阅读说明：本技术 一种对肿瘤具有杀伤作用的拓扑异构酶i抑制剂 (Topoisomerase I inhibitor with tumor killing effect ) 是由 袁健 耿国河 于 2019-12-10 设计创作，主要内容包括：本发明提出了一种对肿瘤具有杀伤作用的拓扑异构酶I抑制剂,通过研究发现(1Z,2E)-1-(甲基亚胺)-4a,5,6,7,8,8a-六氢萘-2(1H)-酮肟(DIA-002)在体内和体外均具有抗肿瘤和诱导DNA损伤的能力,随后的进一步研究发现,DIA-002可以抑制Top1的活性。(The invention provides a topoisomerase I inhibitor with a killing effect on tumors, and researches show that (1Z, 2E) -1- (methylimine) -4a, 5, 6, 7, 8, 8 a-hexahydronaphthalene-2 (1H) -ketoxime (DIA-002) has the capacity of resisting tumors and inducing DNA damage in vivo and in vitro, and further researches show that DIA-002 can inhibit the activity of Top 1.)

1. A compound of the general formula (I) or a pharmaceutically acceptable salt thereof:

2. a pharmaceutical composition comprising a compound of formula (I) as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

Technical Field

The invention relates to the technical field of medicines, in particular to a topoisomerase I inhibitor with a tumor killing effect.

Background

Breast cancer is the most common cancer in women, with about 246660 new cases and 40450 deaths in the united states in 2016. Triple Negative Breast Cancer (TNBC) is an aggressive breast cancer, accounting for approximately 15% of all breast cancers. TNBC is defined as a lack of expression and activation of the Estrogen Receptor (ER), the Progestogen Receptor (PR) and the human epidermal growth factor receptor (HER 2). Currently, treatment options for TNBC patients are still very limited because HER2 targeted therapy or hormonal therapy is ineffective. In addition, the TNBC has high drug resistance, early metastasis, poorer prognosis and the like, and in addition, the conventional chemotherapy is still the main treatment means of the TNBC but the toxic and side effects of the treatment are difficult to tolerate. Therefore, there is an urgent need to develop new methods to treat this challenging disease.

Disclosure of Invention

In view of the problems identified in the background art, the present invention proposes a topoisomerase I inhibitor having a killing effect on tumors.

Treatment of Triple Negative Breast Cancer (TNBC) remains a major challenge in clinical practice and new therapeutic approaches are urgently needed. In this application, (1Z, 2E) -1- (methylimine) -4a, 5, 6, 7, 8, 8 a-hexahydronaphthalene-2 (1H) -ketoxime was found to have antitumor and DNA damage-inducing abilities both in vivo and in vitro, (hereinafter "DIA-002" means "(1Z, 2E) -1- (methylimine) -4a, 5, 6, 7, 8, 8 a-hexahydronaphthalene-2 (1H) -ketoxime"). Subsequent further studies found that DIA-002 could inhibit the activity of Top1, thereby achieving the effect of killing tumor cells.

Drawings

FIG. 1A is a general formula (I);

FIG. 1B is a schematic representation of the anti-proliferation of DIA-002 against various tumor cells;

FIG. 1C is a schematic diagram showing the inhibition of DIA-002 on proliferation of breast cancer cell lines;

FIG. 2A schematic representation of MDA-MB-231 cells treated with 2. mu. mDIA-002;

FIG. 2B Effect of treatment with DIA-002 on the expression of γ H2AX, p-chk1, and p-chk 2;

FIG. 2C Effect of using DIA-002 treatment on Top I cc dot (Foci) formation;

FIG. 3A is a schematic representation of DIA-002 induced MDA-MB-231 cells to develop G2/M cell cycle arrest;

FIG. 3B Effect of DIA-002 treatment on MDA-MB-231 cell pH3 staining;

FIG. 3C Effect of DIA-002 treatment on caspase-3 expression cleaved from MDA-MB-231 cells;

FIG. 4A is a graph of DIA-002 inhibiting the activity of Top I;

FIG. 4B is a graph II of the ability of DIA-002 to inhibit the activity of Top I;

FIG. 5A Effect of using DIA-002 and camptothecin on the volume of MDA-MB-231 transplanted tumors;

FIG. 5B Effect of using DIA-002 and camptothecin on the volume of MDA-MB-231 transplanted tumors;

FIG. 5C is a graph showing the change in body weight of mice in each group.

Detailed Description

The following experiments are combined to demonstrate the effect of the novel type I topoisomerase inhibitors provided by the present invention in inhibiting topoisomerase activity and killing tumor cells.

(1) DIA-002 inhibits the activity of various tumor cell lines including MDA-MB-231 cells.

The chemical structure of DIA-002 is shown in FIG. 1A.

The antitumor effect of DIA-002 was examined on 2 normal cell lines as well as 14 tumor cell lines.

DIA-002 was found to exhibit potent antiproliferative effects on most human cancer cell lines tested (FIG. 1B), with IC50 values as shown in Table 1.

Watch (1)

The clonogenic assay results further confirmed the inhibitory effect of DIA-002 on proliferation of breast cancer cell lines (including MDA-MB-231, MCF-7, SKBR-3, and BT474) (FIG. 1C).

(2) DIA-002 induced DNA damage in MDA-MB-231 cells.

The degree of DIA-002-induced DNA damage was assessed by determining the formation of the nuclear punctum (Foci) gamma H2AX, a marker for DNA damage. The results show that MDA-MB-231 cells treated with 2. mu. mDIA-002 for 15min induced formation of gamma H2AX nuclear Foci (Foci) that gradually increased with increasing treatment time until reaching the maximum level for 1 hour, which lasted for more than 12 hours (FIG. 2A).

In addition, Western Blot results showed that DIA-002 treatment significantly increased the expression levels of γ H2AX, p-chk1, and p-chk2 (FIG. 2B).

In addition, DIA-002 treatment also induced the formation of Top I cc dots (Foci) (FIG. 2C).

These data indicate that DIA-002 treatment induced DNA damage in MDA-MB-231 cells.

(3) DIA-002 induced MDA-MB-231 cells to undergo G2/M cell cycle arrest and apoptosis.

Since DIA-002 has a killing effect on cells and can induce DNA damage, we further investigated its effects on cell cycle progression and apoptosis. By analyzing the cell cycle distribution by flow cytometry, we found that DIA-002 was able to induce G2/M cell cycle arrest in MDA-MB-231 cells (FIG. 3A).

In addition, DIA-002 treatment significantly attenuated pH3 staining (FIG. 3B), indicating that cells mitotically decreased after DIA-002 treatment.

Western Blot results showed that DIA-002 treatment significantly increased cleaved caspase-3 expression (FIG. 3C), indicating that DIA-002 induced apoptosis in MDA-MB-231 cells.

(4) DIA-002 inhibits the activity of Top I.

We investigated the effect of DIA-002 on the activity of TopI and TopII by experiments in which supercoiled PBR322322DNA was converted into a relaxed state. 50. 100nm of DIA-002 and camptothecin (50 μm) showed significant inhibition of Top I activity (FIG. 4A),

however, 50-100 nm DIA-002 had no significant inhibition of Top II activity, and Etoposide from the positive control group had significant inhibition of Top II activity (FIG. 4B).

(5) DIA-002 showed antitumor effect against transplanted tumor in nude mouse.

Based on the in vitro anti-tumor effect of DIA-002, we further tested the therapeutic effect of DIA-002 on transplanted tumors of human TNBCMDA-MB-231 cell nude mice, and camptothecin (a well-known TOP I inhibitor) was used as a positive control.

As shown in FIGS. 5A-B, DIA-002 and camptothecin significantly reduced the volume of MDA-MB-231 transplanted tumors.

In addition, there was no significant change in body weight in each group of mice (FIG. 5C), indicating that the dose of DIA-002 used was not severely systemically toxic to the mice.

The materials and methods that will be used in the above experiments are described below:

cell culture and antibodies:

mammary epithelial cell line MCF-10A, normal human fibroblast cell line IMR90, human breast cancer cell lines MDA-MB-231, SKBR-3, BT474, MCF-7, pancreatic cancer cell line BxPC-3, PANC-1, glioma cell lines U251, T98G, non-small cell lung cancer cell line A549, ovarian cancer cell lines OVCAR8, OVCAR10, prostate cancer cell line L NCAP, PC-3 and colon cancer cell line HCT116 were purchased from the American type culture Collection (ATCC, USA). cells were cultured using an appropriate medium containing 10% fetal bovine serum.

Antibodies against CHK1, p-CHK1(SER345), CHK2, p-CHK2(THR68) and Caspase-3 were purchased from CellSignaling Inc. antibodies against gamma H2AX (05-636) were purchased from Millipore and anti- β -actin antibodies were purchased from Sigma.

Cytotoxicity and clone viability assays:

cells were seeded into 96-well plates at 4000 cells per well. After 24 hours, cells were treated with varying concentrations of DIA-002 for 72 hours. Then, the cell viability was measured using MTS according to the manufacturer's instructions (Promega). Briefly, 20 ml of MTS was added to each well and 3 hours later, the absorbance at 490nm was measured using a microplate reader (Tecan Infinite M1000 Pro).

In six-well plate spread 500 cells, with the indicated DIA-002 concentration treatment of cells. After 10 days, cells were washed with PBS, fixed with methanol, stained with 0.1% crystal violet, and the number of colonies was counted.

Western blotting:

the cells were lysed using cell lysates (0.5% NP40,50mM Tris,150mM NaCl, and 1mM EDTA (NETN) buffer with 10mM Na F,50mM β -glycerophosphate, and 1mg/ml aprotinin, 1mg/ml pepstatin A). proteins were separated by SDS-PAGE gels, then transferred to PVDF membranes, and the proteins were detected with appropriate primary and secondary antibodies.finally the proteins were detected by detection with EC L-Western blot detection reagent (thermofisher).

Immunofluorescence detection of nuclear Foci (Foci):

cells were seeded on cover slips. After treatment, cells were washed with PBS, fixed with 3% paraformaldehyde for 15 minutes, followed by 0.5% Triton-X permeabilized cells diluted with PBS for 5 minutes and blocked with 5% goat serum for 1 hour at room temperature. Then, the cells were incubated with the primary antibody overnight at 4 ℃; the secondary antibody bound to Alexa Fluor 594 or Alexa Fluor488 was then incubated for 20 minutes at 37 ℃. After PBS wash, nuclei were counterstained with DAPI. Signals were detected using confocal microscopy.

Cell cycle analysis:

cells were harvested and fixed with 70% ethanol overnight at-20 ℃ and then washed twice with PBS cells were stained with RNase-containing Propidium Iodide (PI) in the dark for 30 minutes, followed by cell cycle analysis with facs (beckman coulter) data were analyzed by ModFit L T software.

TopI and II mediated DNA relaxation experiments

In brief, a 20. mu.l reaction system was used to study the activity of Top1, including 1. mu.l of human Top1, variable volumes of DIA-002 (final concentration: 50, 100nm) or camptothecin (50. mu.m), 4. mu.l of 5 × complete assay buffer, 1. mu.l of PBR322DNA, and variable volumes of water (to final volume). The mixture was incubated at 37 ℃ for 30 minutes and then stopped using 2. mu.l of 10% SDS. the sample was then run on a 1% agarose gel (containing 0.5. mu.g/ml ethidium bromide), 50V. the electrophoresis gel was placed in water before UV imaging.

In vivo antitumor study:

MDA-MB-231 cells (1 × 106) were suspended in 100. mu.l of PBS and injected subcutaneously into the flanks of 6-week-old female nude mice, and the tumor volume was calculated as V ═ 2 (L× W2)/2(V, volume; L, length; W, width). when the tumor volume reached about 120mm3, the tumor volume was randomly divided into 4 groups (1-4), 5 of each group, control group 1 was intraperitoneally injected with PBS0.1ml, 2 group was intraperitoneally injected with MDA-MB-2312mg/kg every 4d, 3 group was intraperitoneally injected with MDA-MB-2315 mg/kg every 4d, and 4 group was intraperitoneally injected with camptothecin 5 mg/kg. every 4 days to measure body weight and tumor volume.

Counting:

all data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad 5 software (graphpadinc. usa). Statistical significance between groups was assessed using the two-tailed Student's st-tes test or the x 2 test. p <0.05 is a significant difference. The statistical significance in the figure is: no significance, p < 0.05; p <0.01, p < 0.001.