Alkylamine phosphate derivative, and preparation method and application of lipidosome containing alkylamine phosphate derivative

Abstract

The invention discloses a preparation method and application of an alkylamine phosphoric acid derivative and lipidosome containing the alkylamine phosphoric acid derivative, and the alkylamine phosphoric acid derivative has a structure as shown in the following formula (I) or a pharmaceutically acceptable salt, a stereoisomer, a deuterated substance or a prodrug of the alkylamine phosphoric acid derivative. The alkylamine phosphate derivative liposome prepared from the alkylamine phosphate derivative disclosed by the invention has the characteristic of zeta potential transformation under the action of alkaline phosphatase. The liposome initially carries negative charges, and can overcome anion endogenous obstacles (such as a mucus gel layer and the like) on the way to reach a targeted position. When reaching a targeted position, the alkylamine phosphate derivative removes phosphate groups on the surface by using the overexpressed alkaline phosphatase of colorectal cancer tumor, so that the alkylamine phosphate derivative is converted into the cationic liposome, and the cellular uptake rate is improved through cationic charges.

Description

Technical Field

[0001] The present application relates to the fields of biomedicine and medicinal chemistry, and specifically to an alkylamine phosphate derivative, and a preparation method and application of liposomes containing the alkylamine phosphate derivative. Background Art

[0002] Colorectal cancer is one of the most common gastrointestinal malignancies and has become the fifth leading cause of cancer death worldwide and the fifth most common malignant tumor. Liposomes are widely used in the field of tumor treatment. As drug carriers, they have the advantages of reducing drug toxicity and improving drug stability. When liposomes are used to deliver anti-colorectal cancer drugs, they need to overcome different types of obstacles, such as the mucus gel layer and the epithelial cell layer. Since the mucus gel layer is negatively charged, neutral or anionic liposomes can smoothly penetrate the mucus gel layer, while cationic liposomes have a much lower permeability in the mucus gel layer than neutral or anionic liposomes due to charge interactions. However, compared with cationic liposomes, when neutral or anionic liposomes enter the intestinal epithelial cell layer, cellular uptake through endocytosis is not obvious. Therefore, the existing traditional liposomes are not satisfactory in terms of clinical efficacy, especially the improvement of survival rate, due to their poor effect of targeting and binding with colorectal cancer cells. Summary of the invention

[0003] Alkaline phosphatase is an enzyme widely distributed in human liver, bones, intestines, kidneys, placenta and other tissues, and is excreted from the liver to the bile. It is usually used as a marker for the differentiation of colorectal cancer cells and has been shown to be overexpressed at the tumor site. In response to the problem that traditional liposome drug targeted delivery is not effective, this application provides an alkylamine phosphate derivative, a preparation method and application of liposomes containing alkylamine phosphate derivatives according to the characteristics of the colorectal cancer tumor microenvironment. Liposomes containing alkylamine phosphate derivatives can penetrate the mucus gel layer smoothly due to their initial negative charge. When they reach the intestinal epithelial cell layer, under the action of alkaline phosphatase, the alkylamine phosphate derivatives can remove the phosphate groups on the surface and transform into cationic liposomes, which promote the endocytosis of colorectal cancer cells, thereby improving the cellular uptake rate and clinical efficacy of the drug.

[0004] In order to achieve the above technical objectives, this application mainly adopts the following technical solutions:

[0005] 1. An alkylamine phosphate derivative having a structure as shown in the following formula (I) or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof;

[0006]

[0007] m and n are integers greater than or equal to 1.

[0008] 2. The alkylamine phosphoric acid derivative according to item 1, wherein m and n are independently integers of 1-25, preferably integers of 8-20.

[0009] 3. A method for preparing an alkylamine phosphoric acid derivative, comprising the following steps:

[0010] The alkylamine and tert-butyl acrylate are subjected to Michael addition reaction to obtain intermediate 1;

[0011] Using trifluoroacetic acid to react with the intermediate 1 to remove its tert-butyl protecting group to obtain the intermediate 2;

[0012] The carboxyl group of intermediate 2 is reduced to hydroxyl group using lithium aluminum hydride to obtain intermediate 3;

[0013] Using diphenyl phosphite to carry out a phosphorylation reaction with the intermediate three, thereby obtaining an intermediate four;

[0014] The phosphorous acid group of the intermediate tetrakis oxidized to a phosphoric acid group using iodine to give the alkylamine phosphoric acid derivative.

[0015] 4. The preparation method according to item 3, wherein:

[0016] The alkylamine, tert-butyl acrylate and N,N-diisopropylethylamine are dissolved in toluene, and then condensed and refluxed for a certain period of time, and then vacuum dried under reduced pressure, and then dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain a crude product of intermediate 1;

[0017] The crude product of the intermediate one is separated and purified by chromatography to obtain the intermediate one.

[0018] 5. The preparation method according to item 4, wherein the molar ratio of the alkylamine, tert-butyl acrylate and N,N-diisopropylethylamine is (1-2):(2-4):(6-12).

[0019] 6. The preparation method according to item 4, wherein the condensation reflux temperature is 80-100°C and the time is 65-75h.

[0020] 7. The preparation method according to item 3, wherein the alkylamine is of the following formula (II):

[0021]

[0022] m and n are integers greater than or equal to 1.

[0023] 8. The preparation method according to item 7, wherein m and n are independently integers of 1-25, preferably integers of 8-20.

[0024] 9. The preparation method according to item 3, wherein:

[0025] The intermediate 1 is dissolved in dichloromethane and stirred evenly, and then trifluoroacetic acid is added thereto. After the reaction temperature rises to room temperature, the reaction is continued, and then the reduced pressure is reduced and vacuum-dried, and then it is dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain the intermediate 2.

[0026] 10. The preparation method according to item 9, wherein the molar ratio of the intermediate 1 to trifluoroacetic acid is (1-3): (55-165).

[0027] 11. A preparation method according to item 3, wherein the intermediate 2 is dissolved in tetrahydrofuran, and an excess of lithium aluminum hydride is slowly added in a nitrogen atmosphere, and the reaction is continued after the reaction temperature rises to room temperature; after the reaction is completed, the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, the reaction solution is dried under reduced pressure and vacuum, then dissolved in ethyl acetate, and the pH of the solution is adjusted to neutral, the organic phase is washed with water, and finally dried to obtain the intermediate 3.

[0028] 12. The preparation method according to item 11, wherein the temperature during the addition of lithium aluminum hydride is controlled at 0-4°C, and after the addition of lithium aluminum hydride is completed, the reaction temperature rises to room temperature and the reaction time is continued for 6-18 hours.

[0029] 13. The preparation method according to item 3, wherein the intermediate three is dissolved in anhydrous pyridine, diphenyl phosphite is added under an ice bath, after a certain reaction time, the ice bath is removed, the reaction is continued, after the reaction is completed, triethylamine and water are added, the reaction is continued, and then the reduced pressure is reduced and vacuumed to obtain a crude product of the intermediate four;

[0030] The crude product of the intermediate four is separated and purified by chromatography to obtain the intermediate four.

[0031] 14. The preparation method according to item 13, wherein the molar ratio of the intermediate tris(III) to diphenyl phosphite is (1-2):(3-6); and / or

[0032] The molar ratio of the intermediate tris(III), triethylamine and water is (1-2):(10-20):(7-15).

[0033] 15. The preparation method according to item 13, wherein the reaction time of the intermediate tris and diphenyl phosphite in an ice bath is 5 to 15 minutes, and after removing the ice bath, the reaction is continued for 0.5 to 2 hours, and then triethylamine and water are added and the reaction is continued for 0.5 to 2 hours.

[0034] 16. The preparation method according to item 3, wherein the intermediate tetra is dissolved in anhydrous pyridine, triethylamine and iodine are added thereto in an ice bath to form a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued, and then water is added. After a certain reaction time, a saturated sodium thiosulfate solution is added until the reaction solution turns yellow and then the reaction is stopped, the reduced pressure is vacuum-dried, and then dissolved with dichloromethane, the organic phase is washed with water, and allowed to stand and dry to obtain the target crude product;

[0035] The target crude product is separated and purified by chromatography to obtain the target product alkylamine phosphoric acid derivative.

[0036] 17. The preparation method according to item 16, wherein the molar ratio of the intermediate tetraethylamine, triethylamine and iodine is (0.5-1): (2.5-5): (1.5-3).

[0037] 18. The preparation method according to item 16, wherein the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10 to 20 minutes, and then 10 to 20 mmol of water is added and the reaction is carried out for 5 to 10 minutes.

[0038] 19. According to the preparation method described in any one of items 3 to 18, the alkylamine phosphoric acid derivative prepared is the alkylamine phosphoric acid derivative described in item 1.

[0039] 20. An alkylamine phosphate derivative liposome, comprising an alkylamine phosphate derivative prepared by the preparation method of any one of items 1 or 2, or items 3-19, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0040] 21. The alkylamine phosphate derivative liposome according to item 20, wherein the alkylamine phosphate derivative liposome further comprises phospholipid, and the mass ratio of the alkylamine phosphate derivative to the phospholipid is (5-15):1;

[0041] Preferably, the phospholipid is selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0042] 22. A method for preparing alkylamine phosphate derivative liposomes, comprising the following steps:

[0043] Dissolve phospholipids and alkylamine phosphate derivatives in an organic solvent, remove the organic solvent by rotary evaporation under reduced pressure in a water bath, and form a lipid film on the inner wall of the bottle;

[0044] The film is placed in a drying oven to completely remove the organic solvent, and then deionized water is added thereto, hydrated for a certain period of time under water bath conditions, and subjected to probe ultrasound to obtain the alkylamine phosphate derivative liposome.

