CN116848094A

CN116848094A - Tertiary amine lipid compounds and uses thereof

Info

Publication number: CN116848094A
Application number: CN202280005653.1A
Authority: CN
Inventors: 熊梦华; 张厚兵; 鲍燕; 罗智毅; 王均
Original assignee: South China University of Technology SCUT; Sun Yat Sen Memorial Hospital Sun Yat Sen University
Current assignee: South China University of Technology SCUT; Sun Yat Sen Memorial Hospital Sun Yat Sen University
Priority date: 2021-12-29
Filing date: 2022-12-21
Publication date: 2023-10-03
Also published as: WO2023125184A1

Abstract

A tertiary amine lipoid compound with a structure shown in a formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof and application thereof are provided. The compound can efficiently and highly selectively destroy cell membranes or lysosome membranes, thereby killing tumor cells.

Description

Tertiary amine lipid compounds and uses thereof

Technical Field

The invention relates to the technical field of materials and medicines, in particular to a tertiary amine lipoid compound and application thereof.

Background

Malignant tumors have become a large killer that threatens human life and health. The existing anti-tumor therapies such as chemotherapy, radiotherapy and immunotherapy can inhibit the growth of tumors to a certain extent, prolong the life cycle of patients and even completely cure the tumors, but all the anti-tumor therapies face the problems of low specificity and large toxic and side effects, such as normal tissue damage caused by the chemotherapy and the radiotherapy and immune-related toxic and side effects caused by immune check point therapy.

Some cationic polymers can kill pathogens such as tumor cells and bacteria by destroying cell membranes or organelle membranes, have the advantages of broad-spectrum killing effect, difficult drug resistance generation and the like, and have wide application prospects in the treatment of diseases such as tumors. For example, LTX-315 cationic polypeptide can be applied to tumor treatment by intratumoral injection, can cause massive necrosis of tumor cells, and has good effects in combination with medicines such as Yipulimumab, pamammab and the like in melanoma, head and neck cancer, lymphoma and breast cancer, and related researches are in clinical first-phase experiments (https:// clinical. The study of LL-37 polypeptides for the treatment of melanoma by intratumoral injection is also a phase of clinical study (https:// clinicaltrias. Gov /). However, because the medicines can not only destroy tumor cells and pathogenic bacteria with high efficiency, but also have strong cytotoxicity to normal tissue cells, and polypeptide medicines are easy to be degraded by protease, most medicines stop in clinical researches, and most of clinically used medicines are currently treated by local injection.

Therefore, development of drugs for tumor treatment which have no toxic or side effects on normal tissues and cells and can specifically kill tumor cells is urgently needed.

Disclosure of Invention

Based on the above, the invention provides a tertiary amine lipoid compound which is hydrophobic and neutral at normal physiological pH, can be self-assembled with amphiphilic auxiliary lipids such as DSPE-PEG and the like to form lipid nano particles with PEG as a shell layer, and has low cytotoxicity; under the pH condition of the micro-acid, the cell membrane can be protonated to form an amphipathic structure consisting of a hydrophobic structural domain and a cationic structural domain, and the amphipathic structure has strong interaction with a cell membrane or a cell organelle membrane, so that stronger cytotoxicity is generated, and tumor cells are specifically killed.

The invention comprises the following technical scheme.

The application of tertiary amine lipoid compound with a structure shown in formula (I) or stereoisomer or pharmaceutically acceptable salt thereof in preparing pH responsive membrane rupture material:

wherein each R ₁ Each independently selected from: alkyl, unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

each R is ₂ Each independently selected from:or none;

R ₃ and R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic group, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 3-20 membered heterocyclyl; and when R is ₃ When directly connected with N, R ₃ Is not H; when R is ₄ When directly connected with N, R ₄ Is not H;

each R is ₅ Each independently selected from: alkylene, cycloalkyl, aryl;

each R is ₆ Each independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated alkyl, alkenyl, aryl-substituted alkyl,

Each R is ₇ Each independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl-substituted alkyl,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: alkyl, unsaturated alkyl, cycloalkyl, heterocyclyl, alkyl-substituted heterocyclyl, alkenyl, alkynyl, aryl-substituted alkyl, cycloalkyl-substituted alkyl,

R ₉ Selected from: hydroxy, aryl, cycloalkyl, heterocyclyl, R ₈ Substituted heterocyclyl, heteroaryl;

Each X is independently O or NH;

each q and each q' are each independently 0, 1 or 2;

a is 0 or 1;

b is 1, 2, 3, 4 or 5;

c is 1, 2, 3, or 4;

d is an integer between 2 and 12.

The tertiary amine lipid compound may have a structure represented by the following formula (II):

the tertiary amine lipid compound may also have a structure represented by the following formula (III):

R ₄ selected from: r is R ₉ Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic group, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl,

The tertiary amine lipid compound may also have a structure represented by the following formula (IV):

wherein n is 1, 2, 3, 4 or 5;

R ₃ and R is ₄ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₁₀ Alkyl group、C ₁ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, or R ₃ 、R ₄ And the nitrogen atom to which it is attached form a 3-8 membered heterocyclic group.

The tertiary amine lipid compound may also have a structure represented by the following formula (V):

n ₁ 、n ₂ 、n ₃ 、n ₄ each independently selected from: 0. 1, 2, 3 or 4.

In another aspect, the present invention further provides a lipid nanoparticle, which is formed by self-assembling the tertiary amine lipid compound or a stereoisomer or a pharmaceutically acceptable salt thereof, and an amphiphilic molecule in an aqueous medium, wherein the amphiphilic molecule is: an amphiphilic polyethylene glycol lipid molecule, or a combination of an amphiphilic polyethylene glycol lipid molecule and a phospholipid.

On the other hand, the invention also provides a preparation method of the lipid nanoparticle, which comprises the following steps:

dissolving the tertiary amine lipoid compound or stereoisomer or pharmaceutically acceptable salt thereof in an organic solvent to obtain tertiary amine lipoid compound solution;

dissolving the amphiphilic molecules in an organic solvent to obtain amphiphilic molecule solution;

mixing the tertiary amine lipoid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

adding an aqueous medium into the mixed solution, and repeatedly blowing with a pipetting gun to obtain the lipid nanoparticle; or slowly dripping the mixed solution into an aqueous medium under the stirring condition, and continuously stirring to obtain the lipid nanoparticle.

On the other hand, the invention also provides the tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof, or the application of the lipid nanoparticle, which comprises the following technical scheme.

The tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof is applied to the preparation of medicines for preventing and/or treating tumors.

The application of the lipid nanoparticle in preparing a medicament for preventing and/or treating tumors.

The tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof is combined with an anti-tumor drug to prepare the drug for preventing and/or treating tumors.

The application of the lipid nanoparticle combined anti-tumor drug in preparing the drug for preventing and/or treating tumor.

In another aspect, the invention also provides a medicament for preventing and/or treating tumors, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the tertiary amine lipid compound or stereoisomer or pharmaceutically acceptable salt thereof, and/or the lipid nanoparticle.

In another aspect, the present invention also provides a combination for preventing and/or treating tumors, the active ingredients of which include:

component 1: said tertiary amine lipid compound or a stereoisomer or a pharmaceutically acceptable salt thereof, and/or said lipid nanoparticle; the method comprises the steps of,

component 2: antitumor drugs other than component 1;

the component 1 and the component 2 are each independent administration units, or the component 1 and the component 2 together form a combined administration unit.

Based on the technical scheme, the invention has the following beneficial effects:

The invention provides an anti-tumor lipid tertiary amine small molecular compound with activatable acidity, which consists of a tertiary amine head part with convertible hydrophilic and hydrophobic properties and a tail part containing a hydrophobic group. Under normal physiological pH, tertiary amine in the lipid material is hydrophobic and neutral, the head of tertiary amine is in a hydrophobic conformation, and the lipid material can self-assemble to form an anti-tumor lipid nanoparticle with the PEG as a shell layer and activatable acidity under the assistance of auxiliary lipids such as DSPE-PEG, and the interaction with a cell membrane is weak, so that the lipid material has the advantage of low toxicity to normal tissues during in vivo circulation, and the PEG shell can improve the biocompatibility of the lipid material and prolong the blood circulation time of the lipid material; under the condition of slightly acidic pH of tumor tissues (a large number of researches show that tumor cells usually adopt a glycolytic metabolism mode to generate more lactic acid and carbon dioxide, but metabolic waste cannot be normally discharged due to lymphatic reflux blockage in the solid tumor tissues, so that the pH of the peripheral environment of the solid tumor is lower than that of normal body fluid, and is usually between 6.4 and 7.0), the tertiary amine part of the lipid material of the molecular compound can be protonated, so that the nanoparticles form an amphiphilic structure consisting of a hydrophobic domain and a cationic domain, and the amphiphilic structure has extremely strong interaction with cell membranes or organelle membranes, thereby killing the tumor cells with high efficiency and high selectivity. The tertiary amine small molecular compound and/or the antitumor lipid nano particles with activatable acidity can be used for preparing antitumor drugs, and have the advantages of good treatment effect, high selectivity and low toxicity.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of n-decyl acrylate (C10).

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of 9-octadecenylacrylate (C181).

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of cis-9, 12-octadecadienylacrylate (C182).

FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of phenylbutyl acrylate (Cp 4).

FIG. 5 is a thermal graph of the hemolysis rate of lipid nanoparticles prepared in example 1 after incubation with 2% sheep blood at 400. Mu.g/mL for 4 h.

FIG. 6 is a graph showing the relationship between cell activity and pH of lipid nanoparticles of different carbon chain lengths prepared in example 2 and mouse colorectal cancer MC38 cells incubated for 24h at pH 7.4 and 6.8.

