalA25 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0052] Figure 12 The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles C10OHexA25, C10OOctA25, C10ODecA25, and C10ODodA25 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0053] Figure 13 The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, and C12OHex2A25 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0054] Figure 14 The graph shows the relationship between cell viability and pH after co-incubating the lipid nanoparticles of C14OA25, C14OEtA25, and C14OBuA25 prepared in Example 2 with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 h.

[0055] Figure 15 The particle size (a) and potential (b) of the CnOA25 lipid nanoparticles prepared in Example 2, and the particle size (c) and potential (d) of the CnOA27 lipid nanoparticles are shown.

[0056] Figure 16 The particle size (ac) and potential (df) of the CnORA25 lipid nanoparticles prepared in Example 2 are shown.

[0057] Figure 17 The particle size (ab) and potential (cd) of the CnORA25 lipid nanoparticles prepared in Example 2 are shown.

[0058] Figure 18The protonation rate of lipid nanoparticles C10OA25, C12OA25, C14OA25, C16OA25, C18OA25, C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 prepared in Example 2 as a function of pH and their pKa.

[0059] Figure 19 The protonation rate of the lipid nanoparticles C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C14OEtA25, and C14OBuA25 prepared in Example 2 was analyzed as a function of pH and their pKa values.

[0060] Figure 20 The liposome leakage rate of the lipid nanoparticles prepared in Example 2 at different pH values.

[0061] Figure 21 The hemolysis rate of the lipid nanoparticles prepared in Example 2.

[0062] Figure 22 To investigate the toxicity of C14OEtA25 lipid nanoparticles to different tumor cells at the characteristic pH of tumor tissue.

[0063] Figure 23 Localization of C14OEtA25 lipid nanoparticles in tumor cells at the characteristic pH of tumor tissue.

[0064] Figure 24 The uptake of C14OEtA25 lipid nanoparticles by Panc02 tumor cells at pH 6.8 and pH 7.4.

[0065] Figure 25 The effects of endocytosis inhibitors (Wollman penicillin, chlorpromazine hydrochloride, methyl-β-cyclodextrin, sodium azide) and temperature on the cytotoxicity of C14OEtA25 lipid nanoparticles at tumor-specific pH conditions were investigated.

[0066] Figure 26 The in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on Panc02 tumors was studied.

[0067] Figure 27 The in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on EO771 tumors was studied.

[0068] Figure 28 The in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on MC38 tumors was studied. Detailed Implementation

[0069] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0070] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0071] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0072] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0073] In the compounds of this invention, when any variable (e.g., R1, R2, etc.) appears more than once in any component, the definition of each occurrence is independent of the definition of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. A line drawn from a substituent into the ring system indicates that the bond referred to can be attached to any substituted ring atom. If the ring system is polycyclic, it means that such a bond is attached only to any suitable carbon atom of a neighboring ring. It is to be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent is substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as the structure is stable. The phrase "optionally substituted by one or more substituents" is considered equivalent to the phrase "optionally substituted by at least one substituent," and in this case, the preferred embodiment will have 0-3 substituents.

[0074] As used herein, the term "alkyl" refers to both branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched chain. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0075] The term "unsaturated chain hydrocarbon group" refers to a branched or straight-chain unsaturated aliphatic hydrocarbon group with a specific number of carbon atoms, i.e., acyclic unsaturated chain hydrocarbon group, and the carbon chain contains one or more carbon-carbon double bonds or carbon-carbon triple bonds, such as: CH2=CHCH2-, -(CH2)8(CH=CH)CH3, -(CH2)7CH=CH2, -(CH2)8CH=CH2, cis-9,12-octadecadienyl, etc.

[0076] The term "heterocyclic group" refers to cyclic substituents such as monocyclic, fused, spirocyclic, and bridged rings that are saturated or partially unsaturated, wherein one or more ring atoms are selected from heteroatoms of N, O, or S(O)m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon, such as piperidinyl, pyrrolidinyl, etc.

[0077] The "acidity-activated antitumor lipids" described in this invention refer to lipids that can specifically activate antitumor activity to kill tumor cells under specific acidity conditions.

[0078] The "pH-responsive membrane-permeable material" described in this invention refers to a material that exhibits membrane-permeable activity only under specific acidity conditions. For example, it has membrane-permeable activity at pH less than 7, but no or very weak membrane-permeable activity at pH greater than 7. "Membrane-permeable activity" refers to binding to, disturbing, or directly damaging the cell membrane or organelle membrane through adhesion, insertion, or membrane fusion.

[0079] The terms "tertiary amine lipid compound", "lipid", "small molecule compound", "small molecule lipid compound" or "tertiary amine small molecule compound" used in this invention refer to compounds within the structural range shown in formulas (I) to (V) of this invention.

[0080] In one embodiment of the present invention, the use of a tertiary amine lipid compound having the structure shown in formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material is provided:

[0081]

[0082] Each R1 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, alkenyl, alkynyl, aryl, aryl-substituted alkyl, and cycloalkyl-substituted alkyl;

[0083] Each R2 is independently selected from: H, R1; where R is selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0084] R3 and R4 are each independently selected from: H, R9-substituted or unsubstituted alkyl groups, unsaturated chain hydrocarbon groups, cycloalkyl groups, heterocyclic groups, R8-substituted heterocyclic groups, alkenyl groups, alkynyl groups, aryl groups, etc. Alternatively, R3, R4, and the nitrogen atom attached to them can form an R8-substituted or unsubstituted 3-20 membered heterocyclic group; and when R3 is directly attached to N, R3 is not H; when R4 is directly attached to N, R4 is not H.

[0085] Each R5 is independently selected from: alkylene, cycloalkyl, and aryl;

[0086] Each R6 is independently selected from: alkyl, hydroxylated alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl-substituted alkyl,

[0087] Each R7 is independently selected from: alkyl, hydroxyl-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R8-substituted heterocyclic, alkenyl, alkynyl, aryl, aryl-substituted alkyl, Alternatively, R6, R7, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-8 membered heterocyclic groups;

[0088] Each R8 group is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl-substituted heterocyclic, alkenyl, alkynyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl.

[0089] R9 is selected from: hydroxyl, aryl, cycloalkyl, heterocyclic, R8-substituted heterocyclic, and heteroaryl;

[0090] Each q and each q' is independently 0, 1, or 2;

[0091] a can be 1, 2, 3, 4 or 5.

[0092] In some preferred embodiments, the tertiary amine lipid compound has the structure shown in formula (II):

[0093]

[0094] In some preferred embodiments, the tertiary amine lipid compound has the structure shown in formula (III):

[0095]

[0096] R4 is selected from: R9-substituted or unsubstituted alkyl groups, unsaturated chain hydrocarbon groups, cycloalkyl groups, heterocyclic groups, R8-substituted heterocyclic groups, alkenyl groups, alkynyl groups, aryl groups, etc.

[0097] In some preferred embodiments, R4 is selected from: C1-C 22 Alkyl, R9-substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic groups, R8-substituted 3-22 membered heterocyclic groups, C2-C 22 alkenyl, C2-C 22 alkynyl group, C6-C 22 Aryl,

[0098] In some preferred embodiments, R4 is selected from: C1-C 10 Alkyl, R9-substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclic groups, R8-substituted 3-8 membered heterocyclic groups, C2-C 10 alkenyl, C2-C 10 alkynyl, phenyl, naphthyl, anthraceneyl

[0099] In some preferred embodiments, each R5 is independently selected from: C1-C 22 Alkylene, C3-C8 cycloalkyl, C6-C 14 Aryl.

[0100] In some preferred embodiments, each R5 is independently selected from: C1-C 10 Alkylene, C3-C8 cycloalkyl, phenyl, naphthyl, anthracene.

[0101] In some preferred embodiments, each R5 is independently selected from: C1-C6 alkylene and C3-C8 cycloalkyl.

[0102] In some preferred embodiments, each R5 is independently selected from: methylene, ethylene, propylene, butylene, ...

