A new protein tyrosine phosphatase SHP-1 inhibitor and its application

Abstract

The present invention discloses a novel protein tyrosine phosphatase SHP-1 inhibitor and its application. The application of the compound F065-0410 shown in formula (I) in the preparation of a protein tyrosine phosphatase shp-1 specific inhibitor. The compound has a strong inhibitory effect on the activity of SHP-1, but has almost no effect on the activity of SHP-2. Experiments have shown that F065-0410 can regulate the blood glucose concentration of type II diabetes model mice. After injecting the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 into tumor-bearing mice, tumor growth is strongly inhibited and the survival rate of mice is significantly improved. It can be seen that F065-0410 can be used in the preparation of protein tyrosine phosphatase shp-1 inhibitors, and can be used in the preparation of drugs for diseases related to excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1.

Description

Technical Field

[0001] The invention belongs to the field of medicines and relates to a new protein tyrosine phosphatase SHP-1 inhibitor and application thereof. Background Art

[0002] SHP-1 (also known as HCP, SHPTP1, PTP1C or PTPN6) is a protein tyrosine phosphatase that is mainly expressed in hematopoietic cells and epithelial cells, and only expressed in small amounts in cells of other tissues. In addition to the conserved tyrosine phosphatase catalytic sequence, SHP-1 also contains two tandem Src homology domains (SH2) at its N-terminus. These two domains are binding domains for phosphorylated protein tyrosine, mediating the interaction between PTP and its substrate, and the C-terminal tail contains two phosphorylation sites. The enzymatic activity of SHP-1 is regulated by its SH2 domain. In the absence of an activation signal, the N-terminal SH2 domain forms an intramolecular interaction with the phosphatase domain and puts the protein in a self-inhibited non-activated state; when a ligand binds to the two SH2 domains, the N-terminal SH2 domain of SHP-1 is transferred to the other side of the C-terminal SH2 domain, and the C-terminal SH2 domain rotates counterclockwise, resulting in the exposure of the phosphatase domain, thereby binding to the substrate and phosphorylating the substrate. SHP-1 plays a very important role in the intracellular signal regulation process. Abnormal changes in its activity can lead to disorders in the intracellular environment, thus causing a variety of diseases including cancer and diabetes.

[0003] Therefore, identifying specific inhibitors of SHP-1 may not only be used to treat a variety of cancers, but also have the potential to be applied to the treatment of metabolic diseases such as diabetes. Summary of the invention

[0004] The purpose of the present invention is to provide the use of F065-0410 as a protein tyrosine phosphatase SHP-1 inhibitor in view of the above-mentioned deficiencies in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] Use of the compound F065-0410 represented by formula (I) in the preparation of a protein tyrosine phosphatase shp-1 specific inhibitor,

[0007]

[0008] Use of F065-0410 in the preparation of drugs for treating diseases associated with excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1.

[0009] As a preferred embodiment of the present invention, the diseases associated with excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1 include, but are not limited to, any one of diabetes, obesity, immunodeficiency, and cancer.

[0010] As a preferred embodiment of the present invention, the cancer is leukemia or solid cancer, and the solid cancer is preferably melanoma, lung cancer, and colon cancer.

[0011] As a preferred embodiment of the present invention, the dosage form of the drug includes injection, tablet or capsule.

[0012] A protein tyrosine phosphatase shp-1 specific inhibitor composition comprises the compound F065-0410 and pharmaceutical excipients.

[0013] The pharmaceutical excipients are preferably pharmaceutical excipients required for preparing injections, tablets or capsules using compound F065-0410 as an active ingredient.

[0014] Beneficial effects:

[0015] The present invention screens out a compound F065-0410 having specific inhibitory activity of protein tyrosine phosphatase shp-1 from 2.5 million compounds by a specific method. The compound has a strong inhibitory effect on the activity of SHP-1, but has almost no effect on the activity of SHP-2. Experiments have shown that F065-0410 can regulate the blood glucose concentration of type II diabetes model mice, and can play a role in lowering blood glucose and treating diabetes in vivo. And after injecting the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 into tumor-bearing mice, tumor growth is strongly inhibited, and the survival rate of mice is significantly improved. This shows that the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 can play a role in inhibiting tumor growth in vivo. It can be seen that F065-0410 can be used in the preparation of protein tyrosine phosphatase shp-1 inhibitors, and can be used in the preparation of drugs for diseases related to excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Inhibitory effect of compound F065-0410 on the activities of SHP-1 and SHP-2.

[0017] Figure 2 Synthetic route of compound F065-0410.

