Application of TRK inhibitor LYS-8 in preparation of medicine for treating malignant tumors

Abstract

The invention belongs to the field of molecular biology, particularly relates to the field of tumor treatment, and particularly relates to application of a TRK inhibitor LYS-8 in preparation of a medicine for treating malignant tumors, particularly pancreatic cancer. LYS-8 is a small molecule TRK inhibitor with a novel structure, and research finds that LYS-8 can significantly inhibit TRK kinase activity and down-regulate downstream PI3K-AKT and other signal channels. Experimental results show that the inhibition effect of LYS-8 in pancreatic cancer cells is obviously superior to that of colorectal cancer cells, and the IC50 value of LYS-8 is lower; in an animal model, the tumor inhibition rate of LYS-8 on pancreatic cancer reaches about 70%, and the inhibition rate on colorectal cancer is about 56%, which is obviously superior to that of a colorectal cancer model. The invention reveals that LYS-8 has an unexpected curative effect advantage on pancreatic cancer, and a new drug choice is provided for targeted therapy of pancreatic cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, specifically to the field of tumor treatment, and particularly relates to the application of TRK inhibitor LYS-8 in the preparation of a drug for treating malignant tumors, especially pancreatic cancer. BACKGROUND

[0002] Pancreatic cancer is a highly lethal invasive malignancy, and its clinical symptoms often lack specificity. The median survival of patients is about 4 months, and the 5-year survival rate is only 13%. Patients with a family history of pancreatic cancer, carrying specific gene mutations, and high-risk intraductal papillary mucinous neoplasms have a significantly increased risk of developing pancreatic cancer, so they are recommended to be regularly monitored. Surgical resection combined with chemotherapy is the cornerstone of the treatment of pancreatic cancer, but only a small number of patients are suitable for surgery. Multi-drug combination chemotherapy regimens can prolong the survival of patients with metastatic pancreatic cancer, while (neo) adjuvant therapy and induction therapy can improve the survival rate of patients with borderline resectable and locally advanced pancreatic cancer. It is estimated that by 2030, pancreatic cancer will become the second leading cause of cancer-related death, so there is an urgent need for new treatment methods.

[0003] In recent years, molecular targeted therapy has made important progress in the field of solid tumors. The TRK (tropomyosin receptor kinase) family, including TRKA, TRKB, and TRKC, has been found to be abnormally activated or to have gene fusions in various tumors, and is involved in tumor cell proliferation, migration, invasion, and anti-apoptosis. TRK inhibitors have shown some efficacy in lung cancer, thyroid papillary cancer, and other tumors. However, the application of existing TRK inhibitors in pancreatic cancer is relatively limited, and their efficacy and safety have not been fully verified, and the clinical demand is still huge.

[0004] Pancreatic cancer and colorectal cancer have significant differences in molecular mechanisms. Pancreatic cancer cells have a higher dependence on the TRK signaling pathway, while KRAS, BRAF, and other downstream signaling pathways may dominate tumor proliferation in colorectal cancer cells, which limits the anti-tumor effect of existing TRK inhibitors in pancreatic cancer.

[0005] Existing literature has not reported TRK inhibitors with significantly better efficacy in pancreatic cancer than in colorectal cancer, and there is also a lack of specific targeted drug development cases for pancreatic cancer.

[0006] Therefore, the development of a new TRK inhibitor that can exert a significant anti-tumor effect in pancreatic cancer and is superior to the efficacy in colorectal cancer not only has important clinical application value, but also can meet the long-term unmet technical needs in the field of pancreatic cancer treatment. This provides a technical background and research and development basis for the present application. SUMMARY

[0007] The application restructures a known compound, and provides application of a novel TRK inhibitor LYS-8 and a derivative thereof in preparation of a drug for treating pancreatic cancer. The compound shows better antitumor activity than existing TRK inhibitors in in-vivo and in-vitro experiments, and has outstanding curative effect in a pancreatic cancer model.