[0103]

[0104] In some preferred embodiments, each R6 is independently selected from: C1-C 22 Alkyl, hydroxyl-substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C2-C 22 alkenyl, C6-C 14 Aryl, C6-C14 Aryl-substituted C1-C8 alkyl groups

[0105] Each R7 is independently selected from: C1-C 22 Alkyl, hydroxyl-substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic groups, R8-substituted 3-22 membered heterocyclic groups, C2-C 22 alkenyl, C2-C 22 alkynyl group, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl groups Alternatively, R6, R7, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-8 membered heterocyclic groups;

[0106] 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 groups, C1-C8 alkylalkyl-substituted 3-22 membered heterocyclic groups, C2-C 22 alkenyl, C2-C 22 alkynyl group, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl groups

[0107] In some preferred embodiments, each R6 is independently selected from: C1-C 10 Alkyl, hydroxyl-substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C2-C 10 Alkenyl, phenyl, naphthyl, anthraceneyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthraceneyl-substituted C1-C8 alkyl

[0108] Each R7 is independently selected from: C1-C 10 Alkyl, hydroxyl-substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclic groups, R8-substituted 3-8 membered heterocyclic groups, C2-C 10 alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthraceneyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthraceneyl-substituted C1-C8 alkyl Alternatively, R6, R7, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-8 membered heterocyclic groups;

[0109] Each R8 is independently selected from: C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclic groups, C1-C6 alkyl-substituted 3-8 membered heterocyclic groups, C2-C 10 alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthraceneyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthraceneyl-substituted C1-C8 alkyl, C3-C8 cycloalkyl-substituted C1-C8 alkyl

[0110] In some preferred embodiments, each R6 is independently selected from: C1-C6 alkyl groups; hydroxyl-substituted C1-C6 alkyl groups;

[0111] Each R7 is independently selected from: C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, Alternatively, R6, R7, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-8 membered heterocyclic groups;

[0112] Each R8 is independently selected from: C1-C6 alkyl, 5-8 membered heterocyclic, C1-C6 alkyl-substituted 5-8 heterocyclic, phenyl, phenyl-substituted C1-C3 alkyl,

[0113] In some preferred embodiments, R9 is selected from: hydroxyl groups, C6-C... 14 Aryl, C3-C 22 Cycloalkyl, 3-22-membered heterocyclic, R8-substituted 3-22-membered heterocyclic, 5-10-membered heteroaryl.

[0114] In some preferred embodiments, R9 is selected from: hydroxyl, phenyl, naphthyl, anthracene, C3-C8 cycloalkyl, 3-8 membered heterocyclic, R8-substituted 3-8 membered heterocyclic, and 5-8 membered heteroaryl.

[0115] In some preferred embodiments, R4 is selected from: C1-C8 alkyl, R9-substituted C1-C8 alkyl, C3-C8 cycloalkyl, phenyl-substituted C1-C3 alkyl, naphthyl-substituted C1-C3 alkyl, anthracene-substituted C1-C3 alkyl, 3-8 membered heterocyclic groups, R8-substituted 5-8 membered heterocyclic groups, Substituted cyclohexyl;

[0116] R5 is selected from: C1-C4 alkylene groups;

[0117] R6 is selected from: C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl,

[0118] R7 is selected from: C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, Alternatively, R6, R7, and the nitrogen atom attached thereto can form a 5-8 membered heterocyclic group with R8 substitution or no substitution, wherein the heteroatom on the ring in the 5-8 membered heterocyclic group is one or two nitrogen atoms;

[0119] Each R8 is independently selected from: C1-C6 alkyl, phenyl, phenyl-substituted C1-C3 alkyl,

[0120] R9 is selected from: hydroxyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic group, R8-substituted 3-8 membered heterocyclic group, 5-8 membered heteroaryl, wherein the heteroatom on the ring of the 3-8 membered heterocyclic group in R9 is one or two nitrogen atoms.

[0121] In some preferred embodiments, R4 is selected from: C4-C6 alkyl, dimethylamino-substituted butyl, diethylamino-substituted butyl, R9-substituted C1-C3 alkyl, C4-C8 cycloalkyl, benzyl, 5-8 membered heterocyclic group, R8-substituted 5-6 membered heterocyclic group, Substituted cyclohexyl;

[0122] R5 is selected from: C1-C3 alkylene groups;

[0123] R6 is selected from: C3-C4 alkyl groups;

[0124] R7 is selected from: C3-C4 alkyl groups;

[0125] Each R8 is independently selected from: C1-C6 alkyl, benzyl,

[0126] R9 is selected from:

[0127] In some of these embodiments, R4 is selected from: R9-substituted methyl, R9-substituted ethyl, and R9-substituted propyl.

[0128] R9 is selected from:

[0129] In some embodiments, R4 is selected from:

[0130] R5 is selected from: C1-C3 alkylene groups;

[0131] R6, R7, and the nitrogen atom attached to them together form the following groups:

[0132]

[0133]

[0134] In some preferred embodiments, the tertiary amine lipid compound has the structure shown in formula (IV):

[0135]

[0136] Where n is 1, 2, 3, 4 or 5;

[0137] R3 and R4 are independently selected from: C1-C 10 Alkyl and hydroxyl substituted C1-C 10 Alkyl, C1-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, Alternatively, R3, R4, and the nitrogen atom attached to them can form 3-8 membered heterocyclic groups.

[0138] In some preferred embodiments, R3 and R4 are each independently selected from: C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, C1-C6 unsaturated chain hydrocarbon, Alternatively, R3, R4, and the nitrogen atom attached to them can form 5-8 membered heterocyclic groups.

[0139] In some preferred embodiments, each R1 is independently selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon group, C3-C 30 cycloalkyl, C2-C 30 alkenyl, C2-C 30 alkynyl group, C6-C 30 Aryl, C6-C 14 Aryl-substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl.

[0140] In some preferred embodiments, each R1 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 cycloalkyl, C2-C 22 alkenyl, C2-C 22 alkynyl group, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl groups.

[0141] In some preferred embodiments, each R1 is independently selected from: C6-C 20 Alkyl, C6-C 20 Unsaturated chain hydrocarbon group, C5-C 20 cycloalkyl, C6-C 20 alkenyl, C6-C 20 Alkyne, phenyl, naphthyl, anthracene, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthracene-substituted C1-C6 alkyl, C5-C 20 Cycloalkyl-substituted C1-C6 alkyl groups.

[0142] In some preferred embodiments, each R1 is independently selected from: 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, isotriadecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecanyl, n-octadecyl, isooctadecyl, dodecyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclooctyl, cyclodecyl, cyclodecyl, cyclododecylmethyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentyl Methyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, vinyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, docosyl, ethynyl, propynyl, butynyl, penynyl, hexynyl, octynyl, decanynyl, dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, docosyl, linalyl, phenyl, naphthyl, anthraceneyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylhexyl, phenyloctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenemethyl, anthraceneethyl, anthracenepropyl, anthracenebutyl, anthracenehexyl, anthraceneoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

[0143] In some preferred embodiments, each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0144] In some preferred embodiments, each R2 is independently selected from: H,

[0145] In some preferred embodiments, each R2 is not simultaneously hydrogen, that is, at least one R2 is not hydrogen.

[0146] In some preferred embodiments, one R2 is All other R2 molecules are hydrogen.

[0147] In some preferred embodiments, the tertiary amine lipid compound is as shown in formula (V):

[0148]

[0149] Preferably, R2 is

[0150] In some preferred embodiments, each R is independently selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon group, C3-C 30 cycloalkyl, C6-C 30 Aryl, C6-C 14 Aryl-substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl.

[0151] In some preferred embodiments, each R is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 cycloalkyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl groups.

[0152] In some preferred embodiments, each R is independently selected from: methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecal, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexyl, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecyl Methyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, linalyl, phenyl, naphthyl, anthracene, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylhexyl, phenyloctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenemethyl, anthraceneethyl, anthracenepropyl, anthracenebutyl, anthracenehexyl, anthraceneoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

[0153] In some preferred embodiments, the total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, and more preferably 24-26.

[0154] In some preferred embodiments, R3 and R4 are each independently selected from: H, R9-substituted or unsubstituted C1-C. 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic groups, R8-substituted 3-22 membered heterocyclic groups, C2-C 22 alkenyl, C2-C 22 alkynyl group, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl groups Alternatively, R3, R4, and the nitrogen atom attached to them can form 3-10 membered heterocycles with or without R8 substitution.

[0155] In some preferred embodiments, R3 and R4 are each independently selected from: H, R9-substituted or unsubstituted C1-C. 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon groups, C3-C8 cycloalkyl groups, 3-8 membered heterocyclic groups, R8-substituted 3-8 membered heterocyclic groups, C2-C 10 alkenyl, C2-C10 Alkynyl, phenyl, naphthyl, anthraceneyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthraceneyl-substituted C1-C6 alkyl, C3-C8 cycloalkyl-substituted C1-C6 alkyl Alternatively, R3, R4, and the nitrogen atom attached to them can form R8-substituted or unsubstituted 5-10 membered heterocyclic groups.

[0156] In some preferred embodiments, R3, R4, and the nitrogen atom attached thereto form the following groups:

[0157]

[0158] In some preferred embodiments, the tertiary amine lipid compound is selected from the following compounds:

[0159]

[0160]

[0161] Each R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 2-phenylethyl or phenylbutyl;

[0162] Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl, 2-phenylacetyl or phenylbutyryl.

[0163] In some preferred embodiments, the total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, and more preferably 24-26.

[0164] In some preferred embodiments, at least one of R2 is H, and R2 is not simultaneously H.

[0165] In one embodiment of the present invention, a lipid nanoparticle is also provided, which is formed by the self-assembly of the aforementioned tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and an amphiphilic molecule in an aqueous medium, wherein the amphiphilic molecule is an amphiphilic polyethylene glycol polymer, or a combination of an amphiphilic polyethylene glycol polymer and a phospholipid; wherein the amphiphilic polyethylene glycol polymer is an amphiphilic polyethylene glycol lipid molecule and / or a polyethylene glycol-polyester.

