Anti-tumor drug and application thereof

Abstract

The structural formula of the small molecule compound for inhibiting PINK1 is # imgabs0 #, R1 is CH3 or # imgabs1 #, and R2 is # imgabs2 #. The small molecule compound provided by the invention has an obvious inhibiting effect on PINK1, and by inhibiting the activity of PINK1 kinase, the phosphorylation level of Ub-Ser65 is obviously reduced, and mitochondrial damage accumulation is promoted, so that tumor cell proliferation is inhibited. The small molecule compound has an excellent anti-tumor effect and has a remarkable effect of inhibiting cell proliferation on liver cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an anti-tumor drug and application thereof. Background Art

[0002] PINK1 (PTEN-induced kinase 1) is a mitochondrial-associated serine / threonine kinase composed of 581 amino acids. PINK1 is mainly located on the mitochondrial membrane, involved in mitochondrial quality control, and closely related to various biological processes such as cell stress response, energy metabolism, and autophagy regulation. Its N-terminus contains a mitochondrial localization signal (MTS), which can guide PINK1 to the inner membrane of mitochondria. Under normal conditions, PINK1 is imported into the inner membrane space by the mitochondrial transmembrane transport system and processed, and then rapidly degraded. However, when mitochondria are damaged or depolarized, PINK1 accumulates on the outer mitochondrial membrane (OMM) and activates the mitochondrial autophagy process.

[0003] PINK1 plays an important role in a variety of cellular functions, especially in maintaining mitochondrial health, regulating cellular energy metabolism, responding to cellular stress, and regulating apoptosis. The accumulation of PINK1 in damaged mitochondria is a key signal for initiating mitophagy. In normal mitochondria, PINK1 is rapidly transported to the inner membrane of mitochondria and cleaved and degraded, but when the mitochondrial membrane potential is lost (such as mitochondrial damage or depolarization), PINK1 cannot enter the inner membrane and accumulates on the outer membrane of mitochondria. On the outer membrane of mitochondria, PINK1 activates its function by phosphorylating Parkin (an E3 ubiquitin ligase) and promotes the recruitment of Parkin to damaged mitochondria by phosphorylating mitochondrial outer membrane proteins (such as ubiquitin). Activated Parkin further marks damaged mitochondria, marking them as targets for clearance through ubiquitination, and ultimately triggering the autophagy pathway to transport damaged mitochondria to lysosomes for degradation. This process is essential for maintaining mitochondrial quality control, responding to intracellular oxidative stress, and maintaining the stability of cellular energy metabolism.

[0004] In recent years, studies have gradually found that the role of PINK1 in regulating mitochondrial function is closely related to the occurrence and development of tumors. PINK1 can affect the survival, proliferation and tolerance of cancer cells to chemotherapy drugs by regulating mitochondrial autophagy and metabolic function. In some cancers, PINK1 is believed to have the effect of promoting the growth of tumor cells, especially by reducing oxidative stress and promoting the maintenance of mitochondrial function, helping cancer cells adapt to the harsh living environment. In addition, PINK1 can also participate in regulating the metabolism of tumor cells and enhance their ability to adapt to oxidative stress and energy deficiency.

[0005] PINK1 plays a key role in regulating mitochondrial autophagy, maintaining mitochondrial function and responding to oxidative stress, making it a potential drug target for a variety of diseases. In particular, in the field of tumor treatment, by inhibiting PINK1 function, the oxidative stress level of tumor cells can be enhanced, promoting the accumulation of mitochondrial damage, and thus inducing tumor cell death. The development of small molecule inhibitors targeting PINK1 has become an exploratory strategy. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-tumor drug, specifically a small molecule compound that inhibits PINK1, which can inhibit PINK1 from regulating the oxidative phosphorylation function of mitochondria, affect the synthesis efficiency of ATP, and inhibit the hydrolysis of ATP.

[0007] Another object of the present invention is to provide the use of the above compound.

[0008] An anti-tumor drug, specifically a small molecule compound that inhibits PINK1, characterized in that: the structural formula of the compound is: , wherein R1 is CH3 or , R2 is .

[0009] An anti-tumor drug, specifically a small molecule compound that inhibits PINK1, characterized in that the structural formula of the compound is as follows: .

[0010] PINK1 can regulate the oxidative phosphorylation function of mitochondria. The above-mentioned small molecule compounds can effectively inhibit the function of PINK1, enhance the oxidative stress level of tumor cells, promote the accumulation of mitochondrial damage, and thus induce the death of tumor cells.

[0011] The application of the above small molecule compound is characterized by: application in the preparation of anti-tumor drugs.

[0012] More preferably, the tumor is a liver cancer tumor.

