CN107334767A - A kind of application of pyridazinone compound in oncotherapy - Google Patents

Abstract

The present invention relates to the application of a pyridazinone compound in the treatment of tumors. The pyridazinone compound of the present invention has an action mechanism of causing DNA damage by causing DNA double-strand breaks in tumor cells. All of them showed a strong inhibitory effect on proliferation, and in vivo experiments were used to treat transplanted liver cancer in mice, and a moderate tumor inhibitory effect could be obtained. Pyridazinone compound of the present invention, its structural formula is as follows: The compound is a kind of brand-new structural compound that causes DNA double-strand damage in tumor cells, and has a good prospect of being developed as an anti-tumor drug.

Description

Application of a pyridazinone compound in tumor treatment

Technical field:

The invention belongs to the technical field of medicine. More specifically, the present invention relates to the application of a pyridazinone compound in tumor treatment.

Background technique:

Malignant tumors are common diseases that seriously threaten human health and life. The toxicity of anti-tumor drugs in clinical use is a problem that plagues cancer chemotherapy, and finding new anti-tumor drugs is a major task of anti-tumor drug research. The compounds involved in the present invention are pyridazinone compounds. Pyridazinone compounds are a class of heterocyclic compounds with good biological activity, which occupy an important position in the research fields of pesticide and medicine. Pyridazinone compounds can be used as calcium sensitizers for the treatment of cardiac dysfunction and heart failure, and also have certain curative effects in anti-platelet aggregation, antihypertensive, anti-inflammation, anti-shock, and anti-convulsions. Certain pyridazinone derivatives also have good antitumor activity, such as:

Chinese patent 200880017969.2 discloses a pyridazinone derivative with antitumor activity.

Chinese patent 200910127196.X discloses a kind of 6-(3-(trifluoromethyl)phenyl)pyridazin-3(2H)-ketone as the core of the pyridazinone compound shown in the following structural formula I. The use in the preparation of antitumor drugs, especially the use in anti-liver cancer drugs.

Chinese patent 201410018618.0 discloses a series of pyridazinone derivatives and their antitumor effects.

Chinese patent 201310306863.7 discloses the use of pyridazinone compounds as tyrosine kinase inhibitors, especially c-Met inhibitors.

Compounds IMB5036 and IMB5043 are compounds with known structures in existing compound libraries, which can be purchased from the market or synthesized according to corresponding chemical synthesis methods. The two compounds of the present invention were purchased from ENAMINEL Ltd. Its anti-tumor activity has not been reported so far. Although the two compounds belong to pyridazinone compounds, they are significantly different from the prior art in structure. In the process of screening anti-tumor drugs, the inventors unexpectedly found that these two compounds have killing effect on various tumors. effect.

Invention content:

The invention provides a kind of pyridazinone compound, its structural formula is as follows:

Wherein, R1 and R2 are halogen at the same time, preferably Cl at the same time, or Br at the same time; R3 is O, and R4 is selected from the following structures:

The following two pyridazinones are preferred:

Compound I, named IMB5036, has a CAS number of 1090393-42-2.

Compound II, named IMB5043, has a CAS number of 1089995-96-9.

The inventors studied the drug activity of these two compounds and found that IMB5036 and IMB5043 have good antitumor activity, especially the best therapeutic effect on liver cancer. It was further found that it can play an anti-tumor effect by causing DNA double-strand breaks to cause DNA damage.

To this end, the present invention provides an application of the above-mentioned pyridazinone compounds including IMB5036 and IMB5043 in the preparation of antitumor drugs.

The tumors described in the present invention are selected from: liver cancer, colorectal cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, breast cancer, fibrosarcoma and other treatment-resistant or resistant tumors.

The present invention further provides a pharmaceutical composition containing a pyridazinone compound whose structure is as described above.

The beneficial effects of the present invention are illustrated below through experimental data.

Test Example 1: Cytotoxic effect of compounds on cultured cells.

The cytotoxic effect of compounds IMB5036 and IMB5043 on cultured cells was detected by MTT (tetramethylazolazolium salt) method. Cells were incubated with RPMI-1640 medium (Giboco BRL Inc.) containing 10% fetal bovine serum (Giboco BRL Inc.), 2 mM glutamine, 100 μg/ml streptomycin and 100 U/mL penicillin at 37°C with 5% CO ₂ in the incubator.

