CN109303779B - Application of aminopyridine imidazolone derivatives in preparation of antitumor drugs - Google Patents

Abstract

The invention provides an application of aminopyridine imidazolone derivatives in preparation of antitumor drugs, wherein the aminopyridine imidazolone derivatives have a structure shown in a general formula (I):

wherein R is₁Is C_1‑3An alkyl, aromatic, aryl heterocyclic or hydrogen atom; r₂Is C_1‑3An alkyl group, an alkoxy group or a hydrogen atom; r₃Is O-alkylmethyloxime, O-alkylhydroxylamine, an oxadiazole ring, a trifluoromethyl-substituted oxadiazole ring, C_1‑5Alkyl substituted oxadiazole ring, C_1‑5Alkyl-substituted imidazole ring, C_1‑5Alkyl-substituted triazazole ring, C_1‑5Alkyl-substituted thiadiazole rings, N-hydroxyformamides, N-alkoxyformamides; and X is C_1‑4An alkyl group.

Description

Application of aminopyridine imidazolone derivatives in preparation of antitumor drugs

Technical Field

The invention relates to the field of medicinal chemistry, in particular to application of aminopyridine imidazolone derivatives in preparation of antitumor drugs.

Background

Sphingosine phosphate transport protein (spinster homolog 2, Spns2) is a key protein in sphingosine phosphate (S1-phosphate, S1P) transport. S1P is mainly produced by sphingomyelin on the cell membranes of red blood cells, platelets, and endothelial cells, catalyzed by a variety of enzymes. S1P is phosphorylated by Sphingosine (Sph) through Sphingosine kinases (sphingosinases, SPHK1 and SPHK2) in cells, and then transported to the outside of cells by transport proteins to be combined with 1-phosphosphingosine receptors (S1 PRs) of target cells, and S1P can be combined with S1PRs of different subtypes to activate or inhibit different signal pathways, thereby playing an important role in regulating functions of cell proliferation, cell migration, angiogenesis, lymphocyte chemotaxis and the like.

Research shows that the deletion of Spns2 can also obviously reduce the diffusion of various cancer cell lines such as colorectal cancer, breast cancer and the like to the lung of a mouse, and has an inhibiting effect on the diffusion of B16-F10 melanoma cells to the liver. Further findings indicate that loss of all or a specific pattern of lymphatic endothelium by Spns2 results in a decrease in circulating lymphocytes and an increase in the proportion of effector T cells and Natural Killer (NK) cells within the lung, which can serve to provide effective tumor cell killing and reduce the overall metastatic burden. Therefore, Spns2 can become a novel target for the development of antitumor drugs.

Meanwhile, the research and development results of the literature show that the research and development of the medicine based on the S1P signal channel are mainly focused on two targets of sphingosine phosphate kinase SphK and sphingosine 1-phosphate receptor S1 PRs. SphK1 inhibitors mainly include Safingol and Pheritol which enter clinical studies; besides fingolimod, 14S 1PRs modulators enter the clinic globally, indications comprise MS, psoriasis, rheumatoid arthritis, inflammatory bowel disease and the like, and the development of antitumor drugs taking S1PRs as targets is still in the preclinical research stage. In addition, an S1P blocking antibody is also in the process of development, and an anti-tumor drug targeting sphingosine phosphate transporter Spns2 is not reported at present.

Disclosure of Invention

In order to solve the problems, the invention provides an application of an aminopyridine imidazolone derivative in preparing an antitumor drug, wherein the aminopyridine imidazolone derivative has a structure shown in a general formula (I):

wherein R is₁Is C_1-3An alkyl, aromatic, aryl heterocyclic or hydrogen atom;

R₂is C_1-3An alkyl group, an alkoxy group or a hydrogen atom;

R₃is O-alkylmethyloxime, O-alkylhydroxylamine, an oxadiazole ring, a trifluoromethyl-substituted oxadiazole ring, C_1-5Alkyl substituted oxadiazole ring, C_1-5Alkyl-substituted imidazole ring, C_1-5Alkyl-substituted triazazole ring, C_1-5Alkyl-substituted thiadiazole rings, N-hydroxyformamides, N-alkoxyformamides; and

x is C_1-4An alkyl group.

