CN103087031A

CN103087031A - Application of bistetrahydrobenzopyrone dimer compounds in anti-cancer drugs

Info

Publication number: CN103087031A
Application number: CN2011103326606A
Authority: CN
Inventors: 裴月湖; 白皎; 陈刚; 胡小兰
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2011-10-28
Filing date: 2011-10-28
Publication date: 2013-05-08

Abstract

The invention belongs to the technical field of medicine, and relates to an application of a compound 1 and a compound 2 in anti-cancer drugs, wherein the compound 1 and the compound 2 are derived from a special environmental microorganism, and have tumor growth inhibition activities. According to the present invention, the compound 1 and the compound 2 have a structure formula represented by the following figure (I), have stable structure, and can be prepared into various clinically acceptable formulations after matching with appropriate pharmaceutical excipients; in vitro human tumor cell growth inhibition activity test results of the compound 1 and the compound 2 show that the compound 1 and the compound 2 provide tumor growth inhibition activity similar to a positive control drug doxorubicin, and in vivo tumor growth inhibition activity test results show that the compound 2 has characteristics of low toxicity and high tumor growth inhibition rate, and is a novel efficient and low toxicity tumor growth inhibitor; advantages of strong activity, low dosage and the like are provided; and the compound 1 and the compound 2 derived from the special environmental microorganism secondary metabolite can be used for anti-cancer drug preparation.

Description

The application of two tetrahydro benzo pyrone dimer compounds in cancer therapy drug

Technical field

The invention belongs to medical technical field, relate to two and come from special border microorganism Penicillium fungi penicilliumthe application of time two tetrahydro benzo pyrone dimer compounds of this living meta-bolites sp., be specifically related to two tetrahydro benzo pyrone dimer compounds compounds 1and compound 2application at anticancer aspect.

Background technology

WHO is the data presentation of issue recently, and China has become second-biggest-in-the-world cancer state occurred frequently, and annual newly-increased 2,200,000 cancer patientss of China, account for 20% of global cancer patient sum.Wherein especially liver cancer, cancer of the stomach and lung cancer become main Health Killer.Several large Multinational pharmaceutical enterprises in the whole world are all being fallen over each other up-to-date antitumor drug at Discussion on Chinese Listed at present, data according to ADMA, approximately 400 kinds of cancer drugs of existing 178 companies are in clinical experimental stage, so the cancer therapy drug of brand new type closes on social benefit and all can possess huge prospect on economic benefit.

Summary of the invention

The object of the present invention is to provide two kinds to have inhibiting pair of tetrahydro benzo pyrone dimer compounds compound 1 of tumor growth and compound 2 and the application in antitumor drug thereof, the inside and outside tumors inhibition activity shows the novel tumor growth inhibitor that compound 1 and compound 2 are high-efficiency low-toxicity.

The structure of compound 1 and compound 2 is as follows:

test method:

1, the bulk fermentation of bacterial strain (please guarantee that this method does not need other test parameter can prepare compound of the present invention)

by the ordinary method of culturing micro-organisms, get penicilliumsp. appropriate, be inoculated on PDA agar solid slant culture base, carry out activation culture at 28 ℃.Then use the appropriate spore of inoculating needle picking and thalline, be inoculated in the 500mL Erlenmeyer flask containing 150mL seed culture fluid (No. 4 substratum of fungi), be placed in shaking table, under 28 ℃, 180r/m condition, carry out seed culture 48 hours, acquisition penicilliumsp. seed culture fluid.Get this seed culture fluid appropriate, the inoculum size by 5% is inoculated in 500 milliliters of Erlenmeyer flasks of in-built 150mL fermentation culture (No. 4 substratum of fungi), is placed on 28 ℃, the shaking table of 180r/m and produces fermentation 7 days, obtains penicilliumsp. approximately 68 liters of fermentation cultures.

No. 4 substratum of fungi: N.F,USP MANNITOL (mannitol) 2%, glucose (glucose) 2%, yeast extract paste (yeast extract) 0.5%, peptone (poly peptone) 1%, KH ₂p0 ₄0.05%; MgSO ₄7H ₂o 0.03%, corn steep liquor 0.1%, and pH 6.0, old tap water preparation.