FIG. 7 is a graph showing the relationship between cell activity and pH of lipid nanoparticles of different carbon chain lengths prepared in example 2 and mouse colorectal cancer MC38 cells incubated for 24h at pH 7.4 and 6.8.

FIG. 8 is a nuclear magnetic resonance hydrogen spectrum of the C8A45 lipid compound prepared in example 1.

FIG. 9 is a nuclear magnetic resonance hydrogen spectrum of the C8A49 lipid compound prepared in example 1.

FIG. 10 is a nuclear magnetic resonance hydrogen spectrum of the C12A27 lipid compound prepared in example 1.

FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of the C12A33 lipid compound prepared in example 1.

FIG. 12 is a nuclear magnetic resonance hydrogen spectrum of the C14A33 lipid compound prepared in example 1.

FIG. 13 is a nuclear magnetic resonance hydrogen spectrum of the C16A34 lipid compound prepared in example 1.

FIG. 14 is a nuclear magnetic resonance hydrogen spectrum of the C181A17 lipid compound prepared in example 1.

FIG. 15 is a nuclear magnetic resonance hydrogen spectrum of the C181A23 lipid compound prepared in example 1.

FIG. 16 is a nuclear magnetic resonance hydrogen spectrum of the C181A24 lipid compound prepared in example 1.

FIG. 17 is a nuclear magnetic resonance hydrogen spectrum of the C181A28 lipid compound prepared in example 1.

FIG. 18 is a nuclear magnetic resonance hydrogen spectrum of the C182A17 lipid compound prepared in example 1.

FIG. 19 is a nuclear magnetic resonance hydrogen spectrum of the C182A23 lipid compound prepared in example 1.

FIG. 20 is a nuclear magnetic resonance hydrogen spectrum of the C182A24 lipid compound prepared in example 1.

FIG. 21 is a nuclear magnetic resonance hydrogen spectrum of the C182A28 lipid compound prepared in example 1.

FIG. 22 is a nuclear magnetic resonance hydrogen spectrum of the C8A16 lipid compound prepared in example 1.

FIG. 23 is a nuclear magnetic resonance hydrogen spectrum of the C8A19 lipid compound prepared in example 1.

Figure 24 is a concentration-dependent cytotoxicity of a portion of lipid nanoparticles at pH 7.4 and 6.8.

Figure 25 is a concentration-dependent cytotoxicity of a fraction of lipid nanoparticles at pH 7.4 and 6.8.

FIG. 26 is a graph showing the particle size and distribution of lipid nanoparticles in aqueous solution as measured by dynamic light scattering.

FIG. 27 is MTD of tertiary amine lipid nanoparticles detected in ICR mice.

Fig. 28 is a graph of the protonation rate of tertiary amine lipid nanoparticles as a function of pH and their pKa.

FIG. 29 is a graph showing the concentration-dependent cytotoxicity of lipid nanoparticles assembled from C12A27 lipid compounds with different components at pH 7.4 and 6.8; wherein, a is C12A27 and different PEG lipids according to 1:1 cytotoxicity of assembled lipid nanoparticles to MC38 at pH 7.4 and pH 6.8; panel b shows cytotoxicity of DSPE-PEG and C12A27 assembled lipid nanoparticles at different mass ratios to MC38 at pH 7.4 and pH 6.8; panel C and d are cytotoxicity of DSPE-PEG, C12A27 and different helper lipids on MC38 at pH 7.4 and pH 6.8, respectively, assembled lipid nanoparticles at different mass ratios.

FIG. 30 is a mechanism study of cytotoxicity of C12A27 lipid nanoparticles on MC38 cells at pH 6.8; wherein, a is the cytotoxicity of the C12a27 lipid nanoparticle for co-incubation of MC38 at pH 6.8 for 4h under different temperature conditions; panel b shows ATP leakage from the cytoplasm after incubation of C12A27 lipid nanoparticles with MC38 cells for 4h at pH 6.8 at different temperatures; panel C shows the C12A27 lipid nanoparticle with MC38 at pH 6.8 _GFP/mCherry mCherry leakage in the cytoplasm after cell co-incubation and GFP changes on the cell membrane.

FIG. 31 shows C8A16 lipid nanoparticle and C8A19 lipid nanoparticle with MC38 at pH 6.8 _GFP/mCherry mCherry leakage in the cytoplasm after cell co-incubation and GFP changes on the cell membrane.

FIG. 32 shows cell morphology and cell staining of C8A16 lipid nanoparticles, PI and Annexin-FITC incubated with MC38 at pH 6.8.

FIG. 33 is a C8A45 lipid nanoparticle,C8A49 lipid nanoparticle, C12A27 lipid nanoparticle and C12A33 lipid nanoparticle were combined with Panc02 at pH 6.0 _GFP/mCherry mCherry leakage in the cytoplasm after cell co-incubation and GFP changes on the cell membrane.

Fig. 34 is a schematic drug administration and experimental results of therapeutic experiments of C12a27, C14a27, C16a27 lipid nanoparticles to a mouse pancreatic cancer model Panc 02.

FIG. 35 is a schematic and experimental results of therapeutic experimental dosing of C8A16 lipid nanoparticles, C8A19 lipid nanoparticles, C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, C12A27 lipid nanoparticles, C12A33 lipid nanoparticles, and C182A28 lipid nanoparticles to a mouse colorectal cancer model MC 38.

FIG. 36 is a schematic illustration of the therapeutic experimental dosing of C8A45 lipid nanoparticles, C8A49 lipid nanoparticles, C12A27 lipid nanoparticles, C12A33 lipid nanoparticles to a mouse breast cancer model E0771 and experimental results.

Fig. 37 is a schematic administration and experimental results of a therapeutic experiment of C12a27 and C14a27 lipid nanoparticle combined immune checkpoint antibody, agd-1, on a mouse colorectal cancer model, MC 38.

FIG. 38 is a schematic and experimental results of the therapeutic administration of C12A27 lipid nanoparticles in combination with the chemotherapeutic drug liposomal doxorubicin Doxil to EMT-6 in a mouse breast cancer model.

Detailed Description

The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.

In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the compounds of the invention, when any variable (e.g., R ₁ 、R ₂ Etc.) occur more than once in any component, the definition of each occurrence is independent of the definition of each other occurrence. Also, combinations of substituents and variables are permissible provided that such combinations stabilize the compounds. The lines drawn from the substituents into the ring system indicate that the bond referred to may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are only attached to any suitable carbon atom adjacent to the ring. It is to be understood that substituents and substitution patterns of the compounds of this invention may be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that may be readily synthesized from readily available starting materials by techniques in the art and methods set forth below. If the substituent itself is substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, as long as the structure is stabilized. The phrase "optionally substituted with one or more substituents" is considered to be equivalent to the phrase "optionally substituted with at least one substituent" and in this case preferred embodiments will have from 0 to 3 substituents.

The term "alkyl" as used herein is meant to include both branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. For example, "C ₁ -C ₆ Alkyl "medium" C ₁ -C ₆ The definition of "includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example，“C ₁ -C ₆ The alkyl group includes, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl.

The term "unsaturated chain hydrocarbon group" refers to branched and straight unsaturated aliphatic hydrocarbon groups having a specific number of carbon atoms, i.e., acyclic unsaturated chain hydrocarbon groups, and having 1 or more carbon-carbon double bonds in the carbon chain, or having a carbon-carbon triple bond, such as: CH (CH) ₂ ＝CHCH ₂ - _， -(CH ₂ ) ₈ (CH＝CH)CH ₃ ，-(CH ₂ ) ₇ CH＝CH ₂ ，-(CH ₂ ) ₈ CH＝CH ₂ Cis-9, 12-octadecadienyl, etc.

The term "heterocyclyl" is a saturated or partially unsaturated monocyclic, fused, spiro, bridged, or other cyclic substituent 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, for example: piperidinyl, pyrrolidinyl, and the like.

The invention relates to an acidity activatable anti-tumor lipid, which can specifically activate anti-tumor activity under specific acidity conditions to kill tumor cells.

The invention relates to a pH responsive membrane rupture material which can activate the membrane rupture activity under specific acidity conditions.

The tertiary amine lipid compound or lipid or small molecular compound or small molecular lipid compound or tertiary amine small molecular compound refers to a compound in the structural range shown in the formula (I) -formula (V).

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

each R is ₂ Each independently selected from:or none;

each R is ₅ Each independently selected from: alkylene, cycloalkyl, aryl;

Each R is ₇ Each independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R ₈ Substituted heterocyclyl, alkenylAlkynyl, aryl substituted alkyl,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each X is independently O or NH;

each q and each q' are each independently 0, 1 or 2;

a is 0 or 1;

b is 1, 2, 3, 4 or 5;

c is 1, 2, 3, or 4;

d is an integer between 2 and 12.

In some preferred embodiments thereof, the tertiary amine lipid compound has a structure represented by the following formula (II):

In some preferred embodiments thereof, the tertiary amine lipid compound has a structure represented by the following formula (III):

In some preferred embodiments thereof, R ₄ Selected from: c (C) ₁ -C ₂₂ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₂₂ Aryl group,

In some preferred embodiments thereof, R ₄ Selected from: c (C) ₁ -C ₁₀ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracenyl,

In some preferred embodiments, each R ₅ Each independently selected from: c (C) ₁ -C ₂₂ Alkylene, C ₃ -C ₈ Cycloalkyl, C ₆ -C ₁₄ Aryl groups.

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

In some preferred embodiments, each R ₅ Each independently selected from: c (C) ₁ -C ₆ Alkylene, C ₃ -C ₈ Cycloalkyl groups.

In some preferred embodiments, each R ₅ Each independently selected from: methylene, ethylene, propylene, butylene,

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

Each R is ₇ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₁₄ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl group,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, C ₁ -C ₈ Alkyl-substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₁₄ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl, C ₃ -C ₂₂ Cycloalkyl-substituted C ₁ -C ₈ Alkyl group,

In some preferred embodiments, each R ₆ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₂ -C ₁₀ Alkenyl, phenyl, naphthyl, anthryl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl group,

Each R is ₇ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl group,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, C ₁ -C ₆ Alkyl-substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl, C ₃ -C ₈ Cycloalkyl-substituted C ₁ -C ₈ Alkyl group,

In some preferred embodiments, each R ₆ Each independently selected from: c (C) ₁ -C ₆ An alkyl group; hydroxy-substituted C ₁ -C ₆ Alkyl group,

Each R is ₇ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl group,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, 5-8 membered heterocyclyl, C ₁ -C ₆ Alkyl substituted 5-8 heterocyclyl, phenyl substituted C ₁ -C ₃ Alkyl group,

In some preferred embodiments thereof, R ₉ Selected from: hydroxy, C ₆ -C ₁₄ Aryl, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, 5-10 membered heteroaryl.

In some preferred embodiments thereof, R ₉ Selected from: hydroxy, phenyl, naphthyl, anthryl, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, 5-8 membered heteroaryl.

In some of which it is preferred to implementIn the example, R ₄ Selected from: c (C) ₁ -C ₈ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₃ -C ₈ Cycloalkyl, phenyl substituted C ₁ -C ₃ Alkyl, naphthyl substituted C ₁ -C ₃ Alkyl-and anthracenyl-substituted C ₁ -C ₃ Alkyl, 3-8 membered heterocyclyl, R ₈ Substituted 5-8 membered heterocyclyl,A substituted cyclohexyl group;

R ₅ selected from: c (C) ₁ -C ₄ An alkylene group;

each R is ₆ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, -CH ₂ -R ₂ -R ₁ ；

Each R is ₇ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, -CH ₂ -R ₂ -R ₁ ；

Each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, phenyl substituted C ₁ -C ₃ Alkyl group,

R ₉ Selected from: hydroxy, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, 5-8 membered heteroaryl.

In some preferred embodiments thereof, R ₄ Selected from: c (C) ₄ -C ₆ Alkyl group,Dimethylamino-substituted butyl, diethylamino-substituted butyl, piperidinyl-substituted n-propyl, R ₉ Substituted C ₁ -C ₃ Alkyl, C ₄ -C ₈ Cycloalkyl, benzyl, 5-8 membered heterocyclyl, R ₈ Substituted 5-6 membered heterocyclyl,A substituted cyclohexyl group;

each R is ₅ Each independently selected from: c (C) ₁ -C ₃ An alkylene group;

R ₆ selected from: c (C) ₃ -C ₄ An alkyl group;

each R is ₇ Each independently selected from: c (C) ₃ -C ₄ An alkyl group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, benzyl,

R ₉ selected from:

in some preferred embodiments thereof, the tertiary amine lipid compound has a structure represented by the following formula (IV):

wherein n is 1, 2, 3, 4 or 5;

R ₃ and R is ₄ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₁₀ Alkyl, C ₁ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, or R ₃ 、R ₄ And the nitrogen atom to which it is attached form a 3-8 membered heterocyclic group.

In some preferred embodiments thereof, R ₃ And R is ₄ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, C ₁ -C ₆ Unsaturated chain hydrocarbon group, or R ₃ 、R ₄ And the nitrogen atom to which it is attached form a 5-8 membered heterocyclic group.

In some preferred embodiments thereof, the tertiary amine lipid compound has a structure represented by the following formula (V):

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

In some preferred embodiments, each R ₁ Each independently selected from: c (C) ₈ -C ₂₀ Alkyl, C ₈ -C ₂₀ Unsaturated chain hydrocarbon group, C ₅ -C ₂₀ Cycloalkyl, C ₈ -C ₂₀ Alkenyl, C ₈ -C ₂₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₆ Alkyl, naphthyl substituted C ₁ -C ₆ Alkyl-and anthracenyl-substituted C ₁ -C ₆ Alkyl, C ₅ -C ₂₀ Cycloalkyl-substituted C ₁ -C ₆ An alkyl group.

In some preferred embodiments, each R ₁ Each 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, isodecyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isostearyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl methyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylpropyl cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, docosyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, decynyl, dodecenyl, tetradecynyl, hexadecynyl, octadecenyl, docosyl, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylhexyl, phenyloctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, An anthracenyl methyl group, an anthracenyl ethyl group, an anthracenyl propyl group, an anthracenyl butyl group, an anthracenyl hexyl group anthraceneoctyl, cis-9, 12-octadecadienyl, 9-octadecadienyl.

In some preferred embodiments, each R ₁ Each independently selected from: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9, 12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

In some preferred embodiments, each R ₂ Each independently selected from: or none, wherein c is 1 or 2; d is an integer between 2 and 6.

In some preferred embodiments thereof, R ₃ And R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted C ₁ -C ₂₂ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₂₂ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl, C ₃ -C ₂₂ Cycloalkyl-substituted C ₁ -C ₈ Alkyl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ Substituted or unsubstituted 3-10 membered heterocyclic ring.

In some preferred embodiments thereof, R ₃ And R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₆ Alkyl, naphthyl substituted C ₁ -C ₆ Alkyl-and anthracenyl-substituted C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl-substituted C ₁ -C ₆ Alkyl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ Substituted or unsubstituted 5-10 membered heterocyclyl.

In some preferred embodiments thereof, R ₃ 、R ₄ And the nitrogen atom to which it is attached form the following group:

in some preferred embodiments thereof, the tertiary amine lipid compound is selected from the following:

wherein each R ₁ Each independently is: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9, 12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

in one embodiment of the present invention, there is further provided a lipid nanoparticle formed by self-assembling the tertiary amine lipid compound or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and an amphiphilic molecule in an aqueous medium, wherein the amphiphilic molecule is: an amphiphilic polyethylene glycol lipid molecule, or a combination of an amphiphilic polyethylene glycol lipid molecule and a phospholipid.

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

The phospholipids may be selected from, but are not limited to: cholesterol, soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, high purity egg yolk lecithin, dilauroyl lecithin, dithiin phosphatidylcholine, dioleoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1, 2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylserine, lysophosphatidylethanolamine, palmitoyl lysophosphatidylcholine, myristoyl lysophosphatidylcholine, stearoyl lysophosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, (2, 3-dioleoyl-propyl) -trimethylammonium chloride, 3- [ N- (N ']; N' -dimethylaminoethyl-carbamoyl ] cholesterol hydrochloride, 1, 2-dioleyl-3-dimethylamino-propane, 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester, 1, 2-dioctadecyloxy-3-methylammonium propane, (R) -2, 3-bis (oleoyloxy) propyl hydrogen phosphate 2- [ (2-aminoethyl) amino ] ethyl ester.

In some preferred embodiments thereof, the lipid nanoparticle is formed by self-assembly of the tertiary amine lipid compound or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, an amphiphilic polyethylene glycol lipid molecule and 1, 2-palmitoyl phosphatidylglycerol in an aqueous medium; or is formed by self-assembling the tertiary amine lipoid compound or a stereoisomer or a pharmaceutically acceptable salt thereof and amphiphilic polyethylene glycol lipoid molecules in an aqueous medium; preferably, the amphiphilic polyethylene glycol lipid molecule is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

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

in some preferred embodiments, the mass ratio of the tertiary amine lipid compound or stereoisomer or pharmaceutically acceptable salt thereof, to the 1, 2-palmitoyl phosphatidylglycerol is 1:0.5 to 1.5, preferably 1:0.8-1.2.

In an embodiment of the present invention, there is also provided a preparation method of the lipid nanoparticle, including the steps of:

In some preferred embodiments, the organic solvent is selected from ethanol, methanol, dimethylsulfoxide, N-dimethylformamide, or tetrahydrofuran.

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

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

In one embodiment of the invention, the application of the tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing medicines for preventing and/or treating tumors is also provided.

In one embodiment of the invention, the application of the lipid nanoparticle in preparing a medicament for preventing and/or treating tumor is also provided.

In one embodiment of the invention, the application of the tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof in preparing the medicine for preventing and/or treating tumor is also provided.

In one embodiment of the invention, the application of the lipid nanoparticle combined anti-tumor drug in preparing a drug for preventing and/or treating tumors is also provided.

In some preferred embodiments, the anti-tumor agent is an immune checkpoint inhibitor and/or a chemotherapeutic agent.

In some preferred embodiments thereof, the immune checkpoint inhibitor is a PD-1 inhibitor.

In some preferred embodiments, the chemotherapeutic agent is doxorubicin.

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

In one embodiment of the present invention, a medicament for preventing and/or treating tumor is also provided, which is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the tertiary amine lipid compound or stereoisomer or pharmaceutically acceptable salt thereof, and/or the lipid nanoparticle.

In one embodiment of the present invention, there is also provided a combination for preventing and/or treating tumors, the active ingredients of which include:

component 2: antitumor drugs other than component 1;

In some preferred embodiments thereof, the component 2 is an immune checkpoint inhibitor and/or a chemotherapeutic agent.

In some preferred embodiments, the chemotherapeutic agent is doxorubicin.

The compounds of formula (I) -formula (V) according to the invention can be used in combination with other known antitumor agents. When administered in combination, the compounds of formula (I) -formula (V) and the known agent may each be a separate administration unit or together form a combined administration unit; the compounds of formulae (I) - (V) may be administered simultaneously with or separately from other known antitumor agents. When the compounds of formula (I) -formula (V) are administered simultaneously with one or more other drugs, it is preferable to use a pharmaceutical composition containing one or more known drugs together with the compounds of formula (I) -formula (V). Drug combinations also include administration of the compounds of formulas (I) - (V) with one or more other known drugs over overlapping time periods. When the compounds of formula (I) -formula (V) are administered in combination with one or more other known agents, the dosage of the compounds of formula (I) -formula (V) or the known agents may be the same as the dosage administered alone or may be lower than the dosage at which they are administered alone.