In the above application, R₁Is C_1-3An alkyl group or a hydrogen atom;

R₂is C_1-3An alkyl group or a hydrogen atom;

R₃is O-alkylmethyloxime, O-alkylhydroxylamine, an oxadiazole ring, a trifluoromethyl-substituted oxadiazole ring, C_1-5Alkyl substituted oxadiazole ring, C_1-5Alkyl-substituted imidazole ring, C_1-5An alkyl-substituted triazole ring,C_1-5Alkyl-substituted thiadiazole rings, N-hydroxyformamides, N-alkoxyformamides; and

x is C_1-4An alkyl group.

In the above application, the aminopyridine imidazolone derivatives are the following compounds:

in the above application, the aminopyridine imidazolone derivatives are:

in the above applications, the tumor includes breast cancer, colon cancer, liver cancer, stomach cancer, prostate cancer, kidney cancer, bone cancer, lung cancer, ovarian cancer, leukemia, esophageal cancer, cervical cancer, bladder cancer, melanoma, pancreatic cancer, and lymph cancer.

In the application, the IC50 value of the aminopyridine imidazolone derivative is 1-10 mu mol/L.

The invention also provides an anti-tumor medicament, which comprises: aminopyridine imidazolone derivatives or pharmaceutically acceptable salts thereof and auxiliary materials.

In the above antitumor drugs, the pharmaceutically acceptable salts of the aminopyridine imidazolone derivatives include organic salts and inorganic salts.

In the above antitumor drugs, the organic salts include methanesulfonate, formate, acetate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, p-toluenesulfonate, naphthalenesulfonate, lactate and benzoate, and the inorganic salts include hydrochloride, hydrobromide, sulfate and phosphate.

In the above antitumor drugs, the dosage form of the antitumor drug is selected from tablets, capsules, pills, aerosols, oral liquid preparations, granules, powders, injections, syrups, medicated liquors, tinctures, sustained-release preparations or combinations thereof.

The invention discovers the aminopyridine imidazolone derivatives through virtual screening, the derivatives can target Spns2 protein, have obvious anti-tumor effect, and can be used for preparing anti-tumor medicaments.

Drawings

FIGS. 1A to 1D show the effect of aminopyridinoimidazolone derivatives on apoptosis of PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cells, respectively;

FIGS. 2A to 2D show the effect of aminopyridinoimidazolone derivatives on PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cell invasion, respectively;

FIGS. 3A and 3B show the effect of aminopyridinoimidazolone derivatives on PLC cells and MDA-MB-231 cell transfer, respectively.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

The invention provides an application of aminopyridine imidazolone derivatives in preparation of antitumor drugs, wherein the aminopyridine imidazolone derivatives have the following general formula (I):

wherein R is₁Is C_1-3An alkyl, aromatic, aryl heterocyclic or hydrogen atom;

R₂is C_1-3Alkyl, alkaneAn oxy group or a hydrogen atom;

R₃is O-alkylmethyloxime, O-alkylhydroxylamine, an oxadiazole ring, a trifluoromethyl-substituted oxadiazole ring, C_1-5Alkyl substituted oxadiazole ring, C_1-5Alkyl-substituted imidazole ring, C_1-5Alkyl-substituted triazazole ring, C_1-5Alkyl-substituted thiadiazole rings, N-hydroxyformamides, N-alkoxyformamides; and

x is C_1-4An alkyl group.

In the above applications, the aminopyridine imidazolone derivatives are preferably the following compounds:

the experimental materials and sources thereof used in the present invention include:

PLC (liver cancer cell line) cells, MCF-7 (breast cancer cell line) cells, MDA-MB-231 (breast cancer cell line) cells, HCT-8 (colon cancer cell line) cells were purchased from Keji organisms;

RMPI-1640 medium, high-sugar DMEM medium, and Fetal Bovine Serum (FBS), all purchased from Invitrogen;

tetramethyl azoazolium salt (MTT), crystal violet, Matrigel and Annexin/PI apoptosis detection kit are all purchased from Shanghai Biotechnology engineering Co., Ltd.