[0013] Furthermore, the small molecule compound is used in the preparation of PINK1 inhibitor drugs.

[0014] PINK1 inhibitors can block the kinase activity of PINK1, thereby preventing it from initiating mitochondrial autophagy, anti-oxidation and anti-regulatory signal transduction, thereby inhibiting the growth and survival of tumor cells.

[0015] The present invention has the following technical effects: The small molecule compound of the present invention has a significant inhibitory effect on PINK1. By inhibiting PINK1 and inhibiting ATP hydrolysis, the oxidative stress level of tumor cells can be enhanced, and mitochondrial damage accumulation can be promoted, thereby inducing tumor cell death. The small molecule compound has an excellent anti-tumor effect and has a significant effect of inhibiting cell proliferation in liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Identification of small molecule compounds targeting the inhibition of PINK1 kinase.

[0017] Figure 2 :ADP-Glo ​​in vitro kinase assay was used to test the effects of small molecule compounds on inhibiting PINK1 kinase activity in vitro.

[0018] Figure 3 : H NMR spectrum of small molecule compound 5f.

[0019] Figure 4 : H NMR spectrum of small molecule compound 5h.

[0020] Figure 5 : H NMR spectrum of small molecule compound 5j.

[0021] Figure 6 :Effects of various small molecule compounds on the proliferation activity of liver cancer cell Hep3B. DETAILED DESCRIPTION

[0022] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0023] Example 1 The preparation routes of small molecule compounds 5a-5h are as follows:

[0024] The specific preparation steps are as follows: (1) A mixture of methyl 2,4-dioxo-4-(pyridin-3- or 4-yl)butanoate (1 mmol, 0.207 g) and phenylhydrazine hydrochloride (1 mmol, 0.15 g) was heated under reflux in methanol for 5 h. The reaction mixture was cooled, basified to pH 10-12 with K2CO3 solution, diluted with water, and stirred for 10-20 minutes, during which time the product precipitated. The reaction mixture was then filtered, washed with water, dried, and crystallized from water to obtain compound 3.

[0025] (2) Compound 3 was dissolved in 1:1 THF / water (8 mL), and LiOH was then added to the reaction, and the reaction was heated at 100°C for 15 minutes under microwave irradiation. The reaction was then acidified to pH 3-4 and extracted with 4:1 chloroform. / IPA, and the organic layer was dried over MgSO4 and concentrated to remove the solvent to obtain compound 4.

[0026] (3) Compound 4 was added to a 5 mL vial, anhydrous DCM was added to the vial, and then EDC, HOAt and DIPEA were added in sequence. Amine was added and the reaction was sealed under N2. After 12 hours of reaction, the reaction was quenched with water, extracted with 4:1 chloroform / isopropanol, and washed with saturated NaHCO3. The organic layer was collected, dried with MgSO4, concentrated, and purified by Hex / EA flash chromatography to obtain compound 5.

[0027] According to the difference between the R1 group and the R2 group, amines with different substituents were added to prepare different small molecule compounds 5a, 5b, 5c, 5d, 5e, 5f, 5g and 5h, and the corresponding structures are as follows:

[0028] Example 2 The preparation route of small molecule compounds 5i-5p is as follows:

[0029] The specific steps are as follows: (1) 1,3-Diphenylpropane-1,3-dione (448 mg, 2.0 mmol) and 5-amino-1H-pyrazole-3-carboxylic acid ethyl ester (310 mg, 2 mmol) were added to acetic acid. The mixture was subjected to microwave reaction at 120°C for 10 minutes. After cooling to room temperature, acetic acid was removed, diluted with EtOAc (30 mL), and washed with saturated NaHCO3 and brine in sequence. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography using an ethyl acetate / hexane (0-20%) gradient to obtain the relative target product 3 (5,7-diphenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester); (2) Add 5,7-diphenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid ethyl ester (686 mg, 2.0 mmol) and NaOH (6 equivalents) to MeOH:H2O=1:1 (20 mL) and react at 60°C for 5 hours. After the reaction is cooled to room temperature, methanol is removed under reduced pressure, the pH value of the solution is adjusted with 1 mol / L hydrochloric acid, and the residue is filtered to obtain the relative target product 4 (5-7-diphenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid).

[0030] (3) 5-7-Diphenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid (158 mg, 0.5 mmol) and amines with different substituents (0.5 mmol) were dissolved in DCM (2 mL), EDCI (3 eq.) and DIPEA (3 eq.) were added, and stirred at room temperature overnight. The solvent was removed, then diluted with EtOAc (15 mL), washed with saturated NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography using an ethyl acetate / hexane (30-100%) gradient to give the relative target product 5.

