Amino acid derivative, preparation method and application thereof, anti-tumor micelle and preparation method thereof

文档序号：561928 发布日期：2021-05-18 浏览：18次中文

阅读说明：本技术 氨基酸衍生物及其制备方法和应用、一种抗肿瘤胶束及其制备方法 (Amino acid derivative, preparation method and application thereof, anti-tumor micelle and preparation method thereof ) 是由王玉记冯琦琦高洁贾翌江李元明卢燃于 2021-01-06 设计创作，主要内容包括：本发明提供了氨基酸衍生物及其制备方法和应用、一种抗肿瘤胶束及其制备方法,属于抗肿瘤药物技术领域。本发明提供的氨基酸衍生物具有长脂肪链结构,能够增加活性药物的入胞性,从而使氨基酸衍生物具有逆转抗肿瘤药物耐药作用。本发明提供了一种抗肿瘤胶束,包括氨基酸衍生物和紫杉醇。此胶束具有良好的抗肿瘤以及逆转耐药作用。实施例结果表明,本发明提供的抗肿瘤胶束对于A549/TAX细胞的IC-(50)值可达0.56±0.12μM,说明其具有良好的逆转耐药作用。(The invention provides an amino acid derivative, a preparation method and application thereof, an anti-tumor micelle and a preparation method thereof, and belongs to the technical field of anti-tumor drugs. The amino acid derivative provided by the invention has a long aliphatic chain structure, and can increase the cellularity of an active drug, so that the amino acid derivative has the effect of reversing the drug resistance of an anti-tumor drugAnd (4) acting. The invention provides an anti-tumor micelle, which comprises an amino acid derivative and paclitaxel. The micelle has good antitumor and drug resistance reversing effects. The results of the examples show that the anti-tumor micelle provided by the invention has IC for A549/TAX cells 50 The value can reach 0.56 +/-0.12 mu M, which indicates that the compound has good effect of reversing drug resistance.)

1. An amino acid derivative comprises ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine, ((S) -3- (naphthalene-1-yl) -2-stearamidopropionyl) -leucyl-valine, and 2 ((N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine, ((S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine or ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecanamido) propionyl) -leucyl-valine.

2. The process for producing an amino acid derivative according to claim 1, wherein the ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine is produced by a process comprising the steps of:

(1)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to a condensation reaction with oleic acid to give ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester;

(2) the benzyl ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine is subjected to debenzylation under the action of NaOH to obtain ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine;

the preparation method of ((S) -3- (naphthalene-1-yl) -2-stearamidopropionyl) -leucyl-valine comprises the following steps:

(a)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to a condensation reaction with oleic acid to give ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester;

(b) (S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester is subjected to hydrogenolysis reaction under the catalysis of Pd/C to obtain ((S) -3- (naphthalene-1-yl) -2-stearamidopropionyl) -leucyl-valine;

the above 2 ((N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine comprising the steps of:

(I)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester with N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²Condensation of-oleoyl-L-lysine to give 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester;

(II)2(N⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²Hydrogenolysis of-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester under Pd/C catalysis to obtain 2 (N)⁶-(N²- (tert)butoxycarbonyl-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine;

(III)2(N⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²Deprotection of stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine to give 2 ((N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine;

the preparation method of the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine comprises the following steps:

(i)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester and 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid are subjected to condensation reaction to obtain (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester;

(ii) carrying out hydrogenolysis reaction on the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester under the catalysis of Pd/C to obtain (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine;

the preparation method of ((S) -3- (naphthalene-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propionyl) -leucyl-valine comprises the following steps:

①HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to condensation reaction with N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid to give ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester;

(vii) subjecting ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester to deprotection reaction to obtain ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester;

(iii) condensation reaction of decanoic acid with ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucine-valine benzyl ester to give ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propionyl) -leucyl-valine benzyl ester;

(iv) hydrogenolysis of ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine benzyl ester under Pd/C catalysis to give ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine.

3. The method of claim 2, wherein the HCl-NH is₂The preparation method of the (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester comprises the following steps:

carrying out condensation reaction on the L-Val-OBzl and Boc-Leu to obtain L-Boc-Leu-Val-OBzl;

carrying out deprotection reaction on the L-Boc-Leu-Val-OBzl to obtain HCl & Leu-Val-OBzl;

carrying out condensation reaction on the HCl, Leu-Val-OBzl and Boc-3- (1-naphthyl) -L-alanine to obtain Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester;

the Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to deprotection reaction to obtain HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester.

4. The method according to claim 2, wherein N is⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²The preparation method of the oleoyl-L-lysine comprises the following steps:

condensation reaction of oleic acid with HCl-Lys (Boc) -OBzl to obtain N⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester;

said N is⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester is subjected to deprotection reaction to obtain N²-oleoyl lysine benzyl ester;

said N is²-oleoyl lysine benzyl ester with Boc-Arg (NO)₂) Condensation reaction is carried out to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester;

said (N)⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester is subjected to debenzylation reaction to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oleoyl-L-lysine.

5. The method of claim 2, wherein the 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid is prepared by the steps of:

carrying out condensation reaction on decanoic acid and 1-amino-3, 6,9, 12-tetraoxapentadecane pentadecanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza hexacosanoic acid tert-butyl ester;

and carrying out deprotection agent reaction on the 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid.

6. The use of the amino acid derivative of claim 1 or the amino acid derivative prepared by the preparation method of any one of claims 2 to 5 in the preparation of antitumor drugs.

7. An antitumor drug comprising an antitumor active ingredient and the amino acid derivative of claim 1 or the amino acid derivative prepared by the preparation method of any one of claims 2 to 5.

8. An anti-tumor micelle comprising paclitaxel and the amino acid derivative of claim 1 or the amino acid derivative prepared by the preparation method of any one of claims 2 to 5.

9. The anti-tumor micelle of claim 8, wherein the mass ratio of the paclitaxel to the amino acid derivative is 1:10 to 30.

10. The method for preparing an anti-tumor micelle of claim 8 or 9, comprising the steps of:

(1) mixing an amino acid derivative, paclitaxel and an organic solvent to obtain a mixed solution, and preparing the mixed solution into a film;

(2) mixing the film with a polar organic solvent, and carrying out first ultrasonic treatment to obtain a first ultrasonic solution;

(3) mixing the first ultrasonic solution with water, and performing second ultrasonic to obtain a second ultrasonic solution;

(4) and freeze-drying the second ultrasonic dissolving solution to obtain the anti-tumor micelle.

Technical Field

The invention relates to the technical field of antitumor drugs, in particular to an amino acid derivative, a preparation method and application thereof, an antitumor micelle and a preparation method thereof.

Background

Malignant tumors have become a major cause of harm to human health. According to the display of the Global Cancer observer Observation 2018, about 1808 ten thousand new cases of malignant tumors and about 956 ten thousand death cases of the malignant tumors exist in the world, and the 5-year survival rate is 40.5 percent. Tumor heterogeneity causes tumor invasion and drug resistance, and chemotherapy drug resistance and metastasis are important reasons for treatment failure of most tumor patients.

Paclitaxel is a common chemotherapeutic drug, belongs to plant drugs, and can be used for treating cancers such as lung cancer, breast cancer, carcinoma of large intestine, etc. However, paclitaxel is susceptible to development of resistance during use, which may be due to a dose-response nonlinear dose-effect relationship or to reduced tumor sensitivity as a result of chemotherapy.

Disclosure of Invention

In view of the above, the present invention aims to provide an amino acid derivative, a preparation method and an application thereof, and an anti-tumor micelle.

In order to achieve the purpose of the invention, the invention provides the following technical scheme:

the invention provides an amino acid derivative, which comprises ((S) -3- (naphthalene-1-yl) -2-oleamido propionyl) -leucyl-valine, ((S) -3- (naphthalene-1-yl) -2-stearamido propionyl) -leucyl-valine and 2 ((N-N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthalene-propionyl-leucyl-valine, ((S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine or ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecanamido) propionyl) -leucyl-valine.

The present invention provides a method for producing the above amino acid derivative, wherein the method for producing ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine comprises the steps of:

the preparation method of ((S) -3- (naphthalene-1-yl) -2-stearamidopropionyl) -leucyl-valine comprises the following steps:

the above 2 ((N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine comprising the steps of:

(II)2(N⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²Hydrogenolysis of-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester under Pd/C catalysis to obtain 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine;

the preparation method of the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine comprises the following steps:

the preparation method of ((S) -3- (naphthalene-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propionyl) -leucyl-valine comprises the following steps:

①HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester and N-tert-butyloxycarbonyl-heptapolyethylene glycol-carboxylic acid are subjected to condensation reaction to obtain ((S) -2- (28-tert-butyloxycarbonyl-28-aza-4, 7,10, 13)16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester;

Preferably, the HCl & NH₂The preparation method of the (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester comprises the following steps:

carrying out condensation reaction on the L-Val-OBzl and Boc-Leu to obtain L-Boc-Leu-Val-OBzl;

carrying out deprotection reaction on the L-Boc-Leu-Val-OBzl to obtain HCl & Leu-Val-OBzl;

carrying out condensation reaction on the HCl, Leu-Val-OBzl and Boc-3- (1-naphthyl) -L-alanine to obtain Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester;

the Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to deprotection reaction to obtain HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester.

Preferably, said N is⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²Preparation method of-oleoyl-L-lysineThe method comprises the following steps:

condensation reaction of oleic acid with HCl-Lys (Boc) -OBzl to obtain N⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester;

said N is⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester is subjected to deprotection reaction to obtain N²-oleoyl lysine benzyl ester;

said N is²-oleoyl lysine benzyl ester with Boc-Arg (NO)₂) Condensation reaction is carried out to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester;

Preferably, the preparation method of the 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanic acid comprises the following steps:

and carrying out deprotection agent reaction on the 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid.

The invention provides application of the amino acid derivative in preparing antitumor drugs.

The invention provides an anti-tumor medicament, which comprises an anti-tumor active component and the amino acid derivative.

The invention provides an anti-tumor micelle, which comprises paclitaxel and the amino acid derivatives.

Preferably, the mass ratio of the paclitaxel to the amino acid derivative is 1:10 to 30.

The invention provides a preparation method of the anti-tumor micelle, which comprises the following steps:

(1) mixing an amino acid derivative, paclitaxel and an organic solvent to obtain a mixed solution, and preparing the mixed solution into a film;

(2) mixing the film with a polar organic solvent, and carrying out first ultrasonic treatment to obtain a first ultrasonic solution;

(3) mixing the first ultrasonic solution with water, and performing second ultrasonic to obtain a second ultrasonic solution;

(4) and freeze-drying the second ultrasonic dissolving solution to obtain the anti-tumor micelle.

The invention provides an amino acid derivative, which comprises ((S) -3- (naphthalene-1-yl) -2-oleamido propionyl) -leucyl-valine, ((S) -3- (naphthalene-1-yl) -2-stearamido propionyl) -leucyl-valine and 2 ((N-N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine, (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine and ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecanamido) propionyl) -leucyl-valine. The amino acid derivative provided by the invention has a long-chain structure, the long-chain structure is similar to a cell membrane structure, and according to the principle of similarity and intermiscibility, the property of cells can be added by endocytosis, so that the effect of reversing drug resistance is improved, and the amino acid derivative has the effect of reversing the drug resistance of an anti-tumor drug.

The invention provides a preparation method of the amino acid derivative, and the amino acid derivative obtained by the method has high yield, is simple to operate and is easy to realize industrial batch production.

The invention provides an anti-tumor micelle, which comprises the amino acid derivative and paclitaxel. In the present invention, the paclitaxel and the amino acid derivative form micelles by virtue of intermolecular forces, hydrophobic interactions, and hydrogen bonding. The micelle has good antitumor and drug resistance reversing effects.

The invention provides a preparation method of the anti-tumor micelle, which is simple to operate and easy to realize industrial mass production.