Cell culture flasks and 96-well plates were purchased from Saimer Feishale scientific China Co., Ltd. (ii) a

Female SPF grade (specific pathogen free grade) C57BL/6 mice at 5 weeks of age were purchased from the center of laboratory animals at the military medical academy of sciences and housed in an SPF grade double corridor barrier environment.

Hematoxylin, eosin, both purchased from Sigma (Sigma reagent grid).

Compound library from high throughput drug screening center of Tianjin International biomedical Union research institute.

Example 1

The influence of aminopyridine imidazolone derivatives on Spns2 protein is evaluated through virtual screening, and the specific steps are as follows:

1) after a protein structure of Spns2 is downloaded by a PDB database (protein database), a small molecule structure is drawn by using Chemdraw, and then the small molecule structure is respectively introduced into Schrodinger software.

2) Optimizing the structure of the protein and the small molecule, selecting the active center of the protein, and butting to obtain the virtual screening score.

The results of the virtual screening of aminopyridine imidazolone derivatives are shown in table 1 below:

TABLE 1 results of virtual screening of aminopyridinoimidazolidone derivatives

Compound numbering	Docking scoring
			1	3,78
2	4.1
		3	3.65
4	4.5
		5	4.9
6	3.1
		7	5.5
8	5.1
		9	5.43
10	5.87
		11	4.46
12	4.7
		13	3.91
14	4.54
		15	6.06

As can be seen from table 1 above, the compounds 1 to 15 in the aminopyridine imidazolone derivatives have high scores of targeting Spns2, which are all over 3.1 points, and have strong binding capacity, and the compound 15 has the highest score of 6.06, so that the effect is the best.

Example 2

The method for testing the influence of the aminopyridine imidazolone derivatives on the proliferation activities of PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cells comprises the following specific steps:

1) adherent tumor cells were trypsinized into single cells, seeded into 96-well plates at 5000 cells per well in 100. mu.L complete medium per well, and cultured overnight in a cell culture chamber at 37 ℃ and 5% carbon dioxide. After the cells adhere to the wall, the original culture medium is sucked out, 100 mu L of 3.125 mu mol/L, 6.25 mu mol/L, 12.5 mu mol/L, 25 mu mol/L, 50 mu mol/L and 100 mu mol/L cell culture solution of aminopyridine imidazolone derivatives are respectively added as experimental groups, negative control holes (containing MTT, dimethyl sulfoxide and the cells to be measured in the culture medium) and zero adjusting holes (containing MTT and dimethyl sulfoxide in the culture medium) are respectively arranged aiming at the experimental groups of each cell, 5 parallel holes are arranged in each group, and the cells are cultured for 24 hours in a cell culture box with the temperature of 37 ℃ and the carbon dioxide concentration of 5 percent.

2) After 24 hours of incubation, the original medium was aspirated, 100. mu.L of 1mg/mL MTT was added to each well, the incubation was continued for 4 hours in the incubator, and then the MTT was aspirated, 150. mu.L DMSO (dimethyl sulfoxide) was added to each well, and after sufficient dissolution, the absorbance at 570nm was measured with a microplate reader (MR-96A).

3) Cell survival (%) (experimental OD (absorbance) value-zero OD value)/(negative control OD value-zero OD value) × 100%. IC50 values were calculated using Graphpad Prism5, and the results are shown in table 2 below:

TABLE 2 Effect of aminopyridinoimidazolone derivatives on the proliferative Activity of PLC cells, MCF-7 cells, MDA-MB-231 cells, HCT-8 cells

Compound numbering	IC50 μmol/L(PLC)	IC50 μmol/L(MCF-7)	IC50 μmol/L(MDA-MB-231)	IC50 μmol/L(HCT-8)
					1	5.3	4.32	8.24	7.75
2	6.71	6.43	6.74	7.98
					3	7.23	5.78	7.65	5.43
4	7.56	3.21	4.54	6.89
					5	8.67	6.09	7.43	9.65
6	8.1	7.12	8.23	7.43
					7	9.5	7.45	7.65	7.56
8	6.13	6.34	6.23	8.13
					9	5.78	8.98	5.06	11.2
10	8.123	6.77	8.45	9.21
					11	6.12	5.25	6.74	8.55
12	8.12	8.45	5.43	7.12
					13	6.87	5.76	10.23	5.43
14	7.65	4.35	7.56	6.55
					15	1.34	1.02	1.56	1.12