Drugs or active ingredients that may be used in combination with the compounds of formulas (i) - (V) include, but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxin/cytostatics, antiproliferatives, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signaling inhibitors, agents 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 activators, VEGF antibodies, EGF antibodies, and the like.

In some of these embodiments, the drugs or active ingredients that may be used in combination with the compounds of formulas (I) - (IV) include, but are not limited to: albumin, alendronic acid, interferon, al Qu Nuoying, allopurinol sodium, palonosetron hydrochloride, altretamine, aminoglutethimide, amifostine, amrubicin, an Ya pyridine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, minoxin, 5-azacytidine, azathioprine, BCG or tice BCG, betadine, betamethasone acetate, betamethasone sodium phosphate formulation, bexarotene, bleomycin sulfate, british, bortezomib, busulfan, calcitonin, alezomib injection, capecitabine, carboplatin, kang Shide, cefesone, cet Mo Baijie, daunorubicin, chlorambucil, cisplatin, cladribine, clofaxine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, dexamethasone dexamethasone phosphate, estradiol valerate, deniinterleukin 2, dibaume, dulorelin, delazocine, diethylstilbestrol, dafukang, docetaxel, deoxyfluorouridine, doxorubicin, dronabinol, jejunum-166-chitosan complex, eligard, labyrinase, epirubicin hydrochloride, aprepitant, epirubicin, alfuzoxetine, erythropoietin, eplatin, levamisole, estradiol formulations, 17-beta-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxy phosphate, petrolatum, etoposide, fadrozole, tamoxifen formulations, febuxostat, finasteride, feveride, fluorouridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxytestosterone, flusteramine, fotemustine, fludarabine, 1-beta-D-arabinofuranosyl-cytothiadine-5' -stearoyl phosphate, fotemustine, fulvestrant, progastrin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine wafer capsule, goserelin, glatiramer hydrochloride, histrelin, and meflozin, hydrocortisone, erythro-hydroxynonyladenine, hydroxyurea, tetan iso Bei Moshan antibody, idarubicin, ifosfamide, interferon alpha 2A, interferon alpha 2B, interferon alpha nl, interferon alpha n3, interferon beta, interferon gamma la, interleukin 2, intron A, iressa, irinotecan, ketjel, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levamisole calcium levofolinate, sodium levothyroxine formulations, lomustine, lonidamine, dronabinol, nitrogen mustard, mecobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogens, 6-borazine, mesna, methotrexate, methyl aminolevulinate, miltefosine, melomycin, mitomycin C, mitotane, mitoquinone, trospine, doxorubicin citrate liposomes, nedaplatin, pegylated febuxostat, olpreninterleukin, neunogen, nilutamide, tamoxifen, NSC-631570, recombinant human interleukin 1-beta, octreotide, ondansetron hydrochloride, dehydrohydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate formulations, pegine, roxyprogesterone, euphorbia, pernicid, and the like, pennisetum, streptozotocin, pilocarpine hydrochloride, bicubicin, plicamycin, porphin sodium, prednimustine, setprednisolone, prednisone, beclomethamine, procarbazine, recombinant human erythropoietin, raltitrexed, liratio, etidronate rhenium-186, mevalhua, dynamics stretch-A, romidepide, pilocarpine hydrochloride tablet, octreotide, sarustine, semustine, sirolimus, sibutramine, sibutrazol, sodium methylprednisolone, palustric acid, stem cell therapy, streptozocin, strontium chloride-89, levothyroxine sodium, tamoxifen, tamsulosin, testolazine, taxotere, temozolomide, teniposide, testosterone, thioguanine, thiotepa, somatostatin, temozolomide, toldronic acid, topotecan, tolnaftate, tolizumab, toxidan trastuzumab, trocounter, treoshu, tretinoin, methotrexate tablet, trimethamine, trimetraxazole, triptorelin acetate, trastuline pamoate, ulipraline, uridine, valubicin, vislizumab, vincristine, vinblastine, vinamidone, vinorelbine, vitamin Lu Liqin, dexpropimine, net-setamide Ding Sizhi, pivalonine, paclitaxel protein stabilized formulation, acolbifene, interferon r-lb, affinitak, aminopterin, alzoxifene, asorisnil, atomestane, atrasentan, BAY 43-9006, avastin, CCI-779, CDC-501, celecoxib, clenbuterol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dIM, dutasteride, edoxin, efonine, irinotecan, flunixin, valinate, valienamine, difenoxine hydrochloride, dimet al, daphne hydrochloride, and the like, holmium-166 DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, milbexifene, mi Nuoqu acid ester, MS-209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, neovalproate, norlabratex, olimarson, onco-TCS, oside, paclitaxel polyglutamate, sodium silk-miate, PN-401, QS-21, quaternary, R-154, raloxifene, ranpirnase, 13-cis-retinoic acid, satraplatin, orcalcitol, T-138067, tarceva, docosahexaenoic acid paclitaxel, thymol, prostaglandin furin, tipifarnib, tiramimine, TLK-286, toremio, 7R, valproan, prandin, ibvanadn, R-154, raloxifene, MID-100, and combinations thereof.

The medicament for preventing and/or treating tumors of the present invention may be used in non-human mammals or humans.

The pharmaceutically acceptable auxiliary materials used in the medicament for preventing and/or treating tumor of the invention refer to: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity.

"compatible" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention (tertiary amine lipid compounds of formulas I-VII) without significantly reducing the efficacy of the active ingredient.

Pharmaceutically acceptable excipients used in the medicament for preventing and/or treating tumors of the present invention include, but are not limited to, one or more of the following materials: at least one of a solvent, excipient, filler, compatibilizer, binder, humectant, disintegrant, slow solvent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent, and perfume.

Examples of pharmaceutically acceptable adjuvant components include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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.), polyalcohols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (such as tween ) Wetting agents (e.g. sodium lauryl sulphate), colouring agents, flavouring agents, stabilisers, antioxidants, preservatives,Pyrogen-free water, and the like.

The mode of administration of the active ingredient or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and the like.

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

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

(a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

(b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia;

(c) Humectants, for example, glycerin;

(d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

(e) Slow solvents, such as paraffin;

(f) Absorption accelerators, for example quaternary amine compounds;

(g) Wetting agents, for example cetyl alcohol and glycerol monostearate;

(h) Adsorbents, such as kaolin; and

(i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

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

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like. In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances, and the like.

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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The following are specific examples.

Example 1: preparation of tertiary amine lipoid compound obtained by reaction of acrylic ester and amine molecule

Tertiary amine lipid compounds containing different types of hydrophobic groups and tertiary amine structures were prepared in this example. These lipids were synthesized by Michael addition of acrylates (C8, C10i, C12, C14, C16, C18, C181, C182, cp2, cp 4) containing different hydrophobic carbon chains and amine molecules (A1 to A51, 51 total) containing primary and/or secondary amines, wherein C8, C10i, C12, C14, C16, C18, C181, C182, cp2, cp4 were n-octyl acrylate (C8), n-decyl acrylate (C10), isodecyl acrylate (C10 i), n-dodecyl acrylate (C12), n-tetradecyl acrylate (C14), n-hexadecyl acrylate (C16), n-octadecyl acrylate (C18), 9-octadecyl acrylate (C181) in this order Cis-9, 12-octadecadienylacrylate (C182), 2-phenylethylacrylate (Cp 2), phenylbutyl acrylate (Cp 4); a1 to A51 are in turn:

that is, in this example, a series of tertiary amine lipid compounds represented by the formula (VIII) were synthesized by Michael addition reaction of an acrylate and a primary amine-containing molecule. The reaction formula is as follows:

wherein R is ₁ The method comprises the following steps: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9, 12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

Wherein R is ₃ Selected from the following groups:

a series of tertiary amine lipid compounds are synthesized by Michael addition of acrylate and molecules containing secondary amines. An exemplary reaction scheme is as follows:

A series of tertiary amine lipid compounds are synthesized by Michael addition of acrylate and molecules containing both primary and secondary amines. An exemplary reaction scheme is as follows:

The n-decyl acrylate, 9-octadeceneacrylate, cis-9, 12-octadecadienylacrylate and phenylbutyl acrylate required by Michael addition reaction are synthesized by a laboratory, and the nuclear magnetism is shown in figures 1-4; the remaining acrylate and primary or/and secondary amine-containing molecules are obtained as direct purchases and are used without purification.

The method comprises the following specific steps:

100mg (0.20 mmol to 0.60 mmol) of the acrylate (C8, C10i, C10, C12, C14, C16, C18, C181, C182, cp2, cp4, respectively) are placed in a reaction vessel in a molar ratio of 1.1 in terms of the number of reactive sites of the acrylate to the amine molecule (primary amine contains two reactive sites, secondary amine contains one reactive site): 1, respectively adding A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25, A26, A27, A28, A29, A30, A31, A32, A33, A34, A35, A36, A37, A38, A39, A40, A41, A42, A43, A44, A45, A46, A47, A48, A49, A50 and A51, uniformly mixing, and hermetically reacting at 90 ℃ for 3 days. And collecting the product after the reaction is finished and directly using the product.

The tertiary amine lipid compound prepared in this example is abbreviated as C _n A _n Wherein C _n Refers to acrylic ester in raw materials, A _n Refers to amine molecules in the starting material, e.g. C ₈ A ₁ Is defined as R ₁ The tertiary amine lipid compound is prepared by the reaction of acrylic ester (C8) which is n-octyl and amine molecule (A1).

Example 2 preparation of lipid nanoparticles

The tertiary amine lipid compound synthesized in example 1 has a hydrophobic carbon chain moiety and a tertiary amine moiety that is hydrophobic at pH 7.4, and self-assembles in water to form nanoparticles with the aid of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG).

