Glutamine transport inhibitors, pharmaceutical compositions and uses

Abstract

The application discloses a glutamine transport inhibitor and a pharmaceutical composition and application thereof. The structure of the inhibitor is shown as formula I, and the inhibitor also comprises isomers, pharmaceutically acceptable salts or mixtures thereof. The inhibitor and the pharmaceutical composition thereof have ASCT2 inhibiting activity, can effectively inhibit tumor cell glutamine uptake, and realize the inhibiting effect on tumor cells. The application has wide application and can be prepared into a medicine for treating diseases related to ASCT2, and the medicine can exert the pharmacodynamic effect at the molecular level and the cell level. In addition, the compound preparation method is simple and easy to operate.

Description

Technical Field

[0001] This invention relates to a glutamine transport inhibitor, a pharmaceutical composition, and its applications, particularly to a composition that can be prepared for treating sodium-containing substances. + Glutamine transport inhibitors, pharmaceutical compositions and applications of drugs for diseases related to glutamine carrier 2 dependence. Background Technology

[0002] Tumor cells often rely on glutamine for metabolism. Glutamine enters the cell via the glutamine transporter, where it is hydrolyzed by glutaminase to produce glutamate. Glutamate is further converted into α-ketoglutarate, which enters the tricarboxylic acid cycle to provide energy and macromolecular biosynthetic materials for cancer cells.

[0003] Na + Alanine-Serine-Cysteine ​​Transporter 2 (ASCT2) belongs to the SLC1A membrane protein family and is one of the carriers that specifically transport glutamine on the cell membrane. ASCT2 is often highly expressed in tumors and is closely related to the glutamine energy metabolism pathway in tumors.

[0004] Currently, there are no marketed inhibitors targeting ASCT2. PNA is the first ASCT2 inhibitor, but its inhibitory activity is low (IC50). 50 =~1mM); V9302 subsequently showed good antitumor activity in animal xenogeneic tumor models, however its poor selectivity limited its clinical progress. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of existing compounds, such as efficacy but poor target selectivity, this invention aims to provide a glutamine transport inhibitor, pharmaceutical composition and application with excellent inhibitory effect on tumor cell proliferation.

[0006] Technical solution: As a first aspect of the present invention, the glutamine transport inhibitor of the present invention has the structure of Formula I, and the compound further comprises its isomers, pharmaceutically acceptable salts, or mixtures thereof:

[0007]

[0008] in:

[0009] X and Q are

[0010] R a It is hydrogen or methyl, or multiple R a It forms a ring with the carbon atom it is attached to;

[0011] R 1It can be hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy;

[0012] R 2 For 1-4 hydrogens R 2a Substituted 6-10 aryl or 5-10 heteroaryl;

[0013] R 2a It can be hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy;

[0014] R 3 Halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, 1-4 hydrogen atoms bound by R 3b Substituted 6-10 aryl or 5-10 heteroaryl, or R 3a -L 1 -;

[0015] R 3a -L 1 -for R 3a -[C(R b R c )] 0-5 -、R 3a -[C(R b R c )] 0-2 -O-[C(R b R c )] 0-2 -、R 3a -C≡C-[C(R b R c )] 1-2 -O-、R 3a -C=C-[C(R b R c )] 1-2 -O-、R 3a -[C(R b R c )] 0-2 -C(O)-[C(R b R c )] 0-2 -、R 3a -[C(R b R c )] 0-2 -C(O)NH-[C(R b R c )] 0-2 -、R 3a -[C(Rb R c )] 0-2 -NHC(O)-[C(R b R c )] 0-2 -、R 3a -[C(R b R c )] 0-2 -S(O)2-[C(R b R c )] 0-2 -、R 3a -[C(R b R c )] 0-2 -NHS(O)2-[C(R b R c )] 0-2 -or R 3a -[C(R b R c )] 0-2 -S(O)2NH-[C(R b R c )] 0-2 -;

[0016] R 3a For 1-4 hydrogens R 3c Substituted 6-10 aryl or 5-10 heteroaryl;

[0017] R 3b R 3c It can be hydrogen, halogen, cyano, hydroxyl, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy;

[0018] R b R c It is hydrogen, halogen, C1-C3 alkyl or C3-C6 cycloalkyl, or R b R c It forms a cyclopropyl group with the attached carbon atom;

[0019] m can be 0, 1, 2, 3, or 4;

[0020] n is 0, 1, 2, 3 or 4;

[0021] z is 0, 1, or 2;

[0022] The heteroatoms in the 5-10 aryl groups are N, O, or S, and the number of heteroatoms is 1, 2, 3, or 4.

[0023] Preferably, in the above structure:

[0024] X and Q are

[0025] R a It can be hydrogen or methyl, or two Rs a Form with the attached carbon atom

[0026] R 1 It can be hydrogen, bromine, iodine, methyl, isopropyl, tert-butyl, trifluoromethyl, or methoxy;

[0027] R 2 For 1-4 hydrogens R 2a Substituted phenyl, naphthyl, or pyridyl;

[0028] R 2a It can be hydrogen, fluorine, cyano, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, or isopropoxy;

[0029] R 3 It is chlorine, bromine, iodine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, methoxy, R 3a -CH2O-、R 3a -C≡C-CH2O-、R 3a -C(O)NH- or R 3a -S(O)2NH-, or naphthyl, benzoxazolyl or benzothiazolyl groups with 1-4 hydrogen atoms substituted by hydrogen, fluorine, chlorine, bromine, C1-C4 fluoroalkyl, methoxy groups;

[0030] R 3a For 1-4 hydrogens R 3c Substituted phenyl, naphthyl, pyridyl, benzoxazolyl, or benzothiazolyl;

[0031] R 3c It can be hydrogen, fluorine, chlorine, cyano, hydroxyl, methyl, ethyl, isopropyl, trifluoromethyl, methoxy, ethoxy, or isopropoxy;

[0032] z can be 0, 1, or 2.