From the above table 2, after 24h of administration, under the stimulation of aminopyridinimidazolone derivatives with different concentrations, IC50 values of the compounds 1 to 15 in the aminopyridinimidazolone derivatives to four cell lines are all below 10 μmol/L, and the compounds have good effect of inhibiting tumor cell proliferation, wherein IC50 values of the compounds 15 to the four cell lines are all around 1 μmol/L and much smaller than IC50 values of other compounds, and the effect is the best.

Example 3

The method for testing the influence of the aminopyridine imidazolone derivatives on the apoptosis of PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cells comprises the following specific steps:

1) cell collection: the suspension cells were collected directly into 10mL centrifuge tubes at 5X 10 cells per sample⁶Centrifuging at 1000r/min for 5min, and discarding the culture solution.

2) Washing with phosphate buffer solution for 1 time, and centrifuging for 5min at 500-1000 r/min.

3) The cells were resuspended in 100. mu.L of a labeling solution mixed with FITC-Annexin V and PI (both available from Shanghai Biotech, Ltd.) and incubated at room temperature for 15min in the absence of light.

4) The cells were pelleted by centrifugation at 1000r/min for 5min and washed 1 time with phosphate buffer.

5) Adding fluorescence (SA-FLO M S) solution, incubating at 4 deg.C for 20min, and keeping away from light while shaking occasionally.

6) Flow cytometry analysis: FITC (fluorescein isothiocyanate) fluorescence was detected with a band pass filter with a wavelength of 515nm and PI (nuclear fluorescent dye) was detected with another filter with a wavelength of greater than 560nm, in the case of the flow cytometer excitation light wavelength of 488 nm.

7) And (5) judging a result: on the dot plot of the bivariate flow cytometer, the lower left quadrant shows viable cells as (FITC-/PI-); the upper right quadrant is a non-viable cell, i.e., a necrotic cell, and is (FITC +/PI +); whereas the lower right quadrant is apoptotic cells and is (FITC +/PI-).

The experimental results are shown in the following fig. 1A to fig. 1D, and fig. 1A to fig. 1D show the apoptosis rates of four cell lines after administration of 10 μmol/L for 24 hours under stimulation of aminopyridinoimidazolidone derivatives, wherein the abscissa is the compound name and the ordinate is the total apoptosis percentage, compared with the control group, the compounds 1 to 15 all have good effects of promoting tumor cell apoptosis, and the compound 15 has the most obvious effect, and the apoptosis rates are respectively as high as 83.2% (PLC), 79.2% (MCF-7), 81.2% (MDA-MB-231) and 86.5% (HCT-8).

Example 4

The method for testing the influence of the aminopyridine imidazolone derivatives on the migration of PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cells comprises the following specific steps:

1) and (3) soaking the gun head in 75% ethanol for 2 hours, taking out the gun head, and airing the gun head in a biological safety cabinet for later use.

2) Adherent cells were trypsinized into single cells and plated in 24-well plates at 1.0 x 10 cells per well⁶500. mu.L/mL of cell suspension, Inserts were inserted into the well plate and aligned in the same direction so as to be in close contact with the bottom surface of the 24-well plate; the 24-well plate is placed in a cell culture box for culturing for 24 hours, so that the cells are attached to the wall fully.

3) After 24 hours, washing cell fragments and nonadherent cells in the holes by phosphate buffer saline solution, observing under a microscope and taking pictures; each set was set up in triplicate, and the cells were incubated in a cell incubator after administration (aminopyridine imidazolone derivatives), photographed 24 hours and 48 hours after administration, respectively, and observed for the degree of healing of the scratch.

