Eef2k inhibitors, their preparation and use in the preparation of antitumor drugs

Abstract

The application belongs to the field of compound medicines, and particularly relates to an eEF2K inhibitor, which is a compound with the structure of formula 1 and pharmaceutically acceptable crystals or salts thereof. 20 In the formula 1, R1 is O or -NR3, R2 is a carbon chain or an oxacarbon chain with 1-6 carbon atoms, and R3 is H or a carbon chain with 1-6 carbon atoms. 10 The application further includes a preparation method of the eEF2K inhibitor and an application of the eEF2K inhibitor in preparing an anti-tumor medicine. The application provides a novel small-molecule compound with the structure of formula 1, and the compound unexpectedly has excellent eEF2K inhibiting activity, and can obtain excellent pharmacological activity such as anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to the field of small molecule chemical drugs. Background Art

[0002] Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca-dependent protein encoded by the human eEF2K gene. 2+ / Calmodulin-dependent protein kinase. eEF2K can regulate protein synthesis by regulating peptide chain elongation through phosphorylation of eEF2 (Thr 56) or autophosphorylation. Recently, an increasing number of studies have shown that enhanced eEF2K activity is often observed in many types of tumors, including triple-negative breast cancer and melanoma. Activated eEF2K can inhibit protein synthesis, reduce cellular nutrient and energy consumption, thereby helping tumor cells resist adverse environments and promote their proliferation. Consistent with this hypothesis, an increasing number of studies have reported that inhibiting the expression or activity of eEF2K impairs tumor cell proliferation. Therefore, eEF2K is considered a new potential target for cancer treatment, and the development of eEF2K-targeting compounds has important clinical significance. However, there are no drugs targeting eEF2K that can be used in clinical practice to treat tumors or other diseases. Therefore, the development of such compounds has broad research prospects.

[0003] Currently, most reported small molecule inhibitors of eEF2K inhibit its ATP site. Among them, the more representative NH125 has the disadvantage of non-specific inhibition of eEF2K. While A-484954 has a certain degree of selective inhibition of eEF2K, its anti-tumor effect is poor and it is difficult to cause tumor cell death at effective concentrations. Therefore, it is necessary to discover an eEF2K inhibitor that can effectively kill tumor cells. Summary of the Invention

[0004] In response to the problem that existing eEF2K inhibitors have unsatisfactory activity, the first purpose of the present invention is to provide an eEF2K inhibitor with a novel structure, aiming to significantly improve the eEF2K inhibitory activity.

[0005] The second purpose of the present invention is to provide a method for preparing the eEF2K inhibitor and its use in preparing anti-tumor drugs.

[0006] The third object of the present invention is to provide an anti-tumor drug comprising the eEF2K inhibitor.

[0007] An eEF2K inhibitor, which is a compound having the structure of Formula 1 and a pharmaceutically acceptable crystal or salt thereof;

[0008]

[0009] In the formula 1, R1 is O or -NR3, and R2 is C1 to C 20 carbon chain or oxygen-carbon chain; R3 is H or C1~C 10 carbon chain.

[0010] The present invention provides a novel small molecule compound of formula 1, and the compound unexpectedly has excellent eEF2K inhibitory activity, thereby obtaining excellent anti-tumor and other pharmacological activities.

[0011] In the present invention, the R1 is NH.

[0012] The R2 is -(CH2)n-, or -CH2(CH2OCH2)mCH2-, and the n and m are independently integers of 1 to 10.

[0013] Preferably, n is 5 to 11, further 6 to 10, and further 7 to 9. Studies of the present invention have shown that the combined control of the R2 group and the chain segment can unexpectedly further achieve synergy and further enhance the inhibitory activity of the compound on eEF2K and the anti-tumor activity.

[0014] In the present invention, the eEF2K inhibitor is a compound having a structure of Formula 1-A;

[0015]

[0016] The present invention also provides a method for preparing the eEF2K inhibitor, which is obtained by condensing the compound of formula 2 and the compound of formula 3;

[0017]

[0018] In Formula 2 and Formula 3, R1 and R2 are selected to react in the same manner as in Formula 1; and R4 is a hydroxyl group, a halogen group, or an ester group.

[0019] In the present invention, conventional esterification, amidation, etc. can be used to condense Formula 2 and Formula 3 to obtain the compound of Formula 1.

[0020] In the present invention, the molar ratio of Formula 2 to Formula 3 is 1:0.9-1.5. An acid binding agent may be added during the reaction. There are no special requirements for the reaction time and temperature, which can be adjusted according to conventional in-process control methods.

[0021] In the present invention, the formula 2 is formula 2-A, which is formula 2 in which R4 is an ester group, and is obtained by substitution of formula 4 and formula 5.

[0022]

[0023] The R5 is a halogen, and R6 is a C1-C6 alkyl.

[0024] In the present invention, the substitution coupling of Formula 4 and Formula 5 can be achieved using conventional means. For example, the molar ratio of Formula 4 to Formula 5 is 1:0.9-1.5; an acid binding agent is added during the reaction. The reaction temperature is, for example, room temperature, and the reaction time is, for example, 1-10 hours.

[0025] In the present invention, the halogen is, for example, Br or I.

[0026] In the present invention, the formula 2 is formula 2-B, which is formula 2 in which R4 is a hydroxyl group, and is obtained by acid hydrolysis reaction of formula 2-A.

[0027] In the present invention, the formula 2 is formula 2-C, which is formula 2 wherein R4 is a halogen, and is obtained by acylation reaction of formula 2-B with an acylating agent, such as thionyl chloride, NBS, NCS, or NIS.

[0028]

[0029] The R7 is a halogen, and R8 is a C1-C6 alkyl.

[0030] In the present invention, the condensation of Formula 6 and Formula 7 can be achieved based on conventional processes, conditions, and principles. For example, when the halogen is F, Cl, Br, or I, the molar ratio of Formula 6 to Formula 7 can be 1:0.9-1.5. The reaction temperature can be 70-100°C, and the reaction time can be controlled using conventional in-process control methods, for example, 5-8 hours.

[0031] The present invention also provides a use of the eEF2K inhibitor in the preparation of a drug for inhibiting eEF2K.

[0032] In the application of the present invention, the drug inhibiting eEF2K is an anti-tumor drug; more preferably, it is an anti-breast cancer drug.

[0033] The application of the present invention is to combine the eEF2K inhibitor and a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable preparation.

[0034] The present invention also provides an anti-tumor drug comprising a pharmaceutically effective amount of the eEF2K inhibitor;

[0035] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, such as excipients, fillers, stabilizers, flavoring agents, carriers, and the like.

[0036] Preferably, it also has a pharmaceutically acceptable dosage form.

[0037] Beneficial effects

[0038] The present invention provides a novel small molecule compound of formula 1, and the compound unexpectedly has excellent eEF2K inhibitory activity, thereby obtaining excellent anti-tumor and other pharmacological activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram and statistical graph of protein bands after adding a1 to a10 at a dose of 10 μM;

[0040] Figure 2 Statistical graph showing the effects of a5, a6, and a7 at different doses on the intracellular eEF2K protein content;

[0041] Figure 3 Schematic diagram of the effect of a6 at different doses on the intracellular eEF2K protein bands;

[0042] Figure 4 Schematic diagram and statistical graph of eEF2K protein bands in cells treated with 10 μM dose of a6 for different treatment times;

[0043] Figure 5 Schematic diagram and statistical graph of eEF2K protein bands in cells at different times after elution after 48h treatment with 10μM dose of a6;

[0044] Figure 6 Schematic diagram and statistical graph of the intracellular eEF2K protein bands at different doses of a6 and the positive control (VII);

[0045] Figure 7 Schematic diagram and statistical graph of eEF2K protein bands in cells after adding 10 μM dose of a6 in different pretreatments;

[0046] Figure 8 This is a docking simulation diagram of the ternary complex of a6, eEF2K and CRBN;

[0047] Figure 9 Molecular dynamics simulation diagram of the ternary complex of a6, eEF2K and CRBN;

[0048] Figure 10 Figure 2 shows the solid tumor images and tumor growth curves of BALB / c nude mice bearing MDA-MB-231 xenograft tumors after treatment with DMSO, a6, or VII.

[0049] Figure 11 Comparison of tumor weight and body weight changes of BALB / c nude mice model of MDA-MB-231 xenograft tumor after treatment with DMSO, a6 or VII;

[0050] Figure 12 Serum biochemical parameters and H&E staining results of important organs in BALB / c nude mouse models of MDA-MB-231 xenograft tumors after treatment with DMSO, a6, or VII;

[0051] Figure 13 Western blot results of eEF2K expression in BALB / c nude mice model of MDA-MB-231 xenograft tumor after treatment with DMSO, a6, or VII;

[0052] Figure 14 Schematic diagram of the constructed a6@ZIF-8 structure;

[0053] Figure 15 The electron microscopy image, energy dispersive X-ray spectroscopy and X-ray diffraction analysis of a6@ZIF-8;

[0054] Figure 16 The particle size and potential results of a6@ZIF-8;

[0055] Figure 17 Comparison of cellular uptake rates of a6@ZIF-8 and a6;

[0056] Figure 18 Comparison of the anti-cell proliferation ability of a6@ZIF-8 and a6;

[0057] Figure 19 Schematic diagram of eEF2K protein bands in cells treated with a6@ZIF-8 at different doses or for different times; DETAILED DESCRIPTION

[0058] The specific implementation methods of the present invention are described in detail below with reference to the embodiments.

[0059] The present invention provides a novel eEF2K inhibitor having a structure of Formula 1, which can further have the following compound structure based on the structure of Formula 1-A.

[0060]

[0061] An optional eEF2K inhibitor of the present invention, the preparation method comprises the following steps:

[0062] The compounds represented by Formula II and III are co-dissolved in an organic solvent at a molar ratio of 1:1-1.5 (further 1:1.1-1.3), and then a base is added, reacted at 70-100° C. for 5-8 hours, purified by column chromatography, and then an acid is added, and the reaction is continued at room temperature of 25-30° C. for 1-3 hours to obtain a compound represented by Formula IV;

[0063]

[0064] Among them, 1≤n≤9;

[0065] The compounds represented by Formula II and V are co-dissolved in an organic solvent at a molar ratio of 1:1-1.5 (further 1:1.1-1.3), and then a base is added, reacted at 70-100° C. for 5-8 hours, purified by column chromatography, and then an acid is added, and the reaction is continued at room temperature of 25-30° C. for 1-3 hours to obtain the compound represented by Formula VI;

[0066]

[0067] Wherein, R is H or methyl, 1≤n≤3;

[0068] The compounds represented by Formula VII and VIII are co-dissolved in an organic solvent at a molar ratio of 1:1-1.5 (further 1:1.1-1.3), and then a base is added, and the mixture is reacted at room temperature of 25-30°C for 1-10 hours. After purification by column chromatography, an acid is added, and the mixture is reacted at room temperature of 25-30°C for 1-3 hours to obtain a compound represented by Formula IX;

[0069]

[0070] The compounds represented by Formula IV or VI and IX are co-dissolved in an organic solvent at a molar ratio of 1:1-1.5 (further 1:1.1-1.3), and then HOBT, EDCI and TEA are added. The mixture is reacted at room temperature of 25-30° C. for more than 10 hours to obtain a compound represented by Formula X or XI.

[0071]

[0072] Wherein, 1≤n≤9; Example 1:

[0073]

[0074] Among them, 1≤n≤9;

[0075] In the following cases, as an illustrative scheme, the compound II, the F is substituted at the 4-position.

[0076] Synthesis of compounds IV and VI:

[0077] Compound II and III (1≤n≤9) or V (1≤n≤3) are co-dissolved in an appropriate amount of N,N-dimethylformamide solution at a molar ratio of 1:1.1, and N,N-diisopropylethylamine (molar ratio with compound II is 2:1) is added, and the mixture is reacted at 80°C for 6h. After dilution with ethyl acetate, the mixture is washed with water and saturated sodium chloride aqueous solution three times each, dried over anhydrous sodium sulfate, and filtered. The filtrate is rotary evaporated to dryness to obtain a concentrate, which is separated by silica gel column chromatography (eluent is petroleum ether / ethyl acetate in a volume ratio of 1:1). After obtaining the collected product, it is dissolved in a small amount of dichloromethane solution, an appropriate amount of hydrochloric acid ethyl acetate solution is added, and the mixture is reacted at room temperature of 25-30°C for 0.5h. After filtration and drying, the corresponding compound IV or VI is obtained.

[0078] Synthesis of compound 1 (6-hydroxy-2-oxo-2H-dihydrobenzene-3-carboxylic acid):

[0079] Cycloisopropyl malonate (1 eq, 28.96 mmol) and 2,5-dihydroxybenzaldehyde (1 eq, 28.96 mmol) were dissolved in 50 mL of deionized water, reacted at 75° C. for 3 h, filtered, and air-dried to obtain compound 1 as a yellow powder.

[0080] Synthesis of compound VII:

[0081] Compound 1 (1 eq, 14.55 mmol) and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (1.1 eq, 16.01 mmol) were dissolved in an appropriate amount of N,N-dimethylformamide solution, and 1-hydroxybenzotriazole (1.2 eq, 17.46 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 eq, 17.46 mmol) and triethylamine solution (1.2 eq, 17.46 mmol) were added. The reaction was carried out at room temperature of 25-30°C for 6 hours. After dilution with dichloromethane solution, the mixture was washed with water and saturated sodium chloride aqueous solution three times each, dried over anhydrous sodium sulfate and filtered. The filtrate was rotary evaporated to dryness to obtain a concentrate, which was separated by silica gel column chromatography (eluent: dichloromethane / methanol in a volume ratio of 20:1). After obtaining the collected material, it was dissolved in a small amount of dichloromethane solution, and an appropriate amount of ethanolic hydrochloric acid solution was added. The reaction was carried out at room temperature of 25-30°C for 0.5 hours. After filtration and drying, a slightly yellow solid powder of compound VII was obtained.

[0082] Synthesis of compound IX:

[0083] Compound VII (1 eq, 4.25 mmol) and compound VIII (1.1 eq, 4.68 mmol) were co-dissolved in an appropriate amount of N,N-dimethylformamide solution, potassium carbonate (2.2 eq, 9.35 mmol) was added, and the mixture was reacted at room temperature of 25-30 ° C for 8 h. After dilution with dichloromethane solution, the mixture was washed with water and saturated sodium chloride aqueous solution three times each, dried over anhydrous sodium sulfate, and filtered. The filtrate was rotary evaporated to dryness to obtain a concentrate, which was separated by silica gel column chromatography (eluent: dichloromethane / methanol with a volume ratio of 20:1). After obtaining the collected product, it was dissolved in a small amount of dichloromethane solution, an appropriate amount of hydrochloric acid ethanol solution was added, and the mixture was reacted at room temperature of 25-30 ° C for 0.5 h. After filtration and drying, the off-white solid powder compound IX was obtained.

[0084] Synthesis of compounds X and XI:

[0085] Compound IX and IV (1≤n≤9) or VI (1≤n≤3) are dissolved in an appropriate amount of N,N-dimethylformamide solution at a molar ratio of 1:1. 1-Hydroxybenzotriazole (molar ratio to compound IX of 1.2:1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (molar ratio to compound IX of 1.2:1) and triethylamine solution (molar ratio to compound IX of 1.2:1) are added. The mixture is reacted at room temperature of 25-30°C for 12 hours. After dilution with dichloromethane solution, the mixture is washed with water and saturated sodium chloride aqueous solution three times each, dried over anhydrous sodium sulfate, and filtered. The filtrate is rotary evaporated to dryness to obtain a concentrate, which is separated by silica gel column chromatography (eluent: dichloromethane / methanol in a volume ratio of 15:1) to obtain the corresponding compound X or XI, i.e., the final product.

[0086] By the same method as above, changing the structures of R1 and R2 (changing the types of raw materials of compounds III and V), compounds a1 to a10 were prepared respectively.

[0087] The structural characterization results of the synthesized compounds are as follows:

[0088] 6-hydroxy-2-oxo-N-(2-(piperazin-1-yl)ethyl)-2H-chromene-3-carboxamide (Compound VII). Yellow solid. Yield: 85%.

[0089] 1H NMR(500MHz,DMSO-d6)δ9.95(s,1H),8.99(t,J=6.0Hz,1H),8.79(s,1H),7.36(d,J=8.9Hz,1H),7.30(d,J=2.8Hz,1H),7.24(dd,J=8.9,2.9Hz,1H),3.75(t,J=6.2Hz,2H),3.49(s,6H),3.37(t,J=6.4Hz,4H),2.52(t,J=2.1Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ162.63,160.64,154.82,147.99,147.80,123.01,119.33,119.20,117.49,114.28,55.20,48.53,40.46,40.29,34.46.HRMS(ESI,positive):Calcd.for C 16 H 19 N3O4[M+H]+:m / z=318.1454;Found:318.1452.

[0090] N-(2-(4-(2-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)et hyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-ca rboxamide(a1).Yellowish oil,yield:65%.

[0091] 1H NMR(500MHz,DMSO-d6)δ11.10(s,1H),8.94(t,J=5.5Hz,1H),8.79(s,1H),8.05(t,J=5.8Hz,1H),7.89(s,1H),7.63–7.58(m,2H),7.40–7.37(m,1H),7.31–7.29(m,1H),7.22–7.18(m,1H),7.04(d,J=7.1Hz,1H),6.71(t,J=6.1Hz,1H),5.07(dd,J=12.7,5.4Hz,1H),3.62–3.58(m,1H),3.50(q,J=6.0Hz,2H),3.43(q,J=6.4Hz,2H),3.33(t,J=6.0Hz,2H),3.12(t,J=7.4Hz,1H),2.99(s,2H),2.91–2.85(m,1H),2.67(t,J=6.3Hz,4H),2.63–2.61(m,1H),2.60–2.57(m,1H),2.56–2.54(m,2H),2.04(ddd,J=13.2,5.8,3.4Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ173.29,170.55,169.89,169.18,167.76,161.74,161.02,154.80,148.05,147.78,136.68,132.68,119.43,118.99,117.71,117.50,114.25,111.05,109.71,61.15,56.20,53.73,52.48,52.34,49.00,41.80,38.17,36.31,31.45,22.65.HRMS(ESI,positive):Calcd.for C 33 H 35 N7O9[M+H]+:m / z=674.2575;Found:674.2565.

[0092] N-(2-(4-(2-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)p ropyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-c arboxamide(a2).Yellowish oil,yield:55%.

[0093] 1H NMR(500MHz,DMSO-d6)δ11.11(s,1H),8.95(t,J=5.6Hz,1H),8.79(s,1H),8.00(d,J=44.3Hz,1H),7.70–7.48(m,2H),7.38–7.33(m,1H),7.21(dd,J=9.0,2.9 Hz,1H),7.09(d,J=8.6 Hz,1H),7.03(d,J=7.0 Hz,1H),6.72(t,J=6.2 Hz,1H),5.06(dd,J=12.8,5.4 Hz,1H),3.55(q,J=6.1 Hz,4H),3.32(d,J=6.5 Hz,2H),3.20–3.17(m,2H),2.98–2.88(m,2H),2.83–2.77(m,3H),2.65(d,J=30.0 Hz,4H),2.51(dd,J=26.5,4.4 Hz,4H),2.04(dtd,J=13.1,5.6,5.1,2.3 Hz,1H),1.71(p,J=6.7 Hz,2H). 13 C NMR(126 MHz,DMSO-d6)δ173.30,170.58,169.26,167.76,161.90,160.97,154.78,147.93(d,J=35.0Hz),146.72,136.73,127.36,124.75,122.94,119.44(d,J=6.2 Hz),117.55(d,J=10.8Hz),114.26,110.88–109.61(m),60.60,55.97,51.96,49.00,36.16(d,J=39.4 Hz),31.45,29.26,22.64.HRMS(ESI,positive):Calcd.for C 34 H 37 N7O9[M+H]+:m / z=688.2731;Found:688.2744.

[0094] N-(2-(4-(2-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)b utyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-ca rboxamide(a3)

[0095] N-(2-(4-(2-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)p entyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-c arboxamide(a4).Yellowish oil,yield:53%

[0096] 1 H NMR(500 MHz,DMSO-d6)δ11.10(s,1H),8.93(t,J=5.4 Hz,1H),8.78(s,1H),7.87(d,J=8.4 Hz,1H),7.78(t,J=5.9 Hz,1H),7.59–7.56(m,1H),7.39(ddd,J=8.1,6.8,1.0 Hz,1H),7.36–7.32(m,1H),7.29(d,J=2.9 Hz,1H),7.09(d,J=8.6 Hz,1H),7.02(d,J=7.0 Hz,1H),6.52(t,J=5.9 Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),3.47(d,J=5.8Hz,2H),3.29(d,J=6.7 Hz,2H),2.95(s,2H),2.91–2.86(m,2H),2.62–2.57(m,5H),2.53(d,J=4.2 Hz,8H),2.04(ddd,J=10.5,5.9,3.5Hz,1H),1.58(p,J=7.3 Hz,2H),1.46(p,J=7.2 Hz,2H),1.33(ddd,J=12.0,8.7,5.1 Hz,2H). 13C NMR(126 MHz,DMSO-d6)δ173.26,170.53,169.14,167.76,161.62,161.04,154.85,147.98,143.34,136.76,132.64,128.19,126.25,122.92,119.21,117.45,114.25,110.63,61.39,56.33,52.86,52.53,49.02,42.27,38.52,36.57,31.45,29.34,28.82,24.14,22.64.HRMS(ESI,positive):Calcd.forC36H41N7O9[M+H]+:m / z=716.3044;Found:716.3047.

[0097] N-(2-(4-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)h exyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-ca rboxamide(a5).Yellowish oil,yield:52%

[0098] 1H NMR(400 MHz,DMSO-d6)δ11.10(s,1H),9.95(s,1H),8.93(s,1H),8.80(s,1H),7.66(s,1H),7.57(d,J=4.5 Hz,1H),7.35(d,J=8.9 Hz,1H),7.27(d,J=2.8 Hz,1H),7.18(d,J=5.9 Hz,1H),7.08(d,J=8.6 Hz,1H),7.02(d,J=7.3 Hz,1H),6.52(d,J=6.3Hz,1H),5.05(dd,J=12.9,5.4 Hz,1H),3.47(s,2H),3.41(s,1H),3.37(s,2H),3.29(s,1H),3.09(d,J=6.5 Hz,2H),2.89(s,2H),2.62(t,J=3.4Hz,1H),2.59–2.53(m,2H),2.47(d,J=6.5 Hz,6H),2.31–2.23(m,2H),2.04(ddq,J=8.0,5.5,3.2,2.4 Hz,1H),1.56(d,J=7.3 Hz,2H),1.42(d,J=7.0 Hz,2H),1.31(d,J=8.1 Hz,4H). 13 C NMR(101 MHz,DMSO-d6)δ173.28,173.03,170.51(d,J=10.7 Hz),169.38,167.76,161.44,161.11,154.68,148.01,147.81,146.77(d,J=20.2Hz),136.73,132.65,122.83,119.47,118.98,117.61,114.23,110.84,109.48,61.85,56.56,53.44,52.84,52.05,51.68,49.01,42.23,38.56,36.86,31.45,30.99,29.59,29.09,26.57,22.62.HRMS(ESI,positive):Calcd.forC 37 H 43 N7O9[M+H]+:m / z=730.3201;Found:730.3209.

[0099] N-(2-(4-(2-((8-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)o ctyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-ca rboxamide(a6).Yellowish oil,yield:46%

[0100] 1 H NMR(600 MHz,DMSO-d6)δ11.10(s,1H),8.93(t,J=5.4 Hz,1H),8.78(s,1H),7.65(t,J=3.6 Hz,1H),7.57–7.55(m,1H),7.33(dd,J=9.0,1.7 Hz,1H),7.27(d,J=2.4Hz,1H),7.18(d,J=2.9 Hz,1H),7.08(d,J=8.6 Hz,1H),7.02(d,J=8.7Hz,1H),6.97(d,J=7.0 Hz,1H),6.50(dd,J=19.5,5.8 Hz,1H),5.31(s,1H),5.05(dd,J=12.9,5.4 Hz,1H),4.51(dd,J=10.6,4.5 Hz,1H),3.43(s,1H),3.38(s,1H),3.27(d,J=6.8 Hz,2H),3.07(s,1H),2.88(s,2H),2.62–2.52(m,2H),2.51(d,J=1.9 Hz,2H),2.48(t,J=6.5 Hz,5H),2.45(s,1H),2.38(dt,J=12.8,4.2 Hz,1H),2.30–2.26(m,2H),2.08–1.99(m,1H),1.57(s,1H),1.40(s,1H),1.34–1.32(m,2H),1.29(s,2H),1.25(s,2H),1.24(s,4H). 13C NMR(151 MHz,DMSO-d6)δ173.16(d,J=39.2 Hz),170.51(d,J=18.0 Hz),169.35,168.00(d,J=70.0 Hz),161.29(d,J=48.7 Hz),154.94,148.05,147.71,146.78(d,J=30.5 Hz),136.52(d,J=64.4 Hz),132.86(d,J=66.4 Hz),122.93,119.45,118.92,117.62,117.45,117.05,114.24,110.83,110.52,109.83(d,J=114.2 Hz),61.86,56.57,53.45,52.86,52.46,51.69,42.30,38.57,36.86,30.98,29.62,29.13,26.77,24.07,22.62.Calcd.forC 39 H 47 N7O9[M+H]+:m / z=758.3514;Found:758.3519.

[0101] N-(2-(4-(2-((10-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)decyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chromene-3-c arboxamide(a7).Yellowish oil,yield:50%

[0102] 1HNMR(500 MHz,DMSO-d6)δ11.12(s,1H),8.96(t,J=5.6 Hz,1H),8.79(d,J=3.2Hz,1H),7.90(s,1H),7.61(s,1H),7.44(d,J=1.2 Hz,1H),7.34(d,J=1.1Hz,1H),7.30(d,J=3.0 Hz,1H),7.23(dt,J=9.0,2.3 Hz,1H),7.08(d,J=8.6 Hz,1H),7.02(d,J=7.0Hz,1H),6.52(t,J=5.9 Hz,1H),5.06(dd,J=12.8,5.4 Hz,1H),3.55(t,J=5.9 Hz,2H),3.31–3.27(m,2H),3.08(s,2H),3.04(s,2H),2.93–2.88(m,2H),2.81–2.78(m,3H),2.74(s,1H),2.67(s,3H),2.52(p,J=1.8 Hz,4H),2.08–2.01(m,1H),1.56(p,J=7.0 Hz,2H),1.41(q,J=7.0 Hz,2H),1.35–1.26(m,5H),1.24(s,7H).13C NMR(101 MHz,DMSO-d6)δ173.02,170.46,169.97,169.35,168.23,161.44,161.11,154.75,148.03,147.78,146.67,136.29,133.07,122.86,119.45,118.92,117.48,117.03,114.22,110.51,110.20,61.85,56.57,53.43,52.86,52.05,51.69,49.03(d,J=6.9 Hz),42.30,38.58,36.86,30.98,29.63,29.25,29.19,29.14,26.84,24.08,22.63.Calcd.for C41H51N7O9[M+H]+:m / z=786.3827;Found:786.3835.

[0103] N-(2-(4-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chrom ene-3-carboxamide(a8).Yellowish oil,yield:52%

[0104] 1 H NMR(500 MHz,DMSO-d6)δ11.11(s,1H),8.92(t,J=5.4 Hz,1H),8.77(s,1H),7.89(s,1H),7.59(s,1H),7.40(s,1H),7.36–7.33(m,1H),7.32(d,J=1.2 Hz,1H),7.26(d,J=2.9 Hz,1H),7.15(d,J=8.6 Hz,1H),7.04(d,J=7.0 Hz,1H),6.60(t,J=5.8 Hz,1H),5.06(dd,J=12.8,5.4 Hz,1H),3.62(t,J=5.4 Hz,2H),3.50(d,J=6.0 Hz,2H),3.46(s,1H),3.45(s,1H),3.28(q,J=5.9 Hz,2H),3.19(q,J=7.3 Hz,2H),2.97(s,2H),2.90(s,3H),2.74(s,2H),2.63–2.61(m,6H),2.57(d,J=5.5 Hz,2H),2.04(ddd,J=11.5,6.4,4.1Hz,1H). 13 C NMR(101 MHz,DMSO-d6)δ173.27,170.47(d,J=9.8 Hz),169.74(d,J=3.7Hz),169.41,167.73,161.43,161.11,154.68,148.01,147.81,146.87,136.72,132.52,122.83,119.47,118.96,117.87,117.51,114.22,111.20,109.72,69.36,69.06,61.67,56.51,53.36,52.86,51.70,49.02,42.24,38.47,36.83,31.45,31.00,22.63.Calcd.forC 35 H39 N7O 10 [M+H]+:m / z=718.2837;Found:718.2847.

[0105] N-(2-(4-(2-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)am ino)ethoxy)ethoxy)ethyl)amino)-2-oxoethyl)piperazin-1-yl)ethyl)-6-hydroxy-2-ox o-2H-chromene-3-carboxamide(a9).Yellowish oil,yield:55%

[0106] 1 H NMR(400 MHz,DMSO-d6)δ11.13(s,1H),10.00(s,1H),8.95–8.93(m,1H),8.79(s,1H),7.67(s,1H),7.60–7.56(m,1H),7.33(s,1H),7.27(d,J=2.8 Hz,1H),7.19–7.16(m,1H),7.11(d,J=7.5 Hz,1H),7.02(d,J=6.2 Hz,1H),6.61(d,J=6.2 Hz,1H),5.08(dd,J=12.9,5.4 Hz,1H),3.64(s,2H),3.59–3.57(m,2H),3.55(d,J=2.7 Hz,2H),3.49(s,2H),3.46(d,J=2.1 Hz,2H),3.43–3.41(m,2H),3.28(s,1H),3.19(s,1H),2.90(s,2H),2.52(q,J=1.8 Hz,3H),2.46(t,J=5.9 Hz,9H),2.31–2.28(m,1H),2.06(ddd,J=10.7,5.7,3.0 Hz,1H). 13C NMR(101 MHz,DMSO-d6)δ173.27,173.03,170.54,169.65(d,J=2.2Hz),167.73,161.42,161.10,154.69,148.00,147.79,146.81,136.66,132.52,122.83,119.45,118.90,117.49,114.20,111.14,110.83,109.71,70.26,69.99,69.53,69.33,61.67,56.52,53.37,52.88,51.70,49.03,42.13,36.83,31.46,30.99,24.09,22.62.Calcd.for C 37 H 43 N7O 11 [M+H]+:m / z=762.3099;Found:762.3104.

[0107] N-(2-(4-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-o xo-6,9,12-trioxa-3-azatetradecyl)piperazin-1-yl)ethyl)-6-hydroxy-2-oxo-2H-chro mene-3-carboxamide(a10).Yellowish oil,yield:54%

[0108] 1H NMR (500MHz, DMSO-d6) δ11.11(s,1H),8.94(t,J=5.5Hz,1H),8.77(s,1H),7.96(s,1H),7.88(d,J=8.4Hz,1H),7.58(d,J=7.4Hz,1H),7 .43–7.39(m,1H),7.33–7.30(m,1H),7.24(dd,J=9.0,2.9Hz,1H),7.14(d,J=8.6Hz,1H),7.03(d,J=7.0Hz,1H),6.60(t,J=5.8Hz,1H),5 .06(dd,J=12.8,5.4Hz,1H),3.62(s,2H),3.58–3.56(m,2H),3.55–3.54(m,2H),3.51(d,J=2.3Hz,2H),3.47(s,2H),3.42(d,J=5.9Hz, 2H),3.25(d,J=5.8Hz,2H),2.96(s,2H),2.91(s,2H),2.74(s,2H),2.63–2.59(m,5H),2.54(d,J=4.9Hz,2H),2.49(s,6H),2.04(s,1H). 13 C NMR(101MHz,DMSO-d6)δ173.28,173.03,170.45,169.51,168.22,161.53,161.08,1 54.82,148.01,146.61,143.35,136.25,132.99,128.11,125.74,123.98,122.90,11 9.08,117.47,114.23,110.74,70.32,70.26,70.01,69.42,61.45,56.43,53.06,52. 72,52.08,51.70,49.04(d,J=3.7Hz),42.17,38.55,36.68,30.99,24.08.Calcd.for C 39 H 47 N7O 12 [M+H]+:m / z=806.3361; Found:806.3376.

[0109] Example 1: Verification of the eEF2K degradation function and mechanism of compound X or XI

[0110] 1. Cell Culture and Plating

[0111] The experiment used MDA-MB-231 cell lines with a passage number of 12 to 20 and good condition. 5 / mL(2×10 4 pieces / cm 2 ) were seeded at a density of 10 μM in six-well plates, with each well serving as a drug intervention group. When the cells grew to a density of 70% to 80%, the drug-treated group was replaced with serum-free DMEM medium containing 10 μM DMSO solution of compound X or XI (corresponding to the above-mentioned compounds a1-10 and b1-10, respectively); the control group was supplemented with serum-free DMEM medium containing an equal amount of DMSO.

[0112] 2. Cell protein extraction

[0113] After drug addition, the cells were incubated in a 37°C 5% CO2 cell culture incubator for 48 hours, and then cell protein extraction was performed. All operations were performed on ice, specifically including the following steps:

[0114] ① Preparation of protein lysis buffer: use Western and IP cell lysis buffer (Biyuntian, P0013) + 1% PMSF (Biyuntian, ST505) mixed;

[0115] ② Remove the culture medium and rinse the cells in the six-well plate with 1 ml of PBS;

[0116] ③ Add 120 μL of protein lysate to each well and incubate in a 4°C refrigerator for 30 min;

[0117] ④ Use a cell scraper to scrape off the adherent cells and transfer the cells + lysate to a pre-cooled 1.5 mL EP tube at the bottom;

[0118] ⑤ Centrifuge the EP tube containing the collected cells at 4°C, 13,300 rpm, for 15 min.

[0119] ⑥ After centrifugation, transfer the supernatant (protein extract) to another 1.5 mL EP tube with pre-cooled bottom.

[0120] 3. Sample preparation and Western Blot

[0121] The entire process of sample preparation and Western Blot includes the following steps:

[0122] ① Total protein concentration determination: The total protein concentration was determined using the BCA colorimetric method. 2 μL of protein extract was added to a 96-well plate, diluted to 20 μL with PBS, and 200 μL of BCA working solution was prepared and added. After incubation at 37°C for 30 minutes, the absorbance at 562 nm was measured. Three replicates were performed for each sample well. A standard curve was drawn using the protein standard provided in the BCA kit (Thermo, 23225). The average total protein concentration of each sample was calculated based on the absorbance values ​​obtained. The sample volume for each sample was calculated using the standard total protein loading amount.

[0123] ② Sample preparation: Add 5x Loading Buffer (Thermo, NP0008) and cell extract to the protein sample at a volume ratio of 1:4, mix, and heat in a 100°C metal bath for 10 min.

[0124] ③ Electrophoresis: Prepare the electrophoresis solution using 14.4 g glycine, 3.03 g TRIS base, and 1 g SDS, dilute to 1 L with purified water. Add protein sample or 2 μL of protein marker to each well in sequence. Fill the electrophoresis tank with the prepared electrophoresis solution and run the solution at 80 V for 30 min, followed by 120 V for 50 min.

[0125] ④ Transfer: Prepare transfer buffer by adding 14.4g glycine and 3.03g TRIS base to 1L with purified water. Form a sandwich system in Transfer Buffer (Tris-Glycine-SDS-Ethanol) and transfer to a transfer tank filled with pre-prepared transfer buffer. Transfer the protein blot from the gel to the PVDF membrane on ice at 270mA for 95 minutes.

[0126] ⑤ Blocking and cutting: Place the transferred PVDF membrane face down immediately in an incubation box containing 5% skim milk powder, shake at room temperature for 1 hour, then wash three times with TBST solution, and then cut out the bands at the corresponding positions according to the molecular weight of eEF2K and GAPDH.

[0127] ⑥ Primary antibody incubation: The primary antibody for eEF2K was Human-eEF2K antibody (ABcam, ab45168) diluted 1:1000, and the primary antibody for GAPDH was GAPDH (Servicebio, GB11002) diluted 1:2000. Incubate at 4°C for 15-18 hours. After primary antibody incubation, rinse three times with TBST to remove unbound residual antibodies on the membrane.

[0128] ⑦ Secondary antibody incubation: The secondary antibody was a rabbit secondary antibody diluted 1:5000 and incubated at room temperature for 2 hours. After the secondary antibody incubation, the membrane was rinsed three times with TBST to remove the residual unbound antibody on the membrane.

[0129] ⑧ Band exposure: Prepare 2 mL of luminescent solution (New Saimei, P10300) and evenly add it to the surface of the PVDF membrane. Use the automatic exposure mode for imaging to obtain the corresponding protein bands.

[0130] 4. Band analysis and statistics

[0131] Image processing and band abundance analysis were performed using Image Lab software, and data summary and statistics were performed using GraphPad software. Each group of results was independently repeated two to three times.

[0132] At a dose of 10 μM, compounds a1-a10 had different degrees of degradation effects on eEF2K protein in MDA-MB-231 cells ( Figure 1 In MDA-MB-231 and HCC1806 cells, a6 showed the most significant degradation of eEF2K protein, and degraded eEF2K protein in a concentration- and time-dependent manner, with the effect lasting even after a6 was washed away ( Figure 2-5 ). And at different concentrations, a6's degradation effect on eEF2K was far superior to that of the positive control group (VII) ( Figure 6 ). After pre-treatment of MDA-MB-231 cells with NEDD8 activating enzyme inhibitor MLN4924, proteasome inhibitor MG132, eEF2K ligand (VII) or CRBN E3 ubiquitin ligase ligand Thalidomide for 6 hours, a6 was added and incubated for 48 hours, and its effect on eEF2K protein degradation was significantly inhibited ( Figure 7 The degradation rates of eEF2K protein in MDA-MB-231 cells by the above compounds at a concentration of 10 μM are summarized in Table 1.

[0133] Example 2: CCK8 assay to detect the inhibitory effect of compound X or XI on MDA-MB-231 cell proliferation

[0134] (1)Planning

[0135] MDA-MB-231 cell growth was observed, and cells in the logarithmic growth phase were selected for the experiment. Cells were trypsinized and centrifuged at 800 rpm for 5 minutes. 10,000 cells were seeded into a 96-well plate (100 μL cell suspension) per well. The edge wells were filled with 200 μL of PBS and the 96-well plate was incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0136] (2) Drug incubation

[0137] The compound stock solution was prepared in DMSO at a concentration of 100 mM. The stock solution was diluted to the following concentration gradient (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM), with three replicates per gradient. In addition, blank and control groups were set up (the blank group wells contained only DMEM complete medium; the control group wells contained only MDA-MB-231 cells and DMEM complete medium). The 96-well plates were incubated in a 37°C, 5% CO2 incubator for 72 h.

[0138] (3) OD value detection

[0139] The 96-well plate that had been incubated with drugs for 72 h was removed from the incubator, and 20 μL of CCK8 (5 mg / mL) was added to each well in a biosafety cabinet in the dark. The 96-well plate was placed in the incubator and incubated for 1.5 h. The 96-well plate was then removed and the absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0140] Table 1

[0141]

[0142]

[0143] a:eEF2K degradation rate(Dr) was calculated by quantified westernblots after48h in MDA-MB-231cells.

[0144] Survival rate calculation formula: Survival rate (%) = [(OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%

[0145] The summary results of the inhibition rates of a5, a6, and a7 on MDA-MB-231 and HCC1806 cell proliferation are shown in Table 2.

[0146] Table 2

[0147]

[0148] Example 3: Molecular docking simulation and molecular dynamics simulation of the binding of a6 to the ternary complex of eEF2K and CRBN.

[0149] Using the software Molecular docking simulation and molecular dynamics simulation were performed using software (2021-4, LLC, New York, NY, USA). The protein crystal structure files of eEF2K (PDB code: 8gm4) and CRBN (PDB code: 4v2z) were downloaded through the PDB database (Protein Data Bank https: / / www.rcsb.org / ). The Protein preparation component of the molecular docking software Maestro 13.5 was used to prepare the above files, and the Tnip-tolide files were processed using the LigPrep component; the binding sites were defined using the Receptor Grid Gener component, and the Ligand Docking component was used to perform docking simulations on small molecules and proteins, and Desmond was used for molecular dynamics simulations. The results of molecular docking simulations showed that a6 could form key forces with E229, Y236, D274 of eEF2K and F77, W79, and Y101 of CRBN ( Figure 8 ); Molecular dynamics simulation results show that the ternary complex formed after the addition of a6 has higher stability than the binding of eEF2K and CRBN ( Figure 9 ).

[0150] Example 4: Detection of the efficacy of a6 in BALB / c nude mice bearing MDA-MB-231 xenograft tumors

[0151] All animal experiments were approved by the Department of Laboratory Animal Science of Central South University. 6 The 100 μL DMEM medium was then injected into the right side of 4-week-old female nude mice. Each group consisted of 6-7 mice. The tumor-bearing mice were randomly divided into groups. When the tumor size reached 80-90 mm 3 (Volume = length × width 2 ×π / 6) and mice were randomly divided into designated groups and treated for 14 days. DMSO, compound VII, and a6 were injected intraperitoneally every other day. Mice were randomly divided into designated groups and treated with: (1) vehicle (DMSO), (2) compound VII (20 mg / kg), (3) a6 (10 mg / kg), and (4) a6 (20 mg / kg). Tumor volume and mouse body weight were measured every other day.

[0152] Considering that a6 has an unusually strong anti-proliferative and degradative effect in vitro, we further conducted in vivo studies to examine its efficacy in BALB / c nude mice bearing MDA-MB-231 xenograft tumors ( Figure 5After the last treatment, the mice were sacrificed and dissected, and the xenograft tumors were collected for further analysis. Figure 10-11 As shown, compared with the control group, intraperitoneal injection of a6 can significantly inhibit the growth of MDA-MB-231 tumors. The tumor growth inhibition rate (TGI%) of the 10 mg / kg group was 68.2%, and the TGI% of the 20 mg / kg group was 85.3%, while the eEF2K inhibitor VII had little inhibitory effect on tumor growth.

[0153] At the same time, a6 (10 mg / kg) and a6 (20 mg / kg) were well tolerated by nude mice. We tested a series of serum biochemical markers of hepatotoxicity and renal toxicity. At the therapeutic dose, a6 did not observe obvious drug-related toxicity in the xenograft model, and H&E staining showed no obvious pathological damage to important organs such as the heart, liver, spleen, lungs, and kidneys ( Figure 12 ), and no animals died during the treatment period in any group. In conclusion, these data indicate that a6 is effective and safe in vivo in xenograft mouse models.

[0154] To elucidate the molecular changes induced by a6 in tumors in vivo, we then investigated whether a6 could deplete eEF2K protein in xenograft tumor tissues. Immunoblot analysis showed that a6 at doses of 10 mg / kg and 20 mg / kg effectively reduced eEF2K protein levels in MDA-MB-231 tumor tissues, whereas VII did not cause a significant decrease in eEF2K expression ( Figure 13 ).

[0155] Example 5: Construction of a6@ZIF-8 nanoparticles to enhance the therapeutic effect of a6 against triple-negative breast cancer

[0156] Since a6 has a large molecular weight and poor water solubility, a6@ZIF-8 nanoparticles were constructed to enhance the ability of a6 to penetrate the cell membrane, thereby enhancing the anti-triple-negative breast cancer effect of a6.

[0157] (1) Preparation of a6@ZIF-8 nanoparticles

[0158] First, 10 μL of a 10 mM a6 solution in DMSO was rapidly added to 1 mL of a 60 mM 2-methylimidazole solution under vigorous stirring. The mixture was stirred at room temperature for 5 minutes. Next, 1 mL of a 10 mM zinc nitrate hexahydrate solution was added dropwise, with continued stirring at room temperature. After the reaction was complete, the mixture was centrifuged at 16,000 rpm, the supernatant discarded, and the precipitate redispersed with deionized water to obtain a6@ZIF-8 nanoparticles.

[0159] (2) Characterization of a6@ZIF-8 nanoparticles

[0160] The particle size and potential of a6@ZIF-8 NPs were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano series (Nano ZS, Malvern Instruments). The morphology of a6@ZIF-8 NPs was observed using transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS, Titan G260-300, FEI).

[0161] (3) Determination of cellular uptake of a6@ZIF-8 nanoparticles

[0162] MDA-MB-231 cells were cultured at 10 7 Cells were plated in 6-well plates at a density of 1 μM cells / mL and exposed to the same concentrations of a6 or a6@ZIF-8 (1 μM and 2 μM). The cells were incubated at 37°C for 12 hours to reach a steady state. The culture medium was then discarded, and the cells were washed twice with PBS. The cells were then collected in EP tubes and sonicated at 20 Hz for 2 minutes using a cell sonicator. The supernatant was collected and the intracellular accumulation of a6 was determined by HPLC.

[0163] (4) CCK8 assay to detect the cell proliferation inhibitory effect of a6@ZIF-8 nanoparticles

[0164] Please refer to Example 2 for detailed method.

[0165] (5) Detection of the degradation effect of a6@ZIF-8 nanoparticles on eEF2K

[0166] Please refer to Example 1 for detailed method.

[0167] Based on the above experiments showing the anti-tumor effect of a6, we next designed and fabricated a nanocarrier-based system that can deliver a6 to tumor sites ( Figure 14 Transmission electron microscopy images showed that a6@ZIF-8 nanoparticles had an ordered dodecahedral morphology. Energy dispersive X-ray spectroscopy analysis showed that the nanoparticles contained C, O, N, and Zn elements. X-ray diffraction analysis confirmed the structural purity of a6@ZIF-8 nanoparticles ( Figure 15 The average particle size of the prepared a6@ZIF-8 nanoparticles was about 191.5 nm, and the ζ potential was -25.5 mV ( Figure 16 HPLC analysis revealed that the cellular uptake rate of a6@ZIF-8 was 15-20 times higher than that of a6 at the same concentration ( Figure 17 a6@ZIF-8 has a good anti-proliferative ability against MDA-MB-231 and HCC1806, IC 50 Compared with a6, it is reduced by more than 10 times ( Figure 18a6@ZIF-8 can also cause a significant decrease in eEF2K expression in MDA-MB-231 and HCC1806 in a dose- and time-dependent manner, which is significantly higher than that of a6 ( Figure 19 The above results indicate that a6@ZIF-8 nanoparticles significantly enhance the degradation ability of a6 for eEF2K and its anti-triple-negative breast cancer effect.

Claims

1. An eEF2K inhibitor, characterized in that A compound having the structure of Formula 1 and a pharmaceutically acceptable salt thereof; Formula 1 In the formula 1, R1 is NH; The R2 is -(CH2)n-, wherein n is 5 to 11; Alternatively, the R2 is -CH2(CH2OCH2)mCH2-, and the compound of formula 1 of R2 is a compound having structure a9 or a10; 。 2. The eEF2K inhibitor according to claim 1, wherein The n is 6 to 10.

3. The eEF2K inhibitor according to claim 1, wherein The n is 7-9.

4. A method for preparing the eEF2K inhibitor according to any one of claims 1 to 3, characterized in that: The compound of formula 2 and the compound of formula 3 are subjected to condensation reaction to obtain; Formula 2 Formula 3 In Formula 2 and Formula 3, the ranges of R1 and R2 are the same as those in Formula 1; R4 is hydroxyl, halogen or -OR6; Wherein, R6 is a C1~C6 alkyl group.

5. The method for preparing the eEF2K inhibitor according to claim 4, wherein The formula 2 is formula 2-A, which is formula 2 in which R4 is -OR6, and is obtained by substitution reaction of formula 4 and formula 5; Formula 4 Formula 5 The R5 is a halogen, and R6 is a C1~C6 alkyl.

6. The method for preparing the eEF2K inhibitor according to claim 5, wherein The formula 2 is formula 2-B, which is formula 2 wherein R4 is a hydroxyl group, and is obtained by acid hydrolysis of formula 2-A; The formula 2 is formula 2-C, which is formula 2 wherein R4 is a halogen, and is obtained by acylation reaction of formula 2-B with an acylating agent; the acylating agent is thionyl chloride, NBS, NCS or NIS.

7. The method for preparing the eEF2K inhibitor according to claim 4, wherein: Formula 3 is obtained by substitution and acid hydrolysis of Formula 6 and Formula 7; Formula 6 Formula 7 The R7 is a halogen, and R8 is a C1-C6 alkyl.

8. Use of the eEF2K inhibitor according to any one of claims 1 to 3 in the preparation of a drug for inhibiting eEF2K.

9. The use according to claim 8, characterized in that The drug that inhibits eEF2K is an anti-tumor drug.

10. The use according to claim 9, characterized in that The eEF2K inhibition drug is an anti-breast cancer drug.

11. The use according to any one of claims 8 to 10, characterized in that The eEF2K inhibitor is combined with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable preparation.

12. An anti-tumor drug, characterized in that: Comprising a pharmaceutically effective amount of the eEF2K inhibitor according to any one of claims 1 to 3.

13. The antitumor drug according to claim 12, characterized in that: Also contains pharmaceutically acceptable excipients.

14. The antitumor drug according to claim 13, characterized in that It also has a pharmaceutically acceptable dosage form.

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