[0008] To achieve the above object, the technical scheme of the application is as follows:

[0009] In a first aspect, the application provides application of a compound LYS-8 or a pharmaceutically acceptable salt thereof and a derivative thereof in preparation of a drug for treating tumors, wherein the structure of the LYS-8 is shown in formula I:

[0010]

[0011] Further, the tumors are solid tumors.

[0012] Further, the tumors are malignant tumors, and specifically, pancreatic cancer or colorectal cancer.

[0013] Further, the LYS-8 is a TRK (tropomyosin receptor kinase) kinase inhibitor, and can specifically inhibit the kinase activity of TRKA, TRKB and TRKC, thereby inhibiting the proliferation of tumor cells.

[0014] Further, the LYS-8 can induce apoptosis of cancer cells by inhibiting TRK and downstream PI3K-AKT and MAPK / ERK signaling pathways, and reducing the phosphorylation level of TRK.

[0015] Further, the drug preparation is a tablet, a capsule, an injection or a sustained-release preparation.

[0016] Further, the LYS-8 or the derivative thereof and a pharmaceutically acceptable carrier, excipient or solvent together constitute a pharmaceutical composition.

[0017] Further, the administration concentration of the LYS-8 is 10-100 mg / kg body weight / day; preferably, the administration concentration is 20-80 mg / kg body weight / day; and most preferably, the administration concentration is 30-60 mg / kg body weight / day.

[0018] In a second aspect, the application provides application of a compound LYS-8 or a pharmaceutically acceptable salt thereof and a derivative thereof in preparation of a preparation for inhibiting proliferation of tumor cell lines, wherein the inhibition is in-vitro inhibition, and the structure of the LYS-8 is shown in formula I:

[0019]

[0020] Further, the tumor cell line is a colorectal cancer cell line and / or a pancreatic cancer cell line.

[0021] In a third aspect, the present application provides a use of a compound LYS-8 or its pharmaceutically acceptable salt and its derivative in the preparation of a preparation for inhibiting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathway, wherein the structure of LYS-8 is shown in formula I:

[0022]

[0023] Further, LYS-8 can induce apoptosis of cancer cells by inhibiting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathway and reducing the phosphorylation level of TRK.

[0024] In a fourth aspect, the present application provides a use of a substance for specifically detecting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathway in the preparation of a reagent for determining the effect of LYS-8 in the treatment of cancer as claimed in claim 1, characterized in that the substance detects whether the phosphorylation level of TRK is reduced.

[0025] Further, the cancer is colorectal cancer or pancreatic cancer.

[0026] Further, the substance for specifically detecting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathway is an antibody.

[0027] The beneficial effects of the present application include:

[0028] Through the improvement of the known drug indacaterol, a compound LYS-8 is obtained, which can specifically bind and inhibit the kinase activity of TRKA, TRKB and TRKC. Compared with solid tumors such as colorectal cancer, LYS-8 shows stronger inhibitory effect on the proliferation of pancreatic cancer cells and animal models, which proves its specific advantage in the treatment of pancreatic cancer and provides a new targeted treatment idea for pancreatic cancer. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The chemical synthesis route map of the compound LYS-8 of the present application.

[0030] Figure 2 The specific inhibition results of the compound LYS-8 on TRK kinase show that the compound can selectively inhibit the kinase activity of TRKA, TRKB and TRKC, and has no significant effect on other MAK kinases.

[0031] Figure 3The cell viability detection results of compound LYS-8 in pancreatic cancer cell lines CFPAC1 (A), MIA PaCa-2 (B) show that LYS-8 can significantly inhibit the proliferation of pancreatic cancer cells.

[0032] Figure 4 The cell viability detection results of compound LYS-8 in colorectal cancer cell lines HCT-8 (A), SW480 (B) show that LYS-8 can significantly inhibit the proliferation of colorectal cancer cells. Figure 3 The comparison shows that the inhibitory effect of LYS-8 on pancreatic cancer cells is more significant.