[0045] 23. The preparation method according to item 22, wherein the temperature under the water bath conditions is 30 to 70°C, and the hydration time under the water bath conditions is 15 to 60 minutes.

[0046] 24. The preparation method according to item 22 or 23, wherein the prepared alkylamine phosphate derivative liposome is the alkylamine phosphate derivative liposome described in item 20 or 21.

[0047] 25. A pharmaceutical composition, comprising: a pharmaceutically active ingredient and a drug delivery carrier for delivering the pharmaceutically active ingredient, wherein the drug delivery carrier is the alkylamine phosphate derivative liposome described in item 20 or 21 or the alkylamine phosphate derivative liposome prepared in any one of items 22-24 or contains the alkylamine phosphate derivative described in item 1 or 2 or contains the alkylamine phosphate derivative described in any one of items 3-19;

[0048] Preferably, the active ingredient of the drug is selected from at least one of nucleic acids, small molecule drugs, macromolecular drugs, protein drugs, polypeptides, and antibodies, preferably nucleic acids, and the nucleic acids are selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamers.

[0049] 26. Use of the alkylamine phosphate derivative described in item 1 or 2, the alkylamine phosphate derivative described in any one of items 3 to 19, the alkylamine phosphate derivative liposome described in item 20 or 21, or the alkylamine phosphate derivative liposome described in any one of items 22 to 24 in the preparation of a drug for targeted treatment or prevention of colorectal cancer.

[0050] The alkylamine phosphate derivatives of the present application and the alkylamine phosphate derivative liposomes prepared by the alkylamine phosphate derivatives have the characteristics of zeta potential conversion under the action of alkaline phosphatase. The liposomes initially have a negative charge and can overcome the endogenous anion obstacles (such as mucus gel layer, etc.) on the way to the target location. When reaching the target location, the alkylamine phosphate derivatives are made to remove the phosphate groups on the surface by utilizing the alkaline phosphatase overexpressed by colorectal cancer tumors, thereby converting into cationic liposomes, and improving the cell uptake rate by the cationic charge. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0052] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate 1 provided in this application.

[0053] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the intermediate 2 provided in this application.

[0054] Figure 3This is the hydrogen nuclear magnetic resonance spectrum of intermediate 3 provided in this application.

[0055] Figure 4 The hydrogen nuclear magnetic resonance spectrum of intermediate 4 provided in this application.

[0056] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the didodecylamine phosphate derivative provided in this application.

[0057] Figure 6 This is the mass spectrum of the didodecylamine phosphate derivative provided in this application.

[0058] Figure 7 This is a graph showing the Zeta charge changes of liposomes containing didodecylamine phosphate derivatives provided in this application.

[0059] Figure 8 This is a graph of cell viability at different dilution ratios of liposomes containing didodecylamine phosphate derivatives provided in this application.

[0060] Fig. 9 The cumulative release curve of phosphate from liposomes containing didodecylamine phosphate derivatives provided in the present application.

[0061] Fig.10 The in vitro uptake of the liposomes containing didodecylamine phosphate derivatives provided in the present application. (A) is a laser confocal microscopy image of the experimental group (without phosphatase inhibitor); (B) is a laser confocal microscopy image of the control group (containing 1% (v / v) phosphatase inhibitor); (C) is the average fluorescence intensity of the experimental group and the control group. DETAILED DESCRIPTION

[0062] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for the sake of clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0063] the term

[0064] Unless otherwise described, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art. For the purpose of interpreting this specification, the following terminology description will be applied, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. If any description of a term set forth conflicts with any document incorporated by reference into this application, the term description set forth below shall prevail.

[0065] Unless the context requires otherwise, in this specification and claims, the word "comprise" and variations such as "include" and "containing" are to be interpreted in an open and inclusive sense, that is, meaning "including, but not limited to."

[0066] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, and is generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Although lipids are generally weakly water-soluble, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids", including fats and oils, as well as waxes; (2) "compound lipids", including phospholipids and glycolipids (e.g., DMPEPEG2000); and (3) "derivatized lipids", such as steroids. In addition, lipids also include lipid-like compounds. The term "lipid-like compound", also referred to as "lipid-like", refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0067] As used herein and unless otherwise indicated, the term: "cationic lipid" refers to a lipid capable of being positively charged. Exemplary cationic lipids include one or more positively charged amine groups. Preferred cationic lipids are ionizable so that they can exist in a positively charged form or a neutral form depending on the pH value. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. This charge state can affect plasma protein absorption, blood clearance and tissue distribution, and the ability to form an endosomal non-bilayer structure, which is crucial for the intracellular delivery of nucleic acids.

[0068] As used in the present application and unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms. In one embodiment, the alkyl group has, for example, two to fifty carbon atoms (C2-C50 alkyl), four to twenty carbon atoms (C4-C24 alkyl), six to sixteen carbon atoms (C6-C16 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and is connected to the rest of the molecule by a single bond. The example of alkyl includes, but is not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, etc. Unless otherwise indicated, the alkyl group is optionally substituted.

[0069] The term "prodrug" refers to a derivative of a compound or nucleic acid drug of the present application that can be provided directly or indirectly after being applied to a patient. The prodrug of the composition can be prepared by modifying the functional group present in the compound or nucleic acid (DNA, ASO, siRNA, mRNA, tRNA) in such a way that the modification can be cleaved in routine operation or in vivo to obtain the parent compound or nucleic acid. Particularly preferred prodrugs are compounds and nucleic acid drugs (e.g., more easily absorbed into the blood) that can improve the bioavailability of the composition of the present application when applied to a patient, or compounds and nucleic acid drugs that promote the delivery of the parent compound to the site of action (e.g., lymphatic system). Unless otherwise noted, all prodrug forms of the compounds of the present application are within the scope of the present application, and various prodrug forms are well known in the art.

[0070] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

[0071] The term "pharmaceutically acceptable salt" refers to acidic and / or basic salts formed by the compounds of the present application with inorganic and / or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound. It can also be obtained by mixing the above-mentioned compound with a certain amount of acid or base appropriately (e.g., equivalent). The salt described in the present application can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0072] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In one embodiment, the inorganic salt is an ammonium salt, a sodium salt, a potassium salt, a calcium salt, and a magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines, including naturally occurring substituted amines; cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like.

[0073] The compounds provided herein may contain one or more asymmetric centers, and therefore may produce enantiomers, diastereomers and other stereoisomeric forms, which may be defined as (R)- or (S)- or as (D)- or (L)- for amino acids according to absolute stereochemistry. Unless otherwise indicated, the compounds provided herein are intended to include all such possible isomers, as well as racemic and optically pure forms thereof. When the compounds described herein contain olefinic double bonds or other geometric asymmetric centers, unless otherwise indicated, the compounds are intended to include E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0074] As used herein and unless otherwise indicated, the term "isomer" refers to different compounds with the same molecular formula. "Stereoisomers" are isomers that differ only in the way their atoms are arranged in space. "Atropisomers" are stereoisomers obtained by hindered rotation around a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of any ratio of a pair of enantiomers may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are separated into E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0075] The term "nucleic acid" refers to a nucleotide polymer of any length, and includes, for example, DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Nucleic acids may be in single-strand or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimetics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence disclosed in the present application is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5′ to 3′ addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA strand that has the same sequence as the RNA transcript and is located at the 5′ end relative to the 5′ end of the RNA transcript is called the “upstream sequence”; the sequence region on the DNA strand that has the same sequence as the RNA transcript and is located at the 3′ end relative to the 3′ end of the RNA transcript is called the “downstream sequence”.

[0076] The term "coding nucleic acid" or its grammatical equivalents when used to refer to nucleic acid molecules include: (a) nucleic acid molecules that can be transcribed to produce mRNA and then translated into peptides and / or polypeptides when in a natural state or by methods well known to those skilled in the art; and (b) mRNA molecules themselves. The antisense strand is the complementary sequence of such a nucleic acid molecule, and the coding sequence can be inferred from it. The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, the UTR is called a 5'-UTR if it is located at the 5' end of the coding region, and the UTR is called a 3'-UTR if it is located at the 3' end of the coding region.

[0077] As used herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more open reading frames (ORFs), which can be translated by a cell or organism having the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs).

[0078] In certain embodiments, mRNA is a monocistronic mRNA comprising only one ORF. In certain embodiments, monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, mRNA is a polycistronic mRNA comprising two or more ORFs. In certain embodiments, polycistronic mRNA encodes two or more peptides or proteins that may be identical or different from each other. In certain embodiments, each peptide or protein encoded by polycistronic mRNA comprises at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by polycistronic mRNA each comprise at least one epitope of a different antigen. In any one of the embodiments described herein, the at least one epitope may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 epitopes of an antigen.

[0079] The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

[0080] As used herein, the term "peptide" refers to a polymer containing from two to fifty (2-50) amino acid residues linked via one or more covalent peptide bonds. The term is applicable to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs or non-natural amino acids).

[0081] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers having more than fifty (50) amino acid residues linked by covalent peptide bonds. That is, the description of polypeptides is equally applicable to the description of proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., antigens).

[0082] The term "antigen" refers to a substance that can be recognized by a subject's immune system (including an adaptive immune system) and can trigger an immune response (including an antigen-specific immune response) after the subject is contacted with the antigen. In certain embodiments, the antigen is a protein associated with a diseased cell, such as a cell infected with a pathogen or a neoplastic cell (e.g., a tumor-associated antigen (TAA)).