Drawings

FIG. 1 shows the concentration of 5X 10^-4Transmission electron micrograph of M compound 1;

FIG. 2 shows the concentration of 5X 10^-5Scanning electron micrographs of M compound 1;

FIG. 3 shows the concentration of 5X 10^-5Transmission electron micrograph of M compound 1;

FIG. 4 is a Tyndall diagram of Compound 1;

FIG. 5 shows the concentration of 5X 10^-4Transmission electron micrograph of M compound 2;

FIG. 6 shows the concentration of 5X 10^-5Scanning electron micrographs of compound 2;

FIG. 7 shows the concentration of 5X 10^-5Transmission electron micrograph of M compound 2;

FIG. 8 is a Tyndall diagram for Compound 2;

FIG. 9 shows the concentration of 5X 10^-5Transmission electron micrograph of M compound 3;

FIG. 10 shows the concentration of 5X 10^-6Scanning electron micrographs of compound 3;

FIG. 11 shows the concentration of 5X 10^-6Transmission electron micrograph of M compound 3;

FIG. 12 is the Tyndall effect diagram for Compound 3;

FIG. 13 shows the concentration of 5X 10^-5Transmission electron micrograph of M compound 4;

FIG. 14 shows the concentration of 5X 10^-5Scanning electron micrographs of compound 4;

FIG. 15 is the Tyndall effect diagram for Compound 4;

FIG. 16 shows the concentration of 5X 10^-5Transmission electron micrograph of M compound 5;

FIG. 17 shows the concentration of 5X 10^-5Scanning electron micrographs of compound 5;

FIG. 18 is a Tyndall effect plot for Compound 5.

Detailed Description

The invention provides an amino acid derivative, which comprises ((S) -3- (naphthalene-1-yl) -2-oleamido propionyl) -leucyl-valine with a structure shown as a formula 1 and ((S) -3- (naphthalene-1-yl) -2-stearamide with a structure shown as a formula 2Propionyl) -leucyl-valine, 2 ((N) having the structure shown in formula 3⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine, (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine of the structure shown in formula 4 or ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecanylamino) propionyl) -leucyl-valine of the structure shown in formula 5.

In the invention, the amino acid derivative has a long-chain structure and can increase the cell property, so that the amino acid derivative has the effect of reversing the drug resistance of the antitumor drug.

The present invention provides a method for producing the amino acid derivative, wherein the method for producing ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine comprises the steps of:

(2) the benzyl ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine undergoes debenzylation under the action of NaOH to obtain ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine.

In the present invention, the HCl & NH₂The preparation method of the (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester comprises the following steps:

carrying out condensation reaction on the L-Val-OBzl and Boc-Leu to obtain L-Boc-Leu-Val-OBzl;

carrying out deprotection reaction on the L-Boc-Leu-Val-OBzl to obtain HCl & Leu-Val-OBzl;

carrying out condensation reaction on the HCl, Leu-Val-OBzl and Boc-3- (1-naphthyl) -L-alanine to obtain Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester;

the Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to deprotection reaction to obtain HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester.

In the invention, the condensation reaction is carried out on the L-Val-OBzl and the Boc-Leu to obtain the L-Boc-Leu-Val-OBzl. In the present invention, the molar ratio of L-Val-OBzl to Boc-Leu is preferably 1:1 to 1.3. In the present invention, the condensation system of the condensation reaction further preferably comprises EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and HOBt, and the molar ratio of L-Val-OBzl, EDC and HOBt is preferably 1:1: 0.2-1.1, and more preferably 1:1: 1. In the invention, the pH value of the condensation reaction system is preferably 8-10, and more preferably 9. In the present invention, the condensation reaction is carried out in a polar organic solvent, which is preferably tetrahydrofuran.

Preferably, mixing Boc-Leu, EDC and HOBt with a polar organic solvent, and activating under an ice bath condition to obtain an activated mixed solution; and adding L-Val-OBzl into the activated mixed liquid, adjusting the pH value to 8-10, and carrying out a first condensation reaction to obtain Boc-Leu-Val-OBzl. The mixing mode of the invention has no special requirement, and the mixing mode known to the person skilled in the art can be used, such as stirring and mixing; in the present invention, the polar organic solvent is preferably acetonitrile, tetrahydrofuran or dimethylformamide. In the invention, the activation time is preferably 20-30 min; the invention can generate active ester to activate carboxyl through the activation. In the present invention, the pH adjusting agent for adjusting pH is preferably N-methylmorpholine (NMM); the invention can enable amino group to be dissociated by adjusting the pH value.

The temperature of the condensation reaction is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

During the condensation reaction, the present invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting material point disappears. In the invention, the mobile phase of the thin layer chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of the ethyl acetate to the petroleum ether is preferably 1: 2; in the present invention, the Rf value in the thin layer chromatography is preferably 0.3.

After the condensation reaction, the present invention preferably performs a post-treatment on the condensation reaction liquid, and the post-treatment preferably includes the steps of:

and sequentially removing the polar organic solvent from the condensation reaction liquid, extracting and separating, washing an organic phase, drying and carrying out column chromatography separation to obtain the pure Boc-Leu-Val-OBzl product.

The present invention preferably uses a rotary evaporator to remove the polar organic solvent.

In the present invention, the extraction separation preferably comprises the steps of: and adding ethyl acetate into the condensation reaction liquid after the organic solvent is removed, carrying out ultrasonic mixing, then adding distilled water to obtain an upper-layer oil phase and a lower-layer water phase, and separating out the lower-layer water phase through a separating funnel.

In the present invention, the washing detergent is preferably sequentially saturated NaHCO₃Solution, saturated NaCl solution, saturated KHSO₄Solution, saturated NaCl solution, saturated NaHCO₃And a saturated NaCl solution; the number of washing times per detergent is preferably 3.

In the present invention, the drying preferably comprises the steps of: drying the washed oil phase by adding a drying agent, filtering to remove the drying agent, and evaporating the obtained filtrate to dryness. In the present invention, the desiccant is preferably anhydrous Na₂SO₄The drying time of the drying agent is preferably 2-10 hours, and more preferably 4-8 hours. The evaporation to dryness is preferably carried out using a rotary evaporator according to the invention.

In the invention, the stationary phase of the column chromatography separation is preferably silica gel, and the mobile phase is preferably ethyl acetate and petroleum ether; the volume ratio of the ethyl acetate to the petroleum ether is preferably 1: 5.

And after the L-Boc-Leu-Val-OBzl is obtained, carrying out deprotection reaction on the L-Boc-Leu-Val-OBzl to obtain HCl & Leu-Val-OBzl. In the present invention, the deprotection reagent is preferably a hydrogen chloride-ethyl acetate solution; the molar concentration of the deprotection reagent is preferably 2-4 mol/L, and more preferably 3 mol/L; the ratio of the mass of the Boc-Leu-Val-OBzl to the volume of the deprotection reagent is preferably 1 g: 8-14 mL, more preferably 1 g: 10-12 mL. According to the invention, the deprotection reaction is preferably carried out under the condition of ice bath stirring, and the time for the deprotection reaction is preferably 6-10 h, and more preferably 7-8 h.

During the deprotection reaction, the present invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting material spot disappears. In the invention, the mobile phase of the thin layer chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of the ethyl acetate to the petroleum ether is preferably 1: 2; in the present invention, the Rf value in the thin layer chromatography is preferably 0.3.

After the deprotection reaction, the present invention preferably performs a post-treatment on the obtained deprotection reaction solution, and the post-treatment preferably includes the following steps:

and sequentially concentrating, washing and drying the deprotection reaction solution to obtain a pure HCl & Leu-Val-OBzl product.

In the present invention, the concentration is preferably concentration under reduced pressure; in the present invention, the concentration under reduced pressure is not particularly limited, and a concentration under reduced pressure known to those skilled in the art may be used, and the concentrated state is preferably a syrup state. In the present invention, the washing detergent is preferably anhydrous ethyl acetate; the drying mode is preferably decompression and pumping. The present invention preferably repeats the concentration, washing and drying, and the number of repetitions is preferably three.

After obtaining the HCl & Leu-Val-OBzl, carrying out condensation reaction on the HCl & Leu-Val-OBzl and Boc-3- (1-naphthyl) -L-alanine to obtain Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester. In the present invention, the molar ratio of HCl Leu-Val-OBzl to Boc-3- (1-naphthyl) -L-alanine is preferably 1: 1.2. In the invention, EDC and HOBt are preferably included in the condensation system, and the molar ratio of HCl, Leu-Val-OBzl, EDC and HOBt is preferably 1:1: 0.2-1.1, and more preferably 1:1: 1. In the invention, the pH value of the condensation reaction system is preferably 8-10, and more preferably 9. In the present invention, the condensation reaction is carried out in a polar organic solvent, which is preferably acetonitrile.

The temperature of the condensation reaction is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

During the condensation reaction, the present invention preferably monitors the reaction by Thin Layer Chromatography (TLC), and the specific operation is the same as above, and will not be described herein.

After the condensation reaction, the condensation reaction liquid is preferably subjected to post-treatment in the invention, and the post-treatment method is the same as the post-treatment method in the preparation of L-Boc-Leu-Val-OBzl, and is not described in detail herein.

After obtaining the Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester, the Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester is subjected to deprotection reaction to obtain HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester. In the present invention, the type of the deprotecting reagent, the specific operation of the deprotecting and the post-treatment are the same as above, and are not described herein again.

In the present invention, HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester and oleic acid are subjected to condensation reaction to obtain ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester. In the present invention, the HCl & NH₂The molar ratio of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester to oleic acid is preferably 1: 1.26. In the present invention, EDC and HOBt, HCl & NH, are preferably also included in the condensation reaction system₂The molar ratio of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester, EDC and HOBt is preferably 1:1: 1. In the present invention, the condensation reaction is carried out in a polar organic solvent, which is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

After the condensation reaction, the present invention preferably performs a post-treatment on the obtained condensation reaction liquid, and the post-treatment preferably comprises the following steps:

and sequentially removing the polar organic solvent from the first condensation reaction liquid, extracting and separating, washing an organic phase and carrying out column chromatography separation to obtain a pure Boc-Leu-Val-OBzl product.

The present invention preferably uses a rotary evaporator to remove the polar organic solvent.

In the present invention, the extraction separation preferably comprises the steps of: adding ethyl acetate into the first condensation reaction liquid after removing the organic solvent, then adding n-butanol, separating liquid, and reserving n-butanol phase.

After obtaining the benzyl ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine, the benzyl ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine was subjected to debenzylation reaction with NaOH to obtain ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine. In the present invention, the molar ratio of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester to NaOH is preferably 1: 2. In the present invention, the organic solvent added during the debenzylation reaction is methanol and tetrahydrofuran. The invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting material point disappears. According to the invention, the reaction is preferably stopped by adding saturated KHSO4 and adjusting the pH to 7.

In the invention, the temperature of the debenzylation reaction is preferably 36-45 ℃, and the time is preferably 8-24 h, and more preferably 12 h.

In the present invention, after the debenzylation reaction, the present invention preferably performs a post-treatment on the debenzylation reaction solution, and the post-treatment preferably comprises the following steps:

performing rotary evaporation on tetrahydrofuran, adjusting the pH value to 3-4, adding ethyl acetate, transferring to a separating funnel, separating a water layer, keeping an ethyl acetate layer, adding ethyl acetate into the water layer again, performing extraction washing for 2 times, adding saturated NaCl, separating the water layer, keeping the ethyl acetate layer, performing extraction washing for 2 times, adding anhydrous Na₂SO₄And (5) drying. Then, ethyl acetate was removed by filtration and rotary evaporation in this order to obtain a pure product of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine.

In the present invention, the process for preparing ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine is represented by formula A.

In the present invention, the method for preparing ((S) -3- (naphthalen-1-yl) -2-stearamidopropanoyl) -leucyl-valine comprises the steps of:

(b) the benzyl ((S) -3- (naphthalene-1-yl) -2-oleamidopropanoyl) -leucyl-valine is subjected to hydrogenolysis reaction under the catalysis of Pd/C to obtain ((S) -3- (naphthalene-1-yl) -2-stearamidopropanoyl) -leucyl-valine.