[0033] Figure 5 The clonogenic assay results of compound LYS-8 in pancreatic cancer cells (ASPC1) and colorectal cancer cells (SW620) show that LYS-8 has a more significant inhibitory effect on the clonogenicity of pancreatic cancer cells, and the effect is more prominent than that in colorectal cancer cells.

[0034] Figure 6 The anti-tumor effect diagram of compound LYS-8 in a colorectal cancer mouse xenograft tumor model shows A tumor size measurement, B tumor volume, C mouse weight, and D tumor weight. Figure 7 The anti-tumor effect diagram of compound LYS-8 in a pancreatic cancer mouse xenograft tumor model shows A tumor size measurement, B mouse weight, C tumor volume, and D tumor weight.

[0035] Figure 8 The expression change diagram of TRK signaling pathway related proteins in pancreatic cancer after treatment with compound LYS-8 shows that LYS-8 can reduce the phosphorylation level of TRK and inhibit its downstream signaling pathway. DETAILED DESCRIPTION

[0036] The embodiments and technical solutions of the present application are further described below in combination with the drawings and specific examples. It should be understood that those skilled in the art can appropriately adjust or improve the related process parameters on the basis of referring to the content of the present application. In particular, it should be pointed out that any equivalent replacement or obvious change made without departing from the spirit and essence of the present application and within the scope of protection should be considered to fall within the scope of protection of the present application. The methods and applications of the present application have been described in detail through preferred examples, and those skilled in the art can make corresponding modifications, changes or combinations without departing from the concept of the present application, and the technical effects should be covered within the scope of the present application.

[0037] The present application will be described below through specific examples, but the present application is not limited thereto.

[0038] Example 1: Structure of compound LYS-8

[0039] Test method: The compound LYS-8 provided by the application belongs to a TRK inhibitor, and the specific synthesis route is as shown in Figure 1 Specifically:

[0040] Step a:

[0041] 5-acetyl-2,8-dihydroxyquinoline 1 (300.0 g, 1.48 mol) (purchased from Nanjing Fain Chemical Co., Ltd.) was dissolved in acetone (3 L) and water (300 mL), and potassium carbonate (265.9 g, 1.92 mol) was added, and after stirring, it was heated to reflux. Then slowly drop bromobenzene (306.6 g, 1.77 mol), after the drop is completed, continue to reflux and stir. After monitoring the completion of the reaction by HPLC, hot filtration was carried out, and the filter cake was washed with water (1.5 L) and petroleum ether (1.5 L) in turn, and dried to obtain intermediate 2 as a light yellow solid (350 g, yield 80.8%), purity 97.7%. HR-MS (ESI + ): calculated value C 18 H 15 NO3[M+H] + : 294.1085, found 294.1010.

[0042] Step b:

[0043] Intermediate 2 (350 g, 1.19 mol) was added to a 3 L three-necked flask, and then DCM (1050 mL) and MeOH (700 mL) were added, and then tetrabutylammonium tribromide (688 g, 1.43 mol) was added, and the temperature was raised to 37°C, and stirred for 4 h. After monitoring the completion of the reaction by TLC, the filter cake was washed with ice methanol (100 mL) after cooling to 25°C. After drying, intermediate 3 was obtained as a white solid (320 g, yield 72%), purity 97%. HR-MS (ESI + ): calculated value C 18 H 14 BrNO3[M+H] + : 372.0157, found 372.0189.

[0044] Step c:

[0045] Intermediate 3 (120 g, 322.4 mmol) and (S)-2-methyl-CBS-oxazaborolidine (10.7 g, 38.7 mmol) were dissolved in anhydrous THF (1.2 L), warmed to 50 °C, stirred for 0.5 h, then cooled to -10 °C, BH3-THF (1.0 M in THF) (451 mL, 451 mmol) was added dropwise slowly, keeping the internal temperature <-10 °C. After the addition was completed, the reaction was continued at -10 °C for 2 h, after TLC monitoring the reaction was completed, methanol (400 mL) was added dropwise slowly to quench. The reaction solution was concentrated under reduced pressure, the crude product was added with HCL (1 N, 1.4 L), stirred at 25 °C for 8 h, suction filtered. The filter cake was washed with water, dried to give intermediate 14 as a white solid (110 g, yield 91%), purity 92%, e.e.: 90%. HR-MS (ESI + ): calculated for C 18 H 16 BrNO3[M+H] + : 374.0314, found 374.0398.