[0166] The amphiphilic polyethylene glycol lipid molecule may be selected from, but is not limited to, polyethylene glycol-dispalmitoylphosphatidylethanolamine, polyethylene glycol-distearate-phosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, and 2-(polyethylene glycol)-N,N-tetracosylacetamide; the amphiphilic polyethylene glycol-polyester may be selected from, but is not limited to, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, and polyethylene glycol-polylactic acid-glycolate; preferably, the amphiphilic polyethylene glycol The molecular weight of polyethylene glycol in the polymer is 500 g / mol to 20000 g / mol, preferably 1000 g / mol to 3000 g / mol, and more preferably 1800 g / mol to 2200 g / mol; preferably, the molecular weight of polyester in the amphiphilic polyethylene glycol-polyester is 500 g / mol to 20000 g / mol, preferably 1000 g / mol to 3000 g / mol, and more preferably 1800 g / mol to 2200 g / mol.

[0167] The phospholipids may be selected from, but are not limited to: cholesterol, soybean phospholipids, hydrogenated soybean phospholipids, soybean lecithin, egg yolk lecithin, high-purity egg yolk lecithin, dilauroyl lecithin, disqualyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearyl phosphatidylcholine, dipalmitoyl lecithin, distearyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidyl ethylstilbestrol Alkylamine, palmitoyl lysophosphatidyl, myristoyl lysophosphatidyl, stearoyl lysophosphatidyl, dimyristoyl phosphatidyl ethanolamine, distearyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, (2,3-dioleoyl-propyl)trimethylammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleoyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, 1,2-bisoctadecenoxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester.

[0168] In some preferred embodiments, the lipid nanoparticles are formed by the 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 molecules are distearate phosphatidylethanolamine-polyethylene glycol 2000.

[0169] 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, even more preferably 1:0.8-1.2; and most preferably 1:1.

[0170] In one embodiment of the present invention, a method for preparing the lipid nanoparticles is also provided, comprising the following steps:

[0171] The tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt is dissolved in an organic solvent to obtain a tertiary amine lipid compound solution;

[0172] The amphiphilic molecule is dissolved in an organic solvent to obtain an amphiphilic molecule solution;

[0173] The tertiary amine lipid compound solution and the amphiphilic molecule solution are mixed to obtain a mixed solution;

[0174] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing it with a pipette; or, the lipid nanoparticles are obtained by slowly adding the mixed solution dropwise to an aqueous medium under stirring and continuing to stir.

[0175] In some preferred embodiments, the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide, or tetrahydrofuran.

[0176] 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; and the concentration of the amphiphilic molecule solution is 20 mg / mL-200 mg / mL, more preferably 50 mg / mL-100 mg / mL.

[0177] In some preferred embodiments, the volume ratio of the mixed solution to the aqueous medium is 1:1-20, more preferably 1:2-10.

[0178] In one embodiment of the present invention, the use of the aforementioned tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in the preparation of a medicament for the prevention and / or treatment of tumors is also provided.

[0179] In one embodiment of the present invention, the use of the lipid nanoparticles in the preparation of medicaments for the prevention and / or treatment of tumors is also provided.

[0180] 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 carcinoma, nasopharyngeal carcinoma, or brain cancer.

[0181] In one embodiment of the present invention, a medicament for the prevention and / or treatment of tumors is also provided, which is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt described in the present invention, and / or the lipid nanoparticles described therein.

[0182] The compounds of formulas (I)-(V) of the present invention can be used in combination with other known antitumor drugs. When administered in combination, the compounds of formulas (I)-(V) and the known drugs can be independent administration units or together form a combined administration unit; the compounds of formulas (I)-(V) can be administered simultaneously with or separately from other known antitumor drugs. When the compounds of formulas (I)-(V) are taken simultaneously with one or more other drugs, it is preferable to use a pharmaceutical composition containing one or more known drugs and the compounds of formulas (I)-(V). Drug combination also includes taking the compounds of formulas (I)-(V) with one or more other known drugs during overlapping time periods. When the compounds of formulas (I)-(V) are used in combination with one or more other known drugs, the dose of the compounds of formulas (I)-(V) or the known drugs can be the same as the dose of either drug alone, or it can be lower than the dose of either drug alone.

[0183] Drugs or active ingredients that can be used in combination with compounds of formulas (I)-(V) include, but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxins / cell inhibitors, antiproliferative agents, proteotransferase 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.

[0184] In some of these embodiments, the drugs or active ingredients that can be used in combination with compounds of formulas (I) to (V) include, but are not limited to: interleukin, alendronate, interferon, atrazonoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglucopyranoside, amifostine, amrubicin, azithromycin, anatozol, dolasetron, aranesp, arglabin, arsenic trioxide, arnoxin, 5-azacytidine, azathioprine, BCG or TICE BCG, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotine, bleomycin sulfate, bromouridine, bortezomib, busulfan, calcitonin, alectozimumab injection, capecitabine, carboplatin, cephalosporin, CE Fesone, Simomo Interleukin, Daunorubicin, Chlorbutamol, Cisplatin, Cladribine, Cladribine, Chlordroxylphosphate, Cyclophosphamide, Cytarabine, Dacarbazine, Actinomycin D, Daunorubicin Liposome, Dexamethasone, Dexamethasone Phosphate, Estradiol Valerate, Diane-166 Interleukin-2, Depomere, Delorelin, Delazosin, Diethylstilbestrol, Diflucan, Docetaxel, Deoxyfluorouracil, Doxorubicin, Drolactone, Chin-166 Chitosan Complex, Eligard, Raburicase, Epirubicin Hydrochloride, Aprepitant, Epirubicin, Ibertin Alfa, Erythropoietin, Etoposide, Levamisole Tablets, Estradiol Preparations, 17-β-Estradiol, Estrogenus Estibutine Sodium Phosphate, Ethinylestradiol, Amifostine, Hydroxyphosphate, Vanbyl, Etoposide, Fazo Zolpidem, Tamoxifen preparations, Finastatin, Ferastatin, Ferastatin, Fluorouracil, Fluconazole, Fludarabine, 5-Fluorodeoxyuridine monophosphate, 5-Fluorouracil, Flumethasone, Flutamide, Formestan, 1-β-D-arasulofuranylcytidine-5'-stearoyl phosphate, Formustin, Fulvestrant, Gamma globulin, Gemcitabine, Gemtuzumab, Imatinib Mesylate, Carburexa rice paper capsules, Goserelin, Granisilone Hydrochloride, Histamine Relin, Hemifentanil, Hydrocortisone, Erythro-hydroxynonyladenine, Hydroxyurea, Tetan-Isbemumab, Idarubicin, Ifosfamide, Interferon α, Interferon-α2, Interferon α-2A, Interferon α-2B, Interferon α-n1, Interferon α-n3, Interferon Interferon-β, Interferon-γ-1a, Interleukin-2, Intron A, Iressa, Irinotecan, Keterene, Lentinan sulfate, Letrozole, Levofloxacin, Leuprorelin, Levofloxacin acetate, Levotetraimidazole, Levolecithin calcium salt, Levothyroxine sodium, Levothyroxine sodium preparations, Lomustine, Clonidamine, Drowanediol, Nitrogen mustard, Mecobalamin, Medroxyprogesterone acetate, Medroxyprogesterone acetate, Melphalan, Esterified estrogen, 6-Mercaptopurine, Mesna, Methotrexate, Methylaminolevulinate, Mitefocin, Minocycline, Mitomycin C, Mitotane, Mitoxantrone, Tralocysteine, Doxorubicin citrate liposomes, Nedaplatin, Pegylated filgrastim, Olepipril, Interleukin, Neupogen, Nilumethicone, TamoxifenNSC-631570, Recombinant Human Interleukin-1-β, Octreotide, Odanciron Hydrochloride, Dehydrocortisone Oral Solution, Oxaliplatin, Paclitaxel, Prednisone Sodium Phosphate Preparation, Pegaspargase, Pegasys, Pentostatin, Streptomycin Preparation, Pilucarpine Hydrochloride, Pirarubicin, Prucalomycin, Porphyrom Sodium, Prednisone, Steprednisolone, Prednisone, Premarin, Procarbazine, Recombinant Human Erythropoietin, Raltitrexate, Ribeye, Rhenium-186 Etidronate, Rituximab, Rituximab-A, Romotide, Pilocarpine Hydrochloride Tablets, Octreotide, Samosine, Semustine, Cizonan, Sobuzosen, Methylprednisolone Sodium, Paphosphatase, Stem Cell Therapy, Levozocin, Strontium Chloride-89, Levothyroxine Sodium, Tamoxifen Tansulosin, Tastolactone, Dosotericin, Tecithiazide, Temozolomide, Teniposide, Testosterone Propionate, Methyltestosterone, Thioguanine, Thiotepa, Thyroid Stimulating Hormone, Tiludronate, Topotecan, Toremifene, Tosimozab, Trastuzumab, Triostazol, Retinoic Acid, Methotrexate Tablets, Trimethylmelamine, Trimethoprim, Triptorelin Acetate, Triptorelin Dihydroxynaphthyl Naphthyl Acetate, Ufodin, Ureidine, Pentorubicin, Visorcinol, Vincristine, Vincristine, Vincrylein, Vinorelin, Verrucidium, Dextromethorphan, Nettostatin Ester, Scyproterone, Paclitaxel Protein Stabilizer, Acolbifene, Interferon R-LB, Affinitak, Aminopterin, Azoxifen, A Soprisnil, Atamitan, Atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, Cetuximab, Clinatropin, Cyproterone Acetate, Decitabine, DN-101, Doxorubicin-MTC, dSLIM, Dutasteride, Edotecarin, Eflunomide, Ecinotecan, Fenivel-Amine, Histamine Dihydrochloride, Histamine Relin Hydrogel Implant, Holmium-166DOTMP, Ibandronic Acid, Interferon-γ, Intron-PEG, Ixabepilone, Keyhole Hemocyanin, L-651582, Lanleptide, Lasoxifene, Libra, Lonafamib, Miprexifene, Minotriol, MS- 209. Liposomes MTP-PE, MX-6, Nafarelin, Nemorubicin, Neovastatin, Noratropide, Olimerson, Onco-TCS, Osidem, Paclitaxel Polyglutamate, Sodium Pormylate, PN-401, QS-21, Quasi-Yan, R-154, Raloxifene, Leopoldin, 13-cis-retinoic acid, Saplatin, Ciocalcitriol, T-138067, Tarceva, Docosahexaenoic acid paclitaxel, Thymosin α1, Gazofuran, Tipifanib, Tirazamine, TLK-286, Toremifene, Trans-MID-lo7R, Vasoprothiolane, Vatalanib, Vertepofen, Vinpocetine, Z-100, and Zoledronic acid or combinations thereof.