In the present invention, the HCl & NH₂The condensation reaction of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester with oleic acid to give ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester is the same as above and will not be described herein.

After obtaining the benzyl ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine, the benzyl ((S) -3- (naphthalen-1-yl) -2-oleamidopropanoyl) -leucyl-valine is catalyzed by Pd/CThe hydrogenolysis reaction was carried out to give ((S) -3- (naphthalen-1-yl) -2-stearamidopropionyl) -leucyl-valine. In the invention, the mass ratio of ((S) -3- (naphthalene-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester to the Pd/C catalyst is preferably 28:3, in the invention, the hydrogenolysis reaction temperature is preferably room temperature, and the time is preferably 6-12 h; h in the hydrogenolysis reaction₂The pressure of (A) is preferably 1.1 to 1.4 times atmospheric pressure.

During the course of the hydrogenolysis reaction, the present invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting material point disappears. In the invention, the mobile phase of the thin layer chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of the ethyl acetate to the petroleum ether is preferably 1: 2; in the present invention, the Rf value in the thin layer chromatography is preferably 0.3.

After the hydrogenolysis reaction, the hydrogenolysis reaction solution is preferably sequentially filtered and evaporated to dryness to obtain a pure product of ((S) -3- (naphthalene-1-yl) -2-stearamidopropionyl) -leucyl-valine. The invention has no special requirements on the filtration mode, and the filtration mode which is well known by the technicians in the field can be used; according to the invention, the Pd/C catalyst in the hydrogenolysis reaction liquid is removed through the filtration. The evaporation to dryness is preferably carried out using a rotary evaporator according to the invention.

In the present invention, the reaction process of ((S) -3- (naphthalen-1-yl) -2-stearamidopropanoyl) -leucyl-valine is shown in formula B.

In the present invention, 2 ((N) is⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine comprising the steps of:

(I)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester with N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oleoyl-L-lysine to obtain2(N⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester;

(II)2(N⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²Hydrogenolysis of-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester under Pd/C catalysis to obtain 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine;

In the present invention, said N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²The process for the preparation of oleoyl-L-lysine preferably comprises the following steps:

(1) condensation reaction of oleic acid with HCl-Lys (Boc) -OBzl to obtain N⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester;

(2) said N is⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester is subjected to deprotection reaction to obtain N²-oleoyl lysine benzyl ester;

(3) said N is²-oleoyl lysine benzyl ester with Boc-Arg (NO)₂) Condensation reaction is carried out to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester;

(4) said N is⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester is subjected to debenzylation reaction to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oilacyl-L-lysine.

In the present invention, oleic acid was condensed with HCl.Lys (Boc) -OBzl to give N6- (tert-butyloxycarbonyl) -N2-oleoyl lysine benzyl ester. In the invention, the molar ratio of the oleic acid to HCl & Lys (Boc) -OBzl is preferably 1-1.3: 1, more preferably 1.2: 1. In the present invention, EDC and HOBt are also preferably included in the condensation reaction system, and the molar ratio of oleic acid, EDC and HOBt is preferably 1:1: 1. In the present invention, the organic solvent used for the condensation reaction is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

Obtaining the N⁶- (tert-butyloxycarbonyl) -N²After benzyl oleoyl lysine ester, the N⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester is subjected to deprotection reaction to obtain N²-oleoyl lysine benzyl ester. In the invention, the deprotection reagent used in the deprotection reaction is preferably hydrogen chloride-ethyl acetate solution; the molar concentration of the deprotection reagent is preferably 2-4 mol/L, and more preferably 3 mol/L; said N is⁶- (tert-butyloxycarbonyl) -N²The ratio by volume of the mass of oleoyl lysine benzyl ester to the volume of deprotecting reagent is preferably 1 g: 8-14 mL, more preferably 1 g: 10-12 mL. According to the invention, the deprotection reaction is preferably carried out under the condition of ice bath stirring, and the time for the deprotection reaction is preferably 6-10 h, and more preferably 7-8 h.

During the first deprotection reaction, the present invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting point disappears. In the invention, the mobile phase of the thin layer chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of the ethyl acetate to the petroleum ether is preferably 1: 2; in the present invention, R in the thin layer chromatography_fThe value is preferably 0.3.

To obtain N²After benzyl oleoyl lysine ester, the N²-oleoyl lysine benzyl ester with Boc-Arg (NO)₂) Condensation reaction is carried out to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester. In the present invention, said N²-oleoyl lysine benzyl ester with Boc-Arg (NO)₂) The molar ratio of (A) to (B) is preferably 1 to 1.4: 1. In the present invention, EDC and HOBt, and the Boc-Arg (NO) are preferably included in the condensation reaction system₂) The molar ratio of EDC to HOBt is preferably 1:1: 0.2-1.1. In the present invention, the organic solvent used for the condensation reaction is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

Obtaining the N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²After oleoyl-lysine benzyl ester, the N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester is subjected to debenzylation reaction to obtain N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oleoyl-L-lysine. In the present invention, the reagent used for the debenzylation is preferably NaOH, and the N is⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²The molar ratio of oleoyl-lysine benzyl ester to NaOH is preferably 1: 2.

Obtaining the N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²After oleoyl-L-lysine, HCl. NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester with N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²Condensation of-oleoyl-L-lysine to give 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester. In the present invention, the HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester with N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oilThe molar ratio of acyl-L-lysine is preferably 1-1.2: 1. In the present invention, EDC and HOBt, and N are preferably included in the condensation system of the condensation reaction⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-L-arginyl) -N²The molar ratio of oleoyl-L-lysine to EDC and HOBt is preferably 1:1.12: 1.12. In the invention, the pH value of the condensation system is preferably 8-9. In the present invention, the solvent for the condensation reaction is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

During the first condensation reaction, the present invention preferably monitors the progress of the reaction by Thin Layer Chromatography (TLC) and terminates the reaction when the starting material point disappears. In the invention, the mobile phase of the thin layer chromatography is preferably methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is preferably 1: 15; in the present invention, the Rf value in the thin layer chromatography is preferably 0.5.

Obtaining the 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²After oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²Hydrogenolysis of-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester under Pd/C catalysis to obtain 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine.

In the present invention, the 2 (N) is⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²The mass ratio of-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester to Pd/C is preferably 9: 1. In the invention, the hydrogenolysis reaction is preferably carried out at room temperature for 6-12 h; h in the hydrogenolysis reaction₂The pressure of (A) is preferably 1.1 to 1.4 times atmospheric pressure.

Obtained by2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²After stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine, 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²Deprotection of stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine to give 2 ((N)⁶-L-arginyl) -N²-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine. In the invention, the deprotection reagent is preferably a hydrogen chloride-ethyl acetate solution, and the concentration of the deprotection reagent is preferably 2-4 mol/L, and more preferably 3 mol/L. According to the invention, the deprotection reaction is preferably carried out under the condition of ice bath stirring, and the time for the deprotection reaction is preferably 6-10 h, and more preferably 7-8 h.

In the present invention, 2 ((N) is⁶-L-arginyl) -N²The reaction process of-stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine is shown in formula C.

In the present invention, the method for preparing (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine comprises the steps of:

(ii) and carrying out hydrogenolysis reaction on the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester under the catalysis of Pd/C to obtain the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine.

In the present invention, the process for producing 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanic acid preferably comprises the steps of:

(1) carrying out condensation reaction on decanoic acid and 1-amino-3, 6,9, 12-tetraoxapentadecane pentadecanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza hexacosanoic acid tert-butyl ester;

(2) and carrying out deprotection agent reaction on the 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid.

In the invention, capric acid and 1-amino-3, 6,9, 12-tetraoxapentadecane pentadecanoic acid tert-butyl ester are subjected to condensation reaction to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid tert-butyl ester. In the present invention, the molar ratio of the decanoic acid to the 1-amino-3, 6,9, 12-tetraoxapentadecane pentadecanoic acid tert-butyl ester is preferably 1:1. In the invention, EDC and HOBt are preferably included in the condensation system of the condensation reaction, and the molar ratio of the decanoic acid to EDC and HOBt is preferably 1:1: 0.2-1.1, and more preferably 1:1: 1. In the invention, the pH value of the condensation system is preferably 8-9. In the present invention, the solvent for the condensation reaction is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

After the 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid tert-butyl ester is obtained, carrying out deprotection reagent reaction on the 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid tert-butyl ester to obtain 17-oxo-4, 7,10, 13-tetraoxa-16-aza-hexacosanoic acid. In the invention, the deprotection reagent is preferably a hydrogen chloride-ethyl acetate solution, and the concentration of the deprotection reagent is preferably 2-4 mol/L, and more preferably 3 mol/L. According to the invention, the deprotection reaction is preferably carried out under the condition of ice bath stirring, and the time for the deprotection reaction is preferably 6-10 h, and more preferably 7-8 h.

In the present invention, HCl & NH₂Condensation reaction of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester and 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid to obtain (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 1)3-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester. In the present invention, as mentioned, HCl. NH₂The molar ratio of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester to 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid is preferably 1:1, in the present invention, EDC and HOBt are preferably included in the condensation system of the condensation reaction, and the molar ratio of 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid to EDC and HOBt is preferably 1:1: 1. In the invention, the pH value of the condensation system is preferably 8-9. In the present invention, the solvent for the condensation reaction is preferably acetonitrile.

In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

To obtain (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester, and carrying out hydrogenolysis reaction on the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester under the catalysis of Pd/C to obtain the (S) -3- (naphthalene-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine. In the present invention, the mass ratio of (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine benzyl ester to Pd/C is preferably 9.5: 1. In the invention, the hydrogenolysis reaction is preferably carried out at room temperature for 6-12 h; h in the hydrogenolysis reaction₂The pressure of (A) is preferably 1.1 to 1.4 times atmospheric pressure.

In the present invention, the process for preparing (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine is represented by formula D.

In the present invention, the method for preparing ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propionyl) -leucyl-valine comprises the steps of:

In the present invention, HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester and N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid were subjected to condensation reaction to give ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester. In the present invention, the HCl & NH₂The molar ratio of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester to N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid is preferably 1:1, in the present invention, EDC and HOBt are preferably included in the condensation system of the condensation reaction, and the molar ratio of N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid to EDC and HOBt is preferably 1:1: 1.In the invention, the pH value of the condensation system is preferably 8-9. In the present invention, the solvent for the condensation reaction is preferably acetonitrile. In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

To obtain ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester, and then subjecting ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester to deprotection reaction to obtain ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) valine benzyl ester -yl) propionyl-leucyl-valine benzyl ester. In the invention, the deprotection reagent is preferably a hydrogen chloride-ethyl acetate solution, and the concentration of the deprotection reagent is preferably 2-4 mol/L, and more preferably 3 mol/L. According to the invention, the deprotection reaction is preferably carried out under the condition of ice bath stirring, and the time for the deprotection reaction is preferably 6-10 h, and more preferably 7-8 h.

To obtain ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester, decanoic acid was condensed with ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucine-valine benzyl ester to obtain ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamino) propanoyl) -leucyl-valine benzyl ester. In the present invention, the molar ratio of decanoic acid to ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucine-valine benzyl ester is preferably 1:1, in the present invention, EDC and HOBt are preferably included in the condensation system of the condensation reaction, and the molar ratio of decanoic acid to EDC and HOBt is preferably 1:1: 1. In the invention, the pH value of the condensation system is preferably 8-9. In the present invention, the solvent for the condensation reaction is preferably acetonitrile. In the invention, the condensation reaction temperature is preferably 20-35 ℃, and more preferably 25-30 ℃; the time is preferably 12 to 24 hours, and more preferably 16 to 20 hours.

After obtaining the described ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine benzyl ester, ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine benzyl ester was subjected to hydrogenolysis reaction under Pd/C catalysis to obtain ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamino) propanoyl) -leucyl-valine. In the present invention, the mass ratio of ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine benzyl ester to Pd/C is preferably 8.5: 1. In the invention, the hydrogenolysis reaction is preferably carried out at room temperature for 6-12 h; h in the hydrogenolysis reaction₂The pressure of (A) is preferably 1.1 to 1.4 times atmospheric pressure.

