CN110028482B

CN110028482B - 4-position split melphalan nitrogen mustard derivative of brefeldin A and preparation method and application thereof

Info

Publication number: CN110028482B
Application number: CN201810042479.3A
Authority: CN
Inventors: 李达翃; 华会明; 李占林; 田康涛; 韩通; 孙艺珈; 蔡林璇
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2018-01-11
Filing date: 2018-01-17
Publication date: 2021-01-29
Anticipated expiration: 2038-01-17
Also published as: CN110028482A

Abstract

The invention relates to the field of natural medicines and medicinal chemistry, and relates to a preparation method and application of a 4-site split melphalan nitrogen mustard derivative of brefeldin A. In particular to a preparation method of melphalan derivatives introduced with DNA alkylating agent at 4-OH site of brefeldin A and application thereof in preparing antitumor drugs. The 4-site split melphalan nitrogen mustard derivative of brefeldin A and the pharmaceutically acceptable salt thereof have the structure shown in the general formula I, wherein n is shown in the claims and the specification.

Description

4-position split melphalan nitrogen mustard derivative of brefeldin A and preparation method and application thereof

Technical Field

The invention belongs to the field of natural medicines and medicinal chemistry, and relates to a preparation method and application of a 4-position split melphalan nitrogen mustard derivative of brefeldin A, in particular to derivatives modified at a 4-OH site of the brefeldin A, and relates to the brefeldin A derivatives substituted by a DNA alkylating agent melphalan derivative at the 4-OH site, a preparation method thereof and application thereof in preparing antitumor medicines.

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 melphalan derivative with good anti-tumor activity, namely a 4-position split melphalan nitrogen mustard derivative of brefeldin A, and further providing a pharmaceutical composition containing the derivative, wherein the 4-position split melphalan nitrogen mustard derivative of brefeldin A or the composition thereof has anti-tumor activity.

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

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

wherein n is an integer of 1 to 12.

Preferably, n is an integer from 1 to 6.

More preferably, n is 2.

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

the brefeldin A (1) reacts with TBSOTf under the condition of 2, 6-dimethyl pyridine/DMF to obtain TBS protected derivatives (2) of 4-OH and 7-OH of the brefeldin A, and TBS protecting groups of the 4-OH are selectively removed by TBAF/THF to obtain TBS protected derivatives (3) of 7-OH of the brefeldin A.

Melphalan (4) with methanol in SOCl₂In the presence of the catalyst, heating and refluxing to react to obtain melphalan methyl ester (5), and then reacting the (5) with dianhydride under the catalysis of DMAP to obtain melphalan methyl ester carboxylic acid derivative (6).

And reacting the intermediate (3) with a melphalan methyl ester carboxylic acid derivative (6) at room temperature under the EDCI/DMAP condition to obtain a compound (7), and deprotecting to obtain a target compound (8).

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

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

Detailed Description

Example 1

The brefeldin A intermediate 3(32mg,0.08mmol) was dissolved in dichloromethane (2.5ml), melphalan methylbutanoic acid (34mg,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 separated by means of a silica gel column (petroleum ether: ethyl acetate ═ 2:1) to obtain intermediate 7, and further 7 was dissolved in anhydrous THF, and the 7-TBS protecting group was removed by means of a THF solution of TBAF, and then separated by means of a silica gel column (petroleum ether: ethyl acetate ═ 2:1) to obtain 8-1 as a yellow oily substance in 21% yield. HRMS (ESI, M + Na)⁺)m/z calcd for C₃₉H₄₃Cl₂N₅O₆H:703.2523,found703.2451.¹H NMR(400MHz,DMSO-d₆):δ(ppm):7.24(1H,dd,J＝15.8,3.1Hz,C₃-H),7.0(2H,d,J＝8.6Hz,Ar-H),6.65(2H,d,J＝8.7Hz,Ar-H),5.72(1H,m,C₁₁-H),5.62(1H,dd,J＝15.8,1.7Hz,C₂-H),5.15-5.25(2H,m,C₄-H,C₁₀-H),4.72(1H,m,C₁₅-H),4.37(1H,m,-NH),4.05(1H,m,C₇-H),3.69(8H,m,-NCH₂CH₂Cl),3.58(3H,s,OCH₃),0.69-2.89(22H,m,-COCH₂CH₂CO-,-ArCH₂CHNCO-,C₅,2C₆,2C₈,C₉,2C₁₂,2C₁₃,2C₁₄-H,CH₃).¹³C NMR(100MHz,DMSO-d₆):δ(ppm)172.7,171.8,171.3,165.5,149.0,145.4,137.2,130.6,130.6,130.2,125.6,117.5,112.1,112.1,76.6,71.6,70.8,54.4,52.6,52.6,52.2,49.5,43.4,43.3,41.6,41.6,40.7,36.4,33.8,31.8,30.0,29.2,26.9,21.1.

Pharmacological test

Experimental equipment and reagent

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 hole (absolute OD value of drug sensitive hole-absolute OD value of blank control hole)

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 brefeldin A derivative has far less 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 melphalan nitrogen mustard derivative of brefeldin a shown in a general formula I and pharmaceutically acceptable salts thereof:

wherein n is an integer of 1 to 12.

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

wherein n is an integer of 1 to 6.

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

wherein n is 2, namely:

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

5. A pharmaceutical formulation comprising a 4-split melphalan-like nitrogen mustard derivative of brefeldin a 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 4-split melphalan-like nitrogen mustard derivatives of brefeldin a and pharmaceutically acceptable salts thereof, as claimed in claim 1, of the general formula I:

reacting the brefeldin A (1) with TBSOTf under the condition of 2, 6-dimethyl pyridine/DMF to obtain TBS protective derivatives (2) of 4-OH and 7-OH of the brefeldin A, and selectively removing TBS protective groups of 4-OH by TBAF/THF to obtain TBS protective derivatives (3) of 7-OH of the brefeldin A;

melphalan (4) with methanol in SOCl₂Heating and refluxing to react under the existence condition to obtain melphalan methyl ester (5), and then reacting the melphalan methyl ester (5) with dianhydride under the DMAP catalysis condition to obtain melphalan methyl ester carboxylic acid derivative (6);

reacting the intermediate (3) with a melphalan methyl ester carboxylic acid derivative (6) at room temperature under the EDCI/DMAP condition to obtain a compound (7), and deprotecting to obtain a target compound (8);

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

8. Use of the pharmaceutical composition of claim 4 for the preparation of a medicament for the treatment of a neoplastic disease.

9. Use of a pharmaceutical formulation according to claim 5 for the preparation of a medicament for the treatment of a tumor disease.

10. The use according to any one of claims 7 to 9, wherein the tumour is leukaemia, prostate cancer or liver cancer.