CN110028477A

CN110028477A - The preparation method and purposes of the 4- position split nitrogen mustard derivatives of a kind of brefeldin A

Info

Publication number: CN110028477A
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: 2019-07-19
Anticipated expiration: 2038-01-11
Also published as: CN110028477B

Abstract

The present invention relates to natural drug and field of medicinal chemistry, and in particular to the preparation method and purposes of the 4- position split nitrogen mustard derivatives of a kind of brefeldin A with anti-tumor activity.Shown in the following general formula I of 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt structure of brefeldin A of the present invention, wherein n is as described in claims and specification.

Description

The preparation method of the 4- position split nitrogen mustard derivatives of a kind of brefeldin A and Purposes

Technical field

The invention belongs to pharmaceutical technology fields, are related to natural drug and field of medicinal chemistry, and in particular to mine-laying phenanthrene moral bacterium The 4-OH position split nitrogen mustard derivatives of plain A.More particularly to these brefeldin A 4-OH position split DNA alkylating agent nitrogen Derivative of mustard class compound and preparation method thereof and application in preparation of anti-tumor drugs.

Background technique

Brefeldin A (brefeldin A, BFA) is that a kind of cometabolism of Saccharomyces (Ascomycetes) produces Object belongs to macrolide type antibiotic, also referred to as decumbin or ascochitine.It in 1958, by Singleton et al. from It is isolated in the fermentation liquid of Penicillium decumbens.Brefeldin A, crystal are in prismatic, and white does not dissolve in Petroleum ether, chloroform, water are soluble in ethyl acetate, acetone and methanol.It is anti-swollen that pharmacological research shows that brefeldin A has The pharmacological activity such as tumor, antiviral, antimycotic, anti-nematode and antimitotic, wherein anti-tumor activity is especially pronounced.NCI's grinds Study carefully and shows that the brefeldin A property of can choose inhibits and kills human tumor cells.Macrolide antibiotic brefeldin A The number of ways such as cell Proliferation can be inhibited to inhibit simultaneously with induced various types of tumors apoptosis as a kind of er stress agent Tumour cell is killed, including human cervical carcinoma cell, prostate gland cancer cell, chronic lymphocytic leukemia cell and follicular lymphoma Cell etc. has inhibiting effect to kinds of tumor cells.Research shows that brefeldin A by activation mitochondria and it is dead by Body approach and the cell invasion for inhibiting focal adhesion kinase to adjust come apoptosis-induced.For example, brefeldin A can be by swashing Apoptosis occurs for the mitochondrial pathways living and capase-8, Bid dependent form access induction ovarian cancer cell.Brefeldin A withers Dying effect may be to be adjusted by the generation of active oxygen and the consumption of glutathione, and this adjusting will lead to apoptosis correlation The activation of caspase approach.In addition, brefeldin A can inhibit sticking for the OVCAR-3 cell induced by fetal calf serum And transfer.This effect is completed by the activation for the intracellular Related Component for inhibiting focal adhesion kinase to rely on.Mine-laying is luxuriant and rich with fragrance Moral rhzomorph A leads to the apoptosis for activating induction er stress to adjust of caspase-3,6 by the mitochondrial pathways.With right Brefeldin A pharmacological research gos deep into, and brefeldin A causes the great interest of domestic and international scientist, opens The synthetic work to brefeldin A derivative is opened up.Purpose is that acquisition activity is more preferable, toxicity is lower, property is more stable Antitumor candidate compound.It is separated, compared with the report in terms of mechanism of action with extracting, about brefeldin A structural modification Report in terms of the pharmaceutical chemistry such as synthesizing with transformation, derivative is less.

Chlormethine series pharmaceuticals, also referred to as DNA alkylating agent, belong to cytotoxic drug, and mechanism of action is to be formed in vivo The height reactive intermediate of electron deficient or other compounds with active electrophilic groups, and then occur with large biological molecule Covalent Irreversible binding makes DNA molecular loss of activity or is broken.This kind of drug is clinically widely used, but it Toxic side effect is bigger, lacks specificity to cytosis, and with the generation of tumor drug resistance in recent years, therapeutic effect is not Therefore ideal is chemically modified chlormethine series pharmaceuticals, improving its curative effect has critically important value.