The tumor cell lines used are all common cell lines, which are stored in our laboratory, and can also be purchased from commercial channels such as ATCC cell bank (Rockville, MD, USA) and the National Experimental Cell Resource Sharing Platform. The cells in the logarithmic growth phase were digested and counted, and 4,000 cells/well were spread on a 96-well plate. After 24 hours of culture, different concentrations of drugs were added, and 3 parallel wells were set for each drug concentration. After continuing to culture for 48 hours, add 20 μL of 5 mg/mL MTT (Amresco, Ohio, USA) dissolved in PBS to each well. After continuing to culture at 37°C for 4 hours, discard the supernatant, add 150 μL of dimethyl sulfoxide, and store at room temperature. Shake on a shaker for 15 minutes, and measure the absorbance value A at 570 nm on a microplate reader (Thermo Labsystems, Multiskan MK3). For each experiment, 3 wells were set up as control wells without drugs and blank wells without cells. According to the formula: inhibition rate%=(A control group-A administration group)/(A control group-A blank group)×100%, calculate the drug's inhibition rate on cell proliferation and calculate the half inhibitory concentration (IC ₅₀ ). The results are shown in Table 1. The two compounds have good killing effects on tumor cells from different sources. IMB5036 has the strongest killing effect on human liver cancer cell SMMC-7721, with an IC ₅₀ of 1.85 μM; IMB5043 also has the best killing effect on SMMC-7721 cells. has the strongest killing effect, with an IC ₅₀ of 3.56 μM. Human embryonic liver cells L02 were not sensitive to the two compounds.

Table 1. Cytotoxic activity of IMB5036 and IMB5043 on tumor cells and normal cell L02

Experimental Example 2: Detection of the effects of IMB5036 and IMB5043 on DNA damage by indirect immunofluorescence.

Take the SMMC-7721 cells in the logarithmic growth phase and inoculate them in a six-well plate covered with coverslips. After 24 hours, add 1 μM IMB5036 and 2 μM IMB5046 respectively, and set up control wells. After 24 hours of drug treatment, remove the coverslips The slices were washed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% TritonX-100 for 15 minutes, blocked with 1% BSA at room temperature for 30 minutes; AF488-labeled γ-H2AX antibody (EMD Millipore) was added at temperature Incubate at 37°C for 1 hour, observe and take pictures under a fluorescent microscope (Olympus IX81). The results showed that tumor nuclei treated with IMB5036 and IMB5043 significantly increased the formation of phosphorylated H2AX foci compared with the control group (Figure 1).

Experimental Example 3: Animal experimental treatment schemes of IMB5036 and IMB5043.

The in vivo efficacy of the compounds was evaluated using the human liver cancer SMMC-7721 xenograft tumor model. The SMMC-7721 cells were inoculated subcutaneously in the axilla of NIH nu/nu mice at 1×10 ⁷ /0.2mL/only, and two weeks later, the tumor pieces were cut into small pieces of 2mm ³ in normal saline, and then inoculated with a trocar. The tumor mass was transplanted subcutaneously into the axilla of nude mice. On the seventh day, the nude mice were divided into groups according to the size of the tumor mass, with 7 mice in each group, so that the average value of the tumor mass size of the animals in each group was close to each other. The compound was dissolved in dimethyl sulfoxide (Sigma)/polyoxyethylene castor oil (Cremophor EL) (Sigma)/normal saline (1:2:17) mixture, administered intraperitoneally, and the dosage was 12.5 or 25 mg /kg, 5 times/week, 0.2mL/only, for two consecutive weeks. During the experiment, the long axis a and the short axis b perpendicular to it were measured every 2-3 days, and the body weight of the animals was recorded. The tumor volume and inhibition rate were calculated according to the formula V=1/2ab ² (tumor volume of the control group−tumor volume of the test group)/tumor volume of the control group×100%. Animals were sacrificed by dislocation when the tumor volume reached 1000 mm ³ .

The experimental results show that IMB5036 can significantly inhibit the growth of tumors. IMB5036 of 12.5 and 25mg/kg was administered intraperitoneally, and the tumor inhibition rates on the 28th day after administration were 30.6% and 66.2% respectively (Fig. 2). The animals had no obvious weight loss or behavioral abnormalities (Fig. 3), which indicated that the mice could better tolerate the drug at this dose. IMB5043 shows a moderate tumor inhibitory effect. IMB5043 of 12.5 and 25 mg/kg was administered intraperitoneally, and the tumor inhibitory rates on the 28th day after administration were 28.6% and 58.8% respectively (Fig. 4). Animals in all treatment groups had no Obvious weight loss or abnormal behavior (Fig. 5) shows that the mice can better tolerate this dose of drug.

The present invention further includes a compound of the present invention or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from salts formed with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, or acetic acid , trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, and their alkali metal salts, alkaline earth Metal salts, silver salts, barium salts, etc.

The present invention also provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions are in the form of formulations suitable for pharmaceutical use. Pharmaceutical preparations are selected from tablets, capsules, granules, oral liquids, injections and the like.

In the pharmaceutical composition of the present invention, as a preparation form, the effective amount of the compound of the invention contained in each dose is 0.1-1000 mg, and each dose refers to each preparation unit, such as each piece of a tablet, and each capsule of a capsule. , can also refer to each dosage, such as 100 mg each time.