[0031] According to the different substituents of the amine added in step (3), the R groups are different, and the structures of the prepared small molecule compounds 5i, 5j, 5k, 5l, 5m, 5n, 5o and 5p are as follows: , , , , , , , .

[0032] Example 3 Screening of small molecule compounds that target and inhibit PINK1 1. Cellular phosphorylation assay In order to identify whether the synthesized small molecule compounds have the effect of targeted inhibition of PINK1, 3×10 6 Hep3B liver cancer cells were inoculated into 60 mm culture dishes and treated with 2 μM Oligomycin and 2.5 μM antimycin A for 12 h to induce mitochondrial autophagy. Then, the cells were treated with 20 μM small molecule compounds for 24 h. The cells were collected and the phosphorylation level of ubiquitin-Serine 65 (Ub-Ser65) (p-Ub S65) was detected by Western blotting. The phosphorylation level of Ub-Ser65 was detected using anti-p-Ub (S65) antibody.

[0033] The results are as follows Figure 1 As shown in A, compared with the control group, small molecule compounds 5f, 5g, 5h, 5j and 5p can significantly downregulate the phosphorylation level of Ub-Ser65, indicating that these five small molecule compounds have an inhibitory effect on PINK1.

[0034] 2. In vitro phosphorylation assay In order to further analyze the effects of small molecule compounds 5f, 5g, 5h, 5j and 5p on the level of PINK1 phosphorylated Ub-S65 in vitro, we incubated recombinant protein PINK1, Ub (ubiquitin) and small molecule compounds, and then detected the phosphorylation level of Ub-Ser65 using anti-p-Ub (S65) antibody. The results are shown in Figure 2. Figure 1 As shown in B, 5f, 5h and 5j can significantly inhibit the phosphorylation level of Ub-Ser65, confirming that 5f, 5h and 5j are small molecule compounds that target and inhibit PINK1.

[0035] 3. ADP-Glo ​​in vitro kinase assay (1) 2 μM unphosphorylated recombinant protein Pediculus humanus PINK1 (PhPINK1) was incubated with different concentrations of small molecule compounds 5f, 5g, 5h, 5j and 5p in 100 μL kinase buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT) for 30 minutes (30°C). At the same time, a blank control group (without kinase) and a positive control group (no drug addition group, i.e., 0 μM) were set up to verify the effectiveness of the experimental system; (2) Add 10 μM Ub as substrate and then add 10 μM ATP to start the reaction. The final volume is 200 μL and incubate at 30°C for 10 min. (3) Add 200 μL of Glo-ADP detection reagent. After incubation, measure the luminescent signal of each well using a multifunctional microplate reader. The luminescent intensity is proportional to the amount of ADP generated in the reaction.

[0036] The effects of small molecule compounds 5f, 5g, 5h, 5j and 5p on PINK1 kinase activity were analyzed by the above experimental results. Figure 2 As shown, 5f and 5h reduced ATP hydrolysis in a concentration gradient, 5j also reduced ATP hydrolysis but did not change in a concentration gradient, and 5g and 5p had almost no effect on the ATP hydrolysis process. The above results indicate that small molecule compounds 5f, 5h and 5j are new small molecule inhibitors with novel structures for PINK1.

[0037] The NMR spectra of the small molecule compounds 5f, 5h and 5j screened out above that have inhibitory effects on PINK1 were analyzed, and the results are as follows: The yield of small molecule compound 5f (N-(2-chloropyridin-4-yl)-1-methyl-5-(o-tolyl)-1H-pyrazole-3-carboxamide) was 91.7%. Its H NMR spectrum was as follows: Figure 3As shown, 1H NMR (400 MHz, CDCl3) δ 8.92 (s,1H), 8.30 (d, J = 5.4 Hz, 1H), 7.78 (s, 1H), 7.60 – 7.52 (m, 1H), 7.44 – 7.28(m, 3H), 7.22 (d, J = 7.3 Hz, 1H), 6.85 (s, 1H), 3.71 (s, 3H), 2.18 (s, 3H).13C NMR (101 MHz, CDCl3) δ 160.29 (s), 152.50 (s), 150.28 (s), 147.03 (s),145.37 (s), 144.49 (s), 137.52 (s), 130.59 (s), 130.20 (s), 129.84 (s), 128.93 (s), 126.09 (s), 113.30 (s), 112.40 (s), 107.86 (s), 37.36 (s), 19.82(s).

[0038] The yield of small molecule compound 5h (N-(2-chloropyridin-4-yl)-1-(4-methoxyphenyl)-5-(o-tolyl)-1H-pyrazole-3-carboxamide) was 90.4%. Its H NMR spectrum was as follows: Figure 4Shown, 1H NMR (400 MHz, CDCl3) δ9.01 (s, 1H), 8.31 (d, J = 5.4 Hz, 1H), 7.81 (s, 1H), 7.56 (d, J = 5.4 Hz,1H), 7.31 (s, 1H), 7.19 (t, J = 8.3 Hz, 5H), 7.02 (s, 1H), 6.83 (d, J = 8.6Hz, 2H), 3.80 (s, 3H), 2.03 (s, 3H).13C NMR (101 MHz, CDCl3) δ 160.24 (s),159.27 (s), 152.54 (s), 150.30 (s), 146.94 (s), 145.50 (s), 145.13 (s),137.15 (s), 132.42 (s), 130.58 (d, J = 6.6 Hz), 129.56 (s), 129.33 (s),126.00 (s), 125.57 (s), 114.18 (s), 113.40(s), 112.46(s), 109.25(s), 55.51(s), 19.95(s).

[0039] The small molecule compound 5j is a white solid with a yield of 92.0%. Figure 5 As shown: 1 H NMR(400 MHz, CDCl3) δ 8.81 (s, 1H), 8.17 – 8.15 (m, 2H), 8.08-8.06 (m, 2H), 7.68– 7.65 (m, 5H), 7.56-7.54 (m, 3H), 7.48 (s, 1H), 7.41 (s, 1H), 7.06 (t, J = 8.7Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ 159.57, 157.14, 150.84, 150.25, 146.88, 136.95, 133.62, 131.35, 130.97, 129.30, 129.09, 128.88, 127.40, 121.73,115.84, 115.61, 107.22, 98.42. HRMS (ESI)m / z calcdforC 25 H 18 FN4O +(M+H) + 409.1465, found 409.1471.

[0040] Example 4 The novel small molecule compounds 5f and 5h that target and inhibit PINK1 have anti-proliferation effects on liver cancer cells. The steps are as follows: (1) Cell seeding: Cells were seeded into 96-well plates, with 5,000 cells added to each well. The next day, 10 μM small molecule compounds were added to treat the cells for 48 hours. The control group was DMSO-treated cells, and blank wells (no cells) were set up. (2) Add MTT solution: Add 10 μL of 0.5 mg / mL MTT solution to each well, shake gently, and continue to incubate the cells at 37°C, 5% CO2 for 4 hours; (3) After the incubation, discard the supernatant and carefully add 150 μL of DMSO to each well to dissolve the formazan crystals. Shake the 96-well plate to mix the solution evenly and ensure that the formazan crystals are completely dissolved.

[0041] (4) Use a multifunctional microplate reader to measure the absorbance (OD value) of each well at a wavelength of 570 nm. This absorbance value is proportional to the number of living cells.

[0042] (5) Compare the OD value of the experimental group with that of the control group to evaluate the cell survival rate. The formula for calculating the cell survival rate is: Cell survival rate (%) = (OD value of the experimental group / OD value of the control group) × 100%.

[0043] The results are as follows Figure 6 As shown, small molecule compounds 5a, 5b, 5c, 5f, 5g, 5h, 5j and 5i can inhibit the proliferation of Hep3B cells to varying degrees, among which 5h, 5h and 5j have the most significant inhibitory effects on cell proliferation activity.

[0044] The half maximal inhibitory concentration (IC) of small molecule compounds 5f, 5h, and 5j was determined by plotting drug concentration versus cell survival rate curves. 50 ), and the results are shown in Table 1.

[0045] Table 1:

[0046] The results showed that 5f, 5h and 5j inhibited the proliferation of Hep3B cells. 50The values ​​were 5.86 μM, 3.32 μM and 11.84 μM, respectively. This result indicated that 5f, 5h and 5j exerted an anti-liver cancer cell proliferation effect by targeting and inhibiting PINK1, and also confirmed that 5f, 5h and 5j can be used as a new small molecule anti-liver cancer inhibitory drug targeting PINK1.

Claims

1. A small molecule compound that inhibits PINK1, characterized in that: The structural formula of the compound is: , wherein R1 is CH3 or , R2 is .

2. A small molecule compound that inhibits PINK1, characterized in that: The structural formula of the compound is: 。 3. The use of the small molecule compound as claimed in claim 1, characterized in that: Application in the preparation of anti-tumor drugs.

4. Use of the small molecule compound according to claim 1 in the preparation of PINK1 inhibitor drugs.

5. The use of the small molecule compound as claimed in claim 2, characterized in that: Application in the preparation of anti-tumor drugs.

6. Use of the small molecule compound according to claim 2 in the preparation of PINK1 inhibitor drugs.

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