2, the separation of fermented liquid

Fermentation culture is filtered, be divided into supernatant liquor and mycelium.Mycelium 80%(volume fraction) aqueous acetone solution supersound extraction 4 times, after concentrating under reduced pressure, use the equal-volume chloroform, ethyl acetate, propyl carbinol extracts successively, extract respectively 3 times, obtain the chloroform extract (67.5g) of mycelium acetone extract, crude extract (70.9g) after ethyl acetate extract (3.4g) and n-butanol extract (22.8g) merge the chloroform extract of mycelium acetone extract (67.5g) and ethyl acetate extract (3.4g), through silica gel column chromatography, separate, once use sherwood oil-acetone gradient elution, be eluted to sherwood oil: obtain yellow needle crystal (1.2g during acetone (100:50), compound 1).Sherwood oil: acetone (100:50) stream part is used chloroform-methanol gradient elution, chloroform: methyl alcohol (100:1) obtains yellow needle crystal (100.3mg, compound 2).

3, spectral data

Compound 1: yellow needle crystal (acetone), [ a] _d ²⁵=+85 (c 1.2, CHCl ₃), ESI-MS:m/z 638[M]+. ¹H-NMR?(?300?MHz?,CDCl ₃)? δ?:?13.78?(2H,s,OH-8,OH-8’)、11.75(2H,s,OH-1,?OH-1’)、7.46?(2H,d,J=8.4Hz,H-3,H-3’)、6.63?(2H,d,J=8.4Hz,H-4,H-4’)、3.93?(2H,dd,J=10.8,2.7Hz,H-5,H-5’)、3.73(6H,s,H-13,H-13’)、2.81(2H,d,J=2.7Hz,?OH-5,OH-5’)、2.74(2H,dd,J=5.3,18.3Hz,H-7,H-7’)、2.41(2H,t,J=6.3,5.3Hz,H-6,?H-6’)、2.31?(2H,dd,J=10.5,10.8Hz,H-7,H-7’)、1.17?(6H,d,J=6.3Hz,H-11,H-11’);? ¹³C-NMR?(?300?MHz?,CDCl ₃)

?:187.16?(C-9,C-9’)、177.55?(C-8,C-8’)、170.28?(C-12,C-12’)、159.38?(C-1,C-1’)、158.27?(C-4a,C-4a’)、140.18?(C-3,C-3’)、118.23?(C-2,C-2’)、107.56?(C-4,C-4’)、106.86?(C-9a,C-9a’)、101.53?(C-8a,C-8a’)、84.73?(C-10a,C-10a’)、76.58?(C-5,C-5’)、53.28?(C-13,C-13’)、36.26?(C-7,C-7’)、29.71?(C-6,C-6’)、17.99?(C-11,C-11’)

Compound 2: yellow needle crystal (acetone), [ a] _d ²⁵=+16 (c 2.25, acetone), [ a] _d ²⁵=-2.201 (c 0.25, Me ₂cO), ¹h-NMR (300 MHz, CDCl ₃) δ: 13.77 (1H, s, OH-8), 13.71 (1H, s, OH-8 '), 11.65 (1H, s, OH-1), 11.39 (1H, s, OH-1 '), 7.78 (1H, d, J=8.7Hz, H-3), 7.49 (1H, d, J=8.7Hz, H-3 '), 6.63 (1H, d, J=8.7Hz, H-4), 6.61 (1H, d, J=8.7Hz, H-2 '), 3.94 (1H, d, J=9.8Hz, H-5), 3.83 (1H, d, J=9.8Hz, H-5 '), 3.73 (3H, s, H-13), 3.68 (3H, s, H-13 '), 2.78 (2H, m, OH-5, OH-5 '), 2.71 (2H, m, H-7b, H-7b '), 2.43 (2H, m, H-6, H-6 '), 2.33 (2H, m, H-7a, H-7a '), 1.20 (3H, d, J=6.3Hz, H-11), 1.18 (3H, d, J=6.3Hz, H-11 '), ¹³c-NMR (75MHz, CDCl ₃) δ: 187.12 (C-9, C-9 '), 177.64 (C-8), 177.26 (C-8 '), 170.26 (C-12, C-12 '), 161.73 (C-1 '), 159.33 (C-1), 158.25 (C-4a, C-4a '), 140.65 (C-3), 140.39 (C-3 '), 118.01 (C-2), 115.47 (C-4 '), 110.41 (C-2 '), 107.33 (C-4), 106.94 (C-9a, C-9a '), 101.65 (C-8a), 101.49 (C-8a '), 84.81 (C-10a, C-10a '), 76.51 (C-5, C-5 '), 53.22 (C-13, C-13 '), 36.26 (C-7, C-7 '), 29.28 (C-6), 29.04 (C-6 '), 18.00 (C-11), 17.89 (C-11 ').