[0083] The present application provides an alkylamine phosphate derivative, wherein the alkylamine phosphate derivative has a structure as shown in the following formula (I) or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof;

[0084]

[0085] m and n are integers greater than or equal to 1.

[0086] Specifically, m and n are independently integers of 1-25, preferably integers of 8-20.

[0087] For example, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.

[0088] For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.

[0089] m and n may be the same or different.

[0090] In some embodiments, m is 12, n is 12, and the alkylamine phosphoric acid derivative is a didodecylamine phosphoric acid derivative, specifically having the following structure:

[0091]

[0092] In some embodiments, m is 10, n is 12, and the alkylamine phosphoric acid derivative has the following structure:

[0093]

[0094] In some embodiments, m is 14, n is 14, and the alkylamine phosphoric acid derivative has the following structure:

[0095]

[0096] The present application also includes all suitable isotopic variants of the alkylamine phosphate derivatives. An isotopic variant is defined as a compound in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be introduced into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen and oxygen, for example 2 H (deuterium), 3 H (tritium), 11 C. 13 C. 14 C. 15 N. 17 O and 18 O.

[0097] The present application provides a method for preparing an alkylamine phosphoric acid derivative, comprising the following steps:

[0098] Step 1: subjecting an alkylamine to a Michael addition reaction with tert-butyl acrylate to obtain an intermediate 1;

[0099] Step 2: using trifluoroacetic acid to react with the intermediate 1 to remove its tert-butyl protecting group to obtain the intermediate 2;

[0100] Step 3: using lithium aluminum hydride to reduce the carboxyl group of intermediate 2 to hydroxyl group, thereby obtaining intermediate 3;

[0101] Step 4: using diphenyl phosphite to carry out a phosphorylation reaction with the intermediate 3 to obtain the intermediate 4;

[0102] Step 5: using iodine to oxidize the phosphorous acid group of the intermediate 4 into a phosphoric acid group, thereby obtaining the alkylamine phosphoric acid derivative.

[0103] In step 1, the alkylamine, tert-butyl acrylate, and N,N-diisopropylethylamine are dissolved in toluene, and then condensed and refluxed for a certain period of time, and then vacuum-dried under reduced pressure, and then dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain a crude product of intermediate 1;

[0104] The crude product of the intermediate one is separated and purified by chromatography to obtain the intermediate one.

[0105] Furthermore, the molar ratio of the alkylamine, tert-butyl acrylate and N,N-diisopropylethylamine is (1-2): (2-4): (6-12).

[0106] In some embodiments, the molar ratio of the alkylamine to tert-butyl acrylate is 1:(2-4).

[0107] In some embodiments, the molar ratio of the alkylamine to tert-butyl acrylate is 1.2:(2-4).

[0108] In some embodiments, the molar ratio of the alkylamine to tert-butyl acrylate is 1.5:(2-4).

[0109] In some embodiments, the molar ratio of the alkylamine to tert-butyl acrylate is 1.8:(2-4).

[0110] In some embodiments, the molar ratio of the alkylamine to tert-butyl acrylate is 2:(2-4).

[0111] Specifically, the molar ratio of the alkylamine to tert-butyl acrylate can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1.5:2, 1.5:2.5, 1.5:3, 1.5:3.5, 1.5:4, 2:2, 2:2.5, 2:3, 2:3.5, 2:4, etc.

[0112] In some embodiments, the molar ratio of the alkylamine to N,N-diisopropylethylamine is 1:(6-12).

[0113] In some embodiments, the molar ratio of the alkylamine to N,N-diisopropylethylamine is 1.2:(6-12).

[0114] In some embodiments, the molar ratio of the alkylamine to N,N-diisopropylethylamine is 1.5:(6-12).

[0115] In some embodiments, the molar ratio of the alkylamine to N,N-diisopropylethylamine is 1.8:(6-12).

[0116] In some embodiments, the molar ratio of the alkylamine to N,N-diisopropylethylamine is 2:(6-12).

[0117] Specifically, the molar ratio of the alkylamine to N,N-diisopropylethylamine can be 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1.5:6, 1.5:6.5, 1.5:7, 1.5:7.5, 1.5:8, 1.5:8.5, 1.5:9, 1.5:9.5, 1.5:10, 1.5:10.5, 1.5:11, 1.5:11.5, 1.5:12, 2:6, 2:6.5, 2:7, 2:7.5, 2:8, 2:8.5, 2:9, 2:9.5, 2:10, 2:10.5, 2:11, 2:11.5, 2:12, etc.

[0118] Furthermore, the condensation reflux temperature is 80-100°C, for example, 80°C, 82°C, 85°C, 88°C, 90°C, 92°C, 95°C, 98°C, 100°C, etc.

[0119] The condensation reflux time is 65 to 75 h, for example, 65 h, 68 h, 70 h, 72 h, 74 h, 75 h, etc.

[0120] In some embodiments, 1-2 mmol alkylamine, 2-4 mmol tert-butyl acrylate, and 6-12 mmol N,N-diisopropylethylamine are dissolved in 7-14 mL toluene, and then condensed and refluxed at 80-100° C. for 65-75 h, vacuum-dried, and then dissolved with ethyl acetate, the organic phase is washed with water 2-3 times, and then the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and then dried with anhydrous Na2SO4 for 3-5 h to obtain a crude product of intermediate 1. The crude product of intermediate 1 is separated and purified by silica gel column chromatography, and the ratio of eluent PE / EA is (5-15): 1, thereby obtaining intermediate 1.

[0121] Furthermore, the alkylamine is of the following formula (II):

[0122]

[0123] m and n are integers greater than or equal to 1.

[0124] Furthermore, m and n are independently integers of 1-25, preferably integers of 8-20.

[0125] In step 2, the intermediate 1 is dissolved in dichloromethane and stirred evenly, and then trifluoroacetic acid is added thereto. After the reaction temperature rises to room temperature, the reaction is continued, and then the reduced pressure is reduced and vacuum-dried, and then it is dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain intermediate 2.

[0126] Furthermore, the molar ratio of the intermediate 1 to trifluoroacetic acid is (1-3):(55-165).

[0127] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 1:(55-165).

[0128] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 2:(55-165).

[0129] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 3:(55-165).

[0130] Specifically, the molar ratio of the intermediate 1 to trifluoroacetic acid can be 1:55, 1:65, 1:75, 1:85, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:165, 2:55, 2:65, 2:75, 2:85, 2:95, 2:100, 2:110, 2:120, 2:130, 2:140, 2:150, 2:160, 2:165, 3:55, 3:65, 3:75, 3:85, 3:95, 3:100, 3:110, 3:120, 3:130, 3:140, 3:150, 3:160, 3:165, etc.

[0131] In some embodiments, 1-3 mmol of intermediate 1 is dissolved in 4-12 mL of dichloromethane, stirred at 0-4°C for 5-15 min, then 4-12 mL of trifluoroacetic acid is added, and the reaction temperature is continued to react for 2-4 h after rising to room temperature, and then the mixture is dried under reduced pressure and vacuum, then dissolved with ethyl acetate, and the organic phase is washed with water for 2-3 times. The obtained organic phase is then shaken with saturated NaCl to remove water for 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 h to obtain intermediate 2.

[0132] In step 3, the intermediate 2 is dissolved in tetrahydrofuran, and an excess of lithium aluminum hydride is slowly added in a nitrogen atmosphere, and the reaction is continued after the reaction temperature rises to room temperature; after the reaction is completed, the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, the reaction solution is dried under reduced pressure and vacuum, and then dissolved in ethyl acetate, and the pH of the solution is adjusted to neutral.

[0133] The organic phase is washed with water and finally dried to obtain intermediate 3.

[0134] Furthermore, the temperature during the addition of lithium aluminum hydride is controlled at 0 to 4°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, etc. After the addition of lithium aluminum hydride is completed and the reaction temperature rises to room temperature, the reaction time is continued for 6 to 18 hours, for example, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.

[0135] In some embodiments, 1-4 mmol of intermediate 2 is dissolved in 4-17 mL of tetrahydrofuran, and an excess of lithium aluminum hydride (LiAlH4) is slowly added at a temperature of 0-4°C under the protection of a nitrogen atmosphere. After the reaction temperature rises to room temperature, the reaction is continued for 6-18 hours, and then the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, the reaction is stopped and the solution is vacuum-dried. Then it is dissolved in ethyl acetate, the pH of the solution is adjusted to neutral with 1M HCl, the organic phase is washed with water 2-3 times, and the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 hours, thereby obtaining intermediate 3.

[0136] In step 4, the intermediate 3 is dissolved in anhydrous pyridine, diphenyl phosphite is added under an ice bath, and after a certain reaction time, the ice bath is removed and the reaction is continued. After the reaction is completed, triethylamine and water are added and the reaction is continued, and then the reaction is reduced pressure and vacuum spin-dried to obtain a crude product of the intermediate 4;

[0137] The crude product of the intermediate four is separated and purified by chromatography to obtain the intermediate four.

[0138] Furthermore, the molar ratio of the intermediate tris(III) to diphenyl phosphite is (1-2):(3-6).

[0139] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1:(3-6).

[0140] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.2:(3-6).

[0141] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.5:(3-6).

[0142] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.8:(3-6).

[0143] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 2:(3-6).