In the present invention, the process for preparing ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propionyl) -leucyl-valine is represented by formula E.

The invention provides application of the amino acid derivative in the scheme or the amino acid derivative prepared by the preparation method in the scheme in preparation of antitumor drugs. In the present invention, the antitumor agent comprises the amino acid derivative and an active ingredient. In the present invention, the active ingredient is preferably paclitaxel. In the invention, when the amino acid derivative is used for preparing the antitumor drug, the amino acid derivative has the effect of reversing the drug resistance of the antitumor drug.

The invention provides an anti-tumor micelle, which comprises paclitaxel and the amino acid derivative in the scheme. In the invention, the mass ratio of the paclitaxel to the amino acid derivative is preferably 1: 10-30, and more preferably 1: 15-20. In the invention, the anti-tumor micelle has good anti-tumor and reversal drug resistance effects. In the present invention, the paclitaxel and the amino acid derivative form micelles by virtue of intermolecular forces, hydrophobic interactions, and hydrogen bonding.

In the present invention, the preparation method of the anti-tumor micelle preferably comprises the steps of:

(1) mixing an amino acid derivative, paclitaxel and an organic solvent to obtain a mixed solution, and preparing the mixed solution into a film;

(2) mixing the film with a polar organic solvent, and carrying out first ultrasonic treatment to obtain a first ultrasonic solution;

(3) mixing the first ultrasonic solution with water, and performing second ultrasonic to obtain a second ultrasonic solution;

(4) and freeze-drying the second ultrasonic dissolving solution to obtain the anti-tumor micelle.

The invention mixes amino acid derivative, paclitaxel and organic solvent to obtain mixed solution, and the mixed solution is made into film. The present invention does not require any particular mixing means, and mixing means known to those skilled in the art may be used. In the present invention, the organic solvent is preferably dichloroethane; the invention has no special requirements on the dosage of the organic solvent, and the amino acid derivative and the paclitaxel can be completely dissolved. In the invention, the mixed solution is preferably subjected to rotary evaporation to prepare a film; in the invention, the rotary evaporation temperature is preferably 30-40 ℃, and more preferably 35 ℃; the time is preferably 15-60 min, and more preferably 25-40 min; the speed of the rotary evaporation is preferably 100-300 rpm, and more preferably 150-250 rpm.

After the film is obtained, the film is mixed with a polar organic solvent, and first ultrasonic treatment is carried out to obtain a first ultrasonic solution. In the present invention, the polar organic solvent is preferably ethanol, methanol or acetonitrile. The invention has no special requirement on the dosage of the polar organic solvent, and the film can be completely dissolved. In the invention, the power of the first ultrasonic wave is preferably 300-400W, more preferably 350W, and the time is preferably 10-30 min, more preferably 15-25 min. According to the invention, a uniform solution can be obtained by the first ultrasonic treatment.

After the first ultrasonic solution is obtained, the first ultrasonic solution is mixed with water, and second ultrasonic treatment is carried out to obtain a second ultrasonic solution. In the invention, the volume ratio of the first ultrasonic dissolving liquid to water is preferably 1: 15-25, more preferably 1: 20. in the invention, the power of the second ultrasonic wave is preferably 300-400W, more preferably 350W, and the time is preferably 10-30 min, more preferably 15-25 min. According to the invention, through the second ultrasonic treatment, a nano system with the Tyndall phenomenon can be obtained.

After the second ultrasonic dissolving solution is obtained, the second ultrasonic dissolving solution is freeze-dried to obtain the anti-tumor micelle. The present invention does not require any particular means for lyophilization, and those well known to those skilled in the art can be used.

The amino acid derivatives, the preparation method and the use thereof, and an anti-tumor micelle of the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

EXAMPLE 1 preparation of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine

(1) Preparation of L-Boc-Leu-Val-OBzl

1.15g (5mmol) of Boc-Leu, 0.96g (5mmol) of EDC and 0.68g (5mmol) of HOBt were weighed, stirred, added with 20mL of acetonitrile, activated in ice bath for 20min, and 1.35g (5.5mmol) of HCl & NH were weighed₂-Val-OBzl was added to the eggplant flask and NMM 2mL was added to adjust PH to 9, the reaction was carried out at room temperature for 12 hours, the reaction was monitored by TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.3), and the reaction was terminated by disappearance of the starting material spot. Removing organic solvent with rotary evaporator to obtain yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain pale yellow suspension, adding distilled water, and layering. Pouring the mixed solution into a separating funnel, discarding the lower layer liquid, reserving the upper ethyl acetate layer, and using saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And saturated NaCl was extracted and washed 3 times each. Anhydrous Na for ester layer₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness on a rotary evaporator. Separating with silica gel column chromatography using ethyl acetate and petroleum ether, wherein the ratio of ethyl acetate: petroleum ether 1:5 the expected product is obtained as a white waxy solid weighing 1.74g with a yield of 82.86%.

ESI-MS(m/e):455.5[M+Cl]^-，443.2[M+Na]⁺；Mp 88.1-88.5℃；[α]²⁵ _D＝-39.99(c＝0.1,CH₃OH)。¹H-NMR(300MHz，DMSO-d₆):δ/ppm＝7.98(d,J＝8.2Hz,1H),7.36(d,J＝2.9Hz,5H),6.91(d,J＝8.4Hz,1H),5.19(m,2H),4.22(dd,J＝8.2,6.2Hz,1H),4.03(m,1H),2.05(m,1H),1.57(s,2H),1.36(s,10H),0.84(m,12H).

(2) Preparation of HCl Leu-Val-OBzl

Weighing 1.74g (4.14mmol) of Boc-Leu-Val-OBzl in an eggplant bottle, adding a stirrer, slowly dropwise adding 17mL of hydrogen chloride-ethyl acetate solution (4M) under ice-bath stirring, placing the obtained product in a drying tube, reacting for 6 hours under ice-bath stirring, and monitoring by TLC (ethyl acetate/petroleum ether 1:2, Rf is 0.3) to stop the reaction after the raw material point disappears. The reaction solution was concentrated under reduced pressure with a water pump, and the residue was dissolved in 20mL of anhydrous ethyl acetate, then dried under reduced pressure, and repeatedly washed 3 times to give a white solid. Weigh 1.35g, 91.84% yield.

ESI-MS(m/e):321.7[M+H]⁺,355.3[M+Cl]^-；Mp 125.3-126.5℃；[α]²⁵ _D＝-29.99(c＝0.1,CH₃OH)。¹H-NMR(300MHz,CDCl₃):δ/ppm＝8.39(s,2H),7.77(d,J＝7.2Hz,1H),7.32(s,5H),5.13(q,J＝12.2Hz,2H),4.48(t,J＝5.9Hz,1H),4.35(s,1H),2.24(s,1H),1.77(m,1H),1.25(m,2H)0.92(dt,J＝16.2,5.5Hz,12H).

(3) Preparation of Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester

1.00g (3.17mmol) Boc-3- (1-naphthyl) -L-alanine, 0.62g (3.25mmol) EDC, 0.43g (3.19mmol) HOBt in an eggplant flask, adding 30mL acetonitrile to the stirrer, activating for 20min in ice bath, weighing 1.35g (3.79mmol) HCl Leu-Val-OBzl, adding 20mL acetonitrile, dissolving in another eggplant flask, and adding 1mL NMM to adjust pH to 9. Then the two reaction solutions are mixedMix and react for 12 hours, and the reaction is monitored by TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.3) and the starting material spot disappears. Removing organic solvent with rotary evaporator to obtain light yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain light yellow suspension, adding distilled water, and clarifying and layering the solution. Transferring the mixed solution to a separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And saturated NaCl was extracted and washed 3 times each. With anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness on a rotary evaporator. Silica gel column chromatography (ethyl acetate: petroleum ether 1: 5) using ethyl acetate and petroleum ether gave the title product as a white solid, weighing 1.67g, 85.20% yield.

ESI-MS(m/e):640.7[M+Na]⁺,616.6[M-H]^-；Mp 152.9-154.2℃；[α]²⁵ _D＝-59.99(c＝0.1,CH₃OH).

¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.15(d,J＝8.04Hz,1H),7.88(d,J＝7.53Hz,1H),7.78(d,J＝7.86Hz,1H),7.54(m,2H),7.37(m,6H),7.28(s,1H),6.52(d,J＝8.19Hz,1H),6.37(d,J＝7.35Hz,1H),5.18(q,J＝12.21Hz,2H),4.52(m,2H),4.41(m,1H),3.66(m,1H),3.47(m,1H),2.19(m,1H),1.95(m,1H),1.38(m,10H),0.90(m,12H).

(4) Preparation of HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester

To a flask containing 1.67g (2.71mmol) of Boc-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester in eggplant, a stirrer was added, and 16mL of a hydrogen chloride-ethyl acetate solution (4M) was slowly added dropwise with stirring in ice bath, and the mixture was placed in a drying tube, reacted for 6 hours with stirring in ice bath, and then the reaction was terminated. The reaction was stopped by TLC monitoring (ethyl acetate/petroleum ether 1:2, Rf ═ 0.3) disappearance of starting material spot. The reaction solution was dried under reduced pressure with a water pump, and the residue was dissolved in 20mL of dry ethyl acetate, dried under reduced pressure, and washed repeatedly for 3 times. A white solid was obtained, weighing 1.45g, with a yield of 97.32%.

ESI-MS(m/e):518.7[M+H]⁺,552.6[M+Cl]^-；Mp 209.4-210.6℃；[α]²⁵ _D＝-41.72(c＝0.1,CH₃OH).

¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.72(d,J＝8.04Hz,1H),8.42(s,4H),8.32(d,J＝7.17Hz,1H),7.94(d,J＝7.41Hz,1H),7.84(m,1H),7.56(m,2H),7.35(s,7H),5.10(d,J＝3.27Hz,2H),4.50(d,J＝7.47Hz,1H),4.17(t,J＝6.30Hz,2H),3.47(s,2H),3.34(s,2H),3.17(s,1H),2.50(s,4H),2.10(m,1H),1.61(m,2H),1.38(m,2H),0.92(t,J＝8.58Hz,6H),0.84(s,6H).

(5) Preparation of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester

Weighing 150mg (0.53mmol) of oleic acid, 86mg (0.45mmol) of EDC and 61mg (0.45mmol) of HOBt into an eggplant bottle, adding a stirrer, adding 20mL of acetonitrile, and activating for 20min in ice bath; 250mg (0.45mmol) of HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester in another Era bottle was dissolved by adding 20mL acetonitrile and NMM was added to adjust the pH to 9. The two reaction solutions were mixed and reacted for 12 hours, followed by monitoring the reaction by TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.5), and the reaction was terminated after the starting material spot disappeared. Removing organic solvent with rotary evaporator to obtain light yellow viscous sample, adding ethyl acetate, dissolving, adding n-butanol, and clarifying and layering. Pouring the mixed solution into a separating funnel, discarding the lower layer liquid, reserving the upper n-butanol layer, and using saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And washing with saturated NaCl for 3 times. And (3) rotating out n-butanol by a rotary evaporator, adding silica gel, stirring, and performing silica gel column chromatography separation by using ethyl acetate and petroleum ether, wherein the ethyl acetate: petroleum ether 1:3 the expected product is obtained, weighing 280mg, yield 79.67%.

ESI-MS(m/e):782.8[M+H]⁺,Mp 55.5-56.5℃；[α]²⁵ _D＝-20.99(c＝0.1,CH₃OH).

¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.13(m,4H),7.52(tt,J＝6.8,5.3Hz,2H),7.36(m,7H),5.32(m,3H),5.11(m,2H),4.69(td,J＝9.4,9.0,4.0Hz,1H),4.47(q,J＝7.8Hz,1H),4.22(dd,J＝8.0,6.1Hz,1H),3.51(dd,J＝14.3,4.3Hz,1H),3.14(m,1H),2.13(m,2H),1.97(q,J＝7.0,5.9Hz,7H),1.44(dd,J＝8.6,6.0Hz,3H),1.25(m,21H),1.14(m,4H),1.00(s,2H),0.86(m,15H).