[0046] Step d:

[0047] Intermediate 4 (110 g, 293.9 mmol), imidazole (120 g, 1763.4 mmol) and DMAP (3.6 g, 29.4 mmol) were dissolved in DMF (440 mL), cooled to -5 °C, TBSCL (177.2 g, 1175.6 mmol) dissolved in DMF (440 mL) was added dropwise slowly, keeping the temperature <0 °C. After the addition was completed, the reaction was carried out at 25 °C for 8 h. After TLC monitoring the reaction was completed, 2 L ice water was added to quench, a large amount of white solid precipitated after about 15 min. Suction filtered, the filter cake was added with 1.5 L n-heptane, stirred at room temperature for 1 h, suction filtered, the filter cake was washed with heptane (200 mL), dried to give intermediate 5 as a white solid (120 g, yield 83%), purity 98%. HR-MS (ESI + ): calculated for C 24 H 30 BrNO3Si[M+H] + : 488.1178, found 488.1115.

[0048] Step e:

[0049] Intermediate 5 (120 g, 245.7 mmol) was dissolved in DMSO (360 mL), and then 5,6-diethyl-2,3-dihydro-1H-inden-2-amine hydrochloride (61 g, 270.2 mmol) (purchased from Nanjing Fain Chemical Co., Ltd.), sodium carbonate (78 g, 737.1 mmol), and sodium iodide (3.7 g, 24.6 mmol) were added in sequence. The reaction was heated to 110°C under nitrogen protection, and HPLC showed that the raw material was <5% when the reaction was stopped. The reaction was cooled to 60°C, quenched by adding ice water (600 mL), extracted with ethyl acetate (200 mL x 3), the organic phases were combined, washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. A black-brown residue of intermediate 6 was obtained, which was used in the next step without purification. HR-MS (ESI + ): calculated C 37 H 48 N2O3Si[M+H] + :597.3468, found 597.3488.

[0050] Step f:

[0051] Intermediate 6 in the previous step was dissolved in anhydrous ethanol (300 mL), and then 37% HCL (60 mL) was added, and the reaction was heated to 70°C for about 6 h. After HPLC monitoring showed that the reaction was completed, the reaction was cooled to 0°C and stirred for 2 h, filtered, and the filter cake was washed with ice ethanol (50 mL). After drying, intermediate 7 was obtained as a light yellow solid (92 g, two-step yield 70%), with a purity of 93%. HR-MS (ESI + ): calculated C 31 H 34 N2O3[M+H] + :483.2603, found 483.2581.

[0052] Step g:

[0053] To intermediate 7 (92 g, 172 mmol) was added dichloromethane (400 mL), and then a sodium carbonate (23.7 g, 224 mmol) aqueous solution (400 mL) was added to adjust the pH value to about 9.0. The mixture was separated, the water layer was extracted with DCM (200 mL x 2), the organic phases were combined, washed with water (200 mL) and saturated brine (200 mL) in sequence, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in acetone (200 mL) and methanol (300 mL), and then D-tartaric acid (12.9 g, 86 mmol) dissolved in methanol (100 mL) was slowly added dropwise, stirred at 20°C for 2 h, filtered, the filter cake was washed with ice methanol, and dried to obtain intermediate 8 as a white solid (80 g, yield 83.4%), which was a hemitartrate. Purity: 99.6%, e.e.: >99%.