The specific operation is as follows: dissolving tertiary amine lipoid compound in ethanol to obtain a lipid solution with the concentration of 50 mg/mL; taking DSPE-PEG ₂₀₀₀ Dissolving in ethanol to obtain DSPE-PEG with concentration of 50mg/mL ₂₀₀₀ A solution; taking 50 μl of lipid solution and 50 μl of LDSPE-PEG ₂₀₀₀ Mixing the solution in a 1.5mL centrifuge tube to obtain a mixed solution; adding 20 μl of the mixed solution into another 1.5mL centrifuge tube, adding 180 μl deionized water or 1×PBS with a pipette, rapidly adding into the mixed solution, and repeatedly blowing with the pipette to obtain lipid nanoparticles; or 3mL of pure water or 1 XPBS is taken in a 10mL round bottom flask, 1mL of the mixed solution is taken by a liquid-transfering gun and slowly added into water dropwise under the condition of stirring, and lipid nano particles are obtained after stirring for 10 min; transferring into a dialysis bag with molecular weight cut-off of 14000Da, and dialyzing in pure water to remove ethanol.

EXAMPLE 3 haemolytic Activity of lipid nanoparticles

This example examined the hemolytic activity of the series of lipid nanoparticles prepared in example 2 and screened lipid material with a concentration of 400 μg/mL and less than 5% hemolytic activity at 4h incubation.

The specific experimental steps are as follows: washing sheep blood with 1 XPBS for 2 times until supernatant is clear, transparent and colorless after centrifugation, and diluting sheep blood with 1 XPBS to 4%; taking 16 mu L of the lipid nanoparticle prepared by 1 XPBS in example 2, adding 34 mu L of 1 XPBS into 184 Kong Xi cell culture plates, adding 50 mu L of 4% sheep blood, and incubating in a 37 ℃ incubator for 4 hours; centrifuging at 3000rpm for 10min, then sucking 50 μl of supernatant into another 384-well plate, and detecting absorbance at 576nm with a multifunctional enzyme-labeled instrument; in addition, PBS control without lipid nanoparticles (50. Mu.L of 1 XPBS+50. Mu.L of 4% sheep blood), nanoparticle control with lipid nanoparticles only, and positive control with triton X0.2% (50. Mu.L of 0.2% c+50. Mu.L of 4% sheep blood) were set.

The test results are shown in FIG. 5, and it can be seen that the hemolytic activity of most of the lipid nanoparticles is low (< 5%), while the hemolytic activity of the small portion of the lipid nanoparticles is high (. Gtoreq.5%), indicating that the hemolytic toxicity of the large portion of the lipid nanoparticles is low. Lipid nanoparticles with a hemolysis rate of less than 5% were selected for the next experiment.

EXAMPLE 4 cytotoxicity of lipid nanoparticles at pH characteristic of Normal and tumor tissues

This example evaluates the killing effect of drugs on tumor cells by CCK8 method (Cell Counting Kit-8) at pH 7.4 and pH 6.8, and studies the cytotoxicity of the series of lipid nanoparticles prepared in example 2 at pH characteristic of normal and tumor tissues.

The specific experimental steps are as follows: DMEM medium was adjusted to ph=6.8 and 7.4 using 6mol/L HCl solution for later use, and 25mM HEPES was added to maintain pH stable. The mouse colorectal cancer cell line MC38 (purchased from ATCC) was cultured in DMEM medium containing 10% (v/v) fetal bovine serum. The lipid nanoparticle prepared in example 2 (100. Mu.g/mL) was incubated with cells (at a concentration of 1X 10 at pH 7.4 and pH 6.8 ⁶ individual/mL) were incubated in 96-well plates at 37℃with dioxygenAfter 24 hours of culture in a carbon dioxide incubator, the original culture medium is discarded, CCK8 (5 mu L of CCK-8 stock solution and 95 mu L of DMEM culture medium) solution is added, the culture is continued for 2 hours at 37 ℃ in a carbon dioxide incubator, an enzyme-labeled instrument is used for testing the OD 450nm absorbance value and calculating the cell survival rate, and the cell survival rate is calculated as follows:wherein OD _Background To be a blank well containing no cells and only CCK-8, Is a control group containing cells and CCK-8 without drug treatment.

The test results are shown in fig. 6 and 7: the cytotoxicity of the lipid nanoparticles prepared by the invention to tumor cells is basically located below the curve y=x in the figure, which shows that the cytotoxicity of the lipid nanoparticles at ph=7.4 is lower than that at ph=6.8, and shows that the lipid nanoparticles of the invention have pH-responsive cytotoxicity.

EXAMPLE 5 column chromatography purification of preferred tertiary amine lipids

Lipid nanoparticles having cytotoxicity at pH 6.8 50% higher than that at pH 7.4 were selected according to the cytotoxicity results of example 4, and their corresponding tertiary amine lipid compounds were purified and characterized by column chromatography.

Silica gel is used as column packing, and methylene dichloride is used as the raw material: the gradient elution is carried out by taking methanol=2.5 to 10 percent as eluent, the optimized tertiary amine lipoid compound is purified, and the nuclear magnetic characteristics are shown in fig. 8 to 23, and analysis shows that purer target products C8A16, C8A19, C8A45, C8A49, C12A27, C12A33, C14A33, C16A34, C181A17, C181A23, C181A24, C181A28, C182A17, C182A23, C182A24 and C182A28 are obtained.

The purified tertiary amine lipid compound was prepared into lipid nanoparticles as described in example 2. The particle size and distribution of the lipid nanoparticles in the aqueous solution were examined using a dynamic light scattering instrument, and the result showed that the average particle size of the assembled lipid nanoparticles was about 200nm (fig. 26).

The lipid nanoparticles prepared in this example were tested for concentration-dependent cytotoxicity at pH characteristic of normal and tumor tissues as described in example 4: lipid nanoparticles were incubated with MC38 cells at different concentrations for 24h at ph=6.8 and ph=7.4, and their cytotoxicity was detected using MTT method. As shown in fig. 24 and 25, consistent with the results of example 4, lipid nanoparticles prepared with purified tertiary amine lipid compounds have pH-responsive tumor cell killing ability, i.e., cytotoxicity is low at ph=7.4, and cytotoxicity is high at ph=6.8, indicating that such lipid nanoparticles do have pH-activated cytotoxicity, while being dose-dependent.

EXAMPLE 6 protonation of tertiary amine lipid nanoparticles at different pH

The preferred tertiary amine lipid compound of example 4 was prepared into lipid nanoparticles according to the method of example 2, and then the lipid nanoparticles were mixed with buffers of different pH and TNS solution, 100 μl of the solution was added to a black flat bottom 96-well plate, and then the fluorescence intensity was measured using a full-wavelength microplate spectrophotometer, and the degree of protonation and pKa thereof were calculated by simulating the change curve thereof, as follows:

Buffer preparation A buffer solution containing 20mM HEPES, 20mM MES, 20mM ammonium acetate and 260mM NaCl was prepared with ultrapure water, four 25mL portions of the 2 Xbuffer solution were then used, 2mL of 1M HCl solution (Nos. 1 and 2) and 2mL of 1M NaOH solution (Nos. 3 and 4) were added thereto, respectively, and the mixture was made up to 50mL with ultrapure water. Finally, respectively using solutions 1 and 4 and solutions 2 and 3 to prepare buffer solutions with pH values of 6.5-10 and 2.5-6.5 by mutual titration;

the measuring method comprises the following steps: 980. Mu.L of buffers with different pH values were added to a 1.5mL EP tube, 10. Mu.L of 2.5mg/mL lipid nanoparticles and 100. Mu.M TNS aqueous solution were sequentially added thereto, and 100. Mu.L of the solution was added to a black flat bottom 96-well plate after mixing uniformly, and fluorescence intensities at excitation wavelength and emission wavelength of 321nm and 445nm, respectively, were measured using a full-wavelength microplate spectrophotometer.

The calculation method comprises the following steps: the measured data are imported into Origin, boltzmann function in S-type function simulation is used for analysis, the maximum point of fluorescence intensity in a curve is defined as 100% of protonation rate, the minimum point of fluorescence intensity in the curve is defined as 0% of protonation rate, and then the curve is normalized to obtain a simulation graph, and the pH value corresponding to 50% of protonation rate in the graph is the pKa of the lipid nanoparticle.

The test results are shown in fig. 27: the results show that the carbon chain length and the type of tertiary amine significantly influence the protonation rate and pKa of the lipid at different pH values; meanwhile, the other lipid nanoparticles, except for C8a45, had a pka of between 6.0 and 7.0, indicating that these lipid nanoparticles can achieve protonation in this pH range, thereby activating cell membrane disruption-mediated antitumor activity.

EXAMPLE 7 maximum tolerated dose of lipid nanoparticles in ICR mice

This example studies the toxicity of lipid nanoparticles of the present invention by testing the Maximum Tolerated Dose (MTD) in mice, the preferred lipid nanoparticles of example 4 were injected into ICR mice via the tail vein, each starting from 100mg/kg body weight until death of the experimental mice occurred, the maximum dose of mice that did not die after the continuous administration of 5 mice was recorded as the maximum tolerated dose of mice, and plotted as shown in fig. 28: the MTDs of C8A45, C8A49, C12A33 and C14A33 are all greater than 200mg/kg, while the MTDs of C16A34, C181A17, C181A23, C181A24, C181A28, C182A17, C182A23, C182A24 are less than 100mg/kg, and further the MTDs of C12A27 and C182A28 are 120 and 140mg/kg, respectively.