[0144] Specifically, the molar ratio of the intermediate tris to diphenyl phosphite can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1.5:3, 1.5:3.5, 1.5:4, 1.5:4.5, 1.5:5, 1.5:5.5, 1.5:6, 1.8:3, 1.8:3.5, 1.8:4, 1.8:4.5, 1.8:5, 1.8:5.5, 1.8:6, 2:3, 2:3.5, 2:4, 2:4.5, 2:5, 2:5.5, 2:6, etc.

[0145] Furthermore, the molar ratio of triethylamine to water is (10-20):(7-15).

[0146] In some embodiments, the molar ratio of triethylamine to water is 10:(7-15).

[0147] In some embodiments, the molar ratio of triethylamine to water is 12:(7-15).

[0148] In some embodiments, the molar ratio of triethylamine to water is 15:(7-15).

[0149] In some embodiments, the molar ratio of triethylamine to water is 20:(7-15).

[0150] Specifically, the molar ratio of triethylamine and water can be 10:7, 10:7.5, 10:8, 10:8.5, 10:9, 10:9.5, 10:10, 10:10.5, 10:11, 10:11.5, 10:12, 10:12.5, 10:13, 10:13.5, 10:14, 10:14.5, 10:15, 15:7, 15:7.5, 15:8, 15:8.5, 15:9, 15:9.5, 15:10, 15:10. 5. 15:11, 15:11.5, 15:12, 15:12.5, 15:13, 15:13.5, 15:14, 15:14.5, 15:15, 20:7, 20:7.5, 20:8, 20:8.5, 20:9, 20:9.5, 20:10, 20:10.5, 20:11, 20:11.5, 20:12, 20:12.5, 20:13, 20:13.5, 20:14, 20:14.5, 20:15, etc.

[0151] Furthermore, the molar ratio of the intermediate tris(II) to triethylamine is (1-2):(10-20).

[0152] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1:(10-20).

[0153] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.2:(10-20).

[0154] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.5:(10-20).

[0155] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.8:(10-20).

[0156] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 2:(10-20).

[0157] Specifically, the molar ratio of intermediate tris(III) to triethylamine can be 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1.5:10, 1.5:12, 1.5:14, 1.5:16, 1.5:18, 1.5:20, 2:10, 2:12, 2:14, 2:16, 2:18, 2:20, etc.

[0158] Furthermore, the molar ratio of intermediate 3 to water is (1-2):(7-15).

[0159] In some embodiments, the molar ratio of intermediate 3 to water is 1:(7-15).

[0160] In some embodiments, the molar ratio of intermediate 3 to water is 1.2:(7-15).

[0161] In some embodiments, the molar ratio of intermediate three to water is 1.5:(7-15).

[0162] In some embodiments, the molar ratio of intermediate three to water is 1.8:(7-15).

[0163] In some embodiments, the molar ratio of intermediate 3 to water is 2:(7-15).

[0164] Specifically, the molar ratio of intermediate three to water can be 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1.5:7, 1.5:8, 1.5:9, 1.5:10, 1.5:11, 1.5:12, 1.5:13, 1.5:14, 1.5:15, 2:7, 2:8, 2:9, 2:10, 2:11, 2:12, 2:13, 2:14, 2:15, etc.

[0165] Furthermore, the molar ratio of the intermediate tris(III), triethylamine and water is (1-2):(10-20):(7-15).

[0166] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1:(10-20):(7-15).

[0167] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.2:(10-20):(7-15).

[0168] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.5:(10-20):(7-15).

[0169] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.8:(10-20):(7-15).

[0170] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 2:(10-20):(7-15).

[0171] Specifically, the molar ratio of the intermediate tris(III), triethylamine and water can be 1:10:7, 1:15:7, 1:20:7, 1.5:10:7, 1.5:15:7, 1.5:20:7, 2:10:7, 2:15:7, 2:20:7, 1:10:15, 1:15:15, 1:20:15, 1.5:10:15, 1.5:15:15, 1.5:20:15, 2:10:15, 2:15:15, 2:20:15, etc.

[0172] Furthermore, the reaction time of the intermediate tris and diphenyl phosphite in the ice bath is 5 to 15 min, for example, 5 min, 8 min, 10 min, 12 min, 15 min, etc. After removing the ice bath, the reaction is continued for 0.5 to 2 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, etc., and then triethylamine and water are added and the reaction is continued for 0.5 to 2 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, etc.

[0173] In some embodiments, 1-2 mmol of intermediate three is dissolved in 5-10 mL of anhydrous pyridine to form a mixed solution, 3-6 mmol of diphenyl phosphite is added to the mixed solution under ice bath conditions, the ice bath is removed after the reaction for 5-15 min, and the reaction is continued for 0.5-2 h. Then 10-20 mmol of triethylamine and 7-15 mmol of water are added, the reaction is continued for 0.5-2 h, and then the crude product of intermediate four is obtained by vacuum drying under reduced pressure. The crude product of intermediate four is separated and purified by silica gel column chromatography, and the eluent DCM / MeOH ratio is (15-25): 1, thereby obtaining intermediate four.

[0174] In step 5, the intermediate 4 is dissolved in anhydrous pyridine, triethylamine and iodine are added thereto under ice bath conditions to form a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued, and then water is added. After a certain reaction time, a saturated sodium thiosulfate solution is added until the reaction solution turns yellow and then the reaction is stopped, the reduced pressure is vacuum-dried, and then dissolved with dichloromethane, the organic phase is washed with water, and allowed to stand and dry to obtain the target crude product;

[0175] The target crude product is separated and purified by chromatography to obtain the target product alkylamine phosphoric acid derivative.

[0176] Furthermore, the molar ratio of the intermediate tetrakis, triethylamine and iodine is (0.5-1): (2.5-5): (1.5-3), for example, it can be 0.5:2.5:1.5, 0.5:3:1.5, 0.5:4:1.5, 0.5:5:1.5, 0.5:2.5:2, 0.5:3:2, 0.5:4:2, 0.5:5:2, 0.5:2.5:3, 0.5:3:3, 0.5:4:3, 0.5:5:3, 1:2.5:1.5, 1:3:1.5, 1:4:1.5, 1:5:1.5, 1:2.5:2, 1:3:2, 1:4:2, 1:5:2, 1:2.5:3, 1:3:3, 1:4:3, 1:5:3, etc.

[0177] Furthermore, the molar ratio of the intermediate tetrakis(II) to triethylamine is (0.5-1):(2.5-5).

[0178] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 0.5:(2.5-5).

[0179] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 0.8:(2.5-5).

[0180] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 1:(2.5-5).

[0181] Specifically, the molar ratio of the intermediate tetrakis to triethylamine can be 0.5:2.5, 0.5:3, 0.5:3.5, 0.5:4, 0.5:4.5, 0.5:5, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0182] Furthermore, the molar ratio of intermediate tetrakis to iodine is (0.5-1):(1.5-3).

[0183] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 0.5:(1.5-3).

[0184] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 0.8:(1.5-3).

[0185] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 1:(1.5-3).

[0186] Specifically, the molar ratio of intermediate tetraki to iodine is 0.5:1.5, 0.5:2, 0.5:2.5, 0.5:3, 1:1.5, 1:2, 1:2.5, 1:3, etc.

[0187] Furthermore, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10 to 20 minutes, and then 10 to 20 mmol of water is added and the reaction is carried out for 5 to 10 minutes.

[0188] In some embodiments, 0.5-1 mmol of the intermediate tetra is dissolved in 3-7 mL of anhydrous pyridine to form a mixed solution, the mixed solution is placed in an ice bath, and then 2.5-5 mmol of triethylamine and 1.5-3 mmol of iodine are added to the mixed solution to obtain a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10-20 min, and then 10-20 mmol of water is added and reacted for 5-10 min. Then a saturated sodium thiosulfate solution is added until the reaction solution turns yellow and the reaction is stopped, and the reduced pressure is vacuumed and dried. Then it is dissolved with dichloromethane, the organic phase is washed with water 2-3 times, and the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 hours to obtain the target crude product. The target crude product is separated and purified by silica gel column chromatography, and the eluent DCM / MeOH ratio is (5-15): 1, thereby obtaining the target alkylamine phosphoric acid derivative.

[0189] The present application also provides a method for preparing a didodecylamine phosphoric acid derivative, and the technical route thereof is as follows:

[0190]

[0191] Furthermore, the preparation method of the didodecylamine phosphoric acid derivative comprises the following steps:

[0192] Step 1: subjecting didodecylamine to Michael addition reaction with tert-butyl acrylate to obtain intermediate 1;

[0193] Step 2: using trifluoroacetic acid to react with the intermediate 1 to remove its tert-butyl protecting group to obtain the intermediate 2;

[0194] Step 3: using lithium aluminum hydride to reduce the carboxyl group of intermediate 2 to hydroxyl group, thereby obtaining intermediate 3;

[0195] Step 4: using diphenyl phosphite to carry out a phosphorylation reaction with the intermediate 3 to obtain the intermediate 4;

[0196] Step 5: using iodine to oxidize the phosphorous acid group of the intermediate four into a phosphoric acid group, thereby obtaining the didodecylamine phosphoric acid derivative.

[0197] In step 1, the didodecylamine, tert-butyl acrylate, and N,N-diisopropylethylamine are dissolved in toluene, and then condensed and refluxed for a certain period of time, and then vacuum-dried under reduced pressure, and then dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain a crude product of intermediate 1;

[0198] The crude product of the intermediate one is separated and purified by chromatography to obtain the intermediate one.