Summary of the invention

It is derivative the technical problem to be solved by the present invention is to find the good brefeldin A split mustargen of anti-tumor activity Object, and a kind of pharmaceutical composition comprising the derivative is further provided for, the 4- position split mustargen of the brefeldin A Derivative or combinations thereof object has antitumor action.

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

General formula I is the 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt of shown brefeldin A:

Wherein, n is the integer of 0-12.

Preferably, n is the integer of 0-6.

It is highly preferred that n is 0 or 3.

The derivative of general formula I of the present invention can be prepared with following method:

Brefeldin A (1) reacts under the conditions of 2,6- lutidines/DMF with TBSOTf, then selects through TBAF/THF The TBS blocking group for sloughing 4-OH of selecting property, obtains brefeldin A hydroxy derivatives (3).

Brefeldin A hydroxy derivatives (3) are respectively from the aryl nitrogen mustard of different side chain lengths (4) in EDCI/DMAP Under the conditions of react at room temperature and obtain target compound (5), then slough the TBS blocking group of 7-OH through TBAF/THF, obtain mine-laying phenanthrene moral Rhzomorph A hydroxy derivatives (6).

The present invention also provides a kind of pharmaceutical compositions, the 4- position split mustargen comprising brefeldin A shown in general formula I Derivative and its pharmaceutically acceptable salt and its pharmaceutically acceptable salt and pharmaceutically acceptable excipient.

The 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt of brefeldin A of the invention or its drug Composition can be prepared into clinically acceptable preparation with clinically acceptable carrier, and the preparation is tablet, particle Agent, capsule etc..

The present invention, using principle of hybridization, selects the preferable mustargen of activity to spread out using brefeldin A as lead compound It is connected on the position 4-OH of its molecular structure by biology by linking group, has designed and synthesized the mine-laying phenanthrene moral that general formula is I The 4- position split nitrogen mustard derivatives of rhzomorph A.Compound after split has preferable pharmaceutical active.

Specific embodiment

Embodiment 1

Brefeldin A intermediate 3 (32mg, 0.08mmol) is taken, is dissolved in methylene chloride (2.5ml), sequentially adds The DMAP of benzoic acid nitrogen mustard (21mg, 0.08mmol), EDCI (29mg, 0.15mmol) and catalytic amount, is stirred at room temperature reaction, TCL Reaction process is monitored, terminates reaction afterwards for 24 hours.Reaction solution is poured into 20ml mixture of ice and water, methylene chloride extraction (30ml × 3), saturated common salt aqueous solution washs, and anhydrous sodium sulfate is dry, recycles methylene chloride, through silicagel column (petroleum ether: ethyl acetate= 5:1), intermediate 5a is obtained, then 5a is dissolved in anhydrous THF, 7- TBS protecting groups are sloughed with the THF solution of TBAF, through silicon Rubber column gel column (petroleum ether: ethyl acetate=2:1) separation, obtains yellow oil 6a, yield 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.

Embodiment 2

Compound 6b is prepared into referring to the synthetic method of embodiment 1.Yellow oily, 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 testing

Experimental facilities and reagent

Instrument superclean bench (safe and sound company, Su Jing group)

Constant incubator (Thermo electron Corporation)

Microplate reader (BIO-RAD company)

Inverted biologic microscope (Chongqing optical instrument factory)

Agent cell culture medium RPMI-1640, DMEM (high sugar) (GIBCO company)

Fetal calf serum (the Hangzhou four seasons clear Co., Ltd)

CCK-8 (Biosharp Products)

Trypan blue (Solarbio Products)

DMSO (Sigma company)

Cell strain people in loop HL-60, human prostate cancer cell line PC-3,

Human hepatoma cell strain Bel-7402 and HepG-2, human liver cancer cell strain

Bel-7402/5-FU, Human normal hepatocyte strain L-O2

Experimental method

Cell inhibitory activity experimental method

Cell is in 37 DEG C, 5%CO₂Routine culture in the incubator of saturated humidity.Culture solution is containing 10% heat inactivation tire ox The RPMI1640 cell culture medium of serum, penicillin 100U/mL and streptomysin 100U/mL.48h replaces culture solution, and cell is adherent Afterwards, it is passed on 0.25% trypsin digestion.Experiment is in logarithmic growth phase with cell, and trypan exclusion stain shows that cell is living Power > 95%.