Example 8 Effect of lipid nanoparticle Components on cytotoxicity thereof at pH characteristic of Normal and tumor tissues

(1) Effect of polyethylene glycol lipid species on cytotoxicity of lipid nanoparticles

Taking C12A27 as an example, 3 clinically approved polyethylene glycol lipids were selected to study the effect of PEG lipid species and their ratio to C12A27 on cytotoxicity of lipid nanoparticles at different pH. Lipid nanoparticles were prepared with DSPE-PEG, 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG), 2- [ (polyethylene glycol) -2000] -N, N-tetracosacetamide (ALC-0159), respectively, in the amounts of the components and experimental procedures described in example 2, and the cytotoxicity of the lipid nanoparticles at pH characteristic of normal and tumor tissues was studied as described in example 5.

The results are shown as a in fig. 28: three PEG lipids had no effect on cytotoxicity of the resulting lipid nanoparticle at pH 6.8, but on cytotoxicity thereof at pH 7.4; specifically, the lipid nanoparticle containing DSPE-PEG has minimal cytotoxicity at pH 7.4, and DMG-PEG has maximal toxicity to ALC-0159.

(2) Effect of DSPE-PEG to C12a27 ratio on cytotoxicity of the resulting lipid nanoparticles

DSPE-PEG and C12A27 are mixed according to the mass ratio of 0.2: 1. 0.5: 1. 1: 1. 2: 1. 4: 1. 8:1 and lipid nanoparticles were prepared according to the experimental procedure in example 2, and then the cytotoxicity of the lipid nanoparticles was examined with reference to the method described in example 4.

The results are shown in b in fig. 29: the mass ratio of DSPE-PEG to C12A27 is less than or equal to 4:1, no significant effect on cytotoxicity of the lipid nanoparticle at pH 6.8; the mass ratio of DSPE-PEG to C12A27 is 8:1, the cytotoxicity of the lipid nanoparticle at pH 6.8 is significantly reduced; the mass ratio of DSPE-PEG to C12A27 is 0.2:1, the cytotoxicity of the lipid nanoparticle at pH 7.4 is increased, and the mass ratio of DSPE-PEG to C12A27 is more than or equal to 0.5:1, there is no significant effect on the cytotoxicity of the lipid nanoparticle at pH 7.4.

(3) Effects of other helper lipid species on cytotoxicity of lipid nanoparticles

Lipid nanoparticles were prepared by adding the above auxiliary lipids to tertiary amine lipid compound and DSPE-PEG, respectively, using cholesterol (Chol), di-oleoyl lecithin (DOPC), DSPE, and 1, 2-palmitoyl phosphatidylglycerol (DPPG) as other auxiliary lipids, according to the procedure described in example 2, as follows: dissolving tertiary amine lipoid compound C12A27 in BObtaining lipid solution with the concentration of 50mg/mL in alcohol; taking DSPE-PEG ₂₀₀₀ Dissolving in ethanol to obtain DSPE-PEG with concentration of 50mg/mL ₂₀₀₀ A solution; dissolving other auxiliary lipid in ethanol to obtain auxiliary lipid solution with concentration of 50 mg/mL; taking 50. Mu.L of lipid solution and 50. Mu.L of DSPE-PEG ₂₀₀₀ Mixing the solution and 25 mu L (or 50 mu L) of auxiliary lipid solution in a 1.5mL centrifuge tube to obtain a mixed solution; adding 20 mu L of the mixed solution into another 1.5mL centrifuge tube, adding 180 mu L of deionized water by using a liquid-transferring gun, rapidly adding the mixed solution into the mixed solution, and repeatedly blowing by using the liquid-transferring gun to obtain lipid nano particles; transferring into a dialysis bag with molecular weight cut-off of 14000Da, and dialyzing in pure water to remove ethanol.

Cytotoxicity of the resulting lipid nanoparticle was then tested as described in example 4. The results are shown in fig. 29 as c and d: the incorporation of different helper lipids had no significant effect on the cytotoxicity of the lipid nanoparticles at pH 6.8, but the addition of Chol, DOPC, DSPE, which in turn increased the cytotoxicity of the lipid nanoparticles at pH 7.4, had no significant effect on the cytotoxicity of the lipid nanoparticles at pH 7.4.

Example 9 mechanism of lipid nanoparticles to kill tumor cells at pH characteristic of tumor tissue

The C12a27 lipid nanoparticle was incubated with MC38 tumor cells at ph=6.8, at 37 ℃ or 4 ℃ and the activity of the cells at 4h was examined with CCK8 and it was found that the low temperature did not inhibit the killing of the tumor cells by the C12a27 lipid nanoparticle (panel a in fig. 30), indicating that the killing process was energy independent. It was also found that C12a27 could cause tumor cells to release ATP in the cytoplasm rapidly (panel b in fig. 30), suggesting that C12a27 could kill tumor cells by disrupting tumor cell membranes.

Further combining the C12A27 lipid nanoparticle with MC38 expressing GFP and cytoplasmic mCherry _GFP/mCherry Co-incubation in medium at pH 6.8, observed under a laser confocal microscope, the red fluorescence (mCherry) of the cells gradually decreased over time, was almost completely lost at 3h, and was bright-fieldThe phenomenon that the cell membrane of a large number of cells swells and bubbles are spitted can be observed (c diagram in fig. 30), which shows that the lipid nanoparticle provided by the invention can damage the cell membrane of tumor cells, so that the cell membrane is damaged, and the cell content is released, thereby killing the tumor cells. Using C8A16 lipid nanoparticles, C8A19 lipid nanoparticles and MC38 _GFP/mCherry Similar phenomena were also found when cells were co-incubated in medium at pH 6.8 and observed with a high content imaging system (fig. 31). C8a16 lipid nanoparticles were incubated with Propidium Iodide (PI), annexin-V-FITC at pH 6.8 with MC38 cells and observed using a high content imaging system, which resulted in rapid cell death after 2 hours incubation of C8a16 lipid nanoparticles with MC38, with double positive PI and Annexin-V-FITC staining (fig. 32). Furthermore, the lipid nanoparticle pair Panc02 of the present invention _GFP/mCherry Cells also have a similar killing mechanism that results in rapid release of cytoplasmic contents (fig. 33).

Example 10 in vivo tumor suppression experiments with lipid nanoparticles

The tumor inhibition effect of the lipid nanoparticles was verified by in vivo experiments of a C57 mouse pancreatic cancer tumor subcutaneous model (Panc-02 cells), a C57 mouse colorectal cancer tumor subcutaneous model (MC 38 cells), a C57 mouse breast cancer model (E0771 cells). The operation is as follows:

female mice (C57 BL/6,6-8 weeks) were subcutaneously injected 1X 10 in the back ⁷ individual/mL Panc02 cell suspension (100 μl) to establish a pancreatic cancer subcutaneous tumor model; female mice (C57 BL/6,6-8 weeks) were subcutaneously injected 1X 10 in the back ⁷ A single/mL MC38 cell suspension (100. Mu.L) was used to model colorectal carcinoma subcutaneous tumors by injecting 1X 10 in a second breast pad on the right side of female mice (C57 BL/6,6-8 weeks) ⁷ individual/mL E0771 cell suspension (100 μl) to establish breast cancer in situ tumor models. When the tumor grows to 50-100 mm ³ (tumor volume = length x width/2), by tail vein administration, the doses given were as follows, panc02 model: C12A27 (20 mg/kg), C14A27 (50 mg/kg), C16A27 (67 mg/kg); MC38 model: C8A16 (100 mg/kg), C8A19 (100 mg/kg), C8A45 (100 mg/kg), C8A49 (100 mg/kg), C12A27 (30 mg/kg); E0E 0 771 model: C8A45 (80 mg/kg), C8A49 (80 mg/kg), C12A27 (30 mg/kg), C12A33 (80 mg/kg). In addition, a negative control group was set, and only an equal volume of PBS solution was injected. Tumor size was measured using vernier calipers and mouse body weight was recorded.

The in vivo tumor inhibiting effect of the Panc02 model is shown in fig. 34, and it can be seen from the tumor growth curve that the tumor volume of the three administration groups is inhibited to some extent compared with that of the PBS group, wherein the increase of the tumor volume of the C12a27 lipid nanoparticle administration group is significantly slowed down. At the same time, after the mice underwent the whole treatment period, the experimental group had no difference in weight from the PBS control group. In the MC38 tumor model (FIG. 35), the C8A16 lipid nanoparticle, the C8A19 lipid nanoparticle, the C8A45 lipid nanoparticle, the C8A49 lipid nanoparticle and the C12A27 lipid nanoparticle all have a certain inhibition effect on tumor growth, wherein the treatment effect of the C8A49 lipid nanoparticle is the best, and the treatment scheme has no significant effect on the weight of mice. The lipid nanoparticles also have better therapeutic effect on mouse breast cancer E0771, wherein the C8a49 lipid nanoparticles, C12a27 lipid nanoparticles can cause tumor regression in all mice, and the C8a45 lipid nanoparticles, C12a33 lipid nanoparticles can cause tumor regression in some mice (fig. 36).

Example 11 in vivo tumor inhibition experiments with lipid nanoparticles in combination with immune checkpoint inhibitors

In vivo experiments with a C57 mouse colorectal cancer tumor subcutaneous model (MC 38 cells) demonstrated the tumor inhibitory effect of lipid nanoparticles in combination with immune checkpoint antibodies, antibodies against apoptosis receptor 1 (αpd-1). The operation is as follows:

female mice (C57 BL/6,6-8 weeks) were subcutaneously injected 1X 10 in the back ⁷ individual/mL MC38 cell suspension (100 μl) to establish a subcutaneous tumor model of colorectal cancer. When the tumor grows to 50-100 mm ³ (tumor volume = length x width/2), by tail vein administration, the doses were as follows: C12A27 (20 mg/kg), C14A27 (50 mg/kg), alpha PD-1 (0.75 mg/kg). In addition, a negative control group was set, and only an equal volume of PBS solution was injected. Tumor size was measured using vernier calipers and mouse body weight was recorded.

