CN110028477B

CN110028477B - Preparation method and application of 4-site split nitrogen mustard derivatives of brefeldin A

Info

Publication number: CN110028477B
Application number: CN201810026295.8A
Authority: CN
Inventors: 李达翃; 华会明; 李占林; 李佳; 李昊楠; 王茗莹; 焦润伟
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2018-01-11
Filing date: 2018-01-11
Publication date: 2021-02-26
Anticipated expiration: 2038-01-11
Also published as: CN110028477A

Abstract

The invention relates to the fields of natural medicines and medicinal chemistry, in particular to a preparation method and application of a 4-site split nitrogen mustard derivative of brefeldin A with anti-tumor activity. The 4-site split nitrogen mustard derivative of brefeldin A and the pharmaceutically acceptable salt thereof have the following general formula I, wherein n is described in the claims and the specification.

Description

Preparation method and application of 4-site split nitrogen mustard derivatives of brefeldin A

Technical Field

The invention belongs to the technical field of medicines, relates to the fields of natural medicines and medicinal chemistry, and particularly relates to a 4-OH site mosaic nitrogen mustard derivative of brefeldin A. In particular to derivatives of the nitrogen mustard compounds spliced with DNA alkylating agent at the 4-OH position of brefeldin A, a preparation method thereof and application thereof in preparing anti-tumor drugs.

Background

Brefeldin a (BFA) is a secondary metabolite of Ascomycetes (Ascomycetes), belonging to the macrolide type of antibiotics, also known as clindamycin or ascosporin. It was isolated from Penicillium decumbens fermentation broth by Singleton et al in 1958. Brefeldin A has prismatic crystal, is white, is insoluble in petroleum ether, chloroform and water, and is easily soluble in ethyl acetate, acetone and methanol. Pharmacological research shows that brefeldin A has the pharmacological activities of resisting tumor, virus, fungus, nematode, mitosis, etc., wherein the antitumor activity is particularly obvious. NCI studies have shown that brefeldin A selectively inhibits and kills human tumor cells. The macrolide antibiotic brefeldin A serving as an endoplasmic reticulum stress agent can induce a plurality of tumor cells to generate apoptosis, inhibit cell proliferation and other ways to inhibit and kill the tumor cells, wherein the macrolide antibiotic brefeldin A has an inhibiting effect on the tumor cells, such as human cervical cancer cells, prostate cancer cells, chronic lymphocytic leukemia cells, follicular lymphoma cells and the like. Studies have shown that brefeldin a induces apoptosis by activating the mitochondrial and death receptor pathways as well as inhibiting focal adhesion kinase-mediated cell invasion. For example, brefeldin A can induce apoptosis in ovarian cancer cells by activating the mitochondrial pathway and the caspase-8, Bid-dependent pathway. The apoptotic effects of brefeldin a may be mediated by the production of reactive oxygen species and the depletion of glutathione, which leads to the activation of apoptosis-related caspase pathways. In addition, brefeldin A inhibits OVCAR-3 cell adhesion and metastasis induced by fetal bovine serum. This effect is achieved by inhibiting focal adhesion kinase-dependent activation of intracellular related components. Brefeldin A leads to activation of caspase-3, 6 through mitochondrial pathways and thus induces endoplasmic reticulum stress-regulated apoptosis. With the research on the pharmacological action of brefeldin A, brefeldin A has attracted great interest of domestic and foreign scientists and has developed the synthesis of brefeldin A derivatives. Aims to obtain candidate antitumor compounds with better activity, lower toxicity and more stable properties. Compared with reports on extraction separation and action mechanism, the reports on pharmaceutical chemistry aspects such as brefeldin A structure modification and modification, derivative synthesis and the like are less.

The nitrogen mustard type medicine, also called DNA alkylating agent, belongs to the cytotoxic medicine, and its action mechanism is that in vivo it can form electron-deficient highly active intermediate or other compound with active electrophilic group, and then can produce covalent irreversible combination with biological macromolecule so as to make DNA molecule lose activity or produce breakage. The medicine is widely used clinically, but has large toxic and side effects, lacks specificity on cell action, and has unsatisfactory treatment effect along with the occurrence of tumor resistance in recent years, so that the chemical modification of the nitrogen mustard medicine has important value in improving the treatment effect.

Disclosure of Invention

The invention aims to solve the technical problem of finding a brefeldin A split nitrogen mustard derivative with good anti-tumor activity and further providing a pharmaceutical composition containing the derivative, wherein the 4-site split nitrogen mustard derivative of the brefeldin A or the composition thereof has an anti-tumor effect.

In order to solve the technical problems, the invention provides the following technical scheme:

the general formula I is 4-site split nitrogen mustard derivative of brefeldin A and pharmaceutically acceptable salt thereof:

wherein n is an integer of 0 to 12.

Preferably, n is an integer from 0 to 6.

More preferably, n is 0 or 3.