[0033] Preferably, in the above structure:

[0034] X is -C(O)-;

[0035] Q is R a It can be hydrogen or methyl;

[0036] R 1 It can be bromine, iodine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl or methoxy;

[0037] R 2 For 1-4 hydrogens R 2a Substituted phenyl or naphthyl groups;

[0038] R 2a It can be hydrogen, fluorine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy;

[0039] R 3 The compounds are bromine, iodine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, methoxy, and R. 3a -CH2O- or R 3a -C≡C-OCH2-, or phenyl or naphthyl groups with 1-4 hydrogens replaced by hydrogen, fluorine, bromine, C1-C4 fluoroalkyl, or methoxy groups;

[0040] R 3a For 1-4 hydrogens R 3c Substituted phenyl or naphthyl groups;

[0041] R 3c It can be hydrogen, fluorine, chlorine, cyano, hydroxyl, methyl, ethyl, isopropyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy;

[0042] z can be 0, 1, or 2.

[0043] Preferably, in the above structure:

[0044] X and Q are

[0045] R a It can be hydrogen or methyl;

[0046] R 1 The compounds are bromine, iodine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, and methoxy.

[0047] R 2 For 1-4 hydrogens R 2a Substituted phenyl or naphthyl groups;

[0048] R 2a It can be hydrogen, fluorine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy;

[0049] R 3 It is bromine, iodine, methyl, isopropyl, trifluoromethyl, methoxy, or R. 3a -C≡C-CH2O-, or a naphthyl group with 1-4 hydrogens replaced by hydrogen, fluorine, bromine, C1-C4 fluoroalkyl, or methoxy groups;

[0050] R 3a For 1-4 hydrogens R 3c Substituted phenyl, naphthyl, pyridyl, benzoxazolyl, or benzothiazolyl;

[0051] R3c It can be hydrogen, fluorine, chlorine, cyano, methyl, ethyl, isopropyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy;

[0052] z is 0 or 1.

[0053] Preferably, the above-mentioned inhibitor has any of the following general formula structures:

[0054]

[0055] Where k and y are 0, 1, 2, 3, or 4; R 1 R 2a R 3 R 3a The definitions of m and n are as described above.

[0056] More preferably, the inhibitor is selected from any of the following compounds:

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] The pharmaceutically acceptable salt of the above inhibitor is a salt formed by the above compound with an acid or base, wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid, and the base is an inorganic base containing a basic metal cation, an alkaline earth metal cation, or an ammonium cation salt.

[0063] The aforementioned inhibitors, along with pharmaceutically acceptable carriers, form a pharmaceutical composition to produce common pharmaceutical preparations such as tablets, capsules, syrups, suspensions, or injections. Common pharmaceutical excipients such as flavorings, sweeteners, liquid / solid fillers, and diluents may be added to the preparation.

[0064] The above-mentioned inhibitors and their pharmaceutical compositions can be prepared as treatments for Na+ and Na+. + Drugs for diseases related to glutamine carrier 2 dependence, specifically for the treatment of cancer or viral infections.

[0065] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0066] (1) This class of glutamine transport inhibitors and their drug compositions have ASCT2 inhibitory activity, which can effectively inhibit glutamine uptake by tumor cells, thereby inhibiting tumor cells and IC50. 50 The optimal value is less than 10 μM;

[0067] (2) These inhibitors and their drug compositions have wide applications and can be prepared as drugs for treating diseases related to ASCT2; the drugs can exert their effects at both the molecular and cellular levels, reaching molar concentration levels;

[0068] (3) The compound preparation method is simple and easy to operate. Detailed Implementation

[0069] The technical solution of the present invention will be further described below with reference to the embodiments.

[0070] Example 1: Compound L-1

[0071]

[0072] Step 1: Dissolve salicylaldehyde (2.00 g, 16.38 mmol) in 30 mL of acetone, add 3-bromopropyne (1.95 g, 16.38 mmol) and potassium carbonate (4.53 g, 32.76 mmol), heat to 60 °C and react for 1.5 hours. After the reaction is complete, cool to room temperature, filter to remove potassium carbonate, remove acetone under reduced pressure, and then beat with isopropyl ether at low temperature to obtain 2.42 g of white solid, yield 92%. 1 H NMR (300MHz, CDCl3): δ10.49 (s, 1H), 7.87 (dd, J = 7.5Hz, 1H), 7.58 (td, 1H), 7.07-7.14 (m, 2H), 4.84 (d, J = 2.4Hz, 2H), 2.58 (t, J = 2.4Hz, 1H) ppm.

[0073] Step 2: Dissolve 2-((propyl-2-ynyl-1-oxy)benzaldehyde (2.00 g, 12.50 mmol) in 30 mL of tetrahydrofuran, add iodobenzene (2.55 g, 12.50 mmol), triethylamine (6.4 g, 62.48 mmol), cuprous iodide (0.24 g, 1.25 mmol), and Pd(PPh3)4 (0.58 g, 0.50 mmol). Under nitrogen protection, the reaction was carried out at 60 °C for 3 hours. After cooling to room temperature, the reaction solution was poured into