PGK1-targeted small molecular compound and application thereof

Abstract

The invention belongs to the technical field of tumor targeted therapy, and particularly relates to a PGK1-targeted anti-liver cancer small molecule compound and application thereof. The invention provides a PGK1-targeted anti-liver cancer small molecule compound or a pharmaceutically acceptable salt thereof. The invention provides a spiro alkaloid compound with antitumor activity. The spiro alkaloid compound can be applied to research and development of a PGK1 targeted inhibitor for resisting liver cancer. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeted therapy, and specifically relates to an anti-liver cancer small molecule compound targeting PGK1 and an application thereof. Background Art

[0002] Phosphoglycerate kinase 1 (PGK1) is the first ATP-generating enzyme in the glycolysis process. The reaction process catalyzed by it is an important part of cell metabolism, providing the necessary energy and material basis for cell growth and proliferation. The overexpression and high activity of PGK1 play an important role in the occurrence, development and poor prognosis of tumors. It is an important target in the field of tumor metabolism drug research and development, but there are currently no PGK1 inhibitory drugs on the market.

[0003] Liver cancer is one of the most common malignant tumors in the world. Among all types of cancer, the incidence and mortality of liver cancer are at the forefront. Due to the characteristics of liver cancer such as easy recurrence, poor prognosis of patients, and easy drug resistance, the clinical treatment effect is not ideal. From the perspective of mechanism of action, systemic therapy for liver cancer can be divided into two categories, one is immunotherapy and the other is molecular targeted therapy. Molecular targeted therapy refers to the use of specific monoclonal antibodies or kinase inhibitors to target key molecules in the liver cancer signaling pathway, specifically blocking liver cancer signal transduction, thereby inhibiting the growth and proliferation of cancer cells and exerting an anti-cancer effect. Among them, molecular targeted drugs represented by sorafenib have become the first-line drugs for the treatment of liver cancer. However, the side effects of drugs and tumor resistance limit many patients from benefiting from the above therapies, and the discovery of new therapeutic targets and the research of corresponding targeted drugs are imminent. Summary of the invention

[0004] The invention provides a spiro alkaloid compound.

[0005] The present invention provides the use of the above compound.

[0006] According to a specific embodiment of the present invention, the spiroalkaloid compound is selected from

[0007]

[0008] or a pharmaceutically acceptable salt thereof.

[0009] Among them, compound II is the acetylated precursor of compound I, which is metabolized in plasma in vivo and hydrolyzed into the active form of compound I.

[0010] The present invention provides the use of the above compound in preparing a drug for treating cancer. Preferably, the cancer is liver cancer.

[0011] The present invention also provides a pharmaceutical composition, which comprises the above compound and a pharmaceutically acceptable carrier.

[0012] The present invention also provides a pharmaceutical composition for treating cancer, which comprises the above compound and a pharmaceutically acceptable carrier. The cancer is liver cancer.

[0013] The spiroalkaloid compounds of the present invention inhibit cancer cell growth by targeting the metabolism-related kinase PGK1. In addition to being related to a variety of tumors, the metabolism-related kinase PGK1 is also related to vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, and viral infection. Therefore, the spiroalkaloid compounds of the present invention can be used to prepare drugs for treating vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, amyotrophic lateral sclerosis, inflammation, viral infection and other related diseases.

[0014] Preferably, the present invention also provides the use of the above-mentioned compounds in the preparation of PGK1 targeted inhibitory drugs for the treatment of metabolic-related kinase PGK1-related diseases, such as vascular intimal hyperplasia, Kawasaki disease, chronic mountain sickness, polycystic ovary syndrome, amyotrophic lateral sclerosis, inflammation, and viral infection.

[0015] A PGK1 targeted inhibitory drug according to a specific embodiment of the present invention comprises the above compound and a pharmaceutically acceptable carrier.

[0016] Therefore, the present invention also provides an analysis method for the compound of formula I targeting PGK1.

[0017] The above-mentioned drugs can be prepared into oral preparations or injections; the oral preparations include but are not limited to capsules, tablets, granules, and oral liquids; the injections include but are not limited to sterile powder for injection, water injection, sodium chloride or glucose intravenous infusion.

[0018] In the above-mentioned medicine, the oral preparation includes additives, and the additives are selected from at least one of fillers, diluents, disintegrants, binders, lubricants, glidants, surfactants, solvents, flavoring agents, stabilizers, colorants, and preservatives.

[0019] The fillers or diluents include sugars such as lactose, sucrose, glucose, mannitol, sorbitol, and dextrin; starches such as starch, pregelatinized starch, α-starch, and dextrin; celluloses such as microcrystalline cellulose, gum arabic, fenugreek gum, and dextran; and inorganic salts such as calcium sulfate, calcium hydrogen phosphate, medicinal calcium carbonate, light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminosilicate.

[0020] The lubricant, glidant or anti-adhesive agent includes stearic acid; metal stearate such as calcium stearate or magnesium stearate; talc; colloidal silica; micro powder silica gel, hydrogenated vegetable oil; polyethylene glycol, lauryl sulfate such as sodium lauryl sulfate or magnesium lauryl sulfate; silicate such as silicic anhydride or silicate hydrate, etc.

[0021] The adhesive comprises distilled water, ethanol of different concentrations, starch slurry, hydroxypropyl methylcellulose, hydroxypropyl cellulose, povidone, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol and compounds similar to the above excipients.

[0022] The disintegrants include cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or cross-linked carboxymethyl cellulose sodium; cross-linked polyvinyl pyrrolidone; and chemically modified starch / cellulose, such as carboxymethyl starch or carboxymethyl starch sodium.

[0023] The surfactant includes Tween-80, sodium lauryl sulfate, sodium stearate sulfonate and the like.

[0024] The antioxidants include sodium bisulfite, sodium pyrosulfite, sodium sulfite, dried sodium sulfite, sodium thiosulfate, ascorbic acid, methionine (methionine), thiourea, phosphoric acid, citric acid and the like.

[0025] The preservatives or antibacterial agents include benzoic acid and sodium benzoate, sorbic acid, ethanol, parabens (parabens), benzalkonium bromide, o-phenylphenol, benzyl alcohol, phenylethyl alcohol, sodium propionate, sorbic acid, eucalyptus oil, cinnamon oil and peppermint oil.

[0026] The flavoring agents include sweeteners such as saccharin sodium, aspartame, syrup, stevioside, mannitol, sorbitol, mannose, galactose, maltose, fructose, glucose, sucrose, etc.; sour flavoring agents such as citric acid, malic acid or tartaric acid; and aromatics such as fennel oil, peppermint oil, menthol, peppermint water, cinnamon oil, lemon essence, lemon oil and spices of various flavors.

[0027] The method for using the above-mentioned drug includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral, intravenous injection or transdermal permeation, and the drug is applied to a patient in need of treatment.

[0028] Pharmaceutically effective amount refers to a reasonable benefit / risk ratio that can be obtained by applying drug treatment, and a quantity sufficient to treat the disease. The level of effective dose can be determined depending on some factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, application time, route of administration, excretion rate, treatment cycle, drugs used simultaneously, and other factors known in the medical field. The drug of the present invention can be used as an independent therapeutic agent, or combined with other therapeutic agents. In addition, the composition of the present invention can be added with typical therapeutic agents continuously or simultaneously, and the composition can be applied once or multiple times. It is important to consider all the above factors and apply the minimum dose that can produce the maximum effect without side effects, and the dosage can be determined by the physician according to the patient's condition, age, etc.

[0029] The present invention also provides a method for preparing the above compound:

[0030]

[0031] The preparation method of compound I according to a specific embodiment of the present invention comprises the following steps:

[0032] (1) With 2-methoxybenzoquinone A and 3-substituted isatin derivative B as reactants and chiral β-ICD as catalyst, an asymmetric Michael addition reaction takes place in an organic solvent to obtain intermediate C.

[0033] (2) Using methanol as solvent, under acidic conditions, intermediate C undergoes acetalization reaction with methanol to obtain compound I.

[0034] The preparation method of compound II according to a specific embodiment of the present invention comprises the following steps:

[0035] (1) With 2-methoxybenzoquinone A and 3-substituted isatin derivative B as reactants and chiral β-ICD as catalyst, an asymmetric Michael addition reaction takes place in an organic solvent to obtain intermediate C.

[0036] (2) Using methanol as solvent, under acidic conditions, intermediate C undergoes acetalization reaction with methanol to obtain compound I.

[0037] (3) Compound I is reacted with acetic anhydride using dichloromethane as solvent and triethylamine as acid binding agent to obtain the target compound II.

[0038] Beneficial effects of the present invention:

[0039] The compound I of the present invention has a significant inhibitory effect on liver cancer cells in vitro.

[0040] The compound I of the present invention can directly bind to PGK1 and inhibit its kinase activity.

[0041] The compound I of the present invention exerts an anticancer effect by inhibiting the activity of PGK1.

[0042] The spiroalkaloid compound II of the present invention can be hydrolyzed into its active form I after incubation with plasma, which provides a feasible solution for drug development that takes into account both in vitro stability and in vivo activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 Compound I inhibits the proliferation of various liver cancer cells in vitro. (Left) Proliferation curve of liver cancer cells after the action of compound I (HepG2 cells, 72h as an example); (Right) IC values ​​of compound I on various liver cancer cells and normal liver cells at different time points 50 .

[0045] Figure 2 The results show that compound I inhibits the kinase activity of PGK1. (Left) The curve of NADH consumption over time within 30 minutes after the action of different concentrations of compound I; (Right) The inhibition rate of different concentrations of compound I on PGK1 calculated by the OD value at the end of the reaction (30 minutes).

[0046] Figure 3 Shows CETSA binding analysis of compound I and cell PGK1 protein molecules. (Left) Western-blot analysis of PGK1 protein expression; (Right) Grayscale analysis of PGK1 protein relative expression, with GAPDH protein grayscale value as internal reference, and normalized to the grayscale values ​​of the two groups at 37.4°C.

[0047] Figure 4 Shows the SPR binding analysis of compound I and PGK1 protein molecules.

[0048] Figure 5 The results show that the liver cancer inhibitory activity of compound I is dependent on the expression of PGK1. (Left) Western-blot analysis of the PGK1 knockdown efficiency of HepG2 and Huh7 cells; (Right) SRB analysis of the 72h cytotoxicity of compound I on HepG2, Huh7, HepG2-sgPGK1, and Huh7-sg PGK1 cells.

[0049] Figure 6The results show that compound II plays a therapeutic role in liver cancer in vivo. (A) Tumor tissue image; (B) Tumor growth curve; (C) Tumor weight; (D) Body weight curve. *p<0.05, **p<0.01, ns No significant difference compared with the control group.

[0050] Figure 7 It was shown that compound II could be hydrolyzed into compound I after incubation with plasma. DETAILED DESCRIPTION

[0051] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0052] The experimental materials involved in the embodiments of the present invention are:

[0053] Fetal bovine serum was purchased from Yisheng Biotechnology Co., Ltd.;

[0054] MEM medium was purchased from Wuhan Punosai Life Science Technology Co., Ltd.;

[0055] DMEM medium was purchased from Gino Biopharmaceutical Technology Co., Ltd.;

[0056] HepG2 and Huh7 cells were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.;

[0057] Calcium phosphate virus packaging kit (Biyuntian), BCA protein quantification kit (Biyuntian), ECL chemiluminescence detection kit, protein-free rapid blocking solution (Yazyme), and HRP-labeled secondary antibody (Yazyme) were purchased from Qingdao Aifit Biotechnology Co., Ltd.;

[0058] Balb / c nude mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0059] His-PGK1-pEGFP recombinant plasmid, LentiCRISPRv2 sg PGK1 recombinant plasmid, psPAX2 plasmid, and pMD2.G plasmid were constructed and preserved in our laboratory;

[0060] Human PGK1 monoclonal antibody, Nickel-Beads nickel magnetic agarose beads protein purification kit (Beaver), PMSF, 3-phosphoglyceraldehyde, ATP, NADH, and puromycin were purchased from Shanghai Union Biotechnology Co., Ltd.

[0061] Example 1 Synthesis of Spiroalkaloid Compound I

[0062]

[0063] To the reaction flask, 3-substituted isatin derivative B (115.7 mg, 0.3 mmol), β-ICD (1 mg, 0.003 mmol), and dichloromethane (2 mL) were added in sequence, and stirred at -40 °C for 10 minutes. Subsequently, 2-methoxybenzoquinone A (41.4 mg, 0.3 mmol) was added, and the reaction was continued at -40 °C for 5 hours. The raw material B disappeared according to TCL detection. The mixture was transferred to room temperature and dichloromethane was removed by vacuum concentration to obtain a crude product C.

[0064] C was redissolved in methanol (2 mL), p-toluenesulfonic acid (5.1 mg, 0.03 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The intermediate C disappeared by TCL detection. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 6:1) to obtain white solid compound I (96.7 mg, yield 60%).

[0065] 1 H NMR (400MHz, CDCl3) δ7.93(d,J=8.2Hz,1H),7.68(d,J=7.3Hz,1H),7.64(s,1H),7.53-7.47(m,1H),7.38-7.28(m,3H),7.16(t,J=7.6Hz ,1H),6.64(s,1H),6.13(s,1H),5.21(s,1H),3.88(s,3H),3.15(s,3H),2.70(d,J=14.3Hz,1H),2.43(d,J=14.3Hz,1H),1.61(s,9H)ppm.

[0066] 13 C NMR (125MHz, CDCl3) δ178.19,149.32,147.28,144.67,142.44,140.99,139.22,134.69,134.63,129.93,128.80,128.59 ,127.06,126.85,125.14,124.65,115.27,114.83,112.55,101.56,99.55,84.65,56.20,50.33,50.22,45.11,28.19ppm.

[0067] HRMS:[M+Na] + calcd.For C 29 H 28 35 ClNaO7+ 560.1447,found:560.1445;[M+Na] + calcd.For C 29 H 28 37 ClNaO7 + 562.1417;found:562.1418.[α] D 20 +16.28 (c = 0.86 in CHCl3).

[0068] HPLC analysis: DAICEL CHIRALPAK IA, n-hexane / isopropanol=90 / 10, 1.0 mL / min, λ=240 nm, t major =6.45min,t minor =9.77min,ee=98%.

[0069] Example 2 Synthesis of Spiroalkaloid Compound II

[0070]

[0071] Compound I (96.7 mg, 0.18 mmol) was dissolved in dichloromethane (2 mL), and triethylamine (37 μL, 0.27 mmol), acetic anhydride (21 μL, 0.22 mmol) and DMAP (2 mg, 0.018 mmol) were added under stirring at 0°C, and stirred at 0°C for 1 hour until compound I disappeared. Water (2 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (3X 3 mL). The organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure. The crude product was chromatographed on a silica gel column (eluent: petroleum ether: ethyl acetate = 5:1) to obtain compound II (88.6 mg, yield 85%) as a white solid.

[0072] 1 H NMR (400MHz, CDCl3) δ7.85(dd,J=8.2,1.1Hz,1H),7.59(dd,J=7.6,1.4Hz,1H),7.56(q,J=1.2Hz,1H),7.44-7.39(m,1H),7.30-7.23(m,3H),7.09(td, J=7.6,1.1Hz,1H),6.65(s,1H),6.17(s,1H),3.76(s,3H),3.10(s,3H),2. 62(d,J=14.3Hz,1H),2.38(d,J=14.4Hz,1H),2.12(s,3H),1.54(s,9H)ppm.

[0073] 13C NMR (100MHz, CDCl3) δ177.8,168.6,151.8,149.8,149.2,142.0,139.0,135.0,134.6,134.1,129.9,128.8,128 .6,127.2,126.7,125.1,124.5,121.3,114.7,114.6,102.7,99.7,84.7,56.1,50.5,50.0,45.1,28.0,20.6ppm.

[0074] HRMS:[M+H] + Calculate for C 31 H 31 35 ClNO8 + 580.1733,found:580.1730;[M+H] + calcdfor C 31 H 31 37 ClNO8 + 582.1704,found:582.1707.[α] D 20 +24.35 (c=2.30in CHCl3).

[0075] HPLC analysis: DAICEL CHIRALPAK AD-H, n-hexane / isopropanol=90 / 10, 1.0 mL / min, λ=210 nm, t major =4.16min,t minor =4.91min,ee=94%.

[0076] Example 3

[0077] 1. SRB method to detect the proliferation rate of liver cancer cells

[0078] (1) Digest liver cancer cells with trypsin and resuspend in culture medium to form a single-cell suspension.

[0079] (2) After counting, the cells were plated in a 96-well plate with a single-well volume of 90 μL and a cell density of 4×10 3 / well, placed in a 37°C, 5% CO2 incubator, and cultured until completely adhered.

[0080] (3) Different concentrations of 1557-Ac were added to the compound group (final concentrations were 100, 50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05 μmol / L, respectively), and DMSO diluted in equal proportion was added to the blank control group, 10 μL / well, and the action time was 24, 48, 72, and 96 h.

[0081] (4) SRB method to detect OD515 value:

[0082] a. Discard the culture medium in the 96-well plate, add 10% pre-cooled TCA (100 μL per well) to fix the cells, and place in a 4°C refrigerator for more than 1 hour.

[0083] b. Wash with ddH2O for more than 5 times and dry naturally at room temperature.

[0084] c. Add 100 μL of 4 mg / mL SRB solution to each well, stain for 15 min at room temperature in the dark, and discard the SRB.

[0085] d. Wash with 1% glacial acetic acid for more than 5 times and dry naturally at room temperature.

[0086] e. Add 150 μL of 10 mmol / L Tris-base solution to each well until the stain is completely dissolved.

[0087] (5) Place the well plate on an ELISA reader and measure the OD value at 515 nm.

[0088] The cell proliferation rate was calculated according to the following formula:

[0089] Cell proliferation inhibition rate (%) = (OD 对照组 -OD 给药组 ) / OD 对照组 ×100%

[0090] (6) GraphPad was used to draw a graph and calculate the half inhibitory concentration (IC) 50 .

[0091] The results are as follows Figure 1 The results showed that compound I inhibited the proliferation of various liver cancer cells in vitro, and the effect was time- and concentration-dependent. The IC 50 The inhibitory rate of compound I on normal liver cells L-02 was 2.24 μM, and the inhibition rate was weak, with IC50>20 μM. This indicates that the anticancer effect of compound I has certain safety.

[0092] 2. Analysis of PGK1 inhibitory effects of compounds using PGK1 catalytic reaction kinetic model

[0093] (1) The reaction system contained: 5 mM 3-phosphoglyceraldehyde; 1 mM ATP; 5 U GAPDH protein; 0.1 mM NADH and 5 ng purified PGK1 protein, with a total volume of 100 μL. The reaction buffer was 50 mM Tris-HCl (pH 7.5) containing 1 mM MgCl2.

[0094] (2) All reactants except PGK1 protein were added to a 96-well transparent culture plate, and the remaining volume was filled with reaction buffer, including gradient concentrations of compound I (diluted with reaction buffer), and finally purified PGK1 protein was added, marking the start of the catalytic reaction.

[0095] Place the plate on a microplate reader at 340 nm and observe for 30 minutes. Analyze the catalytic activity of PGK1 through the OD value change curve and calculate the compound inhibition rate:

[0096] Inhibition rate (%) = (OD 对照组 -OD 给药组 ) / OD 对照组 ×100%

[0097] (3) Plot using Graphpad and calculate the half inhibitory concentration IC 50 .

[0098] The results are as follows Figure 2 The results showed that compound I directly inhibited the catalytic activity of PGK1 at the molecular level, IC 50 is 3.2μM.

[0099] 3. Cellular Thermal Shift Assay (CETSA) of the Interaction between Compounds and PGK1 Protein

[0100] (1) HepG2 cells (5×10 5 Each well) was inoculated in a 6-well plate and allowed to adhere for 24 hours. After 24 hours, compound I was added at 20 μM and incubated for 3 hours.

[0101] (2) The cells treated with the compound were resuspended in PBS and divided into several equal portions. DMSO was added to 4 equal portions, and 20 μM compound I was added to the other 4 equal portions.

[0102] (3) Use the gradient heating program of the PCR instrument to heat the cell sample at different temperatures.

[0103] (4) The heated cells were frozen and thawed three times with liquid nitrogen, each time for 3 min, and centrifuged at 20,000 g for 20 min at 4°C.

[0104] (5) Collect the supernatant, add protein loading buffer, and boil for 15 min. Detect PGK1 protein level by Western blotting.

[0105] The interaction between compounds and proteins can help PGK1 have stronger stability in high temperature environments. By comparing the degradation of PGK1 protein in the DMSO group and the compound treatment group as the temperature increases, it can be determined whether there is an interaction between compound I and PGK1.

[0106] The results are as follows Figure 3 As shown in the figure, it can be seen that the addition of compound I delayed the degradation of PGK1 caused by temperature increase, indicating that compound I can bind to the PGK1 protein of the cell.

[0107] 4. Surface Plasmon Resonance (SPR) Analysis of the Interaction between Compounds and PGK1 Protein

[0108] (1) SPR was measured using a Biacore X-100plus instrument (GE). PGK1 protein was immobilized on a sensor chip (CM5) using the amine coupling method according to the standard protocol.

[0109] (2) Compound I was diluted with PBSP. Binding experiments were performed at 25°C and a flow rate of 30 μL / min using PBSP buffer as the mobile phase. The 1:1 Langmuir binding model was used to obtain the binding affinity constant using BIA evaluation software.

[0110] The results are as follows Figure 4 As shown, the results confirmed at the molecular level that compound I can bind to PGK1 protein with a binding strength (KD value) of 3.7 μM.

[0111] 5. Analysis of the anti-hepatocellular carcinoma activity of PGK1-mediated compounds

[0112] (1) PGK1 was knocked out using CRISPR / Cas9 technology to construct a stable liver cancer cell line, and the PGK1 knockout virus was packaged and concentrated using a three-plasmid (LentiCRISPRv2 sg PGK1, psPAX2, and pMD2.G) lentiviral vector system.

[0113] (2) The PGK1 knockout virus was packaged by calcium phosphate transfection according to the instruction manual, and the ratio of LentiCRISPRv2sg PGK1, psPAX2, and pMD2.G plasmids was 4:3:1.

[0114] (3) Virus infection and screening of stable transfectants

[0115] HepG2 and Huh7 cells were inoculated in 24-well plates, 15 μl of the virus packaged in the above experiment was added, and 2 μL of Polybrene (4 mg / ml) was added to mix well and placed in a cell culture incubator for infection. After 12 hours, the cells were replaced with medium and cultured continuously. After 48 hours of culture, the infected cells were screened for stable strains. Since the viral vector carries puromycin resistance, puromycin 4 μg / mL was continuously added to the cell culture system for continuous screening. After two weeks, the cells were observed to no longer die, and a stable cell line with successful knockout of PGK1 was obtained. The expression level of PGK1 protein in the screened stable strain was detected by Western Blot experiment to determine the knockout efficiency, and then used in subsequent experiments.

[0116] (4) The SRB method was used to analyze the cytotoxic effect of compound I on PGK1 knockout stable transgenic lines (Hun7-sgPGK1, HepG2-sgPGK1) and the corresponding parental cells.

[0117] The results are as follows Figure 5 As shown in the figure, after PGK1 knockout, the tumor inhibitory effect of compound I was significantly weakened, among which the 72h cytotoxicity of compound I on HepG2 cells increased from 2.24μM to 20.39μM, and the 72h cytotoxicity of compound I on Huh7 cells increased from 1.16μM to 24.93μM, and the cell proliferation inhibitory effect of compound I disappeared, indicating that PGK1 plays a key role in the anti-cancer activity of compound I. Based on the above studies, it can be concluded that compound I exerts anti-liver cancer function by directly targeting PGK1.

[0118] Analysis of the therapeutic effect of compound Ⅱ on liver cancer in vivo

[0119] (1) Nude mouse Huh7 cell xenograft tumor model

[0120] Huh7 cells were subcutaneously inoculated into 6-week-old Balb / c-Nude mice (3×10 6 Each) and monitor tumor growth. When the tumor volume reaches 100 mm 3 Start treatment when

[0121] (2) The treatment group was given Compound I (low, medium and high doses, once a day), and the control group was given normal saline (solvent), and the medication was given for 15 days.

[0122] (3) Analysis of treatment effects:

[0123] a. Monitor tumor volume and body weight changes. The volume is calculated according to the following formula:

[0124] Tumor volume = long diameter × short diameter 2 ×1 / 2;

[0125] b. At the termination time, the mice were killed and dissected to obtain the tumor volume and weigh it;

[0126] c. Tumor volume and tumor weight directly reflect the in vivo therapeutic effect of compound I. The tumor inhibition rate was calculated according to the following formula:

[0127] Inhibition rate (%) = (tumor volume 对照组 -Tumor volume 治疗组 ) / tumor volume 对照组 ×100%

[0128] Changes in animal weight and organ indexes can be used to determine whether there are obvious toxic side effects and show its safety.

[0129] The results are as follows Figure 6 As shown, compound I significantly inhibited the growth of Huh7 xenograft tumors in vivo at medium and high doses, and the volume and weight of the tumors were significantly reduced, with a tumor inhibition rate of 52% at 50 mg / kg and 79% at 100 mg / kg. At the same time, there was no difference in the body weight of mice in the treatment group and the control group, indicating that the treatment at this dose was relatively safe.

[0130] 7. LC-MS study of the hydrolysis of compound II in plasma

[0131] (1) Preparation of reference substance stock solution

[0132] Compound I and Compound II were accurately weighed, dissolved in DMSO, prepared into a stock solution with an initial concentration of 10 mmol / L, and stored at -20°C.

[0133] (2) Sample pretreatment

[0134] a. Plasma collection: Take one SPF nude mouse, collect blood from the heart and place it in an anticoagulant tube. Centrifuge at 3000×g for 10 min at 4°C, collect the upper plasma, and freeze it at -80°C for later use.

[0135] b. Plasma sample processing: Add 1 μL of compound I or compound II to 100 μL of blank plasma, incubate the stock solution at room temperature for 3 hours, and prepare a plasma sample with a mass concentration of 50 μg / mL. Take 100 μL of plasma sample, add 400 μL of methanol, vortex mix for 5 minutes, centrifuge at 12000×g for 10 minutes, centrifuge temperature 4°C, absorb the upper organic layer, freeze-dry, add 100 μL of methanol to dissolve the residue. Centrifuge at 12000×g for 10 minutes, centrifuge temperature 4°C, take the supernatant and filter it through a 0.22μm filter membrane for testing.

[0136] c. On-machine testing

[0137] The treated plasma samples were placed in injection bottles and analyzed using a liquid chromatography-mass spectrometry analyzer (Acquity UPLC H-Class coupled to a SQ Detector 2mass spectrometer (Waters).

[0138] Chromatographic conditions: chromatographic column: ACQUITY UPLC BEH C18 (1.7 mm, 2.1×50 mm); flow rate: 0.50 mL / min; injection volume: 2.00 μL; column temperature: 30° C.; mobile phase A: water + 0.1% formic acid, mobile phase B: acetonitrile, gradient elution, elution program (see Table 1).

[0139] Table 1 ACQUITY UPLC H-Class automatic gradient elution program

[0140]

[0141] Mass spectrometry conditions: MS system: SQ Detector 2; ionization mode: ESI+ / ESI-; capillary voltage: 3000 V; ion source temperature: 150°C; desolvation gas temperature: 500°C; desolvation gas flow rate: 1000 L / Hr; curtain gas flow rate: 50 L / Hr; MassLynx V4.1 software was used to collect and process data.

[0142] The LC-MS results showed that compound I could inhibit the proliferation of liver cancer cells at the cellular level. LC-MS detected the ratio of compound I hydrolyzed to compound II after incubation with plasma, and integrated the peak area under the chromatographic curve, such as Figure 7 As shown, the conversion rate of the process of hydrolyzing acetyl groups to hydroxyl groups was 88.66%, and the proportion of no hydrolysis was 11.01%.

[0143] Table 2. Area integration under the chromatographic curve of compounds

[0144]

[0145] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A compound selected from or a pharmaceutically acceptable salt thereof.

2. Use of the compound according to claim 1 in the preparation of a drug for treating cancer.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition for treating cancer, characterized in that: The pharmaceutical composition comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

5. Use of the compound according to claim 1 in the preparation of PGK1 targeted inhibitory drugs.

6. A PGK1 targeted inhibitory drug, characterized in that: The targeted inhibition drug comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

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