The derivative of the general formula I can be prepared by the following method:

reacting brefeldin A (1) with TBSOTf under the condition of 2, 6-dimethyl pyridine/DMF, and selectively removing TBS protective group of 4-OH by TBAF/THF to obtain brefeldin A hydroxyl derivative (3).

The brefeldin A hydroxyl derivative (3) reacts with aromatic nitrogen mustard (4) with different side chain lengths at room temperature under the EDCI/DMAP condition to obtain a target compound (5), and TBS protective groups of 7-OH are removed by TBAF/THF to obtain the brefeldin A hydroxyl derivative (6).

The invention also provides a pharmaceutical composition which comprises the 4-site split nitrogen mustard derivative of the brefeldin A shown in the general formula I, pharmaceutically acceptable salts thereof and pharmaceutically acceptable excipients.

The 4-site split nitrogen mustard derivative of the brefeldin A, the pharmaceutically acceptable salt thereof or the pharmaceutical composition thereof can be prepared into clinically acceptable preparations with clinically acceptable carriers, and the preparations are tablets, granules, capsules and the like.

The invention takes brefeldin A as a lead compound, utilizes a split principle, selects a nitrogen mustard derivative with better activity, connects the nitrogen mustard derivative to the 4-OH position of the molecular structure of the nitrogen mustard derivative through a connecting group, and designs and synthesizes the 4-position split nitrogen mustard derivative of the brefeldin A with the general formula I. The compound after being combined has better pharmaceutical activity.

Detailed Description

Example 1

Brefeldin A intermediate 3(32mg,0.08mmol) was dissolved in dichloromethane (2.5ml), nitrogen benzoate (21mg,0.08mmol), EDCI (29mg,0.15mmol) and catalytic amount of DMAP were added in sequence, the reaction was stirred at room temperature, the reaction progress was monitored by TCL, and the reaction was terminated after 24 h. The reaction mixture was poured into 20ml of an ice-water mixture, extracted with dichloromethane (30ml × 3), washed with a saturated aqueous solution of brine, dried over anhydrous sodium sulfate, and dichloromethane was recovered, and then passed through a silica gel column (petroleum ether: ethyl acetate ═ 5:1) to obtain intermediate 5a, and 5a was dissolved in anhydrous THF, and the 7-TBS protecting group was removed with a THF solution of TBAF, and then separated with a silica gel column (petroleum ether: ethyl acetate ═ 2:1) to obtain yellow oily substance 6a with a yield of 23%. HR-MS (ESI, M + Na)⁺m/z calcd for C₃₅H₃₈Cl₂N₄O₃H:546.1784,found 546.1745.¹H NMR(400MHz,DMSO-d₆):δ(ppm)7.85(2H,d,J＝8.9Hz,Ar-H),7.35(1H,dd,J＝15.7,3.2Hz,C₃-H),6.88(2H,d,J＝8.91Hz,Ar-H),5.76(1H,m,C₁₁-H),5.55(1H,dd,J＝15.7,1.6Hz,C₂-H),5.43(1H,m,C₁₀-H),5.28(1H,dd,J＝15.11,9.70Hz,C₄-H),4.74(1H,m,C₁₅-H),4.57(1H,d,J＝3.4Hz,OH),4.09(1H,m,C₇-H),3.78-3.84(8H,m,-NCH₂CH₂Cl),0.76-2.62(15H,m,C₅,2C₆,2C₈,C₉,2C₁₂,2C₁₃,2C₁₄-H,CH₃).¹³C NMR(100MHz,DMSO-d₆):δ(ppm)165.4,165.1,151.0,149.8,137.3,131.7,131.7,130.2,117.2,117.0,111.8,111.8,76.4,71.8,70.9,52.2,52.2,50.0,43.4,43.2,41.3,41.3,41.0,33.89,31.89,26.85,21.07.

Example 2

Compound 6b was prepared according to the synthetic method of example 1. Yellow oil, yield 32%. HR-MS (ESI, M + H) M/z calcd for C₃₆H₄₀Cl₂N₄O₃H:566.2435,found 566.2391.¹H NMR(400MHz,DMSO-d₆):δ(ppm)7.27(1H,dd,J＝15.8,3.3Hz,C₃-H),7.04(2H,d,J＝8.6Hz,Ar-H),6.68(2H,d,J＝8.6Hz,Ar-H),5.71(1H,m,C₁₁-H),5.59(1H,dd,J＝15.8,1.6Hz,C₂-H),5.20-5.26(2H,m,C₄,C₁₀-H),4.75(1H,m,C₁₅-H),4.56(1H,s,OH),4.07(1H,m,C₇-H),3.70(8H,m,-NCH₂CH₂Cl),0.74-2.49(21H,m,-CH₂CH₂CH₂-,C₅,2C₆,2C₈,C₉,2C₁₂,2C₁₃,2C₁₄-H,CH₃).¹³C NMR(100MHz,DMSO-d₆):δ(ppm)172.4,165.4,149.3,145.0,137.2,130.2,129.9,129.8,129.8,117.4,112.4,112.4,76.4,71.7,70.8,52.7,52.7,49.6,43.4,43.3,41.6,41.6,40.9,33.9,33.8,33.4,31.8,27.2,26.8,21.1.