It takes in good condition one bottle of cell of logarithmic growth phase, digestive juice (0.125% trypsase+0.01% is added EDTA it) digests, counts 2~4 × 10⁴Cell suspension inoculation is made on 96 orifice plates in cell/mL, and constant temperature CO is set in 100 holes μ L/₂Training It supports and is cultivated 24 hours in case.Liquid is changed, test medicine is added, 100 holes μ L/ are cultivated 72 hours.CCK-8 is added in 96 orifice plates, 50 The hole μ L/ is incubated for 4 hours in incubator.Supernatant is sucked, adds DMSO, 200 holes μ L/ are shaken 10 minutes on plate shaker.It is tested Object investigates 3 concentration (0.25 μM, 0.5 μM, 1 μM), with enzyme linked immunological monitor in the extinction that wavelength is the every hole of measurement at 450nm Degree, calculates separately the cell inhibitory rate under each concentration.

Inhibiting rate calculation method:

Susceptibility hole is with respect to the absolute absolute OD value of OD value ﹣ blank control wells in OD value=susceptibility hole

Experimental result

IC of 1 embodiment of table to 5 kinds of human cancer cells strains and a kind of normal liver cell strain antiproliferative activity₅₀It is worth (μM)

Pharmacological testing proves that the 4- position split nitrogen mustard derivatives of brefeldin A of the invention are to normal cell strain Toxicity is much smaller than tumor cell line, has selectivity, can be further used for preparing anti-tumor drug.

Claims

1. the 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt of brefeldin A shown in general formula I:

Wherein, n is the integer of 0-12.

2. the 4- position split nitrogen mustard derivatives of brefeldin A shown in general formula I described in claim 1 and its pharmaceutically may be used The salt of receiving:

Wherein, n is the integer of 0-6.

3. the 4- position split nitrogen mustard derivatives and its pharmacy of brefeldin A shown in general formula I of any of claims 1 or 2 Upper acceptable salt:

Wherein, n is 0 or 3.

4. the 4- position split nitrogen mustard derivatives of brefeldin A shown in general formula I described in claim 1 and its pharmaceutically may be used The salt of receiving, is selected from:

5. a kind of pharmaceutical composition, wherein shown in general formula I described in the claim 1-4 any one containing therapeutically effective amount The 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt and pharmaceutically acceptable carrier of brefeldin A.

6. a kind of pharmaceutical preparation, which is characterized in that include the phenanthrene of mine-laying shown in general formula I described in claim 1-4 any one Pharmaceutical composition described in the 4- position split nitrogen mustard derivatives and its pharmaceutically acceptable salt or claim 5 of moral rhzomorph A.

7. 4- position split nitrogen mustard derivatives of brefeldin A shown in general formula I as described in claim 1 and its pharmaceutically The preparation method of acceptable salt, it is characterised in that:

Brefeldin A (1) reacts under the conditions of 2,6- lutidines/DMF with TBSOTf, then is deprotected, and mine-laying is obtained Luxuriant and rich with fragrance moral rhzomorph A intermediate (3)；

Intermediate (3) reacts at room temperature under the conditions of EDCI/DMAP from the aryl nitrogen mustard 4 of different side chain lengths obtain compound respectively 5, then target compound (6) are obtained through deprotection；

8. the 4- position split nitrogen mustard derivatives of brefeldin A shown in general formula I described in claim 1-4 any one and Application of its pharmaceutically acceptable salt in the drug of preparation treatment tumor disease.

9. pharmaceutical composition described in claim 5 or pharmaceutical preparation as claimed in claim 6 are in preparation treatment tumor disease Application in drug.

10. application as claimed in claim 8 or 9, which is characterized in that the tumour is leukaemia, prostate cancer or liver cancer.