[0054] Step h:

[0055] To intermediate 8 (80 g, 143.5 mmol) was added dichloromethane (400 mL), then added sodium carbonate (15.2 g, 143.5 mmol) aqueous solution (400 mL) to adjust pH value ≈ 9.0. Separated, the water layer was extracted with DCM (200 mL x 2), the combined organic phase was washed with water (200 mL) and saturated brine (200 mL) successively, dried over anhydrous sodium sulfate. Then added (Boc)20 (37.6 g, 172.2 mmol), stirred at 25 °C for 4 h, TLC monitored the completion of the reaction, concentrated under reduced pressure, added n-heptane (1 L) to the residue, stirred at 25 °C for 2 h, suction filtered, the filter cake was washed with n-heptane, dried to give intermediate 9 as a white solid (76 g, yield 91%), purity 99%. HR-MS (ESI + ): calculated C 36 H 42 N2O5[M+H] + : 583.3127, found 583.3098.

[0056] Step i:

[0057] Intermediate 9 (76 g, 130.4 mmol) was dissolved in methanol (500 mL), added 10% Pd / C (7.6 g, 10%), under hydrogen atmosphere, stirred at 35 °C for about 4 h, TLC monitored the completion of the reaction, suction filtered, the filtrate was concentrated under reduced pressure to give intermediate 10 as a light yellow solid (55 g, yield 86%), purity 96%. HR-MS (ESI + ): calculated C 29 H 36 N2O5[M+H] + : 493.2658, found 493.2611.

[0058] Step j:

[0059] Intermediate 10 (55 g, 111.6 mmol) was dissolved in acetone (500 mL), then added potassium carbonate (23.1 g, 167.4 mmol), slowly added bromoacetonitrile (14.73 g, 122.8 mmol), stirred at reflux for about 4 h, TLC monitored the completion of the reaction, suction filtered while hot, the filtrate was concentrated under reduced pressure to give intermediate 11, which was used in the next step reaction without purification. HR-MS (ESI + ): calculated C 31 H 37 N3O5[M+H] + : 532.2767, found 532.2787.

[0060] Step k:

[0061] The intermediate 11 in the previous step was dissolved in ethyl acetate (500 mL), p-toluenesulfonic acid monohydrate (42.5 g, 223.2 mmol) was added, and the mixture was stirred at 55 °C for about 4 h. After TLC monitoring of the reaction completion, the mixture was filtered, and the filter cake was washed with ethyl acetate to obtain a bis-p-toluenesulfonic acid salt of LYS-8. The crude product was dissolved in dichloromethane (400 mL), and an aqueous solution of sodium carbonate (29.6 g, 279 mmol) (400 mL) was added to adjust the pH value to about 9.0. The mixture was separated, and the aqueous layer was extracted with dichloromethane (200 mL x 2). The combined organic phase was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The mixture was concentrated under reduced pressure, and the residue was dissolved in acetone (150 mL). The mixture was stirred at 15 °C for 1 h, filtered, and the filter cake was washed with dichloromethane (50 mL) and acetone (100 mL). The mixture was stirred at 15 °C for 1 h, filtered, and the filter cake was washed with acetone and dried to obtain LYS-8 as a white solid (30 g, two-step yield 62%), with a purity of 96.0%.1H NMR (300 MHz, DMSO-d6) δ = 8.25 (d, J = 10.0 Hz, 1H), 7.38-7.28 (m, 2H), 6.95 (s, 2H), 6.59 (d, 1H), 5.60 (s, br, 1H), 5.26 (s, 2H), 5.10 (t, J = 6.0 Hz, 1H), 3.55-3.50 (m, 1H), 3.05-2.94 (m, 2H), 2.77-2.76 (m, 2H), 2.64-2.59 (m, 2H), 2.56 (t, J = 7.5 Hz, 4H), 1.13 (t, J = 7.5 Hz, 6H) ppm. HR-MS (ESI + ): calculated for C 26 H 29 N3O3[M+H] + : 432.2242, found 432.2272.

