CN113876784A

CN113876784A - Novel application of boroleucine compound

Info

Publication number: CN113876784A
Application number: CN202111139521.1A
Authority: CN
Inventors: 徐文方; 马玉倩; 戴曰慧; 沈晓艳; 王学健
Original assignee: Weifang High Tech Biological Park Development Co ltd; WEIFANG BOCHUANG INTERNATIONAL ACADEMY OF BIOTECHNOLOGY AND MEDICINE
Current assignee: Weifang High Tech Biological Park Development Co ltd; WEIFANG BOCHUANG INTERNATIONAL ACADEMY OF BIOTECHNOLOGY AND MEDICINE
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2022-01-04
Anticipated expiration: 2041-09-27
Also published as: CN113876784B

Abstract

The invention discloses application of boroleucine compounds in preparation of aminopeptidase N inhibitors and medicines for inhibiting tumor growth. The boroleucine compound is boroleucine with a structure shown in a formula 1 or boroleucine dinoester with a structure shown in a formula 2;

the invention uses the boroleucine and the boroleucine dinoester in the preparation of the APN inhibitor, and can obviously inhibit the activity of the APN. The compounds can be used as APN inhibitor leads for research and development of new drugs, and can also be used for synthesizing APN-targeting fluorescent probes for early tumor discovery.

Description

Novel application of boroleucine compound

Technical Field

The invention relates to the technical field of aminopeptidase N inhibitors, in particular to application of boroleucine and boroleucine dinonyl ester in preparation of aminopeptidase N inhibitors.

Background

Aminopeptidase N (Aminopeptidase N, APN/CD13, EC 3.4.11.2) is a zinc ion-dependent transmembrane metalloprotease that is localized to the outer surface of the cell membrane by non-covalent binding as a homodimer. APN is closely related to the occurrence, development and metastasis of tumors, and the inhibition of APN can inhibit tumor cell metastasis. In addition, APN is a biomarker of semi-resting liver cancer stem cells, and the enhancement of APN expression can promote the survival of the liver cancer stem cells. The above suggests that targeted inhibition of APN may have a good effect on tumor treatment.

Boroleucine ((S) -1-amino-3-methylbutylboronic acid) and boroleucine dinoester ((S) -1-amino-3-methylbutylboronic acid pinane diol ester) belong to alpha-aminoboronic acid derivatives, and are intermediates for preparing proteasome inhibitor bortezomib. In addition, the compounds can inhibit the activity of the metalloprotease enkephalinase (EC 3.4.24.11). Therefore, the compounds are more and more concerned in drug design.

In the process of researching a small-molecule APN inhibitor, the AHPA-LEU dipeptide boric acid citrate compound with APN and proteasome double-target inhibition activity is found to have good antitumor activity, but citrate is unstable, proteasome inhibition activity is weak, and killing activity to tumor cells is not strong enough. Therefore, the search for small molecule compounds which effectively inhibit the activity of APN and are used for research and development of antitumor drugs is always a research hotspot in the field of medicinal chemistry.

Disclosure of Invention

In view of the prior art, the invention aims to provide a new application of boroleucine compounds. According to the invention, the boroleucine and the boroleucine dinoester are used for preparing an APN inhibitor or synthesizing a fluorescent probe targeting aminopeptidase N, and the boroleucine dinoester can obviously inhibit the activity of APN.

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

the invention provides an application of boroleucine compounds in the following 1) or 2):

1) preparing an aminopeptidase N inhibitor;

2) preparing the medicine for inhibiting the growth of the tumor.

Preferably, the boroleucine is used to inhibit tumor growth by inhibiting HUVEC cell two-dimensional tube production.

Preferably, the boroleucine-like compounds inhibit tumor growth by inhibiting tumor cell migration.

Preferably, the boroleucine is used to inhibit tumor growth by inhibiting tumor cell invasion.

Preferably, the boroleucine compound is boroleucine with a structure shown in formula 1 or boroleucine dinoester with a structure shown in formula 2;

the borrelidin is an intermediate for preparing bortezomib, and the preparation method can be seen in the following steps: in the above-phase approach for the synthesis of a-aminobenzonic acid peptides (Blake E.Daniels and Craig E.Stivala, RSC adv.,2018,8, 3343-: a method for preparing Synthesis of boronic acid 8.

The boroleucine dinoester is an intermediate for preparing bortezomib, and the preparation method can be seen in the following steps: application No. 201210156068.X a method for preparing (1R) - (S) -pinanediol-1-amino-3-methylbutane-1-boronic acid ester disclosed in the method for preparing (1R) - (S) -pinanediol-1-amino-3-methylbutane-1-boronic acid ester and salt thereof, and the compound of formula ii prepared by the method has the same structure as boroleucine dinol ester in the present invention.

The invention has the beneficial effects that:

the invention discovers that the brand-new small molecular compounds of boroleucine and boroleucine dinoester can inhibit the enzymatic activity of APN, the APN is closely related to tumor metastasis, and the APN expressed by tumor cells and APN of organisms can promote the tumor cells to metastasize to a far-end tissue to form a metastatic focus. During the tumor metastasis process, the tumor tissue angiogenesis provides convenience for tumor cell metastasis, so that the inhibition of tumor tissue angiogenesis and tumor cell migration and invasion is helpful for inhibiting tumor metastasis. Experiments show that the boroleucine and the boroleucine dinoester can inhibit the generation of two-dimensional tubes of umbilical vein endothelial cells (HUVEC) and the migration and invasion of tumor cells. Therefore, the compound can be used as an APN inhibitor lead for research and development of new drugs, and can also be used for synthesizing APN-targeting fluorescent probes for early tumor discovery.

Drawings

FIG. 1: schematic diagram of docking boroleucine and boroleucine dinoester with APN;

FIG. 2: photographs of experiments inhibiting HUVEC cell two-dimensional tube production;

FIG. 3: photographs of assays for inhibiting breast cancer cell migration;

FIG. 4: quantifying the value of the test for inhibiting the migration of the breast cancer cells;

FIG. 5: photographs of experiments inhibiting breast cancer cell invasion;

FIG. 6: quantifying the value of an experiment for inhibiting the invasion of the breast cancer cells;

FIG. 7: photos of in vivo lung metastasis inhibition assays;

FIG. 8: quantifying lung metastasis pulmonary nodule number in vivo;

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background section, the AHPA-LEU dipeptide boric acid citrate compound with APN and proteasome double target inhibition activity has good antitumor activity, but the citrate is unstable, the proteasome inhibition activity is weak, and the killing activity to tumor cells is not strong enough.

Based on the above, the invention aims to provide a novel application of the boroleucine compound. On the basis of the background technology, further structure-activity relationship research finds that an ILeu protecting group CBZ and a boric acid protecting group dinenol ester are inserted between AHPA and LEU and are all reserved, and the formed new compound boroleucine dinenol ester can greatly improve the APN inhibitory activity and the anti-tumor activity. The level of APN enzyme activity inhibition of the compound is obviously superior to that of the control drugs ubenimex and bortezomib, and the compound also shows good activity in the aspects of two-dimensional tube generation resistance, migration resistance and invasion resistance. APN is closely related to tumor metastasis, and both APN expressed by tumor cells and APN of organisms can promote tumor cells to metastasize to remote tissues to form metastases. During the tumor metastasis process, the tumor tissue angiogenesis provides convenience for tumor cell metastasis, so that the inhibition of tumor tissue angiogenesis and tumor cell migration and invasion is helpful for inhibiting tumor metastasis. Experiments show that the boroleucine and the boroleucine dinoester can inhibit the generation of two-dimensional tubes of umbilical vein endothelial cells (HUVEC) and the migration and invasion of tumor cells. Therefore, the compound can be used as an APN inhibitor lead for research and development of new drugs, and can also be used for synthesizing APN-targeting fluorescent probes for early tumor discovery.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and commercially available.

Description of the drawings: the boroleucine in the examples was prepared according to the preparation of Synthesis of boronic acid 8 as disclosed in A solid-phase aproach for the Synthesis of a-amino boronic acid peptides.

The compound shown in the formula II, namely the boroleucine dinoester, is prepared by the preparation method of (1R) - (S) -pinanediol-1-amino-3-methylbutane-1-boric acid ester disclosed in the method for preparing (1R) - (S) -pinanediol-1-amino-3-methylbutane-1-boric acid ester and salts thereof according to the application number 201210156068.