Pharmacological test

Experimental equipment and reagent

Instrument clean bench (Sujing group Antai company)

Constant temperature incubator (Thermo electronic Corporation)

Enzyme-linked immunosorbent assay (BIO-RAD company)

Inverted biological microscope (Chongqing optical instrument factory)

Reagent cell culture Medium RPMI-1640, DMEM (high sugar) (GIBCO Co., Ltd.)

Fetal bovine serum (Hangzhou Sijiqing Co., Ltd.)

CCK-8(Biosharp company product)

Trypan blue (Solarbio company product)

DMSO (Sigma Co.)

Cell line human promyelocytic leukemia cell HL-60, human prostate cancer cell line PC-3,

Human hepatoma cell strains Bel-7402 and HepG-2, and human hepatoma drug-resistant cell strains

Bel-7402/5-FU and human normal liver cell strain L-O2

Experimental methods

Cell inhibitory activity test method

Cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator with saturated humidity. The culture medium is RPMI1640 cell culture medium containing 10% heat-inactivated fetal calf serum, penicillin 100U/mL and streptomycin 100U/mL. The culture medium was changed for 48h, and after the cells were attached to the wall, they were digested with 0.25% trypsin for passage. The experimental cells are all in logarithmic growth phase, and trypan blue dye exclusion method shows cell viability>95％。

Taking a bottle of cells in a logarithmic phase, adding a digestive juice (0.125% trypsin and 0.01% EDTA) for digestion, and counting by 2-4 × 10⁴cell/mL, preparing cell suspension, inoculating on 96-well plate, 100 μ L/well, and placing in constant temperature CO₂The culture was carried out in an incubator for 24 hours. The solution was changed, the test drug was added at 100. mu.L/well, and cultured for 72 hours. CCK-8 was added to 96-well plates at 50. mu.L/well and incubated in an incubator for 4 hours. The supernatant was aspirated, DMSO was added at 200. mu.L/well and shaken on a shaker for 10 min. The test substances were examined at 3 concentrations (0.25. mu.M, 0.5. mu.M, 1. mu.M), and the cell inhibition rate at each concentration was calculated by measuring the absorbance at a wavelength of 450nm using an enzyme-linked immunosorbent assay.

The inhibition rate calculation method comprises the following steps:

relative OD value of drug sensitive well (absolute OD value of drug sensitive well) — absolute OD value of blank control well

Results of the experiment

TABLE 1 examples IC for antiproliferative activity against 5 human cancer cell lines and 1 normal liver cell line₅₀Value (μ M)

Pharmacological tests prove that the 4-site split nitrogen mustard derivative of the brefeldin A has far lower toxicity to normal cell strains than tumor cell strains, has selectivity, and can be further used for preparing antitumor drugs.

Claims

1. A 4-split nitrogen mustard derivative of brefeldin a shown in a general formula I and pharmaceutically acceptable salts thereof:

wherein n is an integer of 0 to 12.

2. The 4-split nitrogen mustard derivative of brefeldin a shown in the general formula I of claim 1 and pharmaceutically acceptable salts thereof:

wherein n is an integer of 0 to 6.

3. The brefeldin A4-split nitrogen mustard derivative of the general formula I and the pharmaceutically acceptable salt thereof of claim 1, selected from:

。

4. a pharmaceutical composition comprising a therapeutically effective amount of a brefeldin a 4-split nitrogen mustard derivative of general formula I as described in any of claims 1-3 and its pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

5. A pharmaceutical preparation comprising a brefeldin a 4-split nitrogen mustard derivative of general formula I as described in any of claims 1-3 and pharmaceutically acceptable salts thereof or a pharmaceutical composition as described in claim 4.

6. The process for the preparation of brefeldin a 4-split nitrogen mustard derivatives and their pharmaceutically acceptable salts according to claim 1, characterized by:

reacting brefeldin A (1) with TBSOTf under the condition of 2, 6-dimethyl pyridine/DMF, and deprotecting to obtain a brefeldin A intermediate (3);

the intermediate (3) reacts with aromatic nitrogen mustard 4 with different side chain lengths at room temperature under the EDCI/DMAP condition to obtain a compound 5, and then the target compound (6) is obtained through deprotection;

n is as defined in claim 1.

7. Use of the 4-split nitrogen mustard derivative of brefeldin a shown in the general formula I and the pharmaceutically acceptable salts thereof as claimed in any one of claims 1-3 in the preparation of medicaments for treating tumor diseases.

8. Use of the pharmaceutical composition of claim 4 or the pharmaceutical formulation of claim 5 for the manufacture of a medicament for the treatment of a neoplastic disease.

9. The use of claim 7 or 8, wherein the neoplasm is leukemia, prostate cancer or liver cancer.