[0018] Figure 3 NMR spectrum of compound F065-0410.

[0019] Figure 4The protein tyrosine phosphatase SHP-1 inhibitor F065-0410 regulates blood glucose concentration in type 2 diabetic model mice.

[0020] Figure 5 The protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits melanoma growth (A) and improves mouse survival (B).

[0021] Figure 6 The protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits lung cancer growth (A) and improves the survival rate of mice (B).

[0022] Figure 7 The protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits colon cancer growth (A) and improves the survival rate of mice (B). DETAILED DESCRIPTION

[0023] Example 1 Screening of protein tyrosine phosphatase SHP-1 inhibitors

[0024] 1) The human SHP-1 protein structure data (PDB ID: 2B30) was downloaded from the PDB protein database, and the allosteric sites in its monomer form were defined as molecular docking active sites. The Glide docking algorithm in the Schrodinger software package was used to perform virtual screening of about 2.5 million compounds contained in the ChemDiv commercial compound database. After the calculation was completed, the top 1000 compound molecules were screened based on the Lipinski drug-like rule and cluster analysis, and the top 100 diverse molecules were obtained and chemically synthesized.

[0025] 2) Based on the principle that protein tyrosine phosphatase can catalyze the decomposition of p-nitrophenylphosphate (p-NPP) to form yellow p-nitrophenol, and p-nitrophenol has a specific absorption peak at 405nm, we constructed an experimental system for screening SHP-1 inhibitors. Purified SHP-1 protein (abcam, ab51289) was purchased from abcam and SHP-1 inhibitors were screened according to the following reaction system. Reaction system: Mops (3-(N-morpholino) propanesulfonic acid)-NaOH (25Mm), DTT (1Mm), NaCl (0.1M), EDTA (1mM), BSA (1mg / mL), p-NPP (4Mm), SHP-1 recombinant protein (10ug / ml), and the small molecule compounds to be screened were diluted 2 times according to the solubility (experimental wells). The control wells were without adding small molecule compounds, and the reaction system included Mops (3-(N-morpholino)propanesulfonic acid)-NaOH (25 Mm), DTT (1 Mm), NaCl (0.1 M), EDTA (1 mM), BSA (1 mg / mL), p-NPP (4 Mm), and SHP-1 recombinant protein (10 ug / ml). The blank wells were without adding small molecule compounds and SHP-1 protein, and the reaction system included Mops (3-(N-morpholino)propanesulfonic acid)-NaOH (25 Mm), DTT (1 Mm), NaCl (0.1 M), EDTA (1 mM), BSA (1 mg / mL), and p-NPP (4 Mm). The above reaction system was reacted at 37°C for 30 minutes, and the reaction was terminated with NaHCO3, and the OD value was measured at 405 nm. The inhibitory efficiency calculation formula is Inhibitory rate = [OD value of experimental well - OD value of blank control well] / [OD value of control well - OD value of blank control well].

[0026] 3) Use SHP-2 protein (abcam, ab268899) to screen the potential SHP-1 inhibitors screened in step 2) and observe their inhibitory effects on SHP-2. Reaction system: Mops (3-(N-morpholino) propanesulfonic acid)-NaOH (25Mm), DTT (1Mm), NaCl (0.1M), EDTA (1mM), BSA (1mg / mL), p-NPP (4Mm), SHP-2 recombinant protein (10ug / ml), small molecule compounds were diluted 2 times according to solubility. No small molecule compound was added as the control (control well), reaction system: Mops (3-(N-morpholino) propanesulfonic acid)-NaOH (25Mm), DTT (1Mm), NaCl (0.1M), EDTA (1mM), BSA (1mg / mL), p-NPP (4Mm), SHP-2 recombinant protein (10ug / ml). The blank control (blank control well) was made without adding small molecule compounds and SHP-2 protein. The reaction system was: Mops (3-(N-morpholino) propanesulfonic acid)-NaOH (25 Mm), DTT (1 Mm), NaCl (0.1 M), EDTA (1 mM), BSA (1 mg / mL), p-NPP (4 Mm). The above reaction system was reacted at 37°C for 30 minutes, and the reaction was terminated with NaHCO3. The OD value was measured at 405 nm. The inhibitory efficiency calculation formula is Inhibitory rate = [OD value of experimental well - OD value of blank control well] / [OD value of control well - OD value of blank control well].

[0027] Test Results :Based on virtual screening, we obtained a total of 100 potential small molecule inhibitors that may inhibit the activity of SHP-1. Then, we used the purified SHP-1 protein purchased from abcam (abcam, ab51289) to screen the small molecule inhibitors obtained by virtual screening. The screening results showed that compound F065-0410 could significantly inhibit the activity of SHP-1. Further using SHP-2 protein, we found that compound F065-0410 had a strong inhibitory effect on the activity of SHP-1, while the activity of SHP-2 was almost unaffected ( Figure 1 ). The above results indicate that compound F065-0410 is a specific inhibitor of SHP-1.

[0028] Example 2. Synthesis of Compound F065-0410

[0029] 1) Add 2.4mmol 2-amino-6-methoxybenzothiazole( Figure 2 Compound 1), 3.6mmol N-Bocproline( Figure 2Compound 2) was stirred with 3.6mmol l-ethyl-3-(3-dimethylaminopropyl)carbodiimide), 3.6mmol diisopropylethylamine and 0.5mmol 4-dimethylaminopyridine in 8ml anhydrous N,N-dimethylformamide for 20 hours. After terminating the above reaction with 5ml water, 50ml ethyl acetate was added, and then the water layer was extracted twice with 5ml ethyl acetate. The organic layers were combined and washed with 1NHCL (10ml), 10ml saturated sodium bicarbonate (saturated NaHC03), 20ml water (twice) and 10ml physiological saline respectively. The washed organic layer was filtered through concentrated Na2S04 under reduced pressure, and then purified by flash column chromatography using 20-30% ethyl acetate to obtain a white solid. The white solid was dissolved in 4N HCl containing a 1:1 mixture of 1,4-dioxane:1,4-dioxane and stirred for 3 hours. After stirring, the solvent was removed under reduced pressure, and the precipitate was washed with 5 ml of ethyl acetate and 5 ml of ether respectively. The precipitate was dried to obtain compound 3 ( Figure 2 Compound 3).

[0030] 2) Compound 3 (1.0 equivalent), ethyl 4-(chlorosulfonyl)-1-methyl-1H-pyrrole-2-carboxylatesulfonylchloride (ethyl 4-(chlorosulfonyl)-1-methyl-1H-pyrrole-2-carboxylatesulfonylchloride) ( Figure 2Compound 4) (1.0 equivalent) and acetonitrile. Pyridine (2.5 equivalent) was added to the mixture and stirred for 14 hours. After stirring for 14 hours, the reaction solution was purified by silica gel column chromatography (ethyl acetate: cyclohexane = 1:1 + 0.1% TFA) to obtain compound F065-0410. The synthesized compound was identified by NMR. The NMR data are as follows: 'HNMR (400 MHz, CDCl3) δ7.70 (d, J = 8.9 Hz, 1H), 7.32 (d, J = 17 Hz, 1H), 7.28 (d, J = 25 Hz, 1H), 7.21 (d, J = 20 Hz, 1H), 7.04 (dd, J = 8.9, 2.6 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 3.87 (s, 3H), 3.71-3.61 (m, 1H), 2.39-2.16 (m, 2H), 1.89-1.80 (m, 4H), 1.36 (t, J = 7.1 Hz, 3H) ( Figure 3 ).

[0031] Example 3. Protein tyrosine phosphatase SHP-1 inhibitor F065-0410 regulates blood glucose concentration in type II diabetic model mice

[0032] 1) Take 20 healthy C57BL / 6 mice and give them intraperitoneal injection of 5 mg / ml STZ solution every morning for 5 consecutive days. Set up another 10 control group mice and give them intraperitoneal injection of the same amount of citric acid buffer every day. The mice drink tap water and are fed with regular mouse feed. Observe drinking water and eating conditions every day, test body weight once a day after modeling, and test blood sugar once a week after one week of modeling.

[0033] 2) On the 14th day after the injection of STZ solution, the mice injected with STZ solution were randomly divided into two groups. One group of mice was cultured normally, and the other group of mice was injected with protein tyrosine phosphatase SHP-1 inhibitor F065-0410 (5 mg / kg) once every two days. After the injection of the inhibitor, the body weight was tested once a day, and the blood sugar was tested every 5 days.

[0034] Test results: After STZ modeling, blood sugar was tested once a week. The results showed that after the 14th day of modeling, the average blood sugar of mice increased significantly ( Figure 3). After injection of the protein tyrosine phosphatase SHP-1 inhibitor F065-0410, fasting blood glucose concentration was measured every 5 days. The blood glucose concentration of mice injected with the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 (STZ+F065-0410) was compared with the mouse model without the inhibitor injection (STZ). The blood glucose of untreated STZ mice was maintained at a high blood glucose level of 30mmol / L for a long time, while the blood glucose level of STZ mice injected with the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 gradually decreased and eventually returned to normal levels ( Figure 4 ). This shows that the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 can play a role in lowering blood sugar and treating diabetes in vivo.