Example 1 in vitro inhibition of enzymatic activity by boroleucine dinoester:

the method for preparing APN enzyme source (patent application No. 201810629864.8) was used for the evaluation of the aprotinin activity by boroleucine and boroleucine dinonyl ester, and ubenimex positive control 1 and bortezomib as positive control 2.

The test procedure was as follows:

(1) preparing cell homogenate after ultrasonically crushing K562-APN cells, and inoculating the cell homogenate in a 96-well plate;

(2) adding compounds of different concentration gradients to the wells;

(3) adding APN substrate to 1.6mM after 5min, and incubating for 1 h;

(4) the absorbance value at 405nm was measured and IC50 was calculated using Origin software.

Note: the compounds used in the experiments were boroleucine, boroleucine dinoate, ubenimex or bortezomib.

The result shows that the IC50 value of the boraleucine to the APN enzyme inhibition activity is 0.15 +/-0.003 mu M, the IC50 value of the boraleucine dinoester to the APN enzyme inhibition activity is 0.007 +/-0.001 mu M, the positive control 1 ubenimex is 20.167 +/-1.84 mu M, and the positive control 2 bortezomib is 65.69 +/-5.94 mu M, which indicates that the enzyme inhibition effects of the boraleucine and the boraleucine dinoester to the APN are obviously better than those of the ubenimex and the bortezomib.

The invention predicts the combination of boroleucine dinoester and boroleucine with APN active site through molecular docking. As shown in figure 1, hydrogen bonding and hydrophobic interactions facilitate the binding of boroleucine dinonyl ester and boroleucine to the active site of the APN. The boronic acid group in norleucine dinoate can form multiple hydrogen bonding interactions with surrounding residues such as Ala262 and Glu298, with the amino group hydrogen bonding with Glu121, Met263 and Glu 264. In the boronate of boroleucine, one oxygen atom forms hydrogen bond interaction with the amino group of Ala262, and the amino group of boroleucine also has hydrogen bond interaction with the surrounding Glu264 and Glu 320. The residues Met260, Ala262, Met263, Tyr376 and Tyr381 play an important role in the hydrophobic interaction of the APN with boroleucine dinoate and boroleucine. In zinc binding, the boronic acid group of boroleucine dinoester binds to zinc ions, while the amino group of boroleucine also binds to zinc ions. Thus, differences in zinc binding patterns may lead to different enzyme inhibitory activities.

Example 2 inhibition of HUVEC cells by Boroflurane and Boroflurane Dinopate two-dimensional tube Generation experiments

Tumor tissue angiogenesis can provide convenience for tumor cell metastasis, a two-dimensional tube generation test is carried out by using HUVEC cells, the capacity of a compound for inhibiting tumor tissue angiogenesis can be reflected, the capacity of boroleucine and boroleucine dinoteester for inhibiting HUVEC cell two-dimensional tube generation is respectively examined, DMSO solvent is used as a blank control (marked as Ctrl), and bortezomib is used as a positive control.

The test procedure was as follows:

(1) 50 mul of Matrigel gel diluted by 1: 1M 199 culture medium was plated in a 96-well plate and incubated in an incubator;

(2) adding 100 mul of compound after 30min, and supplementing 50 mul of cells into the hole until the concentration of the compound reaches the working concentration;

(3) treating cells, counting, adding 20000 cells/50 μ l/well;

(4) after incubation for 6h, photographs were taken.

Note: the compounds used in the assay were boroleucine, boroleucine dinoate, DMSO solvent or bortezomib.

The results show that, as shown in fig. 2, norleucine and norleucine dinoester significantly inhibited HUVEC cell two-dimensional tube production compared to the blank control and the positive control.

Example 3 inhibition of Breast cancer cell migration by Boroflurane and Boroflurane Dinoyl esters

The scratch test can reflect the migration capacity of the breast cancer cells, examine the capacity of the boroleucine and the boroleucine dinoester for inhibiting the migration of the breast cancer cells, and take DMSO solvent as a blank control (marked as Ctrl) and bortezomib as a positive control.