When the pharmaceutical composition of the present invention is prepared into solid pharmaceutical preparations in the form of tablets and capsules, a solid carrier can be used. The solid carrier that can be used is preferably one or more substances selected from diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, swelling agents, etc., or can be encapsulated substances. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sucrose, lactose, pectin, dextrin, starch, gelatin, methylcellulose, sodium carboxymethylcellulose, cocoa butter, and the like. Because of their ease of administration, tablets and capsules represent the most advantageous oral solid preparations.

The weight ratio of the compound of the present invention in the composition contained in the pharmaceutical composition is 0.1-99.9%, and the weight ratio of the pharmaceutically acceptable carrier in the composition is 0.1-99.9%.

It is especially advantageous to formulate the aforementioned pharmaceutical preparations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form of formulation refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect. Such dosage unit forms may be in packaged form, eg tablets, capsules.

Although the amount of active ingredient contained in dosage unit forms may vary, it will generally be adjusted within the range of 1 to 800 mg, depending on the potency of the active ingredient chosen.

Those skilled in the art can routinely determine the preferred dosage for a particular situation. Generally, the initial treatment dose is lower than the optimum dose of the active ingredient, and then the dose is gradually increased until the optimum therapeutic effect is achieved. For convenience, the total daily dosage may be divided and administered in divided doses.

Compared with the pyridazinone compounds in the prior art, the present invention has higher antitumor activity and less toxic and side effects. Used as an oral antineoplastic drug.

Description of drawings:

Figure 1 IMB5036 and IMB5043 can increase the formation of γ-H2AX foci in the nuclei of human liver cancer cells SMMC-7721 (Figure 1).

Figure 2 IMB5036 can significantly inhibit tumor growth. IMB5036 at 12.5 and 25 mg/kg was administered intraperitoneally, and the tumor inhibition rates were 30.6% and 66.2% on day 28 after administration (Figure 2).

Fig. 3 Animals in all IMB5036-treated groups had no obvious weight loss or behavioral abnormalities (Fig. 3).

Figure 4 IMB5043 shows the tumor inhibitory effect, 12.5 and 25 mg/kg of IMB5043 were administered intraperitoneally, and the tumor inhibitory rates were 28.6% and 58.8% on day 28 after administration (Figure 4).

Fig. 5 Animals in all IMB5043-treated groups had no obvious weight loss or behavioral abnormalities (Fig. 5).

detailed description

The present invention is further illustrated by the following examples, but not as a limitation of the present invention.

Example 1

Tablet preparation

Take 0.5g of any one compound of IMB5036 and IMB5043, 4.5g of starch, 0.9g of powdered sugar and 1ml of distilled water. After mixing the above components evenly, granulate, granulate, dry, add a small amount of lubricant, compress into tablets and pack separately.

Example 2

Preparation of capsules

Take 0.5g of any compound of IMB5036 and IMB5043, 4.5g of starch, 0.9g of powdered sugar and 1ml of distilled water, mix the above components evenly, granulate, granulate, dry, and pack into capsules.

Example 3

Preparation of granules

Take 0.5g of any one compound of IMB5036 and IMB5043, 4.5g of starch, 0.9g of powdered sugar and 1ml of distilled water, mix the above components evenly, granulate, size the granules, dry, and pack to obtain.

Claims (10)

1. The application of a pyridazinone compound in the preparation of antitumor drugs, the structure of the pyridazinone compound is as follows: Wherein, R1 and R2 are halogen at the same time, R3 is O, and R4 is selected from the following structures: or 2. The application according to claim 1, wherein R1 and R2 in the compound are Cl simultaneously, or Br simultaneously. 3. application according to claim 1, wherein said compound is selected from compound I, named after IMB5036, structural formula is as follows: 4. The application according to claim 1, wherein said compound is compound II named after IMB5043, and its structural formula is as follows: 5. The application according to claim 1, wherein the tumor is selected from: liver cancer, colorectal cancer, pancreatic cancer, esophageal cancer, lung cancer, ovarian cancer, breast cancer, fibrosarcoma and other treatment-resistant or resistant tumors. 6. application according to claim 1, wherein said medicine is selected from tablet, capsule, granule, oral liquid, injection. 7. A pharmaceutical composition containing pyridazinone compounds, the structure of which is as follows: Wherein, R1 and R2 are halogen at the same time, R3 is O, and R4 is selected from the following structures: or 8. The pharmaceutical composition according to claim 7, wherein R1 and R2 in the compound are Cl at the same time, or Br at the same time. 9. The pharmaceutical composition according to claim 7, wherein the compound is selected from compound I, named after IMB5036, and the structural formula is as follows: 10. The pharmaceutical composition according to claim 7, wherein said compound is compound II named after IMB5043, and its structural formula is as follows: