CN102872017B - Use of 6-(4-difluoromethoxy)-3methoxyphenyl)pyridazine-3(2H)-one in preparation of antitumor drugs - Google Patents

Abstract

The present invention relates to the use of 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one represented by formula I in the preparation of antitumor drugs, especially anti-liver cancer drugs the use of.

Description

Use of 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one in the preparation of antitumor drugs

technical field

The invention belongs to the field of pharmacy and relates to the use of 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one, in particular to the use of the compound in the preparation of antitumor drugs , especially the application in anti-liver cancer drugs.

Background technique

6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one, trade name: Zardaverine (Zardaverine) is phosphodiesterase PDE III and PDE Inhibitor of IV, clinically used as anesthesia adjuvant (skeletal muscle relaxant). Patents related to zadaverine (WO 2001094319, DE 4310051, DE4207822, EP 523513, EP 163965, US4665074) mainly relate to its anti-inflammatory or anti-immune applications, but its anti-tumor application has not been reported.

As we all know, liver cancer is the fifth most common cancer in men and the eighth most common cancer in women. In 2007, 80% of the estimated new patients with liver cancer will occur in developing countries, and my country alone accounts for 55% of the total. In developing countries, 59% of liver cancers were attributable to HBV and 33% to HCV. Especially in recent years, due to the rampant hepatitis B virus infection in Asia-Pacific countries, the incidence of liver cancer has been increasing, and there is a huge market demand for drugs for the treatment of liver cancer.

Contents of the invention

An object of the present invention is to disclose that 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-ketone shown in formula I is used in the preparation of antitumor drugs, especially Use in anti-liver cancer drugs.

The present inventors discovered for the first time the pharmacological effect of 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one on antitumor through in vitro and in vivo experiments. Specifically, 6-(4-(difluoromethoxy)-3-methoxyphenyl)pyridazin-3(2H)-one showed strong specificity against liver cancer, and its The inhibitory activity of strains BEL-7402 and SMMC-7721 is nearly 1000 times different from that of insensitive cell lines (such as HepG2, Hep3B or NCI-H460). It was also validated in tumor models.

Description of drawings

Figure 1 shows the dose-dependent activity of zadaverine on human hepatoma Bel-7402 transplanted tumors in nude mice to inhibit tumor growth;

Figure 2 is the effect of zadaverine on body weight of nude mice.

Detailed ways

Further illustrate the present invention with embodiment below, but these embodiments do not limit protection scope of the present invention.

Example 1: In Vitro Antitumor Activity of Zadavirine

RPMI 1640 medium, minimum essential medium (Minimum essential medium, MEM), Ham's F12 medium, Dulbecco's modified Eagle's medium (DMEM) medium, M199 medium, fetal bovine Serum (fetal bovine serum, FBS) were purchased from BRL (Gaithersburg, USA) company. Sulforhodamine B (sulforhodamine B, SRB) was purchased from Sigma-Aldrich (St Louis, Mo, USA). The _CO2 cell incubator was purchased from Thermo Forma Scientific (Marietta, OH, United States); the inverted microscope was purchased from Olympus; the CA-1390-1 ultra-clean workbench was purchased from Shanghai Purification Equipment Co., Ltd.; The ten-thousandth balance is a product of Sartorius; the HF safe 1500 continuous sampler is a product of eppendorf; the SPECTRA MAX 190 microplate reader is a product of MolecularDevices (Sunnyvale CA, USA). Human liver cancer cells BEL-7402 and SMMC-7721 were purchased from the Cell Bank of the Institute of Biochemical Cells, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences. Other human tumor cell lines HepG2, NCI-H460, and Hep3B were purchased from American Type Culture Collection Manassas, VA.

Experimental method: The in vitro antitumor activity of the compound was carried out using the sulforhodamine B method. Tumor cells were cultured in RPMI 1640 or DMEM medium (Gibco) containing 10% fetal bovine serum at 37°C and 5% CO ₂ . BEL-7402, SMMC-7721, HepG2, Hep3B and NCI-H460 cell lines were seeded in 96-well plates (0.4-1.0×10 ⁴ cells/well), and after 24 hours, 10 times diluted target compound Zadawi was added Lin; The target compound contains at least 5 concentrations. After 72 hours of compound treatment, the medium was discarded and the cells were fixed with 10% cold trichloroacetic acid. Then stained with sulforhodamine B solution. After washing away the unbound SRB, dissolve the SRB bound to the protein with Tris, measure the OD value with a microplate reader at a wavelength of 515nm, and calculate the cell growth inhibition rate according to the following formula:

Inhibition rate = (OD value _{control well} - OD value _{administration well} ) / OD value _{control well} × 100%

According to the inhibition rate of each concentration, the half inhibitory concentration IC ₅₀ was calculated by Logit method. The results are shown in Table 1.

Table 1. Antitumor effect of Zadavirine in vitro

Example 2: In vivo anti-tumor effect of zadavirine

Experimental method: BALB/cA-nude nude mice, 6-7 weeks old, ♀, purchased from Shanghai Slack Experimental Animal Co., Ltd. Certificate number: SCXK (Shanghai) 2007-0005. Breeding environment: SPF grade. Both zadavirine and the control drug sorafenib were formulated with 0.5% CMC containing 0.1% Tween-80 to the desired concentration. Nude mice were subcutaneously inoculated with human liver cancer Bel-7402 cells. After the tumors grew to 100-200 mm ³ , the animals were randomly divided into groups (d0), solvent control group: n=12; Zadavirin 60mg/kg group, Zadavirin 60mg/kg group, Zadavirin Lin 200mg/kg group and positive control group: n=6, and began to administer, once a day, for 14 consecutive days, then stopped the drug, observed for 37 days, and measured the size of the tumor. The tumor volume was measured 2-3 times a week, the mice were weighed, and the data were recorded.

The formula for calculating tumor volume (V) is: V=1/2×a×b ² (where a and b represent length and width, respectively).

Relative tumor volume (RTV): tumor volume on the last day/tumor volume at the start of dosing.

Experimental results: see Figure 1. Figure 1 shows the curative effect of zadaverine on human hepatoma Bel-7402 xenografted tumors in nude mice. It can be seen from Figure 1 that Zadaverine, administered orally at 200 mg/kg, exhibited a good antitumor effect on BEL-7402 transplanted tumors. At the same time, it can be seen from Figure 2 that the high dose has little effect on the body weight of mice, indicating that the toxicity is very small.

Zadaverine exhibits effective anti-tumor activity both in vivo and in vitro, especially has strong specificity for liver cancer, and it can be used as an anti-tumor drug in clinical application.

Claims (2)

1. 6-(4-(difluoro-methoxy)-3-methoxyphenyl) pyridazine-3 (2H)-one shown in formula I is preparing the purposes in medicines resistant to liver cancer

2. purposes according to claim 1, wherein, described medicines resistant to liver cancer suppresses the activity of BEL-7402 and SMMC-7721 cell strain specifically.