[0062] Example 2 Inhibition of TRK-specific kinase activity by LYS-8

[0063] The in vitro kinase activity assay was used to evaluate the inhibitory activity of LYS-8 on TRKA, TRKB, TRKC. 200 ng / 50 μL of recombinant human TRKA, TRKB, TRKC, MAK kinase protein was reacted with 0.2 mg / mL Poly(Glu, Tyr) / 10 μM ATP, and all enzymatic reactions were carried out at 30°C for 40 minutes. 50 μL of the reaction system included: 40 mM Tris (pH 7.4), 10 mM MgCL2, 0.1 mg / mL BSA, 1 mM DTT, 10 μM ATP, kinase and its substrate. Compound LYS-8 was dissolved in 10% DMSO, and 5 μL of the preparation liquid was added to 50 μL of the reaction system, so that the final DMSO concentration in all reactions was kept at 1%. The experiment used the Kinase-Glo Plus luciferase kinase detection kit, which reflected the kinase activity by measuring the content of residual ATP in the solution after the kinase reaction. The detected luminescence signal was positively correlated with the content of ATP in the solution, and negatively correlated with the kinase activity. IC 50 values were calculated by non-linear regression analysis of normalized dose-response curves using Prism GraphPad software.

[0064] The results, as shown in Figure 2 , LYS-8 can significantly inhibit the kinase activity of TRKA / B / C, and has almost no obvious inhibitory effect on the non-target MAK kinase, which proves that LYS-8 has high specificity.

[0065] Example 3 Comparison of the proliferation activity of LYS-8 in pancreatic cancer and colorectal cancer cell lines

[0066] Logarithmic growth phase pancreatic cancer cell lines (CFPAC1, MIA PaCa-2) and colorectal cancer cell lines (HCT8, SW480) were selected, 3500 cells were inoculated in each well of a 96-well plate, after the cells adhered, different concentrations of drug medium were added to each well, and the plate was placed in a 37°C, 5% CO2 incubator, and three replicate wells were set up, and three control wells (blank control without cells and with only medium) were set up. After 48 h, 10 μL of CCK8 reagent was added to each well, and after 2 h of incubation in the incubator, the absorbance at 450 nm was recorded according to the manufacturer's instructions of the CCK8 kit. The cell survival rate was calculated with time as the horizontal coordinate and the absorbance value as the vertical coordinate, and the concentration-reaction curve was drawn, and the IC 50 values were calculated by non-linear regression method.

[0067] The results, as shown in Figure 3 A-B and Figure 4 A-B, the results showed that LYS-8 had an IC 50Significantly lower than IC in HCT8 and SW480 cells 50 , suggesting that it has stronger selectivity and inhibitory activity on pancreatic cancer cells.

[0068] Pancreatic cancer cell line (ASPC1) and colorectal cancer cell line (SW620) were selected. The cells in the logarithmic growth phase were counted and inoculated in 12-well plates at a density of 500 cells per well. After adhering, different concentrations of LYS-8 (0.5 μM, 1 μM) were added, and a solvent control group was set. The cells were cultured for 7-10 days until visible colonies were formed in the control group. After the culture ended, the culture medium in each well of the 12-well plate was discarded, and each well was washed with PBS for 2-3 times. The PBS in the plate was aspirated with a pipette, and 1 mL of 4% paraformaldehyde was added to each well at room temperature for 15 min. After fixation, the cells were washed with PBS for 2 times. The residual PBS in the wells was aspirated as much as possible, and 1 mL of 0.4% (0.4 g / 100 mL) crystal violet staining solution was added to each well for 15 min. Image capture and observation of the colony formation ability of the cells treated with LYS-8

[0069] The results are shown in Table 1. Figure 5 Table 1 shows that LYS-8 can significantly reduce the number of colonies of pancreatic cancer cells ASPC1, and the inhibition rate increases with the increase of the concentration. The inhibition effect on colorectal cancer cells SW620 is relatively weak. The above results further prove that LYS-8 has a better anti-proliferation effect on pancreatic cancer cells.

