CN102872017A - Use of 6-(4-difluoromethoxy)-3methoxyphenyl)pyridazine-3(2H)-one in preparation of antitumor drugs - Google Patents

Abstract

The invention relates to a use of 6-(4-difluoromethoxy)-3methoxyphenyl)pyridazine-3(2H)-one with the structure represented by formula I in preparation of antitumor drugs especially liver cancer resistant drugs.

Description

The purposes of 6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone in the preparation antitumor drug

Technical field

The invention belongs to field of pharmacology, relate to the purposes of 6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone, be specifically related to the purposes of this chemical compound in preparation antitumor drug, especially medicines resistant to liver cancer.

Background technology

6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone, trade name: zardaverine (Zardaverine) is the inhibitor of phosphodiesterase PDE III and PDE IV, and is clinical in anesthesia adjuvant (skeletal muscle relaxant).The patent relevant with zardaverine (WO 2001094319, DE 4310051, DE4207822, EP 523513, EP 163965, US4665074) relates generally to the purposes of its antiinflammatory or anti-immunology, and its application at anti-tumor aspect is had no report.

As everyone knows, hepatocarcinoma is the fifth-largest common male tumor disease, is the eighth-largest common female tumor disease.In 2007, the newly-increased liver cancer patient of estimation will have 80% to produce in developing country, and only China has just accounted for 55% of sum.And in developing country, 59% hepatocarcinoma is attributable to HBV, and 33% hepatocarcinoma is attributable to HCV.Especially in recent years, because the Asia-Pacific nations hepatitis B virus infection is wreaked havoc, the onset of liver cancer rate constantly rises, and the Hepatoma therapy medicine is had the huge market demand.

Summary of the invention

An object of the present invention is the purposes of the 6-shown in the open formula I (4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone in preparation antitumor drug, especially medicines resistant to liver cancer.

The inventor finds first that by experiment in external and the body 6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone is in the pharmacological action of anti-tumor aspect.Particularly, 6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone shows very strong specificity at anti-liver cancer, its inhibition to sensitive cells strain BEL-7402 and SMMC-7721 inhibition activity active and to insensitive cell strain (such as HepG2, Hep3B or NCI-H460) differs nearly 1000 times, and its antitumor action has also obtained checking in the Nude Mouse Model in hepatoma carcinoma cell BEL-7402 body simultaneously.

Description of drawings

Fig. 1 is that zardaverine is to the activity of the dose-dependent inhibition tumor bulk-growth of Human hepatoma cell line Bel-7402 Nude Mice;

Fig. 2 is that zardaverine is on the impact of nude mouse body weight.

The specific embodiment

The below further specifies the present invention with embodiment, but these embodiment do not limit protection scope of the present invention.

Embodiment 1: the anti tumor activity in vitro of zardaverine

RPMI 1640 culture medium, minimum essential medium (Minimum essential medium, MEM), Ham ' s F12 culture medium, Dole shellfish Kao Shi improvement Yi Geershi culture medium (Dulbecco ' s modified Eagle ' s medium, DMEM) culture medium, M199 culture medium, hyclone (fetal bovine serum, FBS) all available from BRL (Gaithersburg, USA) company.Sulphonyl rhodamine B (sulforhodamine B, SRB) is available from Sigma-Aldrich (St Louis, Mo, USA) company.CO ₂Cell culture incubator is U.S. Thermo Forma Scientific (Marietta, OH, United States) company's product; Inverted microscope is available from Olympus company; CA-1390-1 type superclean bench is Shanghai cleaning equipment company limited product; BT 124S type 100,000/balance is Sartorius company product; HF safe 1500 type continous sample adding apparatus are eppendorf company product; SPECTRA MAX 190 microplate reader are Molecular Devices (Sunnyvale CA, U.S.A.) company's product.Human liver cancer cell BEL-7402 and SMMC-7721 are available from biochemical cell institute of Shanghai Sheng Ke institute of Chinese Academy of Sciences cell bank.Other people tumor cell line HepG2, NCI-H460, Hep3B are all available from American type culture collection storehouse (American Type Culture Collection Manassas, VA).

Experimental technique: the chemical compound anti tumor activity in vitro adopts sulphonyl rhodamine B method.Tumor cell is cultivated with RPMI 1640 or DMEM culture medium (Gibco), includes 10% hyclone, and condition of culture is 37 ℃, 5%CO ₂BEL-7402, SMMC-7721, HepG2, Hep3B and NCI-H460 cell strain are inoculated in respectively 96 orifice plates (0.4-1.0 * 10 ⁴Cells/well), after 24 hours, add the target compound zardaverine of 10 times of dilutions; Target compound contains 5 concentration at least.After the compound treatment 72 hours, discard culture fluid, with 10% cold trichloroacetic acid fixed cell.Then use sulphonyl rhodamine B solution-dyed.Flush away with Tris dissolving and protein bound SRB, under 515nm wavelength is measured OD value with microplate reader not in conjunction with behind the SRB, calculates as follows inhibitory rate of cell growth:

Suppression ratio=(OD value _{Control wells}-OD value _{Dosing holes})/OD value _{Control wells}* 100%

According to each control of the concentration rate, adopt Logit method calculation of half inhibitory concentration IC ₅₀The results are shown in Table 1.

The extracorporeal anti-tumor function of table 1. zardaverine

Embodiment 2: the anti-tumor in vivo effect of zardaverine

Experimental technique: the BALB/cA-nude nude mouse, in 6-7 week, ♀ is available from Shanghai Slac Experimental Animal Co., Ltd..The quality certification number: SCXK (Shanghai) 2007-0005.Feeding environment: SPF level.Zardaverine and control drug Sorafenib (sorafenib) all are made into desired concn with the 0.5%CMC that contains 0.1%Tween-80.Nude mouse subcutaneous vaccination Human hepatocarcinoma Bel-7402 cell treats that tumor growth is to 100-200mm ³After, with animal random packet (d0), solvent control group: n=12; Zardaverine 60mg/kg group, zardaverine 200mg/kg group and positive controls: n=6, and beginning administration, every day 1 time, gavage is 14 days continuously, then drug withdrawal, observation continues 37 days, and measures tumor size.Survey weekly the tumor volume 2-3 time, claim Mus heavy, record data.

Gross tumor volume (V) computing formula is: V=1/2 * a * b ²(wherein a, b represent respectively length and width).

Relative tumour volume (RTV): the gross tumor volume during last 1 day gross tumor volume/beginning administration.

Experimental result: see Fig. 1.Fig. 1 has shown the curative effect of zardaverine to the Human hepatoma cell line Bel-7402 Nude Mice.As can be seen from Figure 1: zardaverine oral administration 200mg/kg shows the antitumor action good to BEL-7402 transplanted tumor.Simultaneously, as can be seen from Figure 2, the body weight impact on mouse during high dose is little, illustrates that toxicity is very little.

Zardaverine all shows the effective antitumour activity in vivo and in vitro, especially hepatocarcinoma is had stronger specificity, and it can be used as antitumor drug and uses clinical.

Claims (3)

1. the 6-shown in the formula I (4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-ketone is in the purposes of preparation in the antitumor drug

2. purposes according to claim 1, wherein, described antitumor drug is medicines resistant to liver cancer.

3. purposes according to claim 2, wherein, described medicines resistant to liver cancer suppresses the activity of BEL-7402 and SMMC-7721 cell strain specifically.

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