[0199] Furthermore, the molar ratio of didodecylamine, tert-butyl acrylate and N,N-diisopropylethylamine is (1-2): (2-4): (6-12).

[0200] In some embodiments, the molar ratio of didodecylamine to tert-butyl acrylate is 1:(2-4).

[0201] In some embodiments, the molar ratio of didodecylamine to tert-butyl acrylate is 1.2:(2-4).

[0202] In some embodiments, the molar ratio of didodecylamine to tert-butyl acrylate is 1.5:(2-4).

[0203] In some embodiments, the molar ratio of didodecylamine to tert-butyl acrylate is 1.8:(2-4).

[0204] In some embodiments, the molar ratio of didodecylamine to tert-butyl acrylate is 2:(2-4).

[0205] Specifically, the molar ratio of didodecylamine to tert-butyl acrylate can be 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1.5:2, 1.5:2.5, 1.5:3, 1.5:3.5, 1.5:4, 2:2, 2:2.5, 2:3, 2:3.5, 2:4, etc.

[0206] In some embodiments, the molar ratio of didodecylamine to N,N-diisopropylethylamine is 1:(6-12).

[0207] In some embodiments, the molar ratio of didodecylamine to N,N-diisopropylethylamine is 1.2:(6-12).

[0208] In some embodiments, the molar ratio of didodecylamine to N,N-diisopropylethylamine is 1.5:(6-12).

[0209] In some embodiments, the molar ratio of didodecylamine to N,N-diisopropylethylamine is 1.8:(6-12).

[0210] In some embodiments, the molar ratio of didodecylamine to N,N-diisopropylethylamine is 2:(6-12).

[0211] Specifically, the molar ratio of didodecylamine to N,N-diisopropylethylamine can be 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1.5:6, 1.5:6.5, 1.5:7, 1.5:7.5, 1.5:8 , 1.5:8.5, 1.5:9, 1.5:9.5, 1.5:10, 1.5:10.5, 1.5:11, 1.5:11.5, 1.5:12, 2:6, 2:6.5, 2:7, 2:7.5, 2:8, 2:8.5, 2:9, 2:9.5, 2:10, 2:10.5, 2:11, 2:11.5, 2:12, etc.

[0212] Furthermore, the condensation reflux temperature is 80-100°C, for example, 80°C, 82°C, 85°C, 88°C, 90°C, 92°C, 95°C, 98°C, 100°C, etc.

[0213] The condensation reflux time is 65 to 75 h, for example, 65 h, 68 h, 70 h, 72 h, 74 h, 75 h, etc.

[0214] In some embodiments, 1-2 mmol didodecylamine, 2-4 mmol tert-butyl acrylate, and 6-12 mmol N,N-diisopropylethylamine are dissolved in 7-14 mL toluene, and then condensed and refluxed at 80-100° C. for 65-75 h, vacuum-dried, and then dissolved with ethyl acetate, the organic phase is washed with water 2-3 times, and then the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and then dried with anhydrous Na2SO4 for 3-5 h to obtain a crude product of intermediate 1. The crude product of intermediate 1 is separated and purified by silica gel column chromatography, and the ratio of eluent PE / EA is (5-15): 1, thereby obtaining intermediate 1.

[0215] In step 2, the intermediate 1 is dissolved in dichloromethane and stirred evenly, and then trifluoroacetic acid is added thereto. After the reaction temperature rises to room temperature, the reaction is continued, and then the reduced pressure is reduced and vacuum-dried, and then it is dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain intermediate 2.

[0216] Furthermore, the molar ratio of the intermediate 1 to trifluoroacetic acid is (1-3):(55-165).

[0217] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 1:(55-165).

[0218] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 2:(55-165).

[0219] In some embodiments, the molar ratio of the intermediate 1 to trifluoroacetic acid is 3:(55-165).

[0220] Specifically, the molar ratio of the intermediate 1 to trifluoroacetic acid can be 1:55, 1:65, 1:75, 1:85, 1:95, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:165, 2:55, 2:65, 2:75, 2:85, 2:95, 2:100, 2:110, 2:120, 2:130, 2:140, 2:150, 2:160, 2:165, 3:55, 3:65, 3:75, 3:85, 3:95, 3:100, 3:110, 3:120, 3:130, 3:140, 3:150, 3:160, 3:165, etc.

[0221] In some embodiments, 1-3 mmol of intermediate 1 is dissolved in 4-12 mL of dichloromethane, stirred at 0-4°C for 5-15 min, then 4-12 mL of trifluoroacetic acid is added, and the reaction temperature is continued to react for 2-4 h after rising to room temperature, and then the mixture is dried under reduced pressure and vacuum, then dissolved with ethyl acetate, and the organic phase is washed with water for 2-3 times. The obtained organic phase is then shaken with saturated NaCl to remove water for 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 h to obtain intermediate 2.

[0222] In step three, the intermediate two is dissolved in tetrahydrofuran, and an excess of lithium aluminum hydride is slowly added in a nitrogen atmosphere, and the reaction is continued after the reaction temperature rises to room temperature; after the reaction is completed, the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, the reaction solution is dried under reduced pressure and vacuum, and then dissolved in ethyl acetate, and the pH of the solution is adjusted to neutral, the organic phase is washed with water, and finally dried to obtain the intermediate three.

[0223] Furthermore, the temperature during the addition of lithium aluminum hydride is controlled at 0 to 4°C, for example, 0°C, 1°C, 2°C, 3°C, 4°C, etc. After the addition of lithium aluminum hydride is completed and the reaction temperature rises to room temperature, the reaction time is continued for 6 to 18 hours, for example, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.

[0224] In some embodiments, 1-4 mmol of intermediate 2 is dissolved in 4-17 mL of tetrahydrofuran, and an excess of lithium aluminum hydride (LiAlH4) is slowly added at a temperature of 0-4°C under the protection of a nitrogen atmosphere. After the reaction temperature rises to room temperature, the reaction is continued for 6-18 hours, and then the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, the reaction is stopped and the solution is vacuum-dried. Then it is dissolved in ethyl acetate, the pH of the solution is adjusted to neutral with 1M HCl, the organic phase is washed with water 2-3 times, and the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 hours, thereby obtaining intermediate 3.

[0225] In step 4, the intermediate 3 is dissolved in anhydrous pyridine, diphenyl phosphite is added under an ice bath, and after a certain reaction time, the ice bath is removed and the reaction is continued. After the reaction is completed, triethylamine and water are added and the reaction is continued, and then the reaction is reduced pressure and vacuum spin-dried to obtain a crude product of the intermediate 4;

[0226] The crude product of the intermediate four is separated and purified by chromatography to obtain the intermediate four.

[0227] Furthermore, the molar ratio of the intermediate tris(III) to diphenyl phosphite is (1-2):(3-6).

[0228] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1:(3-6).

[0229] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.2:(3-6).

[0230] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.5:(3-6).

[0231] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 1.8:(3-6).

[0232] In some embodiments, the molar ratio of the intermediate tris(III) to diphenyl phosphite is 2:(3-6).

[0233] Specifically, the molar ratio of the intermediate tris to diphenyl phosphite can be 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1.5:3, 1.5:3.5, 1.5:4, 1.5:4.5, 1.5:5, 1.5:5.5, 1.5:6, 1.8:3, 1.8:3.5, 1.8:4, 1.8:4.5, 1.8:5, 1.8:5.5, 1.8:6, 2:3, 2:3.5, 2:4, 2:4.5, 2:5, 2:5.5, 2:6, etc.

[0234] Furthermore, the molar ratio of triethylamine to water is (10-20):(7-15).

[0235] In some embodiments, the molar ratio of triethylamine to water is 10:(7-15).

[0236] In some embodiments, the molar ratio of triethylamine to water is 12:(7-15).

[0237] In some embodiments, the molar ratio of triethylamine to water is 15:(7-15).

[0238] In some embodiments, the molar ratio of triethylamine to water is 20:(7-15).

[0239] Specifically, the molar ratio of triethylamine and water can be 10:7, 10:7.5, 10:8, 10:8.5, 10:9, 10:9.5, 10:10, 10:10.5, 10:11, 10:11.5, 10:12, 10:12.5, 10:13, 10:13.5, 10:14, 10:14.5, 10:15, 15:7, 15:7.5, 15:8, 15:8.5, 15:9, 15:9.5, 15:10, 15:10. 5. 15:11, 15:11.5, 15:12, 15:12.5, 15:13, 15:13.5, 15:14, 15:14.5, 15:15, 20:7, 20:7.5, 20:8, 20:8.5, 20:9, 20:9.5, 20:10, 20:10.5, 20:11, 20:11.5, 20:12, 20:12.5, 20:13, 20:13.5, 20:14, 20:14.5, 20:15, etc.

[0240] Furthermore, the molar ratio of the intermediate tris(II) to triethylamine is (1-2):(10-20).

[0241] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1:(10-20).

[0242] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.2:(10-20).

[0243] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.5:(10-20).

[0244] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 1.8:(10-20).

[0245] In some embodiments, the molar ratio of the intermediate tris(III) to triethylamine is 2:(10-20).

[0246] Specifically, the molar ratio of intermediate tris(III) to triethylamine can be 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1.5:10, 1.5:12, 1.5:14, 1.5:16, 1.5:18, 1.5:20, 2:10, 2:12, 2:14, 2:16, 2:18, 2:20, etc.

[0247] Furthermore, the molar ratio of intermediate 3 to water is (1-2):(7-15).

[0248] In some embodiments, the molar ratio of intermediate 3 to water is 1:(7-15).

[0249] In some embodiments, the molar ratio of intermediate 3 to water is 1.2:(7-15).

[0250] In some embodiments, the molar ratio of intermediate three to water is 1.5:(7-15).

[0251] In some embodiments, the molar ratio of intermediate three to water is 1.8:(7-15).

[0252] In some embodiments, the molar ratio of intermediate 3 to water is 2:(7-15).

[0253] Specifically, the molar ratio of intermediate three to water can be 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1.5:7, 1.5:8, 1.5:9, 1.5:10, 1.5:11, 1.5:12, 1.5:13, 1.5:14, 1.5:15, 2:7, 2:8, 2:9, 2:10, 2:11, 2:12, 2:13, 2:14, 2:15, etc.

[0254] Furthermore, the molar ratio of the intermediate tris(III), triethylamine and water is (1-2):(10-20):(7-15).

[0255] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1:(10-20):(7-15).

[0256] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.2:(10-20):(7-15).

[0257] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.5:(10-20):(7-15).

[0258] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 1.8:(10-20):(7-15).

[0259] In some embodiments, the molar ratio of the intermediate tris(III), triethylamine and water is 2:(10-20):(7-15).

[0260] Specifically, the molar ratio of the intermediate tris(III), triethylamine and water can be 1:10:7, 1:15:7, 1:20:7, 1.5:10:7, 1.5:15:7, 1.5:20:7, 2:10:7, 2:15:7, 2:20:7, 1:10:15, 1:15:15, 1:20:15, 1.5:10:15, 1.5:15:15, 1.5:20:15, 2:10:15, 2:15:15, 2:20:15, etc.

[0261] Furthermore, the reaction time of the intermediate tris and diphenyl phosphite in the ice bath is 5 to 15 min, for example, 5 min, 8 min, 10 min, 12 min, 15 min, etc. After removing the ice bath, the reaction is continued for 0.5 to 2 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, etc., and then triethylamine and water are added and the reaction is continued for 0.5 to 2 h, for example, 0.5 h, 1 h, 1.5 h, 2 h, etc.

[0262] In some embodiments, 1-2 mmol of intermediate three is dissolved in 5-10 mL of anhydrous pyridine to form a mixed solution, 3-6 mmol of diphenyl phosphite is added to the mixed solution under ice bath conditions, the ice bath is removed after the reaction for 5-15 min, and the reaction is continued for 0.5-2 h. Then 10-20 mmol of triethylamine and 7-15 mmol of water are added, the reaction is continued for 0.5-2 h, and then the crude product of intermediate four is obtained by vacuum drying under reduced pressure. The crude product of intermediate four is separated and purified by silica gel column chromatography, and the eluent DCM / MeOH ratio is (15-25): 1, thereby obtaining intermediate four.

[0263] In step 5, the intermediate 4 is dissolved in anhydrous pyridine, triethylamine and iodine are added thereto under ice bath conditions to form a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued, and then water is added. After a certain reaction time, a saturated sodium thiosulfate solution is added until the reaction solution turns yellow and then the reaction is stopped, the reduced pressure is vacuum-dried, and then dissolved with dichloromethane, the organic phase is washed with water, and allowed to stand and dry to obtain the target crude product;

[0264] The target crude product is separated and purified by chromatography to obtain the target product, a didodecylamine phosphoric acid derivative.

[0265] Furthermore, the molar ratio of the intermediate tetrakis, triethylamine and iodine is (0.5-1): (2.5-5): (1.5-3), for example, it can be 0.5:2.5:1.5, 0.5:3:1.5, 0.5:4:1.5, 0.5:5:1.5, 0.5:2.5:2, 0.5:3:2, 0.5:4:2, 0.5:5:2, 0.5:2.5:3, 0.5:3:3, 0.5:4:3, 0.5:5:3, 1:2.5:1.5, 1:3:1.5, 1:4:1.5, 1:5:1.5, 1:2.5:2, 1:3:2, 1:4:2, 1:5:2, 1:2.5:3, 1:3:3, 1:4:3, 1:5:3, etc.

[0266] Furthermore, the molar ratio of the intermediate tetrakis(II) to triethylamine is (0.5-1):(2.5-5).

[0267] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 0.5:(2.5-5).

[0268] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 0.8:(2.5-5).

[0269] In some embodiments, the molar ratio of the intermediate tetrakis(II) to triethylamine is 1:(2.5-5).

[0270] Specifically, the molar ratio of the intermediate tetrakis to triethylamine can be 0.5:2.5, 0.5:3, 0.5:3.5, 0.5:4, 0.5:4.5, 0.5:5, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0271] Furthermore, the molar ratio of intermediate tetrakis to iodine is (0.5-1):(1.5-3).

[0272] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 0.5:(1.5-3).

[0273] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 0.8:(1.5-3).

[0274] In some embodiments, the molar ratio of intermediate tetrakis to iodine is 1:(1.5-3).

[0275] Specifically, the molar ratio of intermediate tetraki to iodine is 0.5:1.5, 0.5:2, 0.5:2.5, 0.5:3, 1:1.5, 1:2, 1:2.5, 1:3, etc.

[0276] Furthermore, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10 to 20 minutes, and then 10 to 20 mmol of water is added and the reaction is carried out for 5 to 10 minutes.

[0277] In some embodiments, 0.5-1 mmol of the intermediate tetra is dissolved in 3-7 mL of anhydrous pyridine to form a mixed solution, the mixed solution is placed in an ice bath, and then 2.5-5 mmol of triethylamine and 1.5-3 mmol of iodine are added to the mixed solution to obtain a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10-20 min, and then 10-20 mmol of water is added and reacted for 5-10 min. Then a saturated sodium thiosulfate solution is added until the reaction solution turns yellow and the reaction is stopped, and the reduced pressure is vacuumed and dried. Then it is dissolved with dichloromethane, the organic phase is washed with water 2-3 times, and the obtained organic phase is shaken with saturated NaCl to remove water 2-3 times, and anhydrous Na2SO4 is left to dry for 3-5 hours to obtain the target crude product. The target crude product is separated and purified by silica gel column chromatography, and the eluent DCM / MeOH ratio is (5-15): 1, thereby obtaining the target didodecylamine phosphoric acid derivative.

[0278] In the present application, the didodecylamine phosphoric acid derivative prepared by the above preparation method is the aforementioned didodecylamine phosphoric acid derivative.

[0279] The present application provides an alkylamine phosphate derivative liposome, which includes the aforementioned alkylamine phosphate derivative or a pharmaceutically acceptable salt or stereoisomer thereof.

[0280] The alkylamine phosphate derivative liposome also includes phospholipids, and the mass ratio of the alkylamine phosphate derivative to the phospholipids is (5-15):1, for example, it can be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, etc.

[0281] Preferably, the phospholipid is selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0282] Preferably, the alkylamine phosphoric acid derivative is a didodecylamine phosphoric acid derivative.

[0283] Herein, the dodecylamine phosphate derivative liposome can be used as a drug delivery carrier or as a component of a drug delivery carrier.

[0284] The present application provides a method for preparing alkylamine phosphate derivative liposomes, comprising the following steps:

[0285] Step 1: Dissolve phospholipids and alkylamine phosphate derivatives in an organic solvent, remove the organic solvent by rotary evaporation under reduced pressure in a water bath, and form a lipid film on the inner wall of the bottle.

[0286] The temperature under the water bath condition is 30-60°C, for example, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, etc.

[0287] Step 2: placing the film in a drying oven to completely remove the organic solvent, then adding deionized water thereto, hydrating it in a water bath for a certain period of time, and obtaining the alkylamine phosphate derivative liposomes through probe ultrasound.

[0288] The temperature under the water bath conditions is 30-70°C, for example, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, etc. The hydration time under the water bath conditions is 15-60min, for example, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc.

[0289] In some embodiments, the alkylamine phosphate derivative is a didodecylamine phosphate derivative, and a thin film dispersion method is used to dissolve phospholipids and didodecylamine phosphate derivatives in an organic solvent, and the organic solvent is removed by rotary evaporation under reduced pressure in a water bath at 30 to 60° C., and a lipid film is formed on the inner wall of the bottle. The film is placed in a drying oven at 30 to 60° C. to dry overnight to completely remove the organic solvent, and deionized water is added, and hydrated for 15 to 60 minutes under a water bath at 30 to 70° C., and probe ultrasound is performed to obtain liposomes containing didodecylamine phosphate derivatives.

[0290] The organic solvent is dichloromethane or anhydrous ethanol.

[0291] The alkylamine phosphate derivative liposome prepared by the preparation method described in the present application is the aforementioned alkylamine phosphate derivative liposome.

[0292] The present application provides a pharmaceutical composition, comprising: a pharmaceutically active ingredient and a drug delivery carrier for delivering the pharmaceutically active ingredient, wherein the drug delivery carrier is the aforementioned alkylamine phosphate derivative liposome or comprises the aforementioned alkylamine phosphate derivative.

[0293] The active ingredient of the drug is selected from at least one of nucleic acids, small molecule drugs, macromolecular drugs, protein drugs, polypeptides, and antibodies, preferably nucleic acids. The nucleic acids are selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamers, preferably mRNA.

[0294] The pharmaceutical composition is a nanoparticle preparation, the average size of the nanoparticle preparation is 40nm to 500nm; the polydispersity coefficient of the nanoparticle preparation is ≤50%, preferably ≤30%, and more preferably ≤25%.