[0035] Example 4. Protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits melanoma growth

[0036] Thirty 8-week-old C57BL / 6 male mice were implanted with 10 6 The melanoma B16 tumor cells of mice were grown, and the tumor size was measured every day. When the tumor grew to 5 mm × 5 mm × 5 mm, the mice were randomly divided into three groups, with 10 mice in each group. The first group was not treated and the tumor growth was continued to be observed; the second group was intraperitoneally injected with normal saline; the third group was intraperitoneally injected with protein tyrosine phosphatase SHP-1 inhibitor F065-0410 (5 mg / kg / day) dissolved in normal saline. The tumor size was measured every two days. When the tumor of a mouse grew to 1000 cubic millimeters, the mouse was killed. The survival rate of the mouse was observed (the mouse was considered dead when the tumor grew to 1000 cubic millimeters).

[0037] Test results: Figure 5 As shown, tumor growth was strongly inhibited after injection of the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 ( Figure 5 A), the survival rate of mice was significantly improved ( Figure 5 B) This indicates that the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 can inhibit tumor growth in vivo.

[0038] Example 5. Protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits lung cancer growth

[0039] Thirty 8-week-old C57BL / 6 male mice were purchased and each mouse was implanted with 10 6LLC cells of mouse lung cancer were cultured, and the tumor size was measured every day. When the tumor grew to 5 mm × 5 mm × 5 mm, the mice were randomly divided into three groups, with 10 mice in each group. The first group was not treated and the tumor growth was continued to be observed; the second group was intraperitoneally injected with normal saline; the third group was intraperitoneally injected with protein tyrosine phosphatase SHP-1 inhibitor F065-0410 (5 mg / kg / day) dissolved in normal saline. The tumor size was measured every two days. When the tumor of a mouse grew to 1000 cubic millimeters, the mouse was killed. The survival rate of the mouse was observed (the mouse was considered dead when the tumor grew to 1000 cubic millimeters).

[0040] Test results: Figure 6 As shown, the growth of lung cancer cells was strongly inhibited after injection of the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 ( Figure 6 A), the survival rate of mice was significantly improved ( Figure 6 B). This indicates that the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 can inhibit the growth of lung cancer in vivo.

[0041] Example 6. Protein tyrosine phosphatase SHP-1 inhibitor F065-0410 inhibits colon cancer growth

[0042] Thirty 8-week-old C57BL / 6 male mice were purchased and each mouse was implanted with 10 6 The mice were inoculated with MC38 colon cancer cells and the tumor size was measured every day. When the tumor grew to 5 mm × 5 mm × 5 mm, the mice were randomly divided into three groups, with 10 mice in each group. The first group was not treated and the tumor growth was continued to be observed; the second group was intraperitoneally injected with saline; the third group was intraperitoneally injected with protein tyrosine phosphatase SHP-1 inhibitor F065-0410 (5 mg / kg / daily) dissolved in saline. The tumor size was measured every two days. When the tumor of a mouse grew to 1000 cubic millimeters, the mouse was killed. The survival rate of the mouse was observed (the mouse was considered dead when the tumor grew to 1000 cubic millimeters).

[0043] Test Results :like Figure 7 As shown, colon cancer growth was strongly inhibited after injection of the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 ( Figure 7 A), the survival rate of mice was significantly improved ( Figure 7 B) This indicates that the protein tyrosine phosphatase SHP-1 inhibitor F065-0410 can inhibit the growth of colon cancer in vivo.

Claims

1. Use of the compound F065-0410 represented by formula (I) in the preparation of a specific inhibitor of protein tyrosine phosphatase shp-1, 。 2. Use of F065-0410 in the preparation of a drug for treating a disease associated with excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1; the disease associated with excessive activity or abnormal overexpression of protein tyrosine phosphatase shp-1 is any one of melanoma, lung cancer, and colon cancer.

3. The application according to claim 2, characterized in that: The dosage form of the drug includes injection, tablet or capsule.

4. A protein tyrosine phosphatase shp-1 specific inhibitor composition, characterized in that The invention comprises the compound F065-0410 described in claim 1 and pharmaceutical excipients.

5. The composition according to claim 4, characterized in that The pharmaceutical excipients are pharmaceutical excipients required for preparing injections, tablets or capsules using compound F065-0410 as an active ingredient.

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