[0070] Case 4 Anti-tumor effect of LYS-8 on pancreatic cancer and colorectal cancer xenograft tumor models

[0071] To further verify the in vivo anti-tumor effect of the compound LYS-8 of the present application on different tumor types, nude mouse subcutaneous xenograft tumor models of human pancreatic cancer cell line CFPAC1 and colorectal cancer cell line HCT116 were established, and the anti-tumor activity of LYS-8 was compared under consistent treatment conditions.

[0072] 4-6-week-old female BALB / c nude mice were selected, and the mice were raised under standard environmental conditions (SPF level conditions) and adaptively fed for one week. The PANC-1 (pancreatic cancer) or HCT116 (colorectal cancer) cells in the logarithmic growth phase were digested and collected into centrifuge tubes, washed twice with PBS, and then resuspended with PBS to obtain a final concentration of 1×10 7 μL of the cell suspension was injected subcutaneously into the back of the nude mouse. 3 When the tumor volume reached about 100-150 mm

[0073] Grouping and administration: Each model was randomly divided into 2 groups (n=5): ① solvent control group, orally administered with equal volume of solvent; ② LYS-8 treatment group, orally administered with LYS-8 (50 mg / kg, once a day, for 21 consecutive days).

[0074] Detection index: The length and width of the tumor were measured every 2 days, the tumor volume (V=0.5 x length x width2) was calculated, and the body weight of the mice was recorded. At the end of the experiment, the tumor was peeled off, weighed, and part of the tissue was saved for subsequent detection.

[0075] The tumor growth curve results are shown in Figure 6 and Figure 7 LYS-8 can significantly inhibit the volume growth of PANC-1 pancreatic cancer xenograft tumor, and the tumor inhibition rate is 70% compared with the control group; while in the HCT116 colorectal cancer model, the inhibitory effect is relatively weak, and the tumor inhibition rate is only 56%.

[0076] Example 5: Regulation of LYS-8 on TRK downstream signaling pathways

[0077] To study the mechanism of the compound LYS-8 in pancreatic cancer cells, human pancreatic cancer cell lines CFPAC1 and PANC1 were selected for experiments, and the regulation of LYS-8 on TRK kinase and downstream MAPK / ERK, PI3K / AKT signaling pathways was evaluated. At the same time, the inhibitory effect of LYS-8 on the above downstream signaling pathways was also verified in the CFPAC1 cell subcutaneous tumor model, so as to further confirm its anti-tumor mechanism in vivo.

[0078] After CFPAC1 and PANC1 cells were cultured in 6-well plates to about 70%-80% density, LYS-8 treatment (0.5 μM, 1 μM, 1.5 μM, 2 μM) or equal volume of solvent control was given, and the treatment time was 24 h.

[0079] Discard the supernatant, collect the centrifuged cells, wash them with PBS once, and then collect the cells by centrifugation, use RIPA lysis buffer to stand on ice for 5 minutes, use contact ultrasonic cell crusher, 25% power, 10s on 10s off 4-6 cycles on ice to break the cells, and use ddH2O to clean the probe every time the sample is changed, and use dust-free paper to dry (the probe is stretched to the bottom of the liquid to avoid air bubbles as much as possible). After ultrasonic, the sample is centrifuged at 4℃ for 15 minutes, and the supernatant is transferred to a new EP tube for protein quantification and sample preparation.

[0080] After the proteins are separated by SDS-PAGE electrophoresis, they are transferred to PVDF membrane. Antibodies are used to detect TRK, p-TRK, ERK, p-ERK, AKT, p-AKT, and β-actin as an internal reference.

[0081] The results are shown inFigure 8 TRK phosphorylation inhibition: After LYS-8 treatment, the p-TRK levels of CFPAC1 and PANC1 were significantly reduced in a dose-dependent manner. There was no significant change in the total TRK expression level, indicating that LYS-8 mainly inhibits kinase activity rather than protein expression.

[0082] Downstream signaling pathway regulation: The levels of p-ERK and p-AKT were significantly reduced as the concentration of LYS-8 increased. The total ERK and AKT expression levels remained relatively stable, further proving that the inhibitory effect is through blocking phosphorylation signaling rather than protein degradation.

[0083] Dose-dependent and selective analysis: Different concentration gradients (0.5 μM, 1 μM, 1.5 μM, 2 μM) showed significant inhibition, and the inhibition effect was most significant at 2 μM, suggesting that LYS-8 has a high dose-dependent effect on pancreatic cancer TRK signaling.

[0084] This example shows that LYS-8 can efficiently inhibit TRK kinase phosphorylation and its downstream MAPK / ERK and PI3K / AKT signaling pathways in pancreatic cancer cells, thereby exerting an anti-tumor effect.

[0085] The above-described embodiments are only some of the embodiments of the present application, rather than all the embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor fall within the scope of protection of the present application.

Claims

1. Use of a compound LYS-8 or its pharmaceutically acceptable salt and its derivatives in the preparation of a medicament for treating a tumor, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; and said tumor is a malignant tumor, in particular a colorectal cancer or a pancreatic cancer. LYS-8 is a TRK (tropomyosin receptor kinase) inhibitor, which can specifically inhibit the kinase activity of TRKA, TRKB and TRKC, thereby inhibiting the proliferation of tumor cells.

2. Use according to claim 1, characterized in that, 4. Use of a compound LYS-8 or its pharmaceutically acceptable salt and its derivatives in the preparation of a medicament for inhibiting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; LYS-8 inhibits TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, reduces the level of TRK phosphorylation, thereby inducing apoptosis of cancer cells. said substance detects whether there is a reduction in the level of TRK phosphorylation.

3. Use according to claim 1, characterized in that, said cancer is a colorectal cancer or a pancreatic cancer. said LYS-8 is administered at a concentration of 10-100 mg / kg body weight / day. said inhibition is in vitro, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; LYS-8 inhibits TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, reduces the level of TRK phosphorylation, thereby inducing apoptosis of cancer cells. said tumor cell line is a colorectal cancer cell line and / or a pancreatic cancer cell line.

5. Use of a compound LYS-8 or its pharmaceutically acceptable salt and its derivatives in the preparation of a medicament for treating a tumor, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; and said tumor is a malignant tumor, in particular a colorectal cancer or a pancreatic cancer.

5. Use according to claim 4, characterized in that, LYS-8 is a TRK (tropomyosin receptor kinase) inhibitor, which can specifically inhibit the kinase activity of TRKA, TRKB and TRKC, thereby inhibiting the proliferation of tumor cells.

6. Use of a compound LYS-8 or its pharmaceutically acceptable salt and its derivatives in the preparation of a medicament for inhibiting TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; LYS-8 inhibits TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, reduces the level of TRK phosphorylation, thereby inducing apoptosis of cancer cells. said substance detects whether there is a reduction in the level of TRK phosphorylation.

6. Use of substances that specifically detect TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways in the manufacture of a reagent for determining the effectiveness of LYS-8 according to claim 1 in the treatment of cancer, characterized in that, said cancer is a colorectal cancer or a pancreatic cancer.

7. Use according to claim 6, characterized in that, said LYS-8 is administered at a concentration of 10-100 mg / kg body weight / day.

8. Use according to any one of claims 1 to 3, characterized in that, said inhibition is in vitro, said LYS-8 has a structure as shown in Formula I: ###0001### Formula I; LYS-8 inhibits TRK and its downstream PI3K-AKT and MAPK / ERK signaling pathways, reduces the level of TRK phosphorylation, thereby inducing apoptosis of cancer cells.

9. Use of a compound LYS-8 or a pharmaceutically acceptable salt thereof and derivatives thereof in the preparation of a formulation for inhibiting proliferation of a tumor cell line, characterized in that, said tumor cell line is a colorectal cancer cell line and / or a pancreatic cancer cell line. ​ 10. Use according to claim 9, characterized in that, ​

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