EEF2K inhibitor, preparation thereof and application of eEF2K inhibitor in preparation of antitumor drugs

Abstract

The invention belongs to the field of compound medicines, and particularly relates to an eEF2K inhibitor which is a compound with a structure # imgabs0 # in a formula 1 and a pharmaceutically acceptable crystal or salt thereof. In the formula 1, R1 is O or-NR3, and R2 is a carbon chain of C1 to C20 or an oxa carbon chain; and R3 is H or a carbon chain of C1 to C10. The invention also comprises a preparation method of the eEF2K inhibitor and application of the eEF2K inhibitor in preparation of antitumor drugs. The invention provides a small molecule compound with a structure of a brand new formula 1, and the compound can accidentally have excellent eEF2K inhibitory activity, so that excellent pharmacological activities such as anti-tumor activity can be obtained based on the compound.

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 Ca2+-binding protein encoded by the human eEF2K gene. 2+ / Calmodulin-dependent protein kinase. eEF2K can regulate protein synthesis by regulating peptide chain elongation rate through phosphorylation of eEF2 (Thr 56) or autophosphorylation. Recently, more and more studies have shown that enhanced eEF2K activity can often be observed in many types of tumors, including triple-negative breast cancer, melanoma, etc. Activated eEF2K can inhibit protein synthesis, reduce the consumption of cell nutrition and energy, thereby helping tumor cells resist adverse environments and promote their proliferation. Consistent with this hypothesis, more and more studies have reported that inhibiting the expression or activity of eEF2K will impair tumor cell proliferation. Therefore, eEF2K is considered to be a new potential target for cancer treatment, and the research and development of eEF2K-targeted compounds has important clinical significance. However, there are no drugs targeting eEF2K that can be used in the clinic to treat tumors or other diseases, so the development of such compounds has a very broad research prospect.

[0003] At present, most of the reported eEF2K small molecule inhibitors inhibit its ATP site. Among them, the more representative NH125 has the disadvantage of non-specific inhibition of eEF2K; and although A-484954 has a certain selective inhibitory effect on eEF2K, its anti-tumor effect is poor and it is difficult to cause tumor cell death at an effective concentration. Therefore, it is necessary to find an eEF2K inhibitor that can effectively kill tumor cells. Summary of the invention

[0004] In view of the problem that the existing eEF2K inhibitors have unsatisfactory activity, the first object of the present invention is to provide an eEF2K inhibitor with a completely new 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 is a compound having a 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-C 20 The carbon chain or oxygen heterocarbon chain; R3 is H or C1~C 10 of 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. The present invention shows 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 a compound of formula 2 and a compound of formula 3;

[0017]

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

[0019] In the present invention, the 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 speed and time, which can be adjusted according to conventional 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 based on 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 process. 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 in which R4 is a halogen, and is obtained by acylation reaction of formula 2-B with an acylating agent, such as thionyl chloride, NBS, NCS, NIS, etc.

[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, the halogen is F, Cl, Br or I, and 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 according to conventional control means, for example, 5-8h.

[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 The schematic diagram and statistical diagram of protein bands after adding a1 to a10 at a dose of 10 μM;

[0040] Figure 2 It is a statistical diagram of the effects of a5, a6, and a7 at different doses on the intracellular eEF2K protein content;

[0041] Figure 3 This is a schematic diagram of the effect of a6 at different doses on the intracellular eEF2K protein bands;

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

[0043] Figure 5 The schematic diagram and statistical diagram of the intracellular eEF2K protein bands at different times after elution for 48 hours at a 10 μM dose of a6;

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

[0045] Figure 7 Schematic diagram and statistical diagram 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] Fig. 9 This is a molecular dynamics simulation diagram of the ternary complex of a6, eEF2K and CRBN;

[0048] Fig.10 The solid tumor images and tumor proliferation curves of mice after treatment of BALB / c nude mice with MDA-MB-231 xenograft tumors with DMSO, a6 or VII;

[0049] Fig.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] Fig.12 The results of serum biochemical indexes and H&E staining of important organs after treatment of BALB / c nude mouse model of MDA-MB-231 xenograft tumor with DMSO, a6 or VII;

[0051] Fig.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] Fig.14 Schematic diagram of the structure of the constructed a6@ZIF-8;

[0053] Fig.15 The electron microscope image, energy dispersive X-ray spectrum and X-ray diffraction analysis of a6@ZIF-8;

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

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

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

[0057] Fig.19 Schematic diagram of the eEF2K protein bands in cells treated with a6@ZIF-8 at different doses or for different time periods; DETAILED DESCRIPTION

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

[0059] The present invention provides a novel eEF2K inhibitor of the 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, and the mixture is reacted at 70-100° C. for 5-8 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 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, and after purification by column chromatography, an acid is added, and the reaction is carried out at room temperature of 25-30° C. for 1-3 hours to obtain a 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, and 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; Embodiment 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] Take compound II, and III (1≤n≤9) or V (1≤n≤3) in a molar ratio of 1:1.1, dissolve in an appropriate amount of N, N-dimethylformamide solution, add N, N-diisopropylethylamine (with a molar ratio of 2:1 to compound II), react at 80°C for 6h, dilute with ethyl acetate, wash with water and saturated sodium chloride aqueous solution 3 times each, then dry with anhydrous sodium sulfate and filter, rotary evaporate the filtrate to dryness, and separate the concentrate by silica gel column chromatography (eluent is petroleum ether / ethyl acetate in a volume ratio of 1:1). After obtaining the collected material, dissolve it in a small amount of dichloromethane solution, add an appropriate amount of hydrochloric acid ethyl acetate solution, react at room temperature 25-30°C for 0.5h, filter and dry to obtain the corresponding compound IV or VI.

[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 naturally 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 mixture was reacted at room temperature of 25-30°C for 6 hours. After dilution with dichloromethane solution, it was washed with water and saturated sodium chloride aqueous solution three times respectively, and then 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 (the eluent was dichloromethane / methanol in a volume ratio of 20:1). After the collected material was obtained, it was dissolved in a small amount of dichloromethane solution, and an appropriate amount of hydrochloric acid ethanol solution was added. The mixture was reacted 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 3 times each, and then 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 (the eluent was dichloromethane / methanol in a volume ratio of 20:1). After obtaining the collected material, 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, an off-white solid powder compound IX was obtained.

[0084] Synthesis of compounds X and XI:

[0085] Take compound IX, and IV (1≤n≤9) or VI (1≤n≤3) in a molar ratio of 1:1 and dissolve them in an appropriate amount of N,N-dimethylformamide solution, add 1-hydroxybenzotriazole (molar ratio with compound IX is 1.2:1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (molar ratio with compound IX is 1.2:1) and triethylamine solution (molar ratio with compound IX is 1.2:1), react at room temperature 25-30°C for 12h, dilute with dichloromethane solution, wash with water and saturated sodium chloride aqueous solution three times respectively, then dry with anhydrous sodium sulfate and filter, the filtrate is rotary evaporated to dryness, and the concentrate is separated by silica gel column chromatography (eluent is 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 raw material types of compounds III and V), compounds a1 to a10 can be 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 good condition and a passage number of 12 to 20. The cells were cultured at a rate of 1×10 5 / mL(2×10 4 Pieces / cm 2 ) were seeded at a density of 10 μM in a six-well plate, with each well as a drug intervention group. When the cells grew to a density of 70% to 80%, the serum-free DMEM medium containing 10 μM DMSO solution of compound X or XI (corresponding to the above compounds a1-10 and b1-10, respectively) was replaced in the drug-treated group; the serum-free DMEM medium containing an equal amount of DMSO was added to the control group.

[0112] 2. Cell protein extraction

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

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

[0115] ② Remove the culture medium and rinse the cells in the six-well plate with 1 ml 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 1.5mL 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 Determination of total protein concentration: The total protein concentration was determined using the BCA colorimetric method. Take 2 μL of protein extract and add it to a 96-well plate, dilute it to 20 μL with PBS, prepare and add 200 μL of BCA working solution, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm. Do three replicates for each sample well. Use the protein standard provided in the BCA kit (Thermo, 23225) to draw a standard curve, calculate the average total protein concentration of each sample based on the absorbance values ​​obtained, and unify the total protein loading to calculate the loading volume of each sample.

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

[0124] ③ Electrophoresis: Use 14.4g glycine, 3.03g TRIS base, 1g SDS, and pure water to make up to 1L to prepare electrophoresis solution. Add protein sample or 2μL protein marker to each well in sequence, fill the electrophoresis tank with pre-prepared electrophoresis solution, perform electrophoresis at 80V for 30min, and then at 120V for 50min.

[0125] ④ Transfer: Use 14.4g glycine, 3.03g TRIS base, add pure water to 1L to prepare transfer buffer. Form a sandwich system in Transfer Buffer (Tris-Glycine-SDS-ethanol) and transfer to the transfer tank. Fill the tank with pre-prepared transfer buffer and transfer on ice for 95 minutes at 270mA to transfer the protein blot on the gel to the PVDF membrane.

[0126] ⑤ Blocking and cutting: Place the PVDF membrane with the front side facing down immediately after transfer into an incubation box containing 5% skimmed milk powder, shake it at room temperature for 1 hour, then wash it 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 15-18h. 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, rinse three times with TBST to remove the unbound residual antibody on the membrane.

[0129] ⑧ Band exposure: Prepare 2 mL of luminescent liquid (New Saimei, P10300), evenly add it on the surface of the PVDF membrane, and 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. Two to three independent replicates were performed for each group of results.

[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 had the most significant degradation effect on eEF2K protein, and degraded eEF2K protein in a concentration- and time-dependent manner, and the effect was prolonged after a6 was washed out ( Figure 2-5 ). And at different concentrations, a6's degradation effect on eEF2K was much better than 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 degradation effect on eEF2K protein was significantly inhibited ( Figure 7 The degradation rates of eEF2K protein in MDA-MB-231 cells at the concentration of 10 μM of the above compounds 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] Observe the growth state of MDA-MB-231 cells and select cells in the logarithmic growth phase for the experiment. The cells were digested with trypsin and centrifuged at 800r / min for 5min. Inoculate into 96-well plates, 10,000 cells per well (100μL cell suspension), fill the edge wells with 200μL PBS, and place the 96-well plates in a 37°C, 5% CO2 incubator for 24h.

[0136] (2) Drug incubation

[0137] The solvent of the compound mother solution is DMSO, and the mother solution concentration is 100mM. The mother solution is diluted to the following concentration gradient (0μM, 10μM, 20μM, 40μM, 80μM, 160μM), and 3 parallels are set for each gradient. In addition, a blank group and a control group are set up (only DMEM complete medium is added to the wells of the blank group; and only MDA-MB-231 cells and DMEM complete medium are added to the wells of the control group). The 96-well plate is placed in a 37°C, 5% CO2 incubator and incubated for 72h.

[0138] (3) OD value detection

[0139] The 96-well plate that had been incubated with drugs for 72 h was taken out of 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, then the 96-well plate was taken out and the absorbance at a wavelength of 450 nm was measured using an ELISA 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 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 Receptor Grid Gener component was used to define the binding site, 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 show that a6 can form key forces with E229, Y236, D274 of eEF2K and F77, W79, and Y101 of CRBN ( Figure 8 ); Molecular dynamics simulation results show that compared with the binding of eEF2K and CRBN, the ternary complex formed after the addition of a6 has a higher stability ( Fig. 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 Experimental Animal Science of Central South University. MDA-MB-231 cells (1×10 6 / mouse) was suspended in 100 μL DMEM medium and then injected subcutaneously into the right side of 4-week-old female nude mice. Each group had 6-7 mice. 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 for 14 days of treatment. 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), (4) a6 (20 mg / kg). Tumor volume and mouse body weight were measured every other day.

[0152] Considering that a6 has unusually strong antiproliferative and degradation effects in vitro, we further performed in vivo studies to examine its efficacy in BALB / c nude mice bearing MDA-MB-231 xenograft tumors ( Figure 5A) After the last treatment, mice were sacrificed and dissected, and xenograft tumors were harvested 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 20 mg / kg 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 hepatorenal toxicity. At the therapeutic dose, no obvious drug-related toxicity was observed in the xenograft model with a6, and H&E staining showed no obvious pathological damage to important organs such as the heart, liver, spleen, lung, and kidney ( Fig.12 ), and no animals died during the treatment period in each group. In conclusion, these data indicate that a6 is effective and safe in vivo in xenograft mouse models.

[0154] To elucidate the molecular changes of a6 in tumors in vivo, we then investigated whether a6 could deplete eEF2K protein in xenograft tumor tissues. Immunoblotting analysis showed that a6 at doses of 10 mg / kg and 20 mg / kg could effectively reduce the level of eEF2K protein in MDA-MB-231 tumor tissues, while VII did not cause a significant decrease in eEF2K expression ( Fig.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 10 mM a6 DMSO solution was quickly added to 1 mL of 60 mM 2-methylimidazole solution under vigorous stirring and stirred at room temperature for 5 minutes. Then, 1 mL of 10 mM zinc nitrate hexahydrate aqueous solution was added dropwise and continued to stir at room temperature. After the reaction was completed, the mixed solution was centrifuged at 16000 rpm, the supernatant was discarded, and the precipitate was 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-8NPs were determined by dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano series (Nano ZS, Malvern Instruments). The morphology of a6@ZIF-8NPs 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 The density of cells / mL was placed in a 6-well plate and exposed to the same concentration of a6 or a6@ZIF-8 (1μM, 2μM). The cells were incubated at 37°C for 12h to reach a steady state. Then, the culture medium was discarded, the cells were washed twice with PBS, and then the cells were collected in an EP tube and sonicated at 20wHZ for 2min using a cell sonicator. Finally, 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 the detailed method.

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

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

[0167] Based on the above experiments showing the antitumor effect of a6, we next designed and fabricated a nanocarrier-based system that can deliver a6 to tumor sites ( Fig.14 ). Transmission electron microscopy images show that a6@ZIF-8 nanoparticles have an ordered dodecahedral morphology. Energy dispersive X-ray spectroscopy analysis shows that the nanoparticles contain C, O, N and Zn elements. X-ray diffraction analysis confirms the structural purity of a6@ZIF-8 nanoparticles ( Fig.15 The average particle size of the prepared a6@ZIF-8 nanoparticles is about 191.5 nm, and the zeta potential is -25.5 mV ( Fig.16 HPLC detection found that the cell uptake rate of a6@ZIF-8 was 15-20 times higher than that of a6 at the same concentration ( Fig.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 ( Fig.18a6@ZIF-8 can also cause a significant decrease in the expression of eEF2K in MDA-MB-231 and HCC1806 in a dose- and time-dependent manner, which is significantly higher than that of a6 ( Fig.19 ). The above results show 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 a structure of Formula 1 and a pharmaceutically acceptable crystal or salt thereof; In the formula 1, R1 is O or -NR3, and R2 is C1-C 20 The carbon chain or oxygen heterocarbon chain; R3 is H or C1~C 10 of carbon chain.

2. The eEF2K inhibitor according to claim 1, characterized in that The R1 is NH; The R2 is -(CH2)n-, or -CH2(CH2OCH2)mCH2-, and the n and m are independently integers of 1 to 10; preferably, the n is 5 to 11; further 6 to 10; further 7 to 9.

3. A method for preparing the eEF2K inhibitor according to claim 1 or 2, characterized in that: The compound of formula 2 and the compound of formula 3 are subjected to condensation reaction to obtain; In Formula 2 and Formula 3, R1 and R2 are selected to react in the same manner as Formula 1; and R4 is a hydroxyl group, a halogen group or an ester group.

4. The method for preparing the eEF2K inhibitor according to claim 3, characterized in that: The formula 2 is formula 2-A, which is formula 2 in which R4 is an ester group, and is obtained by substitution reaction of formula 4 and formula 5; The R5 is a halogen, and R6 is a C1-C6 alkyl.

5. The method for preparing the eEF2K inhibitor according to claim 4, characterized in that: The formula 2 is formula 2-B, which is formula 2 in which 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 in which R4 is a halogen, and is obtained by acylation reaction of formula 2-B and an acylating agent.

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

7. Use of the eEF2K inhibitor according to claim 1 or 2 in the preparation of a drug for inhibiting eEF2K.

8. The use according to claim 7, characterized in that The drug for inhibiting eEF2K is an anti-tumor drug; more preferably, it is an anti-breast cancer drug.

9. The use according to claim 7 or 8, characterized in that The eEF2K inhibitor is combined with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable preparation.

10. An anti-tumor drug, characterized in that: Comprising a pharmaceutically effective amount of the eEF2K inhibitor according to claim 1 or 2; Preferably, it further comprises a pharmaceutically acceptable excipient; Preferably, it also has a pharmaceutically acceptable dosage form.

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