The test procedure was as follows:

(1) spreading breast cancer MDA-MB-231 cells into a 6-well plate, and adding 10 mu M compound for treatment for 2 days;

(2) drawing lines by a white gun head with the assistance of a pipette to ensure that the drawn lines are consistent in thickness;

(3) and taking pictures at 0 and 36h, and measuring the migration distance by a computer.

Note: the compound used in the assay was boroleucine, boroleucine dinoate, DMSO solvent or bortezomib.

The results show that, as shown in fig. 3-4, compared with the blank control and the positive control, the norleucine and the norleucine dinoester can obviously inhibit the migration of breast cancer cells, and the inhibition effect of the norleucine and the dinonyl ester is superior to that of the positive control drug bortezomib.

Note: d is migration distance.

Example 4 inhibition of Breast cancer cell invasion by Borrelidin and Borrelidin

The invasion test can reflect the metastatic capacity of the breast cancer cells, examine the capacity of the boroleucine and the boroleucine dinonyl ester for inhibiting the invasion of the breast cancer cells, and take DMSO solvent as a blank control (marked as Ctrl) and bortezomib as a positive control.

The test procedure was as follows:

(1) metrigel gel was prepared as follows 1: 19, diluting, uniformly mixing, and adding 50 mu l of the mixture into a small chamber;

(2) placing the Transwell plate into a culture box, curing for 1-2h, adding 164030 μ l serum-free hydrated basement membrane, and adding 1 x 10 serum-free culture medium into breast cancer cells after 30min⁵The lower chamber is added with 10% FBS culture medium;

(3) adding 10 μ M compound into the chamber, culturing for 24h, washing the chamber with PBS, and fixing with methanol for 10 min;

(4) after sucking the liquid, immersing the substrate into 0.1% crystal violet for dyeing for 2-3 min;

(5) washing twice with PBS, and wiping off the glue and the cells without membrane penetration from the upper chamber with a cotton swab;

(6) and taking a picture under a microscope and counting.

In the present example, the smaller the number of the transmembrane cells, the better the effect of inhibiting the invasion of cancer cells. The results show that, as shown in fig. 5-6, compared with the blank control and the positive control, the norleucine and the norleucine dinoester can obviously inhibit the invasion of breast cancer cells, and the inhibition effect of the norleucine and the dinonyl ester is superior to that of the positive control drug bortezomib.

EXAMPLE 5 evaluation of Compound anti-metastatic Activity in vivo in tumor-bearing mice

In the experiment, mouse liver cancer H22 cells are injected into a Kunming rat tail vein, blood channel transfer is simulated, the in vivo anti-transfer activity of boroleucine and boroleucine dinonyl ester is evaluated, DMSO solvent is used as a blank control (marked as Ctrl), and bortezomib is used as a positive control.

The test procedure was as follows: h22 cell culture expansion, collecting cells, washing twice with sterile PBS solution, and counting cells (cell count: 2.5X 10)⁷Pieces/ml), mice were injected intravenously at the tail, 200. mu.l of each was inoculated. 1 day after inoculation, the mice were sacrificed by pulling the neck after two weeks, randomly grouping and starting intraperitoneal injection at 20mg/kg/d, dissecting the lung tissue and fixing with picric acid, and observing the number of pulmonary nodules.

The results show that, as shown in fig. 7-8, compared with the blank control, bortezomib, boroleucine and boroleucine dinoester can obviously inhibit tumor cell metastasis, and the in vivo anti-tumor metastasis capacity of the boroleucine and the boroleucine dinoester is superior to that of the positive control bortezomib.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of the boroleucine compounds in the following 1) or 2):

1) preparing an aminopeptidase N inhibitor;

2) preparing the medicine for inhibiting the growth of the tumor.

2. The use of claim 1, wherein the boroleucine is used to inhibit tumor growth by inhibiting HUVEC cell two-dimensional tube production.

3. The use of claim 1, wherein the boroleucine-like compound inhibits tumor growth by inhibiting tumor cell migration.

4. The use of claim 1, wherein the boroleucine-like compound inhibits tumor growth by inhibiting tumor cell invasion.

5. The use according to any one of claims 1 to 4, wherein the boroleucine compound is boroleucine having a structure of formula 1 or boroleucine dinoester having a structure of formula 2;