[0295] The pharmaceutical composition may be administered intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0296] In some embodiments, the pharmaceutical composition is administered intravenously.

[0297] The present application also provides a use of the aforementioned pharmaceutical composition in the preparation of a drug for targeted treatment or prevention of colorectal cancer.

[0298] Example

[0299] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0300] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0301] Example 1 Synthesis and structural identification of didodecylamine phosphate derivatives

[0302] 1. Synthesis of Intermediate 1

[0303] 1.41mmol didodecylamine, 2.83mmol tert-butyl acrylate, 8.48mmol N,N-diisopropylethylamine were dissolved in 10mL toluene, condensed and refluxed at 90°C for 72h, vacuum dried, then dissolved with ethyl acetate, washed with water for 3 times, dehydrated with saturated NaCl for 2 times, and dried with anhydrous Na2SO4 for 4h. The crude product was separated and purified by silica gel column chromatography, with the eluent PE / EA ratio of 10:1, to obtain intermediate 1 (yellow oil, yield: 90%).

[0304] 1HNMR (500MHz, CDCl3) δ2.72(t,J=7.4Hz,2H),2.41-2.27(m,6H),1.43(s,13H),1.25(d,J=4.6Hz,36H),0.87(t,J=6.9Hz,6H)( Figure 1 ).

[0305] 2. Synthesis of Intermediate 2

[0306] 2.08 mmol of intermediate 1 was dissolved in 8 mL of dichloromethane and stirred at 0°C for 10 min. Then 8 mL of trifluoroacetic acid was added. After the reaction temperature rose to room temperature, the reaction was continued for 3 h. The mixture was dried under reduced pressure and vacuum. Then the mixture was dissolved in ethyl acetate. The organic phase was washed with water for 3 times. The organic phase was shaken with saturated NaCl to remove water twice. The organic phase was dried over anhydrous Na2SO4 for 4 h to obtain intermediate 2 (yellow oil, yield: 100%).

[0307] 1HNMR (500MHz, CDCl3) δ3.34 (t, J=7.4Hz, 2H), 3.08-2.96 (m, 4H), 2.91 (t, J= 7.4Hz,2H),1.73(q,J=8.2,7.3Hz,4H),1.24(s,37H),0.86(t,J=6.8Hz,6H)( Figure 2 ).

[0308] 3. Synthesis of Intermediate 3

[0309] Dissolve 2.35mmol of intermediate 2 in 10mL of tetrahydrofuran, under nitrogen protection, slowly add excess LiAlH4 at 0°C, and continue the reaction for 12h after the reaction temperature rises to room temperature. Then put the reaction solution in an ice bath, slowly add water to quench the reaction, stop when no gas is generated, and vacuum dry under reduced pressure. Then dissolve it in ethyl acetate, adjust the pH of the solution to neutral with 1M HCl, wash the organic phase with water 3 times, shake the organic phase with saturated NaCl to remove water 2 times, and let it stand and dry with anhydrous Na2SO4 for 4h to obtain intermediate 3 (yellow oil, yield: 100%).

[0310] 1HNMR(500MHz, CDCl3)δ3.79(t,J=5.1Hz,2H),2.64(t,J=5.5Hz,2H),2.44-2.37(m,4H),1 .67(q,J=5.5Hz,2H),1.47(q,J=7.4Hz,4H),1.26(d,J=8.9Hz,37H),0.88(d,J=6.6Hz,6H)( Figure 3 ).

[0311] 4. Synthesis of Intermediate 4

[0312] Dissolve 1.46mmol of intermediate three in 7mL of anhydrous pyridine, add 4.52mmol of diphenyl phosphite under ice bath, react for 10min under ice bath, remove ice bath, and continue to react for 1h. Then add 15.31mmol of triethylamine and 10.94mmol of water, continue to react for 1h, and then vacuum dry under reduced pressure. The crude product is separated and purified by silica gel column chromatography, the eluent DCM / MeOH ratio is 20:1, and intermediate four (yellow oil, yield: 80%) is obtained.

[0313] 1HNMR (500MHz, CDCl3) δ3.97 (dt, J=10.3, 5.6Hz, 2H), 3.20-3.12 (m, 2H), 2.92 (dd, J=11.0, 6.0Hz, 4H), 2.04(dq,J=11.5,5.9Hz,2H),1.68(dt,J=11.5,5.6Hz,4H),1.32-1.21(m,37H),0.86(t,J=6.8Hz,6H)( Figure 4 ).

[0314] 5. Synthesis of target phosphorus didodecylamine phosphate derivatives

[0315] Dissolve 0.82mmol of intermediate four in 5mL of anhydrous pyridine, and add 4.09mmol of triethylamine and 2.46mmol of iodine respectively under ice bath. The reaction solution is heated to room temperature, and after the color no longer changes, continue to stir for 15min. Add 16.38mmol of water and react for 5min. Add saturated sodium thiosulfate solution until the reaction solution turns yellow and stop the reaction, and vacuum dry under reduced pressure. Then dissolve with dichloromethane, wash the organic phase with water 3 times, shake the organic phase with saturated NaCl to remove water 2 times, and let stand and dry with anhydrous Na2SO4 for 4h. The crude product is separated and purified by silica gel column chromatography, and the eluent DCM / MeOH ratio is 10:1 to obtain the target phosphoric acid derivative (yellow oil, yield: 70%).

[0316] 1HNMR (500MHz, CDCl3) δ4.08 (s, 2H), 3.26 (s, 2H), 3.01 (s, 4H), 2.09 (s, 2H), 1.68 (s, 4H), 1.27 (d, J = 29.9Hz, 37H), 0.87 (t, J = 6.9Hz, 6H) ( Figure 5 );

[0317] ESI-HRMS:calculated for C27H57NO4P﹣([M+H]+)at 491.4097,foundat491.4081( Figure 6 ).

[0318] Example 2 Preparation of liposomes containing didodecylamine phosphate derivatives

[0319] 60 mg of lecithin, 6 mg of didodecylamine phosphate derivative, and 2 mg of coumarin-6 were dissolved in a mixed organic solvent of dichloromethane and anhydrous ethanol (2:1), and the organic solvent was removed by rotary evaporation under reduced pressure in a 45°C water bath, and a lipid film was formed on the inner wall of the bottle. The film was placed in a drying oven at 45°C and dried overnight to completely remove the organic solvent, and deionized water was added and hydrated in a 50°C water bath for 40 minutes. After probe ultrasound, a fluorescent liposome sample was obtained for use in subsequent evaluation experiments.

[0320] Example 3: Evaluation of the formulation of liposomes containing didodecylamine phosphate derivatives

[0321] The liposome sample prepared in Example 2 was diluted with deionized water at a ratio of 1:100 (v / v) and placed in a test dish. The particle size, polydispersity index (PDI) and zeta potential of the liposome were measured using a ZetasizerNano ZS90 nanoparticle size potential analyzer. The test was repeated 3 times.

[0322] The experimental results showed that the average particle size of the liposomes was 317.20±8.87nm, the polydispersity coefficient was 0.35±0.04, and the zeta potential was -43.47±0.86.

[0323] Example 4 Changes in zeta potential of liposomes containing didodecylamine phosphate derivatives under the action of alkaline phosphatase

[0324] Take the liposome sample prepared in Example 2, dilute it to 5 mL with deionized water at a ratio of 1:100 (v / v), add 10 μL of alkaline phosphatase solution (1000 U / mL), incubate at 37°C, 300 rpm for 6 h, and use ZetasizerNano ZS90 nanoparticle size potential instrument to measure the Zeta potential values ​​of the liposomes at 0 h and 6 h.

[0325] The experimental results showed that the zeta potential of the liposomes was -44.07±1.27mV at 0h, and changed to 24.70±8.27mV after 6h of alkaline phosphatase action ( Figure 7 ).

[0326] Example 5 Cell Viability Assay

[0327] Caco-2 cells were plated at 2 × 10 4The cells were inoculated in a 96-well cell culture plate at a density of 1:50, 1:100, 1:500, 1:1000 (v / v) with MEM prepared in Example 2. 100 μL of liposomes of the corresponding dilution concentration was added to each well of the experimental group, and MEM without liposomes was added to the control group, and the cells were cultured with Caco-2 cells for 24 hours. Then, the original culture medium was discarded, and the Caco-2 cells were washed twice with 200 μL PBS, and 100 μL MEM (containing 10% CCK-8 solution) was added to each well, incubated for 2 hours, and the absorbance value at 450 nm was measured with an enzyme marker.

[0328] The experimental results showed that the cell survival rates of liposomes at different dilution ratios [1:50, 1:100, 1:500, 1:1000 (v / v)] were 69.51%, 81.54%, 93.73%, 92.42% respectively. There was no obvious cytotoxicity when the liposome dilution ratio was as high as 1:100 (v / v). Figure 8 ).

[0329] Example 6 Phosphate release from liposomes containing didodecylamine phosphate derivatives under the action of alkaline phosphatase

[0330] Caco-2 cells were plated at 5 × 10 5 The cells were inoculated at a density of 1:500 in a 3.5 cm dish and grown until a confluent monolayer was observed. On the day of the experiment, the cells were washed twice with glucose-HEPES buffer (268 mM glucose, 25 mM HEPES, pH 7.4), and 1.5 mL of glucose-HEPES buffer containing (control) or not containing 1% (v / v) phosphatase inhibitor mixture A was added, respectively, and incubated at 37°C for 1 h. The liposomes prepared in Example 2 were diluted with glucose-HEPES buffer at a ratio of 1:500 (v / v), and 500 μL was added to the above cells, respectively, and incubated at 37°C for 5 h. At the predetermined time points (0, 1, 2, 3, 4, 5 h), 50 μL of the sample was taken out and 5 μL of 3.6 M H2SO4 was added to terminate the enzymatic reaction. The released phosphate was determined by the malachite green assay.