As can be seen from the mouse tumor growth curve (fig. 37), both the C12a27 and C14a27 lipid nanoparticles and the αpd-1 combination therapy can enhance the therapeutic effect of αpd-1, wherein the tumor inhibition effect of the C14a27 combination αpd-1 is the best, the tumors of 4 mice are completely disappeared, the tumors of 2 mice in the C12a27 combination group are completely disappeared, and the therapeutic regimen has no significant effect on the body weight of the mice. The results show that the lipid nanoparticle can enhance the curative effect of the immune checkpoint antibody for clinical use, thereby improving the response rate of tumor patients to the immune checkpoint antibody and improving the anti-tumor effect.

Example 12 in vivo tumor inhibition experiments with lipid nanoparticles in combination with chemotherapeutic drugs

In vivo experiments with Balb/c mouse breast cancer in situ model (EMT-6 cells) verify the tumor inhibition effect of lipid nanoparticles combined with chemotherapeutic drug liposome doxorubicin Doxil. The operation is as follows:

a second breast pad injection 3X 10 on the right side of female mice (Balb/c, 6-8 weeks) ⁶ Each/mL of EMT-6 cell suspension (100. Mu.L) was used to establish a subcutaneous tumor model of breast cancer. When the tumor grows to 50-100 mm ³ (tumor volume = length x width/2), by tail vein administration, the doses were as follows: C12A27 (20 mg/kg), C14A27 (50 mg/kg), doxil (2.5 mg/kg). In addition, a negative control group was set, and only an equal volume of PBS solution was injected. Tumor size was measured using vernier calipers and mouse body weight was recorded.

As can be seen from the growth curve of the mice tumors (fig. 38), the C12a27 lipid material in combination with Doxil significantly enhanced the anti-tumor effect, and the tumors of all mice in the combination group completely disappeared. The above results indicate that the lipid nanoparticle of the present invention can enhance the anti-tumor efficacy of chemotherapeutic drugs.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the following embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

The application of tertiary amine lipoid compound with a structure shown in formula (I) or stereoisomer or pharmaceutically acceptable salt thereof in preparing pH responsive membrane rupture material:

wherein each R ₁ Each independently selected from: alkyl, unsaturated alkyl, cycloalkyl, alkenyl, alkynyl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

each R is ₂ Each independently selected from:or none;

R ₃ and R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic group, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 3-20 membered heterocyclyl; and when R is ₃ When directly connected with N, R ₃ Is not H; when R is ₄ When directly connected with N, R ₄ Is not H;

each R is ₅ Each independently selected from: alkylene, cycloalkyl, aryl;

each R is ₆ Each independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated alkyl, alkenyl, aryl-substituted alkyl,

Each R is ₇ Each independently selected from: alkyl, hydroxy-substituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl-substituted alkyl,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: alkyl, unsaturated alkyl, cycloalkyl, heterocyclyl, alkyl-substituted heterocyclyl, alkenyl, alkynyl, aryl-substituted alkyl, cycloalkyl-substituted alkyl,

R ₉ Selected from: hydroxy, aryl, cycloalkyl, heterocyclyl, R ₈ Substituted heterocyclyl, heteroaryl;

each X is independently O or NH;

each q and each q' are each independently 0, 1 or 2;

a is 0 or 1;

b is 1, 2, 3, 4 or 5;

c is 1, 2, 3, or 4;

d is an integer between 2 and 12.
The use according to claim 1, wherein the tertiary amine lipid compound has a structure represented by the following formula (II):
The use according to claim 2, wherein the tertiary amine lipid compound has a structure represented by the following formula (III):

R ₄ selected from: r is R ₉ Substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic group, R ₈ Substituted heterocyclyl, alkenyl, alkynyl, aryl,
Use according to claim 3, characterized in that R ₄ Selected from: c (C) ₁ -C ₂₂ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₂₂ Aryl group,
The use according to claim 4, wherein R ₄ Selected from: c (C) ₁ -C ₁₀ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracenyl,
The use according to any one of claims 1 to 5, wherein each R ₅ Each independently selected from: c (C) ₁ -C ₂₂ Alkylene, C ₃ -C ₈ Cycloalkyl, C ₆ -C ₁₄ Aryl groups.
The use according to claim 6, wherein each R ₅ Each independently selected from: c (C) ₁ -C ₁₀ Alkylene, C ₃ -C ₈ Cycloalkyl, phenyl, naphthyl, anthracenyl.
The use according to claim 7, wherein each R ₅ Each independently selected from: c (C) ₁ -C ₆ Alkylene, C ₃ -C ₈ Cycloalkyl groups.
The use according to claim 8, wherein each R ₅ Each independently selected from: methylene, ethylene, propylene, butylene,
The use according to any one of claims 1 to 5, wherein each R ₆ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₂ -C ₂₂ Alkenyl, C ₆ -C ₁₄ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl group,

Each R is ₇ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₁₄ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl group,Or R is ₆ 、R ₇ And connected theretoThe nitrogen atoms together form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, C ₁ -C ₈ Alkyl-substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₁₄ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl, C ₃ -C ₂₂ Cycloalkyl-substituted C ₁ -C ₈ Alkyl group,
The use according to claim 10, wherein each R ₆ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₂ -C ₁₀ Alkenyl, phenyl, naphthyl, anthryl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl group,

Each R is ₇ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₈ Alkyl group,C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl group,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, C ₁ -C ₆ Alkyl-substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₈ Alkyl, naphthyl substituted C ₁ -C ₈ Alkyl-and anthracenyl-substituted C ₁ -C ₈ Alkyl, C ₃ -C ₈ Cycloalkyl-substituted C ₁ -C ₈ Alkyl group,
The use according to claim 11, wherein each R ₆ Each independently selected from: c (C) ₁ -C ₆ An alkyl group; hydroxy-substituted C ₁ -C ₆ Alkyl group,

Each R is ₇ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl group,Or R is ₆ 、R ₇ And together with the nitrogen atom to which it is attached form R ₈ A substituted or unsubstituted 5-8 membered heterocyclic group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, 5-8 membered heterocyclyl, C ₁ -C ₆ Alkyl substituted 5-8 heterocyclyl, phenyl substituted C ₁ -C ₃ Alkyl group,
The use according to any one of claims 1 to 5, wherein R ₉ Selected from: hydroxy, C ₆ -C ₁₄ Aryl, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, 5-10 membered heteroaryl.
The use according to claim 13, wherein R ₉ Selected from: hydroxy, phenyl, naphthyl, anthryl, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, 5-8 membered heteroarylA base.
The use according to claim 5, wherein R ₄ Selected from: c (C) ₁ -C ₈ Alkyl, R ₉ Substituted C ₁ -C ₈ Alkyl, C ₃ -C ₈ Cycloalkyl, phenyl substituted C ₁ -C ₃ Alkyl, naphthyl substituted C ₁ -C ₃ Alkyl-and anthracenyl-substituted C ₁ -C ₃ Alkyl, 3-8 membered heterocyclyl, R ₈ Substituted 5-8 membered heterocyclyl,A substituted cyclohexyl group;

R ₅ selected from: c (C) ₁ -C ₄ An alkylene group;

each R is ₆ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, -CH ₂ -R ₂ -R ₁ ；

Each R is ₇ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, -CH ₂ -R ₂ -R ₁ ；

Each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, phenyl substituted C ₁ -C ₃ Alkyl group,

R ₉ Selected from: hydroxy, C ₃ -C ₈ Cycloalkyl, 3-8 memberedHeterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, 5-8 membered heteroaryl.
The use according to claim 15, wherein R ₄ Selected from: c (C) ₄ -C ₆ Alkyl, dimethylamino substituted butyl, diethylamino substituted butyl, piperidinyl substituted n-propyl, R ₉ Substituted C ₁ -C ₃ Alkyl, C ₄ -C ₈ Cycloalkyl, benzyl, 5-8 membered heterocyclyl, R ₈ Substituted 5-6 membered heterocyclyl,A substituted cyclohexyl group;

each R is ₅ Each independently selected from: c (C) ₁ -C ₃ An alkylene group;

R ₆ selected from: c (C) ₃ -C ₄ An alkyl group;

each R is ₇ Each independently selected from: c (C) ₃ -C ₄ An alkyl group;

each R is ₈ Each independently selected from: c (C) ₁ -C ₆ Alkyl, benzyl,

R ₉ selected from:
the use according to claim 1, wherein the tertiary amine lipid compound has a structure represented by the following formula (IV):

wherein n is 1, 2, 3, 4 or 5;

R ₃ and R is ₄ Each independently selected from: c (C) ₁ -C ₁₀ Alkyl, hydroxy substituted C ₁ -C ₁₀ Alkyl, C ₁ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, or R ₃ 、R ₄ And the nitrogen atom to which it is attached form a 3-8 membered heterocyclic group.
The use according to claim 17, wherein R ₃ And R is ₄ Each independently selected from: c (C) ₁ -C ₆ Alkyl, hydroxy substituted C ₁ -C ₆ Alkyl, C ₁ -C ₆ Unsaturated chain hydrocarbon group, or R ₃ 、R ₄ And the nitrogen atom to which it is attached form a 5-8 membered heterocyclic group.
The use according to claim 1, wherein the tertiary amine lipid compound has a structure represented by the following formula (V):

n ₁ 、n ₂ 、n ₃ 、n ₄ each independently selected from: 0. 1, 2, 3 or 4.
The use according to any one of claims 1-5, 17-19, wherein each R ₁ Each independently selected from: c (C) ₁ -C ₂₂ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₂₂ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl, C ₃ -C ₂₂ Cycloalkyl-substituted C ₁ -C ₈ An alkyl group.
The use according to claim 20, wherein each R ₁ Each independently selected from: c (C) ₈ -C ₂₀ Alkyl, C ₈ -C ₂₀ Unsaturated chain hydrocarbon group, C ₅ -C ₂₀ Cycloalkyl, C ₈ -C ₂₀ Alkenyl, C ₈ -C ₂₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₆ Alkyl, naphthyl substituted C ₁ -C ₆ Alkyl-and anthracenyl-substituted C ₁ -C ₆ Alkyl, C ₅ -C ₂₀ Cycloalkyl-substituted C ₁ -C ₆ An alkyl group.
The use according to claim 21, wherein each R ₁ Each 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, isodecyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isostearyl, docosyl, cyclopropyl, cyclobutyl Cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl methyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, vinyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecyl, docosyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, decynyl, dodecenyl, tetradecynyl, hexadecynyl, octadecyl, docosyl, linolenyl, phenyl, naphthyl, anthryl, benzyl, phenethyl, phenylpropyl, phenylbutyl, phenylhexyl, phenyloctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracylmethyl, anthracylethyl, anthracropyl, anthracylhexyl, anthraceneoctyl, cis-9, 12-octadecadienyl, 9-octadecenyl.
The use according to claim 22, wherein each R ₁ Each independently selected from: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9, 12-octadecadienyl, 2-phenylethyl, or phenylbutyl.
The use according to any one of claims 1-5, 17-19, wherein each R ₂ Each independently selected from: or none, wherein c is 1 orPerson 2; d is an integer between 2 and 6.
Use according to any one of claims 1-2, characterized in that R ₃ And R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted C ₁ -C ₂₂ Alkyl, C ₂ -C ₂₂ Unsaturated chain hydrocarbon group, C ₃ -C ₂₂ Cycloalkyl, 3-22 membered heterocyclyl, R ₈ Substituted 3-22 membered heterocyclyl, C ₂ -C ₂₂ Alkenyl, C ₂ -C ₂₂ Alkynyl, C ₆ -C ₂₂ Aryl, C ₆ -C ₁₄ Aryl substituted C ₁ -C ₈ Alkyl, C ₃ -C ₂₂ Cycloalkyl-substituted C ₁ -C ₈ Alkyl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ Substituted or unsubstituted 3-10 membered heterocyclic ring.
The use according to claim 25, wherein R ₃ And R is ₄ Each independently selected from: H. r is R ₉ Substituted or unsubstituted C ₁ -C ₁₀ Alkyl, C ₂ -C ₁₀ Unsaturated chain hydrocarbon group, C ₃ -C ₈ Cycloalkyl, 3-8 membered heterocyclyl, R ₈ Substituted 3-8 membered heterocyclyl, C ₂ -C ₁₀ Alkenyl, C ₂ -C ₁₀ Alkynyl, phenyl, naphthyl, anthracyl, phenyl-substituted C ₁ -C ₆ Alkyl, naphthyl substituted C ₁ -C ₆ Alkyl-and anthracenyl-substituted C ₁ -C ₆ Alkyl, C ₃ -C ₈ Cycloalkyl-substituted C ₁ -C ₆ Alkyl, -R ₂ -R ₁ 、 Or R is ₃ 、R ₄ And together with the nitrogen atom to which it is attached form R ₈ Substituted or unsubstituted 5-10 membered heterocyclyl.
The use according to claim 26, wherein R ₃ 、R ₄ And the nitrogen atom to which it is attached form the following group:
use according to claim 1, characterized in that the tertiary amine lipid compound is selected from the following compounds:

wherein each R ₁ Each independently is: n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9, 12-octadecadienyl, 2-phenylethyl, or phenylbutyl.
Use according to claim 28, wherein the tertiary amine lipid compound is selected from the group consisting of:
a tertiary amine lipid compound as claimed in any one of claims 1 to 29 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof.
A lipid nanoparticle formed from self-assembly of a tertiary amine lipid compound as defined in any one of claims 1 to 29, or a stereoisomer or a pharmaceutically acceptable salt thereof, and an amphiphilic molecule in an aqueous medium, the amphiphilic molecule being: an amphiphilic polyethylene glycol lipid molecule, or a combination of an amphiphilic polyethylene glycol lipid molecule and a phospholipid.
The lipid nanoparticle of claim 31, wherein the phospholipid is selected from cholesterol, soybean phospholipid, hydrogenated soybean phospholipid, soybean lecithin, egg yolk lecithin, high purity egg yolk lecithin, dilauroyl lecithin, dithiin phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1, 2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidylethanolamine, palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, di-palmitoyl phosphatidylethanolamine, 2-oleoyl phosphatidylethanolamine, (2-methyl-3- [ beta-propyl-3-N-trichloro-3 '-; N' -dimethylaminoethyl-carbamoyl ] cholesterol hydrochloride, 1, 2-dioleyl-3-dimethylamino-propane, 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester, 1, 2-dioctadecyloxy-3-methylammonium propane, (R) -2, 3-bis (oleoyloxy) propyl hydrogen phosphate 2- [ (2-aminoethyl) amino ] ethyl ester; and/or the number of the groups of groups,

The amphiphilic polyethylene glycol lipid molecule is selected from polyethylene glycol-dipalmitoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, 1, 2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, 2- (polyethylene glycol) -N, N-tetracosylacetamide, polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone and polyethylene glycol-polylactic acid-glycolate; preferably, the molecular weight of polyethylene glycol in the amphiphilic polyethylene glycol lipid molecule is 500 g/mol-20000 g/mol.
The lipid nanoparticle of claim 31 or 32, wherein the lipid nanoparticle is formed by self-assembly of the tertiary amine lipid compound or a stereoisomer or a pharmaceutically acceptable salt thereof, an amphiphilic polyethylene glycol lipid molecule and 1, 2-palmitoyl phosphatidylglycerol in an aqueous medium; or is formed by self-assembling the tertiary amine lipoid compound or a stereoisomer or a pharmaceutically acceptable salt thereof and amphiphilic polyethylene glycol lipoid molecules in an aqueous medium; preferably, the amphiphilic polyethylene glycol lipid molecule is distearoyl phosphatidylethanolamine-polyethylene glycol 2000.
The lipid nanoparticle of claim 33, wherein the mass ratio of the tertiary amine lipid compound or stereoisomer thereof or pharmaceutically acceptable salt thereof to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8, preferably 1:0.5 to 4, more preferably 1:0.5-2, more preferably 1:0.8-1.2;

The mass ratio of the tertiary amine lipoid compound or the stereoisomer or the pharmaceutically acceptable salt thereof to the 1, 2-palmitoyl phosphatidylglycerol is 1:0.5 to 1.5, preferably 1:0.8-1.2.
A method of preparing a lipid nanoparticle according to any one of claims 31 to 34, comprising the steps of:

dissolving the tertiary amine lipoid compound or stereoisomer or pharmaceutically acceptable salt thereof in an organic solvent to obtain tertiary amine lipoid compound solution;

dissolving the amphiphilic molecules in an organic solvent to obtain amphiphilic molecule solution;

mixing the tertiary amine lipoid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

adding an aqueous medium into the mixed solution, and repeatedly blowing with a pipetting gun to obtain the lipid nanoparticle; or slowly dripping the mixed solution into an aqueous medium under the stirring condition, and continuously stirring to obtain the lipid nanoparticle;

preferably, the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N-dimethylformamide or tetrahydrofuran;

preferably, the tertiary amine lipid compound has a concentration of 10mg/mL to 100mg/mL; the concentration of the amphiphilic molecule solution is 20mg/mL-200mg/mL;

Preferably, the volume ratio of the mixed solution to the aqueous medium is 1:1-20.
Use of a tertiary amine lipid compound according to any one of claims 1 to 29, or a stereoisomer thereof or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the prophylaxis and/or treatment of a tumor.
Use of a lipid nanoparticle according to any one of claims 31-34 in the manufacture of a medicament for the prevention and/or treatment of a tumour.
Use of a tertiary amine lipid compound according to any one of claims 1 to 29 or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in combination with an antitumor drug for the preparation of a medicament for the prophylaxis and/or treatment of tumors.
Use of a lipid nanoparticle according to any one of claims 31-34 in combination with an anti-tumor drug for the preparation of a medicament for the prevention and/or treatment of tumors.
The use according to claim 38 or 39, wherein the anti-tumour agent is an immune checkpoint inhibitor and/or a chemotherapeutic agent.
The use of claim 40, wherein the immune checkpoint inhibitor is a PD-1 inhibitor; and/or, the chemotherapeutic drug is doxorubicin.
The use according to any one of claims 36 to 39, wherein the tumour is pancreatic cancer, melanoma, colorectal cancer, lung cancer, squamous carcinoma of the tongue, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer, nasopharyngeal cancer, brain cancer.
A medicament for preventing and/or treating tumors, characterized in that it is prepared from an active ingredient and pharmaceutically acceptable auxiliary materials and/or carriers, wherein the active ingredient comprises the tertiary amine lipid compound or a stereoisomer thereof or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 29, and/or the lipid nanoparticle as claimed in any one of claims 31 to 34.
A combination for the prophylaxis and/or treatment of tumors, characterized in that the active ingredients thereof comprise:

component 1: the tertiary amine lipid compound of any one of claims 1-29, or a stereoisomer or a pharmaceutically acceptable salt thereof, and/or the lipid nanoparticle of any one of claims 31-34; the method comprises the steps of,

component 2: antitumor drugs other than component 1;

the component 1 and the component 2 are each independent administration units, or the component 1 and the component 2 together form a combined administration unit.
The combination according to claim 44, wherein component 2 is an immune checkpoint inhibitor and/or a chemotherapeutic agent.
The combination for preventing and/or treating tumors of claim 45, wherein said immune checkpoint inhibitor is a PD-1 inhibitor; and/or, the chemotherapeutic drug is doxorubicin.