CN107362166B - Application of tetrahydropyrido [4,5- ] thieno [2,3- ] pyrimidine-4 (3) -ketone compound in pharmacy - Google Patents

Abstract

The invention provides a pharmaceutical composition using tetrahydropyrido[4,5-]thieno[2,3-]pyrimidine-4(3)-one compounds as active ingredients, and the preparation of Wee1 small molecule inhibitor application in medicaments. Wee1 kinase is a candidate drug target for tumor therapy, and its inhibitor combined with some chemotherapeutic drugs can enhance its antitumor activity. So far, only one Wee1 small molecule inhibitor has entered the clinical trial stage. The screening of in vitro anti-tumor activity shows that the compound of the present invention has good inhibitory activity against five different tumor cell lines. It can be used to develop new Wee1 small-molecule inhibitors, providing a new option for the field of tumor therapy, and has good application value.

Description

Tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-ones are prepared in application in medicine

technical field

The present invention relates to the technical field of medicine, in particular to a tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compound, and a pharmaceutical combination using the compound as an active ingredient and the application of the compound and the pharmaceutical composition in the preparation of an anticancer drug for inhibiting the overexpression of Wee1 kinase. The compound has inhibitory effect on tumor cell lines and can be used as a leading compound for developing new anti-tumor drugs.

Background technique

Wee1 protein kinase was first discovered in fission yeast cells and belongs to the serine and threonine protein kinase family, which is highly conserved and widely found in eukaryotes. It is a cell cycle regulatory protein that is mainly activated during the S phase or G2/M phase of the cell cycle. It can regulate the phosphorylation state of cyclin-dependent kinase 1 (CDK1), thereby regulating the activity of the complex of CDK1 and cyclin B (cyclinB) to achieve the regulation of the cell cycle, and is important for DNA damage checkpoints. regulating effect. Wee1 protein kinase regulates mitosis through the action of three cellular checkpoints, namely the cellular G2/M phase checkpoint, the cell size checkpoint and the cellular DNA damage checkpoint. Studies have shown that overexpression of Wee1 kinase has been detected in various types of malignant tumor cells, indicating that Wee1 kinase is a candidate target for cancer therapy.

Currently, there are three main Wee1 kinase-based inhibitors, namely MK-1775, PD0407824, and PD0166285. Among them, MK-1775 combined with other chemotherapy drugs to treat ovarian cancer is currently in the phase II clinical research stage, while PD0166285 and PD0407824 inhibitors have not been tested in patients. Wee1 inhibitors play an anti-tumor effect by inhibiting the activity of Wee1, making tumor cells advance from S phase or G2 phase to M phase, unable to complete DNA synthesis and repair, and manifested as abnormal cell histone synthesis and formation of bipolar spindles. , chromosomes are scattered, and eventually exit mitosis, triggering apoptosis, which is called "mitotic catastrophe". Some Wee1 inhibitors have been applied to the clinical stage and have initially shown broad application prospects in anti-tumor therapy. The combination of Wee1 kinase inhibitors and DNA-inhibiting drugs is likely to be a more effective treatment strategy against cancer. Therefore, the discovery of more efficient and specific Wee1 small molecule inhibitors has great practical significance in tumor therapy. Therefore, it is necessary to find more efficient and specific Wee1 small molecule inhibitors. At present, there is no research report on such compounds in cancer treatment.

At present, there is no report on the pharmaceutical composition of tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compounds as the active ingredient in the prior art, and there is no report Application report of the compound and its pharmaceutical composition in the preparation of Wee1 kinase inhibitor and antitumor drug.

SUMMARY OF THE INVENTION

The present invention aims to provide a tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compound (1), comprising compound 1 in an effective amount for treating cancer and medicinal A pharmaceutical composition of a carrier or an excipient, a preparation method of the compound 1 and the pharmaceutical composition, and the application of the compound 1 or the pharmaceutical composition thereof in the preparation of a Wee1 kinase inhibitor medicine.

In order to achieve the above-mentioned purpose of the present invention, the present invention provides the following technical solutions:

A pharmaceutical composition containing a therapeutically effective amount of a tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compound (1) represented by the following structural formula or a medicine thereof with a salt, and at least one pharmaceutically acceptable carrier,

The pharmaceutical composition as described, wherein the pharmaceutically acceptable salt of the compound tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one (1) is hydrochloric acid Salt, hydrobromide, nitrate, mesylate, sulfate, phosphate, succinate, malate, fumarate, maleate, p-toluenesulfonate, tartrate, citric acid Salt, formate, acetate, oxalate, trifluoroacetate, triflate.

The present invention also provides the compound tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compound (1) or a pharmaceutically acceptable salt thereof in the preparation of Wee1 kinase inhibitor application.

And, the use of the compound tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one compound (1) or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

According to the application, the tumor is leukemia, liver cancer, lung cancer, breast cancer and colon cancer.

In addition, the present invention also provides the application of the pharmaceutical composition in the preparation of Wee1 kinase inhibitor.

And, the application of the pharmaceutical composition in the preparation of antitumor drugs.

According to the application, the tumor is leukemia, liver cancer, lung cancer, breast cancer and colon cancer.

Through the biological activity test, the compounds of the present invention have measured IC50 on five tumor cell lines of leukemia, liver cancer, lung cancer, breast cancer and colon cancer. The results indicated that compound 1 had certain tumor growth inhibitory activity in vitro.

When the compounds of the present invention are used as medicines, they can be used directly or in the form of pharmaceutical compositions. The pharmaceutical composition contains 0.1-99%, preferably 0.5%-95% of the compound of the present invention, and the rest are pharmaceutically acceptable salts, or pharmaceutically acceptable carriers and/or excipients that are nontoxic to humans and animals.