The results of the experiments are shown in table 3,

TABLE 3 relative migration distances (relative Oh) of four cell lines 24h after administration of aminopyridinoimidazolidone derivatives (10. mu. mol/L)

Compound numbering	PLC (relative 0h)	MCF-7 (relative 0h)	MDA-MB-231 (relative 0h)	HCT-8 (relative 0h)
					Control	0.31±0.023	0.29±0.029	0.3±0.023	0.38±0.033
1	0.694±0.032	0.694±0.041	0.694±0.018	0.594±0.028
					2	0.607±0.022	0.702±0.071	0.602±0.045	0.702±0.042
3	0.55±0.0345	0.55±0.051	0.65±0.041	0.65±0.051
					4	0.6010±0.042	0.51±0.043	0.61±0.04	0.51±0.05
5	0.610±0.039	0.62±0.053	0.62±0.051	0.52±0.051
					6	0.690±0.023	0.58±0.051	0.68±0.042	0.54±0.04
7	0.640±0.029	0.64±0.061	0.54±0.031	0.56±0.021
					8	0.580±0.041	0.67±0.063	0.56±0.041	0.61±0.031
9	0.500±0.028	0.45±0.041	0.55±0.021	0.65±0.031
					10	0.600±0.023	0.6±0.051	0.5±0.031	0.56±0.0380
11	0.590±0.027	0.61±0.06	0.51±0.032	0.61±0.042
					12	0.700±0.021	0.59±0.05	0.69±0.034	0.54±0.024
13	0.650±0.023	0.6±0.041	0.62±0.041	0.52±0.021
					14	0.520±0.037	0.53±0.031	0.63±0.023	0.53±0.033
15	0.83±0.045	0.89±0.041	0.75±0.029	0.87±0.043

As can be seen from table 3 above, compounds 1 to 15 all have good effects of inhibiting tumor cell migration, and compound 15 has the most significant inhibitory effect.

Example 5

The method for testing the influence of the aminopyridine imidazolone derivatives on the invasion of PLC cells, MCF-7 cells, MDA-MB-231 cells and HCT-8 cells comprises the following specific steps:

1) precooling the tip, plate and Transwell chamber (available from Weistein Bio-pharmaceutical technology, Inc.) at 4 deg.C, placing the dispensed matrigel in a 4 deg.C freezer until it melts, diluting the matrigel on ice, mixing with the corresponding air culture in a ratio of 1: 1, placing the chamber in a 24-well plate, and then spreading the matrigel in the chamber at 50. mu.L per well. Spreading the glue, and standing at 37 deg.C for about 20min until it is solidified; a medium containing 0.1% FBS (fetal bovine serum) was prepared, 50. mu.L per well was applied to the gel, and then placed in an incubator at 37 ℃ for 30 min.

2) Digesting the adherent cells into single cells with trypsin, resuspending in phosphate buffered saline, centrifuging again to remove the supernatant, resuspending the cells in a medium containing 0.1% FBS, counting, diluting the cells to 2X 10⁵The solution was applied to matrigel at a density of 200. mu.L per chamber, and the cells were cultured overnight in a 37 ℃ cell culture chamber.

3) After 24 hours of culture, the cells and matrigel inside the chamber were wiped off with a cotton swab, then the chamber was fixed in methanol pre-cooled at-20 ℃ for 10min, washed with phosphate buffered saline solution 3 times, then the chamber was stained in crystal violet staining solution for 10min, excess staining solution was washed away with phosphate buffered saline solution, and the results were recorded by taking a photograph under a microscope.

The experimental results are shown in fig. 2A to fig. 2D below, and fig. 2A to fig. 2D show the relative invasion percentages of four cell lines after 24h of aminopyridinoimidazolidone derivatives (10 μmol/L) administration, wherein the abscissa is the compound name, the ordinate is the relative invasion rate, and the control group is set as 100%, it can be seen that compounds 1 to 15 all have good effects of inhibiting tumor cell invasion relative to the control group, and the effect of inhibiting invasion of compound 15 is obvious.

Example 6

The method for testing the influence of the aminopyridine imidazolone derivatives on the transfer of PLC cells and MDA-MB-231 cells comprises the following specific steps:

1) sufficient numbers of PLC cells and MDA-MB-231 cells were cultured to make up 1 x 107 cells/mL of physiological saline cell suspension.