Compound 1 of the present invention or 2 or its mixture can be mixed with acceptable pharmaceutical composition clinically with pharmaceutically acceptable carrier.

The novel tumor growth inhibitor that compound 1 of the present invention and compound 2 are high-efficiency low-toxicity.

The accompanying drawing explanation

Fig. 1 is the impact of HXL-2 on the H22 tumor weight in example 2;

Fig. 2 is the impact of HXL-2 on H22 tumor-bearing mice tumor control rate in example 2;

Fig. 3 is H22 tumor-bearing mice starting weight in example 2;

Fig. 4 is the impact of HXL-2 on the final body weight of H22 tumor-bearing mice in example 2;

Final body weight=final weight-tumor weight.

Embodiment

[0011]the following examples can help those skilled in the art more fully to understand the present invention, but do not limit the present invention in any way

Application example 1:

Mtt assay: it is 4～5 * 10 that the 96 every holes of orifice plate add concentration ⁴the cell suspension 100 of individual/ml l, put 37 ℃, 5% CO ₂in incubator.After 24h, add sample liquid, 10

the l/ hole, establish two multiple holes, and 37 ℃, 5% CO ₂effect 72h.Every hole adds the MTT solution 20 of 5mg/ml

l, add lysate, 100 after effect 4h

the l/ hole, put in incubator, after dissolving, by the full-automatic microplate reader of MK-2, surveys 570nm OD value.

Conclusion: compound 1 is compared in contrast medicine Zorubicin with 2, and human body tumour cell A549 and Colo205 are all had to inhibited proliferation preferably

Application example 2:

Trial drug: commercially available 5 FU 5 fluorouracil injection liquid; Compound 1(HXL-2)

Animal and knurl strain: 91 of the healthy male Kunming of SPF level kind small white mouses, body weight 18～22g, provided license licensed licenser licence numbering: the SCXK-(capital)-2009-004 by Beijing China Fukang bio tech ltd.

Murine hepatocarcinoma cell strain H22, preserved by pharmacology teaching and research room of Shenyang Pharmaceutical University.

The mouse feed of freely drinking water, 12h circulation light photograph, conform four days.Extract the H22 tumour cell (ascites) of the Kunming mouse interior generation (the 8th day) that growth conditions is good, with the stroke-physiological saline solution dilution, adjust cell density and be about 3.03 * 10 ⁷ml ^-1, after mixing, in every mouse right fore oxter, inject 0.2ml.

Mouse, after meeting knurl 24h, is divided into 8 groups at random by the body weight equilibrium, and every group of 11-12 only.Be grouped as follows: given the test agent HXL-2 is divided into 20mg/kg, 10mg/kg, 5mg/kg group; Commercially available 5 FU 5 fluorouracil injection liquid 20mg/kg group; Model control group.All administration group equal every day of gastric infusions once, the isopyknic distilled water of model control group gavage every day.

Observed and recorded is respectively organized body weight and the survival condition (the general activity situation of mouse, fur, urine color and ight soil etc.) of mouse.Animal is put to death in 12 days after administration for the first time, peels off tumor tissue, weighs, and calculates inhibition rate of tumor growth (TIR), and calculation formula is as follows: TIR=100% * (the average knurl weight of the average knurl weight-administration of model group group)/average knurl weight of model group.More than the heavy 1g of the average knurl of model control group, and at least 20% knurl focuses on more than 400 mg and can carry out interpretation of result.

H22 tumor-bearing mice body weight change, result shows, between each group of starting weight without significant difference; Final body weight is compared with model group, and positive drug 5 FU 5 fluorouracil group body weight significantly reduces, and administration group body weight is without considerable change: see Fig. 3, Fig. 4.

As can be seen here, compound 1(HXL-2) very little on the body weight impact of mouse, compare and demonstrate very low toxicity with positive control drug, and the in-vivo tumour inhibiting rate is close with positive control drug, be the efficient tumor growth inhibitor of a kind of low toxicity.

Claims

1. two tetrahydro benzo pyrone dimer compounds as shown in 1 and 2:

。

2. a pharmaceutical composition, comprise claimed in claim 1 pair of tetrahydro benzo pyrone dimer compounds 1 or 2 or its mixture and pharmaceutically acceptable carrier.

3. the application of claimed in claim 1 pair of tetrahydro benzo pyrone dimer compounds in preparing antitumor drug.