CN111423419B

CN111423419B - Small molecular compound cyy-260 and application thereof in preparation of antitumor drugs

Info

Publication number: CN111423419B
Application number: CN202010054258.5A
Authority: CN
Inventors: 刘志国; 赵承光; 黄晓颖; 王良兴; 杨乐和; 赵海洋; 庄严; 胡万乐; 戴璇璇; 周斌; 李继法
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-12-17
Anticipated expiration: 2040-01-17
Also published as: CN111423419A

Abstract

The invention discloses a small molecular compound cyy-260 and application thereof in preparing antitumor drugs, wherein the small molecular compound is a JAKs inhibitor, has a structure shown in a formula (I), and has an inhibiting effect in the occurrence and development of non-small cell lung cancer. Experiments prove that cyy-260 can inhibit the proliferation, clone formation and migration of lung cancer cells in a concentration-dependent manner and induce apoptosis. Lower concentrations achieved antitumor biological activity.

Description

Small molecular compound cyy-260 and application thereof in preparation of antitumor drugs

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a small molecular compound cyy-260 and application thereof in preparation of antitumor drugs.

Background

Lung cancer is a heterogeneous group of tumors consisting of over 50 histomorphological subtypes. Over the past few decades, non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC) have been the most commonly used diagnostic terms for lung cancer, mainly due to the limited treatment options, which usually do not require further differentiation of morphological subtypes.

NSCLC accounts for approximately 80% of all lung cancers, with Adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) being the major histological subtypes of NSCLC. Clinically, only a small fraction of NSCLC patients with early diagnosis (stage I or II) can be surgically resected. At the time of diagnosis, over 60% of lung cancer patients develop locally advanced or metastatic disease (stage III or IV), at which time surgical resection may not be an option. Until now, conventional chemotherapy and radiation therapy have remained the primary means of treatment for lung cancer patients. Therefore, the development of novel anti-lung cancer drugs has great significance for controlling the development of lung cancer.

Disclosure of Invention

The invention provides a small molecular compound cyy-260 and application thereof in preparing an anti-tumor medicament, wherein the small molecular compound cyy-260 can be used for inhibiting the occurrence and development of lung cancer.

A small molecule compound cyy-260 has a structure shown in formula (I):

the molecular formula of the small molecular compound is C₂₃H₂₆N₈。

The invention also provides application of the small molecule compound cyy-260 in preparation of antitumor drugs.

The invention discovers that the small molecular compound cyy-260 has obvious anti-tumor effect by researching the pharmacological effect of the small molecular compound cyy-260, and particularly, the small molecular compound can be used for controlling or treating lung cancer.

Drawings

FIG. 1 is the results of the MTT assay for the cyy-260 compound of example 2;

FIG. 2 is the results of the clonogenic assay for the cyy-260 compound of example 2;

FIG. 3 is the results of the test for inhibiting migration of lung cancer cells with the cyy-260 compound of example 3;

FIG. 4 shows the results of the Hoechst staining test in example 4;

FIG. 5 shows the results of the Annexin V-FITC/PI double staining test in example 4;

FIG. 6 shows the results of inhibition of lung cancer cell JAKs protein expression by Cyy-260 in example 5.

Detailed Description

Example 1

(1) 2, 4-dichloro-5-methylpyrimidine (323.96mg, 2.0mmol) and N, N-Diisopropylethylamine (DIPEA) (516.96mg, 4mmol) were dissolved in DMF (4mL) and cooled to 0 ℃. To the mixture was added 6-aminoindazole (266.3mg, 2mmol) dissolved in DMF (2mL) dropwise. The reaction mixture was stirred at 0 ℃ for about 1 hour. Next, the ice bath was removed, the reaction mixture was stirred at room temperature and monitored by TLC, the resulting mixture was extracted with EtOAc (3X 25mL), washed with saturated brine and over anhydrous Na₂SO₄Dried and concentrated to obtain an intermediate product by silica gel column chromatography. The characterization data are as follows:

N-(2-chloro-5-methylpyrimidin-4-yl)-1H-indazol-6-amine

mixing with dichloromethane: methanol 16: 1 as eluent, to give a yellow solid (163.5mg, 21.0% yield).¹H NMR(400MHz,DMSO)δ13.06(s,1H),8.97(s,1H),8.10(s,1H),8.03(s,2H),7.74(d,J＝8.0Hz,1H),7.35(d,J＝6.8Hz,1H),2.24(s,3H)。

The reaction formula is as follows:

(2) the intermediate product of step (1) (259.7mg, 1mmol) and 4- (4-methylpiperazine) aniline (191.27mg, 1mmol) were dissolved in 4ml of methanol, and TFA (148.56 μ L, 2mmol) was added, warmed to 80 ℃, and the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature, and the resulting mixture was adjusted to neutral with saturated sodium hydrogencarbonate, extracted with ethyl acetate (3X 25mL), washed with saturated brine, and then washed with anhydrous sodium chlorideNa₂SO₄Drying and concentrating, and obtaining the product cyy-260 through silica gel column chromatography. The characterization data are as follows:

4-(4-((4-((1H-indazol-6-yl)amino)-5-methylpyrimidin-2-yl)amino)phe nyl)thiomor-pholine 1,1-dioxide

mixing with dichloromethane: methanol 16: 1 as eluent, to give a grey solid (140.9mg, 81.4% yield).¹H NMR(500MHz,DMSO)δ13.00(s,1H),8.83(s,1H),8.45(s,1H),8.01–7.84(m,3H),7.67(d,J＝8.5Hz,1H),7.57(d,J＝8.0Hz,2H),7.43(d,J＝8.0Hz,1H),6.82(d,J＝8.0Hz,2H),3.62(s,4H),3.11(s,4H),2.14(s,3H).¹³C NMR(126MHz,DMSO)δ159.39,158.85,158.29,155.68,142.01,138.02,134.28,133.10,119.84,119.67,119.11,117.52,116.69,105.32,102.49,49.91,47.86,13.56；ESI-MS m/z:450.17(M﹢H)^﹢,calcd for C₂₂H₂₃N₇O₂S,found 450.14.

Example 2 inhibition of Lung cancer cell proliferation and clonogenic assays

We analyzed the effect of cyy-260 on lung cancer cell proliferation by MTT assay as well as by cloning assay. Selecting three human lung cancer cells PC-9, A549 and H1975, collecting logarithmic phase cells, paving the cells in a 96-well plate according to 3000 cells per well, adding different cyy-260 concentrations (D, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10 and 20 mu M) after the adherent monolayer of the cells is paved on the bottom of the well, adding 25 mu l of MTT solution per well (5mg/ml) after the drug action is 48H, and terminating the culture after the continuous incubator is incubated for 4 hours. After discarding the supernatant, 150ul DMSO/well was added to dissolve the crystals. The light absorption value of each well was measured in a multifunctional microplate reader OD490nm, and the half inhibitory concentration (IC50 value) was calculated using GraphPad Prism. The experimental results are shown in FIG. 1, and show that cyy-260 can inhibit the proliferation of lung cancer cell lines in a dose-dependent manner after 48 hours of action.

Tumor cells can be propagated indefinitely to form cell colonies, and through a clone formation experiment, the cyy-260 can inhibit the clone formation of lung cancer cells. Taking cells in exponential growth phase, and preparing the cells into cell suspension by adopting a conventional digestion passage method. Uniformly paving cells into a 6-hole plate according to 1000 cells/hole, adding cyy-260 drugs with different concentrations after the cells adhere to the wall, respectively setting the final concentrations to be 1,2 and 4 mu M, removing the drugs after acting for 24h, adding a fresh culture medium to continue culturing, stopping culturing after the cells form macroscopic colonies, discarding the culture solution, and carefully washing the cells with PBS for 2 times. Fixing with 4% paraformaldehyde, dyeing with crystal violet solution, recovering crystal violet, and air drying. The size and number of colonies between wells were compared and the results are shown in FIG. 2. The results show that cyy-260 limits the clonogenic formation of lung cancer cells concentration-dependently.

Example 3 test for inhibiting migration of Lung cancer cells

We used the transwell experiment to evaluate the effect of cyy-260 on invasion of cell migration. The lung cancer cells PC-9, H1975 are cultured to the logarithmic growth phase, 600-⁴-4×10⁴Adding medicine every other day, continuing to culture in an incubator for 24 days, terminating the culture, discarding culture solution, fixing with 4% paraformaldehyde, staining with crystal violet solution, and taking pictures under a microscope for recording. The results are shown in FIG. 3. The results show that cyy-260 can significantly and concentration-dependently inhibit lung cancer cell migration and invasion.

Example 4 assay for inducing apoptosis of Lung cancer cells

Hoechst is a fluorescent dye with membrane permeability, so that normal cells and intermediate and early apoptotic cells can be stained by Hoechst, but the Hoechst staining form of normal cell nuclei is circular and light blue, and deeper blue particles are arranged in the Hoechst staining form; while the nucleus of the apoptotic cell appears bright blue due to concentration, or the nucleus appears in the form of leaves, fragments and aggregates. The cell nucleus is stained by using a Hoechst33258 apoptosis staining kit, and the deformation degree of the cell nucleus is judged by observing the fluorescent staining condition of the cell nucleus, so that the apoptosis condition is reflected. Soaking common clean cover glass in 70% ethanol for 5 min or more, placing the cover glass in a six-hole plate, and culturing lung cancer cells overnight to reach about 50% fullness. After cyy-260 stimulated apoptosis, the culture was aspirated, fixed with 4% paraformaldehyde, stained with Hoechst33258, and recorded by taking a photograph under a fluorescent microscope. As a result, as shown in FIG. 4, cyy-260 induced apoptosis of lung cancer cells in a concentration-dependent manner.

Next, we again evaluated the effect of cyy-260 on lung cancer apoptosis using flow cytometry. Lung cancer cells are uniformly paved in a 6-well plate, after cyy-260(1,2,4 mu M) with different concentrations acts for 36 hours, the effect of inducing apoptosis of the cyy-260 cells is detected on a flow cytometer by using Annexin V-FITC/PI double staining method. As shown in FIG. 5, cyy-260 was able to induce apoptosis in lung cancer cells dose-dependently.

Example 5 Cyy-260 inhibition of Lung cancer cell JAKs protein expression

The JAK/STAT3 pathway plays an important role in the processes of cell proliferation, invasion and apoptosis and is highly expressed in various tumors. We assessed the expression of JAKs protein of lung cancer cells by cyy-260 by Western Blot experiment. As shown in FIG. 6, cyy-260 inhibited the expression of JAK and STAT3 proteins in lung cancer cells, and was concentration-dependent.

Claims

1. The application of a small molecular compound cyy-260 in preparing antitumor drugs is characterized in that the structure is shown as the formula (I):

the anti-tumor medicine is used for controlling or treating lung cancer.

2. The use of the small molecule compound cyy-260 as claimed in claim 1 for the preparation of an anti-tumor medicament, wherein said lung cancer is non-small cell lung cancer.

3. The use of the small molecule compound cyy-260 as claimed in claim 1 for the preparation of an anti-tumor medicament for inhibiting the proliferation and clonogenic formation of lung cancer cells.

4. The use of the small molecule compound cyy-260 as claimed in claim 1 for the preparation of an anti-tumor medicament for inhibiting the migration of lung cancer cells.

5. The use of the small molecule compound cyy-260 as claimed in claim 1 for the preparation of an anti-tumor medicament for inducing apoptosis in lung cancer cells.

6. The use of the small molecule compound cyy-260 as claimed in claim 1 in the preparation of an anti-tumor medicament for inhibiting the expression of the JAKs protein in lung cancer cells.