The dosage of the compound of the present invention can be varied according to the route of administration, the age and body weight of the patient, the type and severity of the disease to be treated, etc. The daily dose can be 0.01-10 mg/kg body weight, preferably 0.1-5 mg/kg body weight. It can be administered one or more times.

Oral use of solid or liquid preparations, such as powder, tablet, sugar coating, capsule, solution, syrup, drop pills and the like.

Injection can be used in solid or liquid preparations, such as powder injections, solution injections and the like.

Description of drawings:

Figure 1 is a graph showing the IC50 inhibition of the compound tetrahydropyrido[4,5-]thieno[2,3-]pyrimidin-4(3)-one (1) on five different tumor cell lines.

Detailed ways:

In order to better understand the essence of the present invention, the following describes the application of compound 1 of formula (1) of the present invention and its biological activity results in the field of medicine with reference to the accompanying drawings.

Example 1:

The compound of the present invention was purchased from the Netherlands specs company (website: http://www.specs.com/), and its library number is: AO-476/42169315.

Example 2:

Determination of the half growth inhibitory concentration IC50 of the compound 1 of the present invention on five different tumor cell lines:

1. Experimental principle and steps

Aqueous One Solution reagent直接加入培养板孔的培养基中，孵育1-4h，然后以酶标仪读取490nm的吸光度值。该化合物的光密度OD(490nm)值与活细胞数目成正比。检测以顺铂和紫杉醇作为阳性对照。化合物的IC50值通过浓度效应生长曲线计算确定。MTS is an analog of MTT, and succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble Formazan compound, which can be directly dissolved in the medium. When testing, only a small amount of CellTiter

Aqueous One Solution reagent was directly added to the medium in the wells of the culture plate, incubated for 1-4 hours, and then read the absorbance value at 490nm with a microplate reader. The OD (490 nm) value of the compound is proportional to the number of viable cells. The assay used cisplatin and paclitaxel as positive controls. IC50 values for compounds were determined by concentration-response growth curve calculations.

Aqueous One Solution reagent。选择最适细胞浓度的五种不同肿瘤细胞株，包括HL-60，SMMC-7721，A-549，MCF-7和SW480。分别铺入96孔培养板中，每孔100μL，37℃，5％CO₂的环境下孵育24h，分别加入20μLCellTiter

Aqueous One Solution reagent后继续孵育2h，在490nm读取吸光度值。并设置阳性对照组顺铂和紫杉醇。Room temperature, stand for 90 minutes, fully thaw CellTiter

Aqueous One Solution reagent. Five different tumor cell lines with optimal cell concentration were selected, including HL-60, SMMC-7721, A-549, MCF-7 and SW480. Plated into 96-well culture plates, 100 μL per well, incubated at 37°C, 5% CO ₂ for 24 h, and added 20 μL CellTiter

Continue to incubate for 2h after Aqueous One Solution reagent, and read the absorbance value at 490nm. And set positive control group cisplatin and paclitaxel.

2. Reagents and instruments

AQueous One Solution Cell Proliferation AssayReagent: CellTiter

AQueous One Solution Cell Proliferation Assay

Instruments: CO ₂ incubator (Thermo), ultra-clean bench (Thermo), chemiluminescence microplate reader (Thermo)

3. Experimental results

1) Inhibition rate: Inhibition rate=(1-A _sample /A _{positive control} )*100%

The inhibitory activity of compound 1 is shown in Figure 1 and Table 1, wherein the percentage indicates the inhibitory rate of the compound against five different tumor cell lines when the reaction concentration is 50 μM.

Table 1 IC50 (μM) of compounds on different tumor cell lines

Example 3:

Compound 1 was added with 4% sulfuric acid ethanol solution, pH=4, filtered and dried to prepare sulfate compound 1.

Example 4:

Compound 1 was added with 4% hydrochloric acid solution, pH=4, filtered and dried to prepare compound 1 as hydrochloride salt.

Example 5:

Compound 1 was added with 4% tartaric acid solution, pH=4, filtered and dried to prepare tartrate compound 1.

Example 6:

Compound 1 was added with 4% citric acid solution, pH=4, filtered and dried to prepare citrate compound 1.

Example 7:

Capsule: Compound 1 or the salt obtained in Example 2-5 10 mg, lactose 187 mg, and magnesium stearate 3 mg.

Preparation method: the compound or its salt is mixed with a cosolvent, sieved, and uniformly mixed, and the obtained mixture is packed into hard gelatin capsules, each capsule weighing 200 mg, and the active ingredient content is 10 mg.

Claims (3)

1. The application of the pharmaceutical composition in preparing antitumor drugs is characterized in that the tumors are leukemia, liver cancer, lung cancer, breast cancer and colon cancer, and the pharmaceutical composition contains an effective amount of tetrahydropyrido [4,5- ] thieno [2,3- ] pyrimidine-4 (3) -one compounds (1) or pharmaceutical salts thereof shown in the following structural formula and at least one pharmaceutically acceptable carrier,

2. the use of a pharmaceutical composition according to claim 1 for the preparation of a medicament against tumors, wherein the pharmaceutically acceptable salts of the compounds tetrahydropyrido [4,5- ] thieno [2,3- ] pyrimidin-4 (3) -one (1) are hydrochloride, hydrobromide, nitrate, methanesulfonate, sulfate, phosphate, succinate, malate, fumarate, maleate, p-toluenesulfonate, tartrate, citrate, formate, acetate, oxalate, trifluoroacetate, trifluoromethanesulfonate.

3. The application of the compound tetrahydropyrido [4,5- ] thieno [2,3- ] pyrimidine-4 (3) -ketone compound (1) or the medicinal salt thereof in preparing the antitumor drugs is characterized in that the tumors are leukemia, liver cancer, lung cancer, breast cancer and colon cancer.

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