(6) Preparation of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine

280mg (0.36mmol) of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester are added to 2mL of methanol without dissolution; adding 4mL tetrahydrofuran, heating at 40 deg.C to dissolve, adding 2mL 2N NaOH, stirring at room temperature for 12 hr, monitoring the reaction by TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.5), allowing the starting material spot to disappear, and adding saturated KHSO₄Adjusting pH to 7 to terminate the reaction, removing tetrahydrofuran by rotary evaporator, adding saturated KHSO₄Adjusting pH to 3, adding ethyl acetate, transferring to separating funnel, separating water layer, collecting ethyl acetate layer, washing water layer with ethyl acetate for 2 times, adding saturated NaCl, separating water layer, collecting ethyl acetate layer, washing for 2 times, and adding anhydrous Na₂SO₄Drying for 2 hours. Filtration under reduced pressure using a water pump removed the desiccant and the ethyl acetate was removed on a rotary evaporator to give an oil weighing 180mg with a yield of 72.58%.

ESI-MS(m/e):690.8[M-H]^-；[α]²⁵ _D＝-18.67(c＝0.1,CH₃OH)；IR(cm^-1)：3278.59，3066.12，3005.54，2955.32，2922.69，2852.49，1725.84，1639.10，1597.89，1540.41，1454.70，1396.46，1370.01，1202.26，1164.49，1122.04，1081.17，1020.80，967.28，907.54，886.24，790.54，775.46，722.19，695.97；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝12.47(s,1H),8.27–8.04(m,2H),7.95–7.81(m,1H),7.76(d,J＝7.5Hz,1H),7.52(dt,J＝13.0,6.8Hz,2H),7.45–7.24(m,3H),5.42–5.22(m,2H),4.70(td,J＝9.4,4.0Hz,1H),4.55–4.36(m,1H),4.16(dd,J＝8.4,5.6Hz,1H),3.61–3.35(m,1H),3.16(td,J＝14.3,13.1,9.1Hz,1H),2.41–2.02(m,1H),2.09–1.89(m,6H),1.57(m,3H),1.39–1.21(m,18H),1.21–1.09(m,4H),0.88(m,15H).

¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.20,172.54,172.41,171.69,134.31,133.82,132.12,130.09,128.96,128.47,127.69,127.35,127.06,126.87,126.43,125.91,125.66,124.25,57.49,53.44,51.57,41.32,35.66,34.85,31.72,30.41,29.55,29.27,29.12,29.05,29.00,28.93,28.86,27.09,25.58,24.59,23.55,22.53,22.19,19.51,18.40,14.37.

The overall yield of the product was 36.49% and was designated as compound 1.

Example 2 preparation of ((S) -3- (naphthalen-1-yl) -2-stearamidopropanoyl) -leucyl-valine

(1) Preparation of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester reference example 1;

(2) preparation of ((S) -3- (naphthalen-1-yl) -2-stearamidopropionyl) -leucyl-valine

280mg (0.36mmol) of ((S) -3- (naphthalen-1-yl) -2-oleamidopropionyl) -leucyl-valine benzyl ester are added to methanol without dissolution; 30mL of tetrahydrofuran was added to the reaction mixture,heating to dissolve, adding 30mg of palladium-carbon, connecting a reaction bottle and a hydrogen bag by using a three-way pipe, pumping air in a reaction solution by using a vacuum water pump, introducing hydrogen, repeating the steps for 3 times to ensure that the air in the eggplant bottle is exhausted and is fully distributed with the hydrogen, stirring at room temperature for 6 hours, monitoring the reaction by TLC (ethyl acetate/petroleum ether 1:2 and Rf is 0.5), stopping the reaction when the raw material point disappears, filtering the palladium-carbon, evaporating the filtrate by using a rotary evaporator, weighing 228g, and obtaining the yield of 91.94%.

ESI-MS(m/e):694.7[M+H]⁺，692.9[M-H]^-；[α]²⁵ _D＝-18.67(c＝0.1,CH₃OH)；

IR(cm^-1)：3271.92，3065.54，2954.52，2917.87，2849.64，1706.54，1634.58，1543.11，1468.41，1427.99，1393.68，1291.09，1273.25，1257.45，1222.70，1192.81，1161.12，1117.81，1080.98，1020.85，926.79，852.46，788.55，770.21，720.98，699.28，640.08.

¹H NMR(300MHz,DMSO-d₆):δ/ppm＝12.60(s,1H),8.16(ddd,J＝14.0,8.2,3.5Hz,3H),7.96–7.82(m,2H),7.76(dd,J＝7.8,1.7Hz,1H),7.53(pd,J＝6.8,1.5Hz,2H),7.45–7.29(m,2H),4.70(td,J＝9.5,4.0Hz,1H),4.53–4.36(m,1H),4.15(dd,J＝8.4,5.6Hz,1H),3.53(dd,J＝14.2,4.1Hz,1H),3.13(dd,J＝14.3,10.1Hz,1H),2.17–1.87(m,3H),1.62(dq,J＝12.6,6.4Hz,1H),1.50(dd,J＝8.5,6.0Hz,2H),1.24(s,19H),1.36–1.07(m,9H),1.01(t,J＝4.9Hz,2H),0.88(ddd,J＝11.0,6.5,2.8Hz,15H).

¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.23,172.55,172.39,171.70,134.31,133.81,132.12,128.96,127.70,127.35,126.43,125.91,125.66,124.26,57.51,53.43,51.56,41.32,35.66,34.87,31.75,30.42,29.49,29.46,29.31,29.25,29.15,28.86,25.59,24.58,23.55,22.54,22.19,19.52,18.40,14.39.

The overall yield of the product was 46.22% and was designated as compound 2.

Example 32 ((N)⁶-L-arginyl) -N²Preparation of (E) -stearoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine

(1)HCl·NH₂Preparation of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester reference example 1

(2) Preparation of N⁶- (tert-butyloxycarbonyl) -N²-oleoyl lysine benzyl ester

Weighing 1.62g (5.74mmol) of oleic acid, 1.1g (5.76mmol) of EDC and 0.78g (5.78mmol) of HOBt into an eggplant bottle, adding a stirrer, adding 30mL of acetonitrile, and activating for 20min in ice bath; then, 1.9g (5.11mmol) of HCl & Lys (Boc) -OBzl was added, 2mL of NMM was added to adjust pH to 8-9, the reaction was allowed to stand overnight, the solution was clarified, the reaction was monitored by TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.5), the point of the chromogenic starting material for tritonone disappeared, and the reaction was terminated. Removing acetonitrile by a rotary evaporator to obtain a light yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain a white turbid liquid, adding distilled water, dissolving, clarifying and layering. Transferring to separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And washing with saturated NaCl for 3 times. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄Evaporating the filtrate with rotary evaporatorDrying gave an oil which was chromatographed on silica gel using ethyl acetate and petroleum ether (ethyl acetate: petroleum ether 1: 2) to give the title product as a white wax which was weighed 2.3g, 75.02% yield.

ESI-MS(m/e):601.8[M+H]⁺；Mp 58.1-58.8℃.¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.16(d,J＝7.4Hz,1H),7.35(s,3H),7.35(d,J＝9.9Hz,2H),6.76(t,J＝5.6Hz,1H),5.32(t,J＝4.6Hz,2H),5.10(s,2H),4.23(dt,J＝8.1,4.2Hz,1H),2.86(q,J＝6.5Hz,2H),2.10(t,J＝7.4Hz,2H),1.98(p,J＝6.5,5.6Hz,4H),1.74–1.54(m,2H),1.46(t,J＝6.8Hz,2H),1.36(s,9H),1.24(s,26H),0.91–0.78(m,3H).

(3) Preparation of N²-oleoyl lysine benzyl ester

To a solution containing 2.3g (3.83mmol) of N⁶- (tert-butyloxycarbonyl) -N²Adding a stirrer into an eggplant bottle of oleoyl lysine benzyl ester, slowly dropwise adding 23mL of hydrogen chloride-ethyl acetate solution (4M) under ice-bath stirring, placing the eggplant bottle in a drying tube, reacting for 6 hours under ice-bath stirring, and terminating the reaction. TLC (ethyl acetate/petroleum ether 1:2, Rf ═ 0.5) and the reaction was stopped by disappearance of the starting spot. The reaction solution was dried under reduced pressure with a water pump, and the residue was dissolved in 20mL of ethyl acetate, dried under reduced pressure, and washed repeatedly for 3 times. An oil was obtained weighing 1.73g, 84.23% yield.

ESI-MS(m/e):501.2[M+H]⁺；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.08(s,1H),7.35(s,5H),5.32(t,J＝4.7Hz,2H),5.11(s,2H),4.23(dq,J＝8.3,3.8,3.2Hz,1H),2.71(tt,J＝7.6,3.6Hz,2H),2.11(t,J＝7.4Hz,2H),1.97(td,J＝15.5,12.9,7.1Hz,4H),1.68(dd,J＝11.4,5.5Hz,1H),1.68–1.53(m,1H),1.60–1.40(m,3H),1.31(t,J＝12.6Hz,5H),1.23(s,18H),0.91–0.78(m,3H).

(4) Preparation of N⁶-(N²- (tert-butyloxycarbonyl) -N^ω-nitro-arginyl) -N²-oleoyl-lysine benzyl ester

1g (3.13mmol) of Boc-Arg (NO)₂) Adding 0.6g (3.14mmol) EDC and 0.43g (3.19mmol) HOBt into eggplant bottle, adding 20mL acetonitrile into the stirring bar, and activating for 20min in ice bath; 1.73g (3.23mmol) of HCl & N²Oleoyl lysine benzyl ester in another eggplant bottle10mL of tetrahydrofuran was added to dissolve, and 1mL of NMM was added to adjust the pH to 8-9. The two reaction solutions were mixed and reacted overnight, the solution was cloudy, the reaction was monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.5), the starting material spot disappeared and the reaction was stopped. Removing solvent with rotary evaporator, adding ethyl acetate, performing ultrasonic treatment to obtain white turbid solution, adding distilled water, dissolving, clarifying and layering. Transferring to separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And saturated NaCl 3 times, respectively, with compound loss. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄Evaporating the filtrate by using a rotary evaporator, and performing silica gel column chromatography separation by using methanol and dichloromethane, wherein the mass ratio of methanol: dichloromethane 1:15 the expected product is obtained as a white wax weighing 2.2g with a yield of 87.75%.

(5) Preparation of N⁶-(N²- (tert-butoxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oleoyl-L-lysine

2.2g (2.74mmol) (N)⁶-(N²- (tert-butoxycarbonyl) -N^ω-nitro-arginyl) -N²Oleoyl-lysine benzyl ester was dissolved in 2mL of methanol, 2N NaOH 4mL was added, the pH was adjusted to 13-14, the mixture was stirred at room temperature for 12 hours, the reaction was monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.5), the starting material spot disappeared, and saturated KHSO was added₄Adjusting pH to 7 to terminate the reaction, removing the solvent by rotary evaporator, adding saturated KHSO₄The pH was adjusted to 4, and ethyl acetate was added to dissolve, and salt was precipitated. Filtration under reduced pressure using a water pump removed salts and a rotary evaporator removed ethyl acetate to give a white solid weighing 1.75g, 89.74% yield.

(6) Preparation 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester

1.75g (2.46mmol) N⁶-(N²- (tert-butoxycarbonyl) -N^ω-nitro-L-arginyl) -N²-oleoyl-L-lysine,Adding 0.53g (2.77mmol) EDC and 0.37g (2.74mmol) HOBt into eggplant bottle, adding 20mL dry tetrahydrofuran into stirring rod, and activating for 20min under ice bath; 1.4g (2.53mmol) of HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester in another Era bottle was dissolved by adding 10mL of dry tetrahydrofuran, and then adding 1mL of NMM to adjust the pH to 8-9. The two reaction solutions were mixed and reacted overnight, the solution was cloudy, the reaction was monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.5), the starting material spot disappeared and the reaction was stopped. Removing solvent with rotary evaporator, adding ethyl acetate, performing ultrasonic treatment to obtain white turbid solution, adding distilled water, dissolving, clarifying and layering. Transferring to separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And washing with saturated NaCl for 3 times. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness on a rotary evaporator and subjected to silica gel column chromatography using methanol and dichloromethane (methanol: dichloromethane 1: 15) to give the objective product as a white solid, weighing 2.7g, and giving a yield of 90.71%.

ESI-MS(m/e):1233.7[M+Na]⁺；1246.2[M+Cl]^-；Mp ¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.49(s,1H),8.16(d,J＝8.1Hz,1H),8.07(d,J＝7.8Hz,1H),8.00(s,2H),7.88(t,J＝8.3Hz,3H),7.77(s,2H),7.52(s,2H),7.40–7.26(m,6H),6.78(d,J＝7.9Hz,1H),5.31(d,J＝5.0Hz,2H),5.20–5.01(m,2H),4.64(s,1H),4.52–4.39(m,1H),4.20(t,J＝7.2Hz,1H),4.10–3.99(m,2H),3.85(s,1H),3.17(d,J＝5.2Hz,1H),3.12(s,2H),2.96(s,3H),2.07(s,4H),1.96(s,4H),1.57(s,2H),1.45(s,9H),1.30(d,J＝42.6Hz,33H),0.85(td,J＝9.8,8.4,5.0Hz,15H).¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.11,172.56,172.29,171.52,171.09,155.72,136.30,134.04,133.81,132.14,130.07,128.99,128.82,128.50,128.42,128.19,127.61,127.39,126.49,125.94,125.69,124.12,78.55,66.32,57.95,54.44,53.56,53.41,51.41,49.06,35.58,31.70,30.29,29.58,29.52,29.45,29.25,29.12,29.10,29.01,28.62,27.05,27.01,25.59,24.46,23.46,22.51,22.08,19.33,18.61,14.37.

(7) Preparation 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine

270mg (0.223mmol) of 2 (N)⁶-(N²- (tert-butyloxycarbonyl-N)^ω-nitro-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine benzyl ester was dissolved in 10mL of methanol, 30mg of palladium on carbon was added, the reaction flask and hydrogen bag were connected using a three-way tube, air in the reaction solution was first pumped out with a vacuum pump, then hydrogen was introduced, and this was repeated 3 times to ensure that the air in the eggplant flask was purged and filled with hydrogen, stirred at room temperature for 6 hours, and monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.5), the starting material spot disappeared, the reaction was terminated, palladium on carbon was filtered, the filtrate was evaporated to dryness with a rotary evaporator, 220mg was weighed, and the yield was 91.67%.

ESI-MS(m/e):1078.9[M+H]⁺；[α]²⁵ _D＝-21.11(c＝0.1,CH₃OH)；Mp ¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.45(s,1H),8.42(s,1H),8.17(t,J＝7.9Hz,2H),7.90(d,J＝7.8Hz,2H),7.82–7.70(m,2H),7.53(dq,J＝14.2,7.0Hz,3H),7.35(d,J＝5.6Hz,3H),4.68–4.55(m,2H),4.47(s,1H),4.24(s,1H),3.94(t,J＝13.1Hz,1H),3.76(s,1H),3.64(d,J＝13.6Hz,1H),3.19(d,J＝13.9Hz,1H),3.08(s,2H),2.95(d,J＝12.1Hz,1H),2.01(dd,J＝14.0,7.2Hz,4H),1.65(s,2H),1.55–1.31(m,21H),1.22(s,26H),1.17(s,2H),0.87(p,J＝6.9,5.7Hz,15H).

(8) Preparation 2 ((N)⁶-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine

To a solution containing 220mg (0.20mmol) of 2 (N)⁶-(N²- (tert-Butoxycarbonyl-L-arginyl) -N²-oleoyl-lysyl) -amino-3- (1-naphthyl) -propionyl-leucyl-valine to eggplant flask, adding stirring3mL of a hydrogen chloride-ethyl acetate solution (4M) was slowly added dropwise with stirring in an ice bath, and the mixture was placed in a drying tube and reacted for 6 hours with stirring in an ice bath, followed by terminating the reaction. TLC monitoring (methanol/dichloromethane 1:10, Rf ═ 0.3) stopped the reaction by disappearance of the starting material spot. The reaction solution was dried under reduced pressure with a water pump, and the residue was dissolved in 10mL of ethyl acetate, dried under reduced pressure, and washed repeatedly for 3 times. A white solid was obtained, weighing 200mg, yield 93.46%.

ESI-MS(m/e):978.7[M+H]⁺；976.5[M-H]^-；[α]²⁵ _D＝-19.67(c＝0.1,CH₃OH)；Mp 224.8-226.4℃；IR(cm^-1)：3273.07，3065.55，2920.22，2850.18，1715.58，1640.78，1537.63，1463.60，1455.14，1393.25，1370.94，1263.34，1214.84，1156.06，775.75，718.63，678.41，666.61；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.69(s,1H),8.56(s,1H),8.30(s,3H),8.26–8.13(m,2H),8.09(s,1H),7.94(dd,J＝22.3,7.4Hz,3H),7.86–7.71(m,2H),7.53(t,J＝7.6Hz,2H),7.36(d,J＝5.3Hz,2H),4.64(d,J＝11.1Hz,1H),4.48–4.34(m,1H),4.20–3.97(m,1H),4.08(s,1H),3.78(s,5H),3.57(d,J＝12.6Hz,1H),3.20(td,J＝19.4,16.9,8.1Hz,2H),3.03(s,2H),2.16–1.87(m,5H),1.82–1.56(m,3H),1.56–1.31(m,9H),1.22(d,J＝2.9Hz,26H),1.06(s,1H),0.87(ddt,J＝11.2,6.9,4.0Hz,14H).¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.19,173.15,172.52,172.37,172.25,171.21,168.49,157.30,134.05,133.80,132.10,129.00,127.66,127.40,126.53,125.97,125.71,124.16,57.56,53.53,52.38,52.21,51.56,35.58,31.73,30.32,29.47,29.43,29.39,29.26,29.13,28.95,28.67,25.61,24.60,24.49,23.55,23.18,22.54,22.06,21.51,19.50,18.41,14.40.

The total yield of the product was 24.40% and was designated as compound 3.

Example 4 preparation of (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine

(1)HCl·NH₂Preparation of (E) -3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester reference example 1;

(2) preparation of 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid tert-butyl ester

Weighing 296mg (1.72mmol) of decanoic acid, 328mg (1.72mmol) of EDC and 232mg (1.72mmol) of HOBt into an eggplant bottle, adding a stirrer, adding 10mL of acetonitrile, and activating for 20min in ice bath; 550mg (1.71mmol) of 1-amino-3, 6,9, 12-tetraoxapentadecane pentadecanoic acid tert-butyl ester are dissolved in another eggplant flask by adding 10mL of acetonitrile, and 1mL of NMM is added to adjust the pH to 8-9. The two reaction solutions were mixed and reacted overnight, the solution was clear, the reaction was monitored by TLC (methanol/dichloromethane 1:10, Rf ═ 0.5), the spot of the triketone chromogenic starting material disappeared, the spot of the product was visualized by iodine fumigation, and the reaction was terminated. Removing acetonitrile by a rotary evaporator to obtain a light yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain a white turbid liquid, adding distilled water, dissolving, clarifying and layering. Transferring to separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And saturated NaCl 3 times, respectively, with compound loss. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness using a rotary evaporator to give an oil which was 677mg, weighed, 82.85% yield.

(3) Preparation of 17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanoic acid

To an eggplant flask containing 677mg (1.43mmol) of 17-oxo-4, 7,10, 13-tetraoxa-16-azahexa-tert-butyl ester was added a stirrer, 7mL of a hydrogen chloride-ethyl acetate solution (4M) was slowly dropped under stirring in an ice bath, and the mixture was placed in a drying tube and reacted for 6 hours under stirring in an ice bath, followed by terminating the reaction. TLC monitoring (methanol/dichloromethane 1:10, Rf ═ 0.5) and the reaction was stopped by disappearance of the iodine fumigated material spot. The reaction solution was dried under reduced pressure with a water pump, and the residue was dissolved in 20mL of ethyl acetate, dried under reduced pressure, and washed repeatedly for 3 times. An oil was obtained weighing 560mg, 94.28% yield.

ESI-MS(m/e):420.7[M+H]⁺,418.3[M-H]^-；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝12.10(s,1H),7.86(t,J＝5.6Hz,1H),4.05(q,J＝7.1Hz,1H),3.60(dt,J＝8.1,6.3Hz,2H),3.49(s,4H),3.44–3.31(m,3H),3.17(q,J＝5.8Hz,2H),2.44(t,J＝6.3Hz,1H),2.04(t,J＝7.4Hz,2H),1.91(s,1H),1.46(p,J＝7.0Hz,4H),1.27(s,1H),1.23(s,14H),1.17(t,J＝7.1Hz,1H),0.92–0.78(m,3H).

(4) Preparation of (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) aminopropionyl) -leucyl-valine benzyl ester

560mg (1.34mmol) 17-oxo-4, 7,10, 13-tetraoxa-16-azahexadecanoic acid, 255mg (1.34mmol) EDC, 180mg (1.34mmol) HOBt are put in an eggplant bottle, a stirrer is added, 10mL acetonitrile is added, and activation is carried out for 20min under ice bath; 740mg (1.34mmol) of HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester in another eggplant flask was dissolved by adding 10mL of acetonitrile, and NMM1mL was added to adjust the pH to 8-9. The two reaction solutions were mixed and reacted overnight, the solution was clear, the reaction was monitored by TLC (methanol/dichloromethane 1:10, Rf ═ 0.5), the dot of the chromone chromogenic material disappeared, and the reaction was terminated. Removing acetonitrile by a rotary evaporator to obtain a light yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain a white turbid liquid, adding distilled water, dissolving, clarifying and layering. Transferring to separating funnel, discarding the lower layer liquid, retaining the upper ethyl acetate layer, and adding saturated NaHCO₃Saturated NaCl and saturated KHSO₄Saturated NaCl and saturated NaHCO₃And saturated NaCl 3 times, respectively, with compound loss. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness on a rotary evaporator, and subjected to silica gel column chromatography using methanol and methylene chloride (methanol: methylene chloride 1: 10) to give the objective product as an oil, weighing 757mg, and giving a yield of 61.54%.

ESI-MS(m/e):919.5[M+H]⁺，941.4[M+Na]⁺，953.7[M+Cl]^-；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.24–8.05(m,3H),8.00–7.71(m,3H),7.53(pd,J＝7.0,1.9Hz,2H),7.37(qd,J＝5.2,4.4,2.1Hz,7H),5.21–5.02(m,2H),4.46(s,1H),4.20(ddd,J＝12.2,9.1,6.9Hz,1H),4.04(dq,J＝8.6,7.1Hz,2H),3.69–3.27(m,16H),3.17(qd,J＝6.5,6.0,1.9Hz,2H),2.26(t,J＝6.4Hz,2H),2.17–1.96(m,2H),1.99(s,2H),1.44(t,J＝7.3Hz,4H),1.28–1.10(m,16H),0.87(ddd,J＝13.1,7.5,5.4Hz,15H).

(5) Preparation of (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacosanyl) propionyl) -leucyl-valine

757mg (0.82mmol) of (S) -3- (naphthalen-1-yl) -2- (17-oxo-4, 7,10, 13-tetraoxa-16-azahexacarbamic acid) propionyl) -leucyl-valine benzyl ester was dissolved in 20mL of methanol, 80mg of palladium on carbon was added, the reaction flask and hydrogen bag were connected using a three-way tube, air in the reaction solution was first removed by a vacuum pump, then hydrogen was introduced, this was repeated 3 times to ensure that the air in the flask was purged with hydrogen, the mixture was stirred at room temperature for 6 hours, and the reaction was monitored by TLC (methanol/dichloromethane 1:10, Rf ═ 0.5), the starting material spot disappeared, the reaction was terminated, palladium on carbon was filtered, the filtrate was evaporated to dryness with a rotary evaporator, 634mg was weighed, and the yield was 92.96%.

ESI-MS(m/e):851.7[M+Na]⁺，828.0[M-H]^-；[α]²⁵ _D＝-11.67(c＝0.1,CH₃OH)；IR(cm^-1)：3283.37，3067.15，2955.59，2924.62，2869.30，1732.03，1640.46，1538.27，1465.38，1386.96，1369.58，1349.61，1250.44，1210.59，1110.13，1031.54，945.93，882.96，857.29，791.88，777.89，722.38，666.59；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.21(m,2H),7.83(m,3H),7.54(q,J＝6.7Hz,2H),7.39(td,J＝6.7,6.0,3.5Hz,1H),4.08(m,2H),3.62(m,3H),3.49(m,9H),3.38(dp,J＝8.7,3.0Hz,6H),3.17(s,4H),2.25(m,1H),2.05(q,J＝7.4,6.6Hz,2H),1.65(m,1H),1.46(s,2H),1.21(m,11H),0.88(m,15H).¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.24,172.73,172.40,171.55,170.44,134.15,133.81,132.16,128.97,127.74,127.40,126.45,125.95,125.73,124.27,70.24,69.61,67.08,66.47,60.30,57.58,53.48,51.59,49.06,38.90,36.39,35.77,35.11,31.73,30.40,29.35,29.24,29.12,29.08,25.72,24.58,23.53,22.54,22.18,19.53,18.43,14.52,14.40.

The overall yield of the product was 28.19%, and was designated as compound 4.

Example 5 preparation of ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine

(1)HCl·NH₂-3- (1-naphthyl) -L-propionyl-leucinePreparation of benzyl aminoacyl-valine reference example 1;

(2) preparation of ((S) -2- (28-tert-Butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester

215mg (0.39mmol) of N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid, 75mg (0.39mmol) of EDC and 53mg (0.39mmol) of HOBt are weighed in an eggplant bottle, a stirrer is added, 10mL of acetonitrile is added, and activation is carried out for 20min in ice bath; 217mg (0.39mmol) of HCl & NH₂-3- (1-naphthyl) -L-propionyl-leucyl-valine benzyl ester in another eggplant flask was dissolved by adding 10mL of acetonitrile, and 1mL of NMM was added to adjust the pH to 8-9. The two reaction solutions were mixed and reacted overnight, the solution was clarified, the reaction was monitored by TLC (ethyl acetate/petroleum ether 1:1 or methanol/dichloromethane 1:15, Rf ═ 0.3), the ninhydrin color-developing material spot disappeared, and the reaction was terminated. Removing acetonitrile by a rotary evaporator to obtain a light yellow viscous sample, adding ethyl acetate, performing ultrasonic treatment to obtain a white turbid liquid, adding distilled water, dissolving, clarifying and layering. This was transferred to a separatory funnel, the lower liquid was discarded, and the upper ethyl acetate layer was retained. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄Evaporating the filtrate by using a rotary evaporator, adding silica gel, mixing the silica gel with a sample, and performing silica gel column chromatography separation by using methanol and dichloromethane, wherein the methanol: dichloromethane 1:15 the desired product was obtained as an oil weighing 300mg, 73.53% yield.

ESI-MS(m/e):1063.8[M+Na]⁺；[α]²⁵ _D＝-20.99(c＝0.1,CH₃OH)。¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.16(s,2H),8.23–8.01(m,2H),7.90(dd,J＝7.1,2.2Hz,1H),7.83–7.71(m,1H),7.52(ddt,J＝10.4,6.9,3.6Hz,2H),7.45–7.27(m,7H),6.73(s,1H),5.21–5.02(m,2H),4.70(td,J＝8.8,4.4Hz,1H),4.46(q,J＝7.7Hz,1H),4.22(dd,J＝7.9,6.2Hz,1H),3.59–3.31(m,H),3.24–3.10(m,2H),3.06(q,J＝6.0Hz,2H),2.26(t,J＝6.7Hz,2H),2.09(h,J＝6.8Hz,1H),1.60(dt,J＝13.4,6.9Hz,1H),1.44(t,J＝7.1Hz,2H),1.37(s,9H),0.96–0.79(m,15H).

(3) Preparation of ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propanoyl-leucyl-valine benzyl ester

To a flask containing 300mg (0.29mmol) of ((S) -2- (28-tert-butoxycarbonyl-28-aza-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propionyl-leucyl-valine benzyl ester was added a stirrer, and 5mL of a hydrogen chloride-ethyl acetate solution (4M) was slowly added dropwise with stirring in an ice bath, and the mixture was placed in a dry tube and reacted for 4 hours under stirring in an ice bath, whereupon the reaction was terminated. TLC monitoring (methanol/dichloromethane 1:15, Rf ═ 0.3) stopped the reaction by disappearance of the starting material spot. The reaction solution was dried under reduced pressure with a water pump, and the residue was dissolved in 10mL of ethyl acetate, dried under reduced pressure, and washed repeatedly for 3 times. An oil was obtained, weighing 270mg, 96.08% yield.

ESI-MS(m/e):941.8[M+H]⁺，975.9[M+Cl]^-；[α]²⁵ _D＝-20.99(c＝0.1,CH₃OH)。¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.32(s,1H),8.15(td,J＝12.1,11.2,7.9Hz,3H),8.01–7.70(m,2H),7.66–7.45(m,2H),7.45–7.27(m,7H),5.21–5.01(m,2H),4.70(td,J＝8.9,4.5Hz,1H),4.57–4.37(m,1H),4.29–4.11(m,1H),4.03(q,J＝7.1Hz,3H),3.67–3.46(m,9H),3.55–3.30(m,8H),3.16(dd,J＝14.3,9.4Hz,1H),2.96(q,J＝5.5Hz,1H),2.26(t,J＝6.6Hz,2H),1.95(d,J＝23.6Hz,12H),1.66–1.53(m,1H),1.52–1.32(m,2H),1.18(t,J＝7.1Hz,5H),0.99–0.78(m,12H).

(4) Preparation of ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octyloxy-28-azatrioctadecylamido) propanoyl) -leucyl-valine benzyl ester

Putting 48mg (0.28mmol) decanoic acid, 53mg (0.28mmol) EDC and 37mg (0.28mmol) HOBt into an eggplant bottle, adding a stirrer, adding 10mL acetonitrile, and activating for 20min in ice bath; 270mg (0.28mmol) of ((S) -2- (28-amino-4, 7,10,13,16,19,22, 25-octyloxy-dioctadecyl) amino) -3- (naphthalen-1-yl) propanoyl-leucyl-valine benzyl ester in a separate Ergonococcus bottle were dissolved by addition of 10mL of acetonitrile and adjusted to pH 8 by addition of 1mL of NMM. The two reaction solutions were mixed and reacted overnight, the solution was clear, the reaction was monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.3), the ninhydrin color developing starting material spot disappeared, and the reaction was terminated. Removing acetonitrile by a rotary evaporator to obtainAdding ethyl acetate into the light yellow viscous sample, performing ultrasonic treatment to obtain white turbid liquid, adding distilled water, dissolving, clarifying and layering. This was transferred to a separatory funnel, the lower liquid was discarded, and the upper ethyl acetate layer was retained. Adding anhydrous Na₂SO₄Drying for 2 hr, filtering under reduced pressure with water pump to remove anhydrous Na₂SO₄The filtrate was evaporated to dryness by a rotary evaporator, and silica gel was added to the filtrate and stirred, followed by silica gel column chromatography using methanol and dichloromethane (methanol: dichloromethane ═ 1: 15) to obtain the objective product as an oil, which was weighed 257mg and had a yield of 84.96%.

ESI-MS(m/e):1130.2[M+Cl]^-，1117.9[M+Na]⁺；[α]²⁵ _D＝-20.11(c＝0.1,CH₃OH)。¹H NMR(300MHz,DMSO-d₆):δ/ppm＝8.16(td,J＝8.2,3.2Hz,3H),8.04(dd,J＝17.0,8.1Hz,1H),7.90(dd,J＝7.9,1.7Hz,1H),7.78(q,J＝5.1Hz,2H),7.53(tt,J＝6.8,5.2Hz,2H),7.44–7.27(m,7H),5.21–5.02(m,2H),4.70(td,J＝8.8,4.4Hz,1H),4.46(q,J＝7.7Hz,1H),4.23(dd,J＝8.0,6.2Hz,1H),3.62–3.41(m,18H),3.49–3.32(m,5H),3.32(s,7H),3.18(q,J＝6.0Hz,3H),2.33–2.19(m,1H),2.19–1.87(m,4H),1.76(d,J＝22.5Hz,2H),1.60(dt,J＝13.2,6.6Hz,1H),1.46(d,J＝7.0Hz,4H),1.37–1.19(m,2H),1.23(s,8H),0.87(dt,J＝13.2,6.7Hz,15H).

(5) Preparation of ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octaoxy-28-azatrioctadecanamido) propionyl) -leucyl-valine

257mg (0.23mmol) ((S) -3- (naphthalen-1-yl) -2- (29-oxo-4, 7,10,13,16,19,22, 25-octaoxy-28-azaoctacosanoylamino) propionyl) -leucyl-valine benzyl ester was dissolved in 10mL of methanol, 30mg of palladium carbon was added, the reaction flask and the hydrogen bag were connected by a three-way pipe, air in the reaction solution was first removed by a vacuum pump, then hydrogen was introduced, the reaction flask was kept filled with air, stirring was carried out at room temperature for 6 hours while ensuring that the air in the flask was fully filled with hydrogen was removed, and the reaction was monitored by TLC (methanol/dichloromethane 1:15, Rf ═ 0.3), the reaction was stopped when the starting material had disappeared, palladium carbon was filtered, the filtrate was evaporated by a rotary evaporator, and 220mg was weighed, giving a yield of 93.34%.

ESI-MS(m/e):1004.1[M-H]^-；1027.9[M+Na]⁺；[α]²⁵ _D＝-11.67(c＝0.1,CH₃OH)；IR(cm^-1)：3276.82，3067.92，2954.51，2924.18，2868.62，1722.74，1639.04，1538.67，1464.66，1454.80，1369.48，1349.05，1295.82，1249.54，1212.56，1101.74，947.49，882.27，855.68，779.56，722.41，683.77；¹H NMR(300MHz,DMSO-d₆):δ/ppm＝12.54(s,1H),8.18(td,J＝7.8,5.2Hz,3H),7.96–7.70(m,4H),7.63–7.45(m,2H),7.39(q,J＝3.5Hz,2H),4.70(td,J＝8.8,4.1Hz,1H),4.44(q,J＝7.7Hz,1H),4.15(dd,J＝8.4,5.7Hz,1H),3.56(s,3H),3.53(d,J＝8.9Hz,1H),3.50(s,7H),3.53–3.30(m,16H),3.25–3.06(m,1H),3.17(s,11H),2.25(td,J＝6.7,1.7Hz,2H),2.06(dt,J＝14.5,6.9Hz,2H),1.91(s,1H),1.84–1.61(m,1H),1.67–1.47(m,1H),1.48(d,J＝7.9Hz,3H),1.23(s,8H),1.12(d,J＝8.8Hz,1H),0.97–0.78(m,15H)；¹³C NMR(75MHz,DMSO-d₆):δ/ppm＝173.23,172.67,172.46,171.46,170.41,134.16,133.80,132.16,128.96,127.74,127.39,126.44,125.93,125.72,124.28,70.24,70.19,70.07,70.03,69.84,69.63,67.09,57.54,53.47,51.56,49.06,41.25,38.90,36.40,35.77,35.00,31.74,30.39,29.36,29.26,29.13,29.09,25.72,24.58,23.54,22.55,22.20,18.43,18.40,14.40.

The overall yield of the product was 35.35% and was designated as compound 5.

Performance test 1

(ii) self-assembly Properties of Compound 1

Weighing 5 μmol of compound 1, adding 10mL of 80% ethanol, and performing ultrasonic treatment at 300W for 30min to obtain 5 × 10^-4The transmission electron micrograph of the M solution is shown in figure 1.

Take 1mL 5X 10^-4Adding 9mL of 50% ethanol into the M solution, and performing ultrasonic treatment at 300W for 30min to obtain 5 × 10^-5M solution, measured by a nanometer particle sizer at 5X 10^-5The particle size and Zeta potential of the M solution are shown in Table 1. mu.L of the solution was dropped onto a copper mesh, and dried in an oven at 37 ℃ under a Scanning Electron Microscope (SEM) chart shown in FIG. 2 and a Transmission Electron Microscope (TEM) chart shown in FIG. 3. As can be seen from FIGS. 1 to 3, the particle size of the compound 1 in the ethanol solution is relatively stable, and is about 560 nm.

The tyndall effect produced in each group was observed by irradiating a glass bottle containing the solution with a laser pen (λ ═ 650nm) using water as a control group, and the results are shown in fig. 4; FIG. 4 (a) shows a solution before irradiation, FIG. 4 (b) shows a solution after irradiation, and in the (a) and (b), water and 5X 10 are respectively arranged from left to right^-5M solution, 5X 10^-4And (5) M solution. As can be seen from FIG. 4, 5X 10^-5M solution and 5X 10^-4The M solution has obvious Tyndall effect.

Self-assembly characteristics of (di) Compound 2

The concentration is 5X 10 according to the method^-4Ethanol solution of compound 2 of M and concentration of 5X 10^-5Compound 2 in ethanol.

5×10^-4The transmission electron micrograph of the M solution is shown in FIG. 5, 5X 10^-5The scanning electron micrograph of the M solution is shown in FIG. 6, and the transmission electron micrograph is shown in FIG. 7. Measurement of 5X 10 by means of a Nanoparticometer^-5The particle size and Zeta potential of the M solution are shown in Table 1. As can be seen from FIGS. 5 to 7, the particle size of the compound 2 in the ethanol solution is relatively stable, and is around 780 nm.

The tyndall effect produced in each group was observed by irradiating a glass bottle containing the solution with a laser pen (λ ═ 650nm) using water as a control group, and the result is shown in fig. 8; FIG. 8 (a) shows a solution before irradiation, FIG. 8 (b) shows a solution after irradiation, and in the (a) and (b), water and 5X 10 are respectively arranged from left to right^-5M solution, 5X 10^-4And (5) M solution. As can be seen from FIG. 8, 5X 10^-5M solution and 5X 10^-4The M solution has obvious Tyndall effect.

Self-assembly characteristics of (tri) Compound 3

The concentration is 5X 10 according to the method^-4M Compound 3 in an aqueous solution at a concentration of 5X 10^-5M Compound 3 in aqueous solution and at a concentration of 5X 10^-6M compound 3 in water.

5×10^-4The transmission electron micrograph of the M solution is shown in FIG. 9, 5X 10^-5The scanning electron micrograph of the M solution is shown in FIG. 10, and the transmission electron micrograph is shown in FIG. 11. Measurement of 5X 10 by means of a Nanoparticometer^-5Particle size and Zeta potential of M solutionBits, listed in table 1. As can be seen from FIGS. 9 to 11, the particle size of the compound 3 in water is relatively stable, mainly comprising spherical nanoparticles, and is about 50 nm.

The tyndall effect produced in each group was observed by irradiating a glass bottle containing the solution with a laser pen (λ ═ 650nm) using water as a control group, and the result is shown in fig. 12; FIG. 12 (a) shows a solution before irradiation, FIG. 12 (b) shows a solution after irradiation, and in the (a) and (b), water and 5X 10 are placed in the order from left to right^-6M solution, 5X 10^-5M solution and 5X 10^-4And (5) M solution. As can be seen from FIG. 12, 5X 10^-6M solution, 5X 10^-5M solution and 5X 10^-4The M solution has obvious Tyndall effect.

Self-assembly characteristics of (tetra) Compound 4

The concentration is 5X 10 according to the method^-3Ethanol solution of compound 4 of M at a concentration of 5X 10^-4M Compound 4 in an aqueous solution at a concentration of 5X 10^-5M Compound 4 in aqueous solution and at a concentration of 5X 10^-6M compound 4 in water. Measurement of 5X 10 by means of a Nanoparticometer^-5The particle size and Zeta potential of the M solution are shown in Table 1.

5×10^-5The transmission electron micrograph of the M solution is shown in FIG. 13, and the scanning electron micrograph is shown in FIG. 14. As can be seen from fig. 13 and 14, the particle size of compound 4 in water is relatively stable, and the particle size is around 260 nm.

The tyndall effect produced in each group was observed by irradiating a glass bottle containing the solution with a laser pen (λ ═ 650nm) using water as a control group, and the results are shown in fig. 15; FIG. 15 (a) shows a solution before irradiation, FIG. 15 (b) shows a solution after irradiation, and in the (a) and (b), water and 5X 10 are present in the order from left to right^-6M solution, 5X 10^-5M solution and 5X 10^-4And (5) M solution. As can be seen from FIG. 15, 5X 10^-6M solution, 5X 10^-5M solution and 5X 10^-4The M solution has obvious Tyndall effect.

(V) self-Assembly Properties of Compound 5

The concentration is 5X 10 according to the method^-3Ethanol solution of compound 5 of M, concentration 5X 10^-4M Compound 5 in aqueous solution at a concentration of 5X 10^-5M Compound 5 in aqueous solution and at a concentration of 5X 10^-6M compound 5 in water. Measurement of 5X 10 by means of a Nanoparticometer^-5The particle size and Zeta potential of the M solution are shown in Table 1.

5×10^-5The transmission electron micrograph of the M solution is shown in FIG. 16, and the scanning electron micrograph is shown in FIG. 17. As can be seen from fig. 16 and 17, the particle size of compound 5 in water is relatively stable, and is around 220 nm.

The tyndall effect produced in each group was observed by irradiating a glass bottle containing the solution with a laser pen (λ ═ 650nm) using water as a control group, and the results are shown in fig. 18; FIG. 15 (a) shows a solution before irradiation, FIG. 18 (b) shows a solution after irradiation, and in the (a) and (b), from right to left, water and 5X 10 are respectively^-6M solution, 5X 10^-5M solution and 5X 10^-4And (5) M solution. As can be seen from FIG. 15, 5X 10^-6M solution, 5X 10^-5M solution and 5X 10^-4The M solution has obvious Tyndall effect.

TABLE 1 particle size, Zeta potential and Polymer Dispersion Index (PDI) of Compounds 1 to 5

Performance test 2

Evaluation of cytotoxicity of evaluation Compound on tumor cells by MTT method

1) Semi-suspension cell line (S180)

The S180 cells are semi-suspension cells, adherent growth of the cells can be seen after long-time culture, but cell suspension can be achieved by blowing and beating with a pipette, pancreatin digestion is not needed, and the cells can be passaged when the degree of healing is more than 80%. Gently blowing with a pipette, transferring the cell suspension into a centrifuge tube, setting the centrifuge at 1000rpm, centrifuging for 5min, removing supernatant, and adding new supernatantThe whole medium was transferred to a T25 cell culture flask at 37 ℃ with 5% CO₂The cells were cultured in an incubator, and the growth of the cells was observed.

Taking S180 cells growing in logarithmic phase, counting the cells, placing the cells in plates containing 5000 cells in a volume of 100. mu.l per well at 37 ℃ and containing 5% CO₂Culturing in cell culture box in environment, adding medicine after 2 hr, setting 6 holes for each tested medicine and setting control hole, adding complete culture medium containing 5 ‰ DMSO, placing at 37 deg.C and 5% CO₂The ambient cell incubator was incubated for 48 hours. After 48 hours, 25. mu.L of the prepared MTT solution (5 mg/mL) was added to each well plate, and the mixture was placed in a cell incubator for 4 hours to terminate the incubation. The 96-well plate was taken out and put into a centrifuge for centrifugation at 1000rpm for 10 min. After centrifugation, the 96-well plate is carefully taken out, the culture solution in the well is completely sucked, then 150 mu L of DMSO is added into each well by using the gun head, the 96-well plate is placed on a shaking table to be shaken and shaken uniformly, and the crystal formazan is fully dissolved in the DMSO. Finally, setting wave bands at 490nm and 570nm on an enzyme-linked immunosorbent assay instrument, measuring the absorbance value of each hole, and calculating the survival rate of the cells compared with a control group. IC50 was calculated by linear regression curve fitting of log concentration to percent cell survival using GraphPad Prism 5. The experiment was repeated three times in total and then averaged (results are shown in table 1).

2) Adherent cell line

HCT116 cells, LLC cells, MCF-7 cells, HepG2 cells, A549 cells and A549/TAX cells belong to adherent cells, cells growing in logarithmic phase are taken out of liquid in a culture bottle, 2mL of PBS buffer solution is added for washing twice so as to clean serum remained in the culture bottle, 1mL of pancreatin is added, the culture bottle is placed at 37 ℃ and 5% of CO₂Digesting for 2 minutes in the incubator, observing that cells become spherical flowing, adding 2mL of new complete culture medium to terminate digestion, slightly blowing up by using a suction tube, transferring cell suspension into a centrifuge tube, setting the centrifuge for 1000 revolutions per minute, centrifuging for 5 minutes to remove supernatant, then adding fresh complete culture medium into the tube, blowing and beating by using a sterilized gun headAnd (4) uniformity. Then, cell counting was performed.

Culturing 100 μ l of 5000 cell plates in 37 deg.C cell culture box containing 5% CO2 for 6 hr, adding 6 wells for each drug, adding control well containing 5% DMSO only, adding complete culture medium containing 5% DMSO, and culturing at 37 deg.C cell culture box containing 5% CO₂The ambient cell incubator was incubated for 48 hours. After 48 hours, 25. mu.L of the prepared MTT solution (5 mg/mL) was added to each well plate, and the mixture was placed in a cell incubator for culture for about 4 hours, whereupon the culture was terminated. And (3) taking out the 96-well plate, completely absorbing and removing the solution in the holes, adding 150 mu L of DMSO into each hole by using a gun head, placing the 96-well plate on a shaking table, shaking and shaking uniformly, and fully dissolving the formazan crystal in the DMSO. Finally, setting wave bands at 490nm and 570nm on an enzyme-linked immunosorbent assay instrument, measuring the absorbance value of each hole, and calculating the survival rate of the cells compared with a control group. IC was calculated by linear regression curve fitting of log concentration to percent cell survival using GraphPad Prism5₅₀. The experiment was repeated three times in total and then averaged.

IC of Compounds 1-5 in different cells₅₀The values are shown in Table 2.

(II) evaluation of cytotoxicity of evaluation Compound on Normal liver cell L02 by MTT method

The method is the same as the adherent cell treatment method, and the results are shown in Table 2.

TABLE 2 IC of Compounds 1-5 in different cells₅₀Value of

(III) evaluation of cytotoxicity of micelles against tumor cells by MTT method

Preparing micelles with the mass ratios of the compound 1 to the paclitaxel of 10:1, 20:1 and 30:1 respectively, and recording the micelles as micelle 1;

preparing micelles with the mass ratios of the compound 2 to the paclitaxel of 10:1, 20:1 and 30:1 respectively, and recording the micelles as micelles 2;

preparing micelles with the mass ratios of the compound 3 to the paclitaxel of 10:1, 20:1 and 30:1 respectively, and recording the micelles as the micelles 3;

preparing micelles with the mass ratios of the compound 4 to the paclitaxel of 10:1, 20:1 and 30:1 respectively, and marking the micelles as micelles 4;

micelles with mass ratios of compound 5 to paclitaxel of 10:1, 20:1 and 30:1 were prepared, respectively, and are designated as micelle 5.

Each micelle was tested for cytotoxicity against tumor cells in the same manner as above. The results obtained are shown in Table 3.

TABLE 3 cytotoxicity of the respective micelles against tumor cells

As can be seen from tables 2 and 3, the amino acid derivatives provided by the present invention have an effect of reversing drug resistance of antitumor drugs, and the antitumor micelles prepared using the same have a good antitumor effect and an effect of reversing drug resistance of antitumor drugs.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Amino acid derivative, preparation method and application thereof, anti-tumor micelle and preparation method thereof

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