2) After the mouse is disinfected by a 75% alcohol cotton ball, subcutaneously injecting cell suspension with the volume of 0.1mL into the groin part, wherein 80 cell lines are respectively inoculated on the two cell lines, the survival condition and the tumor growth condition of the mouse are observed and recorded every day, and the volume of the tumor is 100mm after the tumor grows³The mice inoculated with the two tumors were randomly divided into 16 groups of 5 mice each, wherein the mice were divided into a blank control group (given physiological saline) and a group of aminopyridine imidazolone derivatives No. 1-15, and the mice were dissected one week after administration to fix tumor tissues and lungs.

3) Fixing the tumor tissue and lung obtained by dissection in formalin solution, placing the fixed tissue into a dehydrator for dehydration, and performing the following procedures: 45min of 50% ethanol, 45min of 70% ethanol, 1h of 80% ethanol, 1h of 90% ethanol, 1h of 95% ethanol, 50min of 100% ethanol A, 45min of 100% ethanol B, 30min of 50% xylene, 25min of xylene A, 25min of xylene B, 20min of xylene C, 1h of paraffin A, 1h of paraffin B and 1h of paraffin C.

4) And (5) placing the dehydrated tissue in an embedding machine for embedding, and waiting for paraffin solidification.

5) Paraffin pathological tissue section: taking the pathological tissues, preparing slices with the thickness of 4 mu m by using a slicer LeicaRM2245 (a semi-automatic rotary slicer), spreading the slices in a warm water bath at 55 ℃, placing the slices on a glass slide, and drying the slices for later use.

6) And an uplink system: the tissue sections were deparaffinized in xylene and rinsed with gradient ethanol (95% by mass, 80% absolute ethanol) dehydrated distilled water for 2min.

7) Hematoxylin staining was performed for 5min, after which time it was rinsed with tap water.

8) Ethanol hydrochloride was differentiated for 30 s.

9) Soaking in tap water for 15 min.

10) Downlink system and mounting: after being cleaned by distilled water, sequentially carrying out alcohol treatment with gradient concentration (80% and 95% of absolute ethyl alcohol by mass fraction), and then carrying out xylene treatment and dehydration; sealing with a sealing machine, and drying in the sun in a fume hood.

11) And counting the number of lung metastasis foci under a microscope.

The experimental results are shown in the following fig. 3A and fig. 3B, fig. 3A to fig. 3B show the number of cell metastases of the aminopyridinoimidazolidone derivative (50mg/kg) after one week of administration to the breast cancer and lung cancer mice, wherein the abscissa is the compound name and the ordinate is the number of metastasis, and comparing the number of cell metastases of the experimental group with the number of cell metastases of the control group, it can be seen that the compounds 1 to 15 all have good effects of inhibiting tumor lung metastasis, and the effect of the compound 15 is more obvious than that of the other derivatives No. 1 to 14.

The present invention is not limited to the above embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (3)

1. An application of aminopyridine imidazolone derivatives in preparing anti-tumor drugs, wherein the aminopyridine imidazolone derivatives have a structure shown in a general formula (I):

wherein R is₁Is C_1-3An alkyl group or a hydrogen atom;

R₂is C_1-3An alkyl group or a hydrogen atom;

R₃is O-alkylmethyloxime, O-alkylhydroxylamine, an oxadiazole ring, a trifluoromethyl-substituted oxadiazole ring, C_1-5Alkyl substituted oxadiazole ring, C_1-5Alkyl-substituted imidazole ring, C_1-5Alkyl-substituted triazazole ring, C_1-5Alkyl-substituted thiadiazole rings, N-hydroxyformamides, N-alkoxyformamides; and

x is C_1-4An alkyl group, a carboxyl group,

wherein, the aminopyridine imidazolone derivatives are the following compounds:

wherein the tumor is breast cancer, colon cancer or liver cancer.

2. The use according to claim 1, wherein the aminopyridinoimidazolidone derivatives are:

3. the use according to claim 1, wherein the aminopyridinoimidazolidone derivatives have an IC50 value of 1 to 10 μmol/L.

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