[0331] The experimental results showed that when phosphatase inhibitor cocktail A was not added, the phosphate released from the liposomes increased in a time-dependent manner. However, when phosphatase inhibitor cocktail A was added, the phosphate release from the liposomes was relatively slow ( Fig. 9 ).

[0332] Example 7 In vitro cellular uptake

[0333] Caco-2 cells were cultured at 5 × 10 5The cells were inoculated into 3.5 cm culture dishes at a density of 1:500 and grown until a confluent monolayer was observed. Before the experiment, the cells were washed and incubated with 1.5 mL OptiMEM at 37° C. for 30 min. Then, the cells were incubated at 37° C. for 1 h with 1.5 mL OptiMEM containing (control) or without 1% (v / v) phosphatase inhibitor mixture A, 500 μL of liposomes prepared in Example 2 diluted with OptiMEM at a ratio of 1:500 (v / v) were added, and incubated at 37° C. for 5 h. At the end of 5 h, the cells were washed with preheated OptiMEM and the cell nuclei were stained with Hoechst33258. The images were taken using a laser confocal microscope and the relative fluorescence intensity was calculated.

[0334] The experimental results showed that when phosphatase inhibitor cocktail A was not added, the uptake rate of fluorescent liposomes by Caco-2 cells was significantly increased compared with the control cells added with phosphatase inhibitor cocktail A (P<0.05) ( Fig.10 ).

[0335] The above are only preferred embodiments of the present application and are not intended to limit the present application in other forms. Any technician familiar with the profession may use the technical contents disclosed above to change or convert them into equivalent embodiments with equivalent changes and apply them to other fields. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present application without departing from the technical solution of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. An alkylamine phosphoric acid derivative, wherein Having a structure as shown in the following formula (I) or a pharmaceutically acceptable salt, stereoisomer, deuterated substance or prodrug thereof; m and n are integers greater than or equal to 1; preferably, m and n are independently integers of 1-25, more preferably integers of 8-20.

2. A method for preparing an alkylamine phosphoric acid derivative, wherein: The steps include: The alkylamine and tert-butyl acrylate are subjected to Michael addition reaction to obtain intermediate 1; Using trifluoroacetic acid to react with the intermediate 1 to remove its tert-butyl protecting group to obtain the intermediate 2; The carboxyl group of intermediate 2 is reduced to hydroxyl group using lithium aluminum hydride to obtain intermediate 3; Using diphenyl phosphite to carry out a phosphorylation reaction with the intermediate three, thereby obtaining an intermediate four; The phosphorous acid group of the intermediate tetrakis oxidized to a phosphoric acid group using iodine to give the alkylamine phosphoric acid derivative.

3. The preparation method according to claim 2, wherein The alkylamine, tert-butyl acrylate and N,N-diisopropylethylamine are dissolved in toluene, and then condensed and refluxed for a certain period of time, and then vacuum dried under reduced pressure, and then dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain a crude product of intermediate 1; The crude product of the intermediate one is separated and purified by chromatography to obtain the intermediate one.

4. The preparation method according to claim 3, wherein The molar ratio of the alkylamine, tert-butyl acrylate and N,N-diisopropylethylamine is (1-2): (2-4): (6-12); and / or The condensation reflux temperature is 80-100° C. and the time is 65-75 hours.

5. The preparation method according to claim 2, wherein: The alkylamine is of the following formula (II): m and n are integers greater than or equal to 1; preferably, m and n are independently integers of 1-25, more preferably integers of 8-20.

6. The preparation method according to claim 2, wherein: The intermediate 1 is dissolved in dichloromethane and stirred evenly, and then trifluoroacetic acid is added thereto. After the reaction temperature rises to room temperature, the reaction is continued, and then the reduced pressure is reduced and vacuum-dried, and then it is dissolved with ethyl acetate, and then the organic phase is washed with water, and finally allowed to stand and dry to obtain the intermediate 2.

7. The preparation method according to claim 6, wherein: The molar ratio of the intermediate 1 to trifluoroacetic acid is (1-3): (55-165).

8. The preparation method according to claim 2, wherein: The intermediate 2 is dissolved in tetrahydrofuran, and an excess of lithium aluminum hydride is slowly added in a nitrogen atmosphere, and the reaction is continued after the reaction temperature rises to room temperature; after the reaction is completed, the reaction solution is placed in an ice bath, and water is slowly added dropwise to quench the reaction. When no gas is generated, it is dried under reduced pressure and vacuum, then dissolved in ethyl acetate, and the pH of the solution is adjusted to neutral, the organic phase is washed with water, and finally dried to obtain the intermediate 3.

9. The preparation method according to claim 8, wherein: The temperature during the addition of lithium aluminum hydride is controlled at 0-4° C. After the addition of lithium aluminum hydride is completed, the reaction temperature rises to room temperature and the reaction time is continued for 6-18 hours.

10. The preparation method according to claim 2, wherein: The intermediate three is dissolved in anhydrous pyridine, diphenyl phosphite is added under ice bath, after a certain reaction time, the ice bath is removed, and the reaction is continued, after the reaction is completed, triethylamine and water are added, and the reaction is continued, and then the intermediate four is dried under reduced pressure and vacuum to obtain a crude product; The crude product of the intermediate four is separated and purified by chromatography to obtain the intermediate four.

11. The preparation method according to claim 10, wherein: The molar ratio of the intermediate tris(III) to diphenyl phosphite is (1-2):(3-6); and / or The molar ratio of the intermediate tris(III), triethylamine and water is (1-2): (10-20): (7-15); and / or The reaction time of the intermediate tris and diphenyl phosphite in the ice bath is 5 to 15 minutes. After the ice bath is removed, the reaction is continued for 0.5 to 2 hours. After that, triethylamine and water are added and the reaction is continued for 0.5 to 2 hours.

12. The preparation method according to claim 2, wherein: The intermediate 4 is dissolved in anhydrous pyridine, triethylamine and iodine are added thereto under ice bath conditions to form a reaction solution, the reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued, and then water is added. After a certain reaction time, a saturated sodium thiosulfate solution is added until the reaction solution turns yellow, and then the reaction is stopped, the reaction is dried under reduced pressure, and then dissolved with dichloromethane, the organic phase is washed with water, and allowed to stand and dry to obtain the target crude product; The target crude product is separated and purified by chromatography to obtain the target product alkylamine phosphoric acid derivative.

13. The preparation method according to claim 12, wherein: The molar ratio of the intermediate tetrakis-triethylamine and iodine is (0.5-1): (2.5-5): (1.5-3); and / or The reaction solution is heated to room temperature, and after the color no longer changes, stirring is continued for 10 to 20 minutes, and then 10 to 20 mmol of water is added and the reaction is continued for 5 to 10 minutes.

14. The preparation method according to any one of claims 2 to 13, wherein the prepared alkylamine phosphoric acid derivative is the alkylamine phosphoric acid derivative according to claim 1.

15. An alkylamine phosphate derivative liposome, wherein: An alkylamine phosphate derivative prepared by the preparation method of any one of claims 1 and 2 to 14 or a pharmaceutically acceptable salt or stereoisomer thereof; Preferably, the alkylamine phosphate derivative liposome further comprises phospholipids, and the mass ratio of the alkylamine phosphate derivative to the phospholipids is (5-15):1; Preferably, the phospholipid is selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

16. A method for preparing alkylamine phosphate derivative liposomes, wherein: The following steps are included: Dissolve phospholipids and alkylamine phosphate derivatives in an organic solvent, remove the organic solvent by rotary evaporation under reduced pressure in a water bath, and form a lipid film on the inner wall of the bottle; The film is placed in a drying oven to completely remove the organic solvent, and then deionized water is added thereto, hydrated for a certain period of time under water bath conditions, and subjected to probe ultrasound to obtain the alkylamine phosphate derivative liposome; Preferably, the temperature under the water bath condition is 30-70° C., and the hydration time under the water bath condition is 15-60 min.

17. The preparation method according to claim 16, wherein: The prepared alkylamine phosphate derivative liposome is the alkylamine phosphate derivative liposome according to claim 15.

18. A pharmaceutical composition, wherein Comprising: a pharmaceutically active ingredient and a drug delivery carrier for delivering the pharmaceutically active ingredient, wherein the drug delivery carrier is the alkylamine phosphate derivative liposome according to claim 15 or the alkylamine phosphate derivative liposome prepared according to claim 16 or 17, or comprises the alkylamine phosphate derivative according to claim 1, or comprises the alkylamine phosphate derivative according to any one of claims 2 to 14; Preferably, the active ingredient of the drug is selected from at least one of nucleic acids, small molecule drugs, macromolecular drugs, protein drugs, polypeptides, and antibodies, preferably nucleic acids, and the nucleic acids are selected from at least one of DNA, ASO, siRNA, miRNA, mRNA, and aptamers.

19. Use of the alkylamine phosphate derivative according to claim 1, the alkylamine phosphate derivative according to any one of claims 2 to 14, the alkylamine phosphate derivative liposome according to claim 15, or the alkylamine phosphate derivative liposome according to any one of claims 16 or 17 in the preparation of a drug for targeted treatment or prevention of colorectal cancer.

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