[0185] The medicament of the present invention for the prevention and / or treatment of tumors can be used in non-human mammals or humans.

[0186] The pharmaceutically acceptable excipients used in the medicaments for the prevention and / or treatment of tumors of the present invention refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0187] "Compatibility" here refers to the ability of each component in the composition to interact with and blend with the active ingredients of the present invention (tertiary amine lipid compounds represented by Formulas I-V) without significantly reducing the efficacy of the active ingredients.

[0188] The pharmaceutically acceptable excipients used in the medicaments for the prevention and / or treatment of tumors of the present invention include, but are not limited to, one or more of the following materials: solvents, excipients, fillers, compatibilizers, binders, humectants, disintegrants, slow solvents, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents, and fragrances.

[0189] Pharmaceutically acceptable examples of 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, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as...). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0190] There are no particular limitations on the administration of the active ingredients or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), etc.

[0191] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0192] 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 components:

[0193] (a) Fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silica;

[0194] (b) Adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0195] (c) Moisturizers, such as glycerin;

[0196] (d) Disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginate, certain complex silicates, and sodium carbonate;

[0197] (e) Slow solvents, such as paraffin;

[0198] (f) Absorption accelerators, such as quaternary ammonium compounds;

[0199] (g) Wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0200] (h) Adsorbents, such as kaolin; and

[0201] (i) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0202] The solid dosage form can also be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient from this composition can be delayed in a portion of the digestive tract. Examples of suitable encapsulating components are polymers and waxes.

[0203] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0204] In addition to the active ingredient, the suspension may contain suspending agents, such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0205] Compositions for parenteral injection may comprise 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.

[0206] The following are specific examples.

[0207] Example 1: Preparation of tertiary amine lipid compounds and their derivatives obtained by reacting epoxides with amine molecules

[0208] This embodiment prepared tertiary amine lipid compounds containing different types of hydrophobic groups, as well as derivatives of tertiary amine lipid compounds obtained by acylation of their monohydroxy or dihydroxy groups. These lipids were first synthesized by ring-opening reactions of epoxides (C4O, C6O, C8O, C10O, C12O, C14O, C16O, C18O) with N-(2-aminoethyl)piperidine (A25) and N-(2-aminoethyl)hooperidine (A27) containing different hydrophobic carbon chains. C4O, C6O, C8O, C10O, C12O, C14O, C16O, and C18O were, respectively, 1,2-epoxybutane (C4O), 1,2-epoxyhexane (C6O), and 1,2-epoxybutane (C18O). Octane (C8O), 1,2-epoxydecane (C10O), 1,2-epoxydodecane (C12O), 1,2-epoxytetradecane (C14O), 1,2-epoxyhexadecane (C16O), and 1,2-epoxyoctadecane (C18O) were synthesized by nucleophilic substitution reactions of the above-synthesized lipids with acyl chlorides of different hydrophobic carbon chains (acetyl chloride, n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoylamine, n-dodecanoyl chloride, n-tetradecanoyl chloride, n-hexadecanoyl chloride, and n-octadecanoyl chloride).

[0209] Specifically, in this embodiment, a series of tertiary amine lipid compounds, as shown in formulas (A) and (B), were synthesized first through a ring-opening reaction of epoxides with N-(2-aminoethyl)piperidine (A25) and N-(2-aminoethyl)hooperidine (A27). The reaction formulas are as follows:

[0210]

[0211]

[0212] R1 is: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl.

[0213] Subsequently, a series of tertiary amine lipid derivatives, as shown in formulas (C) and (D), were synthesized by nucleophilic substitution reactions of the lipid of formula (A) with acyl chlorides of different hydrophobic carbon chains. The reaction formulas are as follows:

[0214]

[0215] Wherein, R1 is: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl; R is: methyl, n-propyl, n-pentyl, n-heptyl, n-nonyl, n-undecyl, n-tridecyl, n-pentadecanyl, n-heptadecyl.

[0216] The raw materials used in the two-step reaction (except for formula (A)) were all purchased directly and used without purification.

[0217] The specific steps are as follows:

[0218] Preparation of tertiary amine lipid compounds with different types of hydrophobic groups:

[0219] 1 g (3.7 mmol-13.9 mmol) of epoxides (C4O, C6O, C8O, C10O, C12O, C14O, C16O, C18O, respectively) was placed in a reaction vessel. N-(2-aminoethyl)piperidine / N-(2-aminoethyl)hopiperidine was added at a molar ratio of epoxide to N-(2-aminoethyl)piperidine of 2.4:1. After mixing thoroughly, the mixture was reacted in a sealed container at 90°C for two days. The product obtained from the reaction was purified by column chromatography before use.

[0220] Column chromatography: Silica gel was used as the column packing material, and a gradient elution was performed with dichloromethane:methanol = 1%–8% as the eluent to purify the synthesized tertiary amine lipid compounds. The collected products were then subjected to rotary evaporation and lyophilization before use. Its NMR characterization is as follows: Figure 1 , Figure 2 As shown, analysis revealed the following pure target products: C4OA25, C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, C18OA25; C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27, with their corresponding structural formulas shown below.

[0221]

[0222] The tertiary amine lipid compounds prepared above are abbreviated as CnOA25 / CnOA27, where CnO refers to the epoxide in the raw material, and A25 / A27 refers to N-(2-aminoethyl)piperidine / N-(2-aminoethyl)hopiperidine in the raw material. For example, C4OA25 refers to the tertiary amine lipid compound prepared by reacting an epoxide (C4O) with an ethyl group (R1) and N-(2-aminoethyl)piperidine (A25) according to the above reaction.

[0223] Preparation of derivatives of tertiary amine lipid compounds modified with different functional groups:

[0224] 200 mg of CmOA25 was placed in a reaction vessel and dissolved in 5 mL of anhydrous dichloromethane. Then, anhydrous triethylamine was added at a molar ratio of CmOA25 to anhydrous triethylamine of 1:2. Finally, the acyl chlorides (acetyl chloride, n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoylamine, n-dodecanoyl chloride, n-tetradecanoyl chloride, n-hexadecanoyl chloride, n-octadecanoyl chloride) were slowly added dropwise to the vessel under ice bath at a molar ratio of 1:1 or 1:2.5. After the addition was complete, the reaction was allowed to proceed overnight at room temperature. The product obtained from the reaction was purified by column chromatography to separate the product with one hydroxyl hydrogen replaced by an alkanoyl group or the product with two hydroxyl hydrogens replaced by an alkanoyl group before use.

[0225] Column chromatography: Silica gel was used as the column packing material, and dichloromethane:methanol = 4% was used as the eluent to purify the tertiary amine lipid derivatives synthesized above, which have one hydroxyl hydrogen substituted with an alkyl acyl group and two hydroxyl hydrogen substituted with alkyl acyl groups. The collected products were then subjected to rotary evaporation and lyophilization before use. Their NMR characterization is as follows: Figures 3-7 As shown, analysis revealed the pure target products C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, C12OHex2A25, C14OEtA25, C14OBuA25, and C14OHexA25, with the corresponding structural formulas shown below.

[0226]

[0227]

[0228] The derivatives of the tertiary amine lipid compounds prepared above are abbreviated as CmOR. x A25, where CmO refers to the epoxide in the initial raw material, A25 refers to N-(2-aminoethyl)piperidine in the initial raw material, and R... x The term refers to the alkyl group (when x is omitted, it means that one hydroxyl hydrogen in the tertiary amine lipid compound is replaced by an alkyl group (mono-substitution); when x is 2, it means that two hydroxyl hydrogens in the tertiary amine lipid compound are replaced by an alkyl group (di-substitution)). For example, C12OEtA25 refers to a mono-substituted tertiary amine lipid compound derivative prepared by reacting C12OA25 with acetyl chloride according to the above reaction.

[0229] Example 2: Preparation of lipid nanoparticles

[0230] Example 1 synthesized a tertiary amine lipid compound or its derivative having a hydrophobic carbon chain moiety and a hydrophobic tertiary amine moiety at pH 7.4, in distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG). 2000 With the assistance of [unclear], nanoparticles can be self-assembled in water.

[0231] The specific procedure is as follows: Dissolve a tertiary amine lipid compound or its derivative in ethanol to obtain a lipid solution with a concentration of 50 mg / mL; take DSPE-PEG... 2000 Dissolved in ethanol, DSPE-PEG with a concentration of 50 mg / mL is obtained. 2000 Solution; take 10 μL of lipid solution and 10 μL of DSPE-PEG. 2000 The solution was mixed in a 1.5 mL centrifuge tube to obtain a mixed solution; 180 μL of sterile water was added to it with a pipette and quickly added to the above mixed solution, and the mixture was repeatedly pipetted to obtain lipid nanoparticles with a concentration of 2.5 mg / mL.

[0232] Example 3: Cytotoxicity of lipid nanoparticles at characteristic pH levels in normal and tumor tissues

[0233] This embodiment evaluates the killing effect of the drug on tumor cells at pH 7.4 and pH 6.8 using the MTT (thiazolyl blue) method, and studies the cytotoxicity of a series of lipid nanoparticles prepared according to the method of Example 2 at the characteristic pH of normal tissues and tumor tissues.

[0234] The specific experimental steps are as follows: DMEM medium was adjusted to pH 6.8 and 7.4 using 6 mol / L HCl solution, and 25 mM HEPES was added to maintain pH stability. The mouse pancreatic cancer cell line Panc02 (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 prepared in Example 2 were diluted with DMEM medium to different concentrations (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) and added to cells (concentration 1×10⁻⁶). 5 Cells were incubated in 96-well plates at 37°C and CO2 incubator for 24 hours each. The original culture medium was then discarded, and MTT solution (10 μL 5 mg / mL MTT solution (dissolved in PBS) + 90 μL LMEM medium) was added. The plates were then incubated at 37°C and CO2 incubator for another 2 hours. The MTT solution was then discarded, and 100 μL of dimethyl sulfoxide was added. After 20 minutes on a shaker, the absorbance at OD 490 nm was measured using a microplate reader, and cell viability was calculated. Cell viability % was calculated as follows: Cell viability % = (OD 490 nm / (5 mg / mL)) / (5 mg / mL MTT solution dissolved in PBS) / (5 mg / mL LMEM medium). 纳米颗粒组-OD 背景 )÷(OD PBS组 -OD 背景 )×100%, of which OD 背景 For blank wells containing only MTT and no cells, OD PBS组 The control group consisted of cells and MTT, with a drug concentration of 0 μg / mL.

[0235] Test results are as follows Figures 8-14 As shown in the test results, the cytotoxicity of the lipid nanoparticles prepared in this invention against tumor cells at pH 7.4 and pH 6.8 exhibits a certain regularity. For lipid nanoparticles prepared from C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25, with the increase of the hydrophobic alkyl chain, the cytotoxicity of tumor cells at pH 7.4 and pH 6.8 changes from significant toxicity to selective killing and then to no toxicity. This indicates that due to the increase in hydrophobicity, the cytotoxicity of lipid nanoparticles at pH 7.4 and pH 6.8 decreases. The short hydrophobic chain segment of C6OA25 causes it to dissolve in solution at pH 6.8 due to protonation and further hydrophilicity after being prepared into lipid nanoparticles, preventing it from interacting with the cell membrane, resulting in lower cytotoxicity at pH 6.8. Figure 8 For lipid nanoparticles prepared from C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27, with the increase of the hydrophobic alkyl chain, the cytotoxicity of tumor cells at pH=7.4 and pH=6.8 changed from significant toxicity in both cases to some selective killing, and then to a decrease in toxicity in both cases. This indicates that due to the increase in hydrophobicity, the cytotoxicity of lipid nanoparticles at pH=7.4 and pH=6.8 is weakened. Figure 9For C6OMyrA25, C6OPalA25, C6OSetA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, C12OHex2A25, C14OEtA25, C14OB For lipid nanoparticles prepared by uA25, the cytotoxicity at pH 7.4 and pH 6.8 decreased with the substitution of hydroxyl groups and the increase of hydrophobicity of the substituent groups. Furthermore, the cytotoxic selectivity first increased and then decreased with the increase of the carbon chain length of the substituent groups. Simultaneously, monosubstituted lipid materials with one hydroxyl hydrogen replaced by an alkyl group exhibited better cytotoxic selectivity than disubstituted lipid materials with two hydroxyl hydrogens replaced by alkyl groups and unsubstituted lipid materials. That is, monosubstituted lipid materials with one hydroxyl hydrogen replaced by an alkyl group showed lower cytotoxicity at pH 7.4 and higher cytotoxicity at pH 6.8. Figures 10-14 Moreover, according to Figures 8-14 In summary, when the total number of carbon atoms (i.e., carbon atoms removed from A25) on the hydrophobic backbone and hydrophobic side chains of the modified lipid material is 28-30, the resulting lipid nanoparticles exhibit the strongest selective killing ability against PancO2 tumor cells at pH 6.8.

[0236] Example 4: Particle size potential of lipid nanoparticles at pH 7.4 and pH 6.8

[0237] Take 100 μL of lipid nanoparticle solution with a concentration of 10 mg / mL, and dilute it to 1 mL with 0.1×PBS solution with pH values ​​of 7.4 and 6.8 respectively. Then add it to the corresponding dedicated test cell and use nanoparticle size and zeta potential instruments to characterize the particle size and potential of the nanoparticles.

[0238] Test results are as follows Figure 15-17 As shown, the particle size range of CnOA25 and CnOA27 nanoparticles is between 100-270 nm, and the potential range is between 1.6-5.3 mV. With the increase of hydrophobic carbon chain length, the particle size of the nanoparticles increases, while the potential shows no obvious pattern. Furthermore, some nanoparticles have slightly larger particle size and potential at pH 6.8 than at pH 7.4. Figure 15 The particle size distribution of CnORA25 nanoparticles showed no obvious pattern. In terms of the nanoparticle potential, the potential at pH 6.8 was slightly higher than that at pH 7.4. Figure 16 and Figure 17).

[0239] Example 5: Protonation degree of lipid nanoparticles at different pH levels

[0240] The lipid nanoparticles prepared in Example 2 were mixed with buffer solutions and TNS solutions of different pH values. 100 μL of the solution was added to a black flat-bottomed 96-well plate. The fluorescence intensity was then measured using a full-wavelength microplate spectrophotometer, and the degree of protonation and pKa were calculated by simulating the change curve. The specific method is as follows:

[0241] Buffer solution preparation: First, prepare a buffer solution containing 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 20 mM 2-(N-morpholine)ethanesulfonic acid (MES), 20 mM ammonium acetate, and 260 mM NaCl using ultrapure water. Then, take four 25 mL aliquots of the 2x buffer solution and add 2 mL and 1 mL of 1 M HCl solution (Aliquots 1 and 2), and 2 mL and 1 mL of 1 M NaOH solution (Aliquots 3 and 4), respectively. Finally, bring the total volume to 50 mL with ultrapure water. Finally, use solutions 1 and 4, and solutions 2 and 3, respectively, to titrate with each other to prepare buffer solutions with pH values ​​of 6.5–10 and 3–6.5 (each 0.5 pH increment).

[0242] Measurement method: 980 μL of buffer solution with different pH values ​​were added to 1.5 mL EP tubes, followed by 10 μL of 2.5 mg / mL lipid nanoparticles and 100 μM TNS aqueous solution. After mixing well, 100 μL of the solution was added to a black flat-bottomed 96-well plate. The fluorescence intensity at excitation and emission wavelengths of 321 nm and 445 nm, respectively, was measured using a full-wavelength microplate spectrophotometer.

[0243] Calculation method: A sigmoid function was used for simulation analysis. The point of maximum fluorescence intensity in the curve was defined as 100% protonation, and the point of minimum fluorescence intensity was defined as 0% protonation. The values ​​were then normalized to obtain the simulated curve. The pH value corresponding to a protonation rate of 50% in this curve is the pK of this lipid nanoparticle. a .

[0244] Test results are as follows Figure 18 , Figure 19 As shown in the figure, the protonation rate of lipid nanoparticles begins to decrease within a certain range, but the range of change varies for different lipid nanoparticles. For example, C16OA25 shows a sudden jump in protonation rate to 80% between pH 6.8 and pH 7.4, while C10OA25 shows a slow decrease in protonation rate to 80% between pH 7.8 and pH 9.5. Figure 18The analysis yielded the pK values ​​corresponding to C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25. a The values ​​are: 8.27, 7.13, 7.21, 6.82, and 6.93; the corresponding pK values ​​for C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 are respectively. a The values ​​are: 8.36, 7.79, 7.61, 7.07, and 7.78. From this, we can see that pK a The relationship with the length of the hydrophobic carbon chain is roughly as follows: as the length of the hydrophobic carbon chain increases, pK... a It shows a trend of first declining and then rising.

[0245] from Figure 19 The analysis yields the pK values ​​corresponding to C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C14OEtA25, and C14OBuA25. a The values ​​are: 6.27, 6.57, 6.34, 5.74, 6.14, 6.00, 5.94, 6.69, 5.84, 5.70, 5.86, 6.73, 6.14, 6.03, 5.83, 6.42, and 6.10.

[0246] Example 6: Liposome leakage caused by lipid nanoparticles at different pH levels

[0247] This embodiment uses liposomes simulating tumor cell membrane components to study dye-loaded leakage, illustrating the interaction between lipid nanoparticles and tumor cell membranes through dye leakage. Liposomes were prepared using a thin-film hydration method. Given that tumor cells, compared to normal cells, have a high expression of phosphatidylserine on their cell membranes, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (sodium salt) (POPS) and 1,2-bis(cis-9-oleoyl)-sn-glycerol-3-phosphate choline (DOPC) (mass ratio 1:9) were used to simulate the tumor cell membrane.

[0248] The preparation method is as follows: Prepare buffer 1 (10 mM Na2HPO4) and buffer 2 (a mixed solution of 10 mM Na2HPO4 and 90 mM NaCl) with deionized water, and adjust the pH of the solution to 7.0. Prepare a 12.5 mM solution of disodium 8-amino-1,3,6-naphthalenetrisulfonic acid (ANTS) and a 45 mmol / L solution of 1,1'-[1,4-phenylenebis(methylene)]bis(4,4'-bipyridine)dibromide (DPX) using buffer 1. Mix the two solutions thoroughly at a 1:1 (v / v) ratio. Take 2 g of dextran gel G-50, soak it in buffer 2 for 7 h, and pack it into a chromatography column to a height of about 10 cm. In a clean 50 mL round-bottom flask, 840 μL of chloroform solution was added as a solvent. Separately, 144 μL of 25 mg / mL DOPC (dissolved in chloroform) and 16 μL of 25 mg / mL POPS (dissolved in chloroform) were added and mixed thoroughly. This mixture was used to prepare liposomes with a DOPC:POPS ratio of 9:1 (w / w). The water bath temperature was heated to 37 °C, and chloroform was removed by vacuum evaporation (200 rpm). The pressure was gradually reduced to 350 mbar in 100 mbar increments and maintained for 30 min. Then, the pressure was further reduced to 100 mbar and maintained for 30 min. Finally, the residual chloroform was dried under vacuum using an oil pump to obtain a uniform lipid film. 2 mL of the prepared ANTS and DPX mixed solution was added to the 50 mL flask containing the lipid film. The mixture was then hydrated by rotation at atmospheric pressure for 1 h at 37 °C and 200 rpm using a rotary evaporator. The obtained liposome solution was transferred to a 2 mL centrifuge tube. After 10 freeze-thaw cycles using liquid nitrogen and a 37°C water bath, the solution was passed through a dextran gel column to separate the liposomes from free ANTS and DPX. Using buffer 2 as the mobile phase, the liposome solution was collected in a 2 mL centrifuge tube. The obtained liposome solution was stored at room temperature protected from light and used within 48 hours.

[0249] The specific experimental procedure for determining the interaction between lipid nanoparticles and liposomes loaded with fluorescent dyes is as follows: PBS was titrated with 6M hydrochloric acid solution and 6M sodium hydroxide aqueous solution to adjust its pH to obtain PBS solutions with pH values ​​of 6.8 and 7.4. Then, 4 μL of 5 mg / mL lipid nanoparticle solution, 10 μL of liposome solution, and 86 μL of PBS buffer at different pH values ​​were added to a 96-well plate with a black background. Three replicates were set up for each different pH value. A negative control group (90 μL of a mixture of 1×PBS solution at pH 6.2 and 7.4 and 10 μL of liposomes) and a positive control group (90 μL of a mixture of 2% Triton X-100 (prepared with water) and 10 μL of liposomes) were set up. After preparation, the mixture was incubated at room temperature in the dark for 1 h. The fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 530 nm was then measured using a microplate reader. The fluorescence intensity of the experimental group was defined as If. t The fluorescence intensity of the negative control group co-incubated with PBS and liposomes was defined as I. n The fluorescence intensity of the positive control group co-incubated with 2% Triton X-100 and liposomes was defined as I. p Finally, using the formula [(I t -I n ) / (I p -I n The corresponding fluorescence leakage rate is calculated by multiplying 100% by 1.

[0250] Test results are as follows Figure 20 As shown in the figure, the lipid nanoparticles prepared by this invention can cause leakage of dyes loaded in liposomes, and the leakage rate caused at pH 6.8 is significantly higher than that at pH 7.4. This indicates that the lipid nanoparticles of this invention have the function of disrupting the phospholipid bilayer membrane, and the membrane disruption ability at pH 6.8 is stronger than that at pH 7.4, indicating that the membrane disruption function of the lipid nanoparticles is pH responsive.

[0251] Example 7: Hemolytic activity of lipid nanoparticles

[0252] This embodiment tested the hemolytic activity of the lipid nanoparticles prepared in Example 2 and verified in Example 3, which exhibited pH-selective cytotoxicity.

[0253] The specific experimental steps are as follows: Sheep blood was washed twice with 1×PBS until the supernatant was clear and colorless after centrifugation. Then, the sheep blood was diluted to 4% with 1×PBS. 16 μL of lipid nanoparticles (1.25 mg / mL) prepared with 1×PBS was added to a 96-well plate. 34 μL of 1×PBS was added, followed by 50 μL of 4% sheep blood. The plate was incubated at 37°C for 4 hours. After centrifugation at 3000 rpm for 10 minutes, 50 μL of the supernatant was transferred to another 96-well plate. The absorbance at 576 nm was measured using a multi-mode microplate reader. Additionally, a PBS control group (50 μL 1×PBS + 50 μL 4% sheep blood) without lipid nanoparticles, a nanoparticle control group containing only lipid nanoparticles, and a positive control group containing 0.2% Triton X (50 μL 0.2% Triton X + 50 μL 4% sheep blood) were set up. The hemolysis rate was calculated as follows: [(OD...] 实验组 -OD 对照组 )÷(OD triton -OD PBS )]×100%.

[0254] Test results are as follows Figure 21 As shown in the experimental results, the hemolytic activity of the lipid nanoparticles prepared by this invention is generally low, mostly below 5%, with C6OPalA25 and C6OSteA25 exhibiting hemolytic activities of 11% and 7%, respectively.

[0255] Example 8: Maximum Tolerated Dose (MTD) Test of Lipid Nanoparticles in ICR Mice

[0256] In this embodiment, the survival rate of C14OEtA25 lipid nanoparticles at 200 mg / kg was tested in ICR mice to evaluate their toxicity to mice.

[0257] The specific method for testing the maximum tolerated dose (MTD) is as follows: C14OEtA25 lipid nanoparticles were injected into ICR mice via tail vein at a dose of 200 mg / kg. Four ICR mice were tested in each group. After injection, the survival rate of the mice was observed and recorded, and the survival rate at the 200 mg / kg dose was determined after 24 hours. The results showed that at the 200 mg / kg injection dose, the survival rate of C14OEtA25 was 100%, indicating that the C14OEtA25 lipid nanoparticles have very low toxicity and high safety.

[0258] Example 9: Toxicity of lipid nanoparticles to different tumor cells at characteristic pH levels in tumor tissue

[0259] This embodiment uses the MTT (thiazolyl blue) method to evaluate the killing effect of the drug on different tumor cells at pH 6.8, and studies the toxicity of C14OEtA25 lipid nanoparticles prepared according to the method of Example 2 on different tumor cells at the characteristic pH of tumor tissue.

[0260] The specific experimental steps are as follows: Adjust the pH of DMEM medium to 6.8 and 7.4 using 6 mol / L HCl solution, and add 25 mM HEPES to maintain pH stability. The mouse pancreatic cancer cell line Panc02, mouse breast cancer cell line EO771, human non-small cell lung cancer cell line A549, human tongue squamous cell carcinoma cell line Cal27, human tongue squamous cell carcinoma cisplatin-resistant cell line Cal27 / DDP, mouse breast cancer cell line 4T1, human cervical cancer cell line Hela, mouse melanoma cell line B16-F10, mouse breast cancer cell line EMT6, human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, and mouse colon cancer cell line MC38 were cultured in DMEM or 1640 medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, the C14OEtA25 lipid nanoparticles prepared in Example 2 were diluted to different concentrations (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) using DMEM or 1640 medium and added to cells (concentration of 1×10⁻⁶). 5 Cells were incubated in 96-well plates at 37°C and CO2 incubator for 24 hours each. The original culture medium was discarded, and MTT solution (10 μL 5 mg / mL MTT solution (dissolved in PBS) + 90 μL LMEM medium) was added. The plates were then incubated at 37°C and CO2 incubator for another 2 hours. The MTT solution was discarded, and 100 μL dimethyl sulfoxide was added. The plates were then placed on a shaker for 20 minutes. The OD absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated as follows: Cell viability % = (OD...) / (...) 纳米颗粒组 -OD 背景 )÷(OD PBS组 -OD 背景 )×100%, of which OD 背景 For blank wells containing only MTT and no cells, OD PBS组 The control group consisted of cells and MTT, with a drug concentration of 0 μg / mL.

[0261] Test results are as follows Figure 22As shown in the test results, the C14OEtA25 lipid nanoparticles prepared in this invention exhibit significant cytotoxicity against various tumor cells at pH 6.8. In addition to killing sensitive tumor cells, the C14OEtA25 lipid nanoparticles also show a killing effect on drug-resistant tumor cells. The IC50 values ​​of the C14OEtA25 lipid nanoparticles against different tumor cells range from 30 to 70 μg / mL.

[0262] Example 10: Localization of lipid nanoparticles in tumor cells at characteristic pH levels in tumor tissue

[0263] C14OEtA25 lipid nanoparticles (incorporated with DSPE-PEG-Cy5) were co-incubated with Panc02 cells in pH 6.8 medium for 2 hours. Tumor cells were stained with the lysosomal dye Lysotracker and the nuclear dye Hoechst 33342. Observation using a rotating confocal microscope revealed overlap between the fluorescence of the lipid nanoparticles and lysosomal fluorescence, and fluorescence from the lipid nanoparticles was also observed on the cell membrane. Figure 23 This indicates that C14OEtA25 lipid nanoparticles may kill tumor cells by acting on the cell membrane and lysosomes of tumor cells.

[0264] Example 11: Tumor cell uptake of lipid nanoparticles at pH 6.8 and pH 7.4

[0265] C14OEtA25 lipid nanoparticles (incorporated with DSPE-PEG-FITC) were incubated with Panc02 cells at pH 6.8 and pH 7.4 for 1, 2, 4, and 17 hours. After drug removal, cells were digested and collected for flow cytometry analysis. The fluorescence of extracellular FITC was quenched by adding 0.4% trypan blue solution before loading. The results showed that cells took up more lipid nanoparticles at pH 6.8, and the difference in uptake increased with time. Figure 24 The results indicate that C14OEtA25 lipid nanoparticles are protonated at the characteristic pH of tumor tissue and interact with tumor cells through charge interactions, thereby promoting the uptake of lipid nanoparticles.

[0266] Example 12 Effects of endocytosis inhibitors and temperature on the cytotoxicity of lipid nanoparticles at tumor-specific pH levels

[0267] C14OEtA25 lipid nanoparticles, combined with different endocytosis inhibitors (Wollman penicillin, chlorpromazine hydrochloride, methyl-β-cyclodextrin, and sodium azide), were incubated with Panc02 cells in a medium at pH 6.8. Cell viability was measured using the MTT assay. The results are as follows: Figure 25As shown, the three endocytosis inhibitors, woumarin, chlorpromazine hydrochloride, and methyl-β-cyclodextrin, did not inhibit the cytotoxicity of C14OEtA25 lipid nanoparticles at different concentrations. However, the cell activity after combined treatment with sodium azide was higher than that after treatment with lipid nanoparticles alone. This indicates that the entry of the lipid nanoparticles into cells is not mediated by macropinocytosis, clathrin, or caveolin, but is energy-dependent.

[0268] C14OEtA25 lipid nanoparticles were incubated with Panc02 cells at 4°C or 37°C, and cell viability was determined by the MTT assay. The results are as follows: Figure 25 As shown, the toxicity of lipid nanoparticles to tumor cells is significantly reduced in an environment of 4°C, further proving that the tumor-killing effect of the lipid nanoparticles of the present invention is energy-dependent.

[0269] Example 13: In vivo tumor-suppressing experiment of lipid nanoparticles

[0270] The tumor-inhibiting effect of lipid nanoparticles was verified in vivo using three C57 mouse models: subcutaneous pancreatic cancer (Panc02 cells), orthotopic breast cancer (EO771 cells), and subcutaneous colorectal cancer (MC38 cells). The procedure was as follows:

[0271] Female mice (C57BL / 6, 6-8 weeks old) were subcutaneously injected with 1×10⁻⁶ mol / L on their backs. 7 Panc02 cell suspension or MC38 cell suspension (100 μL) per mL was used to construct subcutaneous pancreatic cancer tumor models or colorectal cancer subcutaneous tumor models; 1 × 10⁶ cells / mL were injected orally into the breast. 7 EO771 cell suspension (100 μL) was used to construct an orthotopic breast cancer model. The tumor was allowed to grow to 50–100 mm. 3 (Tumor volume = length × width × width / 2). C14OEtA25 lipid nanoparticles were administered via tail vein at a dose of 50 mg / kg. A negative control group was also included, receiving only an equal volume of PBS solution. Tumor size was measured using calipers, and mouse weight was recorded.

[0272] The in vivo tumor-suppressing effect of the Panc02 model is as follows: Figure 26 As shown in the tumor growth curves, the tumor volume in all treatment groups was inhibited to some extent compared to the PBS group. Furthermore, there was no difference in body weight between the experimental and PBS control groups after the entire treatment cycle. In the EO771 tumor model ( Figure 27C14OEtA25 lipid nanoparticles significantly inhibited tumor growth, with tumors completely disappearing in some treatment groups, while the treatment regimen had no significant effect on mouse body weight. In the MC38 tumor model ( Figure 28 C14OEtA25 lipid nanoparticles can also inhibit tumor growth. In one mouse in the treatment group, the tumor completely disappeared. In addition, the treatment regimen had no significant effect on the weight of the mice.

[0273] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The use of a tertiary amine lipid compound having the structure shown in formula (III) or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a medicament for the prevention and / or treatment of tumors: in, Each R1 is independently selected from: C1-C 22 alkyl; Each R2 is independently selected from: H, Where R is selected from: C1-C 22 alkyl; The total number of carbon atoms in R1 and R2 is 8-32; R4 is selected from: ; R5 is selected from: C1-C6 alkylene groups; R6, R7, and the nitrogen atom attached thereto form a 5-8 membered heterocyclic group with or without R8 substitution, wherein the heteroatom on the ring in the 5-8 membered heterocyclic group is one or two nitrogen atoms; R8 is selected from C1-C6 alkyl groups.

2. The application according to claim 1, characterized in that, R5 is selected from: methylene, ethylene, propylene, and butylene.

3. The application according to claim 1, characterized in that, R4 is selected from: R9-substituted C1-C3 alkyl groups; R9 is selected from: , , , .

4. The application according to claim 1, characterized in that, R4 is selected from: ; R5 is selected from: C1-C3 alkylene groups; R6, R7, and the nitrogen atom attached to them together form the following groups: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 5. The application according to claim 1, characterized in that, Each R1 is independently selected from: C6-C 20 alkyl.

6. The application according to claim 1, characterized in that, Each R1 is independently selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, and docosyl.

7. The application according to claim 6, characterized in that, Each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodel, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, and n-octadecyl.

8. The application according to claim 1, characterized in that, The tertiary amine lipid compounds are shown in formula (V): （V）。 9. The application according to claim 8, characterized in that, R2 is .

10. The application according to any one of claims 1-9, characterized in that, R is selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, dodecyl.

11. The application according to any one of claims 1-9, characterized in that, The total number of carbon atoms in R1 and R2 is 22-28.

12. The application according to claim 11, characterized in that, The total number of carbon atoms in R1 and R2 is 24-26.

13. The application according to claim 1, characterized in that, The tertiary amine lipid compounds are selected from the following compounds: ； Each of R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl; Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl; The total number of carbon atoms in R1 and R2 is 8-32.

14. The application according to claim 13, characterized in that, The total number of carbon atoms in R1 and R2 is 22-28.

15. The application according to claim 14, characterized in that, The total number of carbon atoms in R1 and R2 is 24-26.

16. The application according to claim 13, characterized in that, There exists an R2 with the value H, and R2 is not simultaneously H.

17. The use of tertiary amine lipid compounds or pharmaceutically acceptable salts thereof as active ingredients in the preparation of medicaments for the prevention and / or treatment of tumors, characterized in that, The tertiary amine lipid compounds are selected from the following compounds: 。 18. A lipid nanoparticle, characterized in that, It is formed by the self-assembly of a tertiary amine lipid compound having the structure shown in formula (III) or its pharmaceutically acceptable salt, and an amphiphilic molecule in an aqueous medium, wherein the amphiphilic molecule is an amphiphilic polyethylene glycol polymer, or a combination of an amphiphilic polyethylene glycol polymer and a phospholipid; wherein the amphiphilic polyethylene glycol polymer is an amphiphilic polyethylene glycol lipid molecule and / or a polyethylene glycol-polyester; Each R1 is independently selected from: C1-C 22 alkyl; Each R2 is independently selected from: H, Where R is selected from: C1-C 22 alkyl; The total number of carbon atoms in R1 and R2 is 8-32; R4 is selected from: ; R5 is selected from: C1-C6 alkylene groups; R6, R7, and the nitrogen atom attached thereto form a 5-8 membered heterocyclic group with or without R8 substitution, wherein the heteroatom on the ring in the 5-8 membered heterocyclic group is one or two nitrogen atoms; R8 is selected from C1-C6 alkyl groups.

19. The lipid nanoparticles according to claim 18, characterized in that, R5 is selected from: methylene, ethylene, propylene, and butylene.

20. The lipid nanoparticles according to claim 18, characterized in that, R4 is selected from: R9-substituted C1-C3 alkyl groups; R9 is selected from: , , , .

21. The lipid nanoparticles according to claim 18, characterized in that, R4 is selected from: ; R5 is selected from: C1-C3 alkylene groups; R6, R7, and the nitrogen atom attached to them together form the following groups: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 22. The lipid nanoparticles according to claim 18, characterized in that, Each R1 is independently selected from: C6-C 20 alkyl.

23. The lipid nanoparticles according to claim 18, characterized in that, Each R1 is independently selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, and docosyl.

24. The lipid nanoparticles according to claim 23, characterized in that, Each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodel, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, and n-octadecyl.

25. The lipid nanoparticles according to claim 18, characterized in that, The tertiary amine lipid compounds are shown in formula (V): （V）。 26. The lipid nanoparticles according to claim 25, characterized in that, R2 is .

27. The lipid nanoparticles according to claim 18, characterized in that, R is selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, dodecyl.

28. The lipid nanoparticles according to claim 18, characterized in that, The total number of carbon atoms in R1 and R2 is 22-28.

29. The lipid nanoparticles according to claim 28, characterized in that, The total number of carbon atoms in R1 and R2 is 24-26.

30. The lipid nanoparticles according to claim 18, characterized in that, The tertiary amine lipid compounds are selected from the following compounds: ； Each of R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl; Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl; The total number of carbon atoms in R1 and R2 is 8-32.

31. The lipid nanoparticles according to claim 30, characterized in that, The total number of carbon atoms in R1 and R2 is 22-28.

32. The lipid nanoparticles according to claim 31, characterized in that, The total number of carbon atoms in R1 and R2 is 24-26.

33. The lipid nanoparticles according to claim 30, characterized in that, There exists an R2 with the value H, and R2 is not simultaneously H.

34. A lipid nanoparticle, characterized in that, This compound is formed by the self-assembly of a tertiary amine lipid compound or its pharmaceutically acceptable salt, and an amphiphilic molecule in an aqueous medium. The amphiphilic molecule is an amphiphilic polyethylene glycol polymer, or a combination of an amphiphilic polyethylene glycol polymer and a phospholipid. The amphiphilic polyethylene glycol polymer is an amphiphilic polyethylene glycol lipid molecule and / or a polyethylene glycol-polyester. The tertiary amine lipid compound is selected from the following compounds: 。 35. The lipid nanoparticles according to any one of claims 18-34, characterized in that, The amphiphilic polyethylene glycol lipid molecule is selected from: polyethylene glycol-dispalmitoylphosphatidylethanolamine, polyethylene glycol-distearate phosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, and 2-(polyethylene glycol)-N,N-tetracosylacetamide; the amphiphilic polyethylene glycol-polyester is selected from: polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, and polyethylene glycol-polylactic acid-glycolate.

36. The lipid nanoparticles according to any one of claims 18-34, characterized in that, The molecular weight of the polyethylene glycol in the amphiphilic polyethylene glycol polymer is 500 g / mol to 20000 g / mol.

37. The lipid nanoparticles according to claim 36, characterized in that, The molecular weight of the polyester in the amphiphilic polyethylene glycol-polyester is 500 g / mol to 20000 g / mol.

38. The lipid nanoparticles according to any one of claims 18-34, characterized in that, The phospholipids are selected from: cholesterol, soybean phospholipids, hydrogenated soybean phospholipids, soybean lecithin, egg yolk lecithin, high-purity egg yolk lecithin, dilauroyl lecithin, disqualyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearyl phosphatidylcholine, dipalmitoyl lecithin, distearyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidylethanolamine. Palmitoyl lysophosphatidyl, myristoyl lysophosphatidyl, stearoyl lysophosphatidyl, dimyristoyl phosphatidyl ethanolamine, distearyl phosphatidyl ethanolamine, dipalmitoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl ethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleoyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, 1,2-bisoctadecenoxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester.

39. The lipid nanoparticles according to any one of claims 18-34, characterized in that, The lipid nanoparticles are formed by the self-assembly of the tertiary amine lipid compound or its pharmaceutically acceptable salt and amphiphilic polyethylene glycol lipid molecules in an aqueous medium.

40. The lipid nanoparticles according to claim 39, characterized in that, The amphiphilic polyethylene glycol lipid molecule is distearate phosphatidylethanolamine-polyethylene glycol 2000.

41. The lipid nanoparticles according to claim 39, characterized in that, The mass ratio of the tertiary amine lipid compound or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8.

42. The lipid nanoparticles according to claim 41, characterized in that, The mass ratio of the tertiary amine lipid compound or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.5-4.

43. The lipid nanoparticles according to claim 42, characterized in that, The mass ratio of the tertiary amine lipid compound or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.5-2.

44. The lipid nanoparticles according to claim 43, characterized in that, The mass ratio of the tertiary amine lipid compound or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.8-1.

2.

45. The lipid nanoparticles according to claim 44, characterized in that, The mass ratio of the tertiary amine lipid compound or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:

1.

46. ​​A method for preparing lipid nanoparticles according to any one of claims 18-45, characterized in that, Includes the following steps: The tertiary amine lipid compound or its pharmaceutically acceptable salt is dissolved in an organic solvent to obtain a tertiary amine lipid compound solution; The amphiphilic molecule is dissolved in an organic solvent to obtain an amphiphilic molecule solution; The tertiary amine lipid compound solution and the amphiphilic molecule solution are mixed to obtain a mixed solution; The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing it with a pipette; or, the lipid nanoparticles are obtained by slowly adding the mixed solution dropwise to an aqueous medium under stirring and continuing to stir.

47. The method for preparing lipid nanoparticles according to claim 46, characterized in that, The organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide, or tetrahydrofuran; and / or, The concentration of the tertiary amine lipid compound is 10 mg / mL to 100 mg / mL; and / or, the concentration of the amphiphilic molecule solution is 20 mg / mL to 200 mg / mL; and / or, The volume ratio of the mixed solution to the aqueous medium is 1:1-20.

48. The use of the lipid nanoparticles according to any one of claims 18-45 in the preparation of medicaments for the prevention and / or treatment of tumors.

49. The application according to any one of claims 1-9, or any one of claims 13-17, or claim 48, characterized in that, The tumors mentioned include 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.

50. A tertiary amine lipid compound having the structure shown in formula (III) or a pharmaceutically acceptable salt thereof, in, Each R1 is independently selected from: C1-C 22 alkyl; Each R2 is independently selected from: H, Where R is selected from: C1-C 22 alkyl; The total number of carbon atoms in R1 and R2 is 8-32; R4 is selected from: R9-substituted ethyl groups; R9 is selected from: , .

51. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 50, characterized in that, Each R1 is independently selected from: C6-C 20 alkyl.

52. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 50, characterized in that, Each R1 is independently selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, and docosyl.

53. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 52, characterized in that, Each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodel, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, and n-octadecyl.

54. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 50, characterized in that, R is selected from: 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, heptadecanyl, n-octadecyl, isooctadecyl, dodecyl, dodecyl.

55. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 50, characterized in that, Each R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl; Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl.

56. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to any one of claims 50-55, characterized in that, The total number of carbon atoms in R1 and R2 is 22-28.

57. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to claim 56, characterized in that, The total number of carbon atoms in R1 and R2 is 24-26.

58. The tertiary amine lipid compound or its pharmaceutically acceptable salt according to any one of claims 50-55, characterized in that, There exists an R2 with the value H, and R2 is not simultaneously H.

59. A tertiary amine lipid compound or a pharmaceutically acceptable salt thereof, characterized in that, The tertiary amine lipid compounds are selected from the following compounds: 。 60. A drug for the prevention and / or treatment of tumors, characterized in that, It is prepared from an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises a tertiary amine lipid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 50-59, and / or lipid nanoparticles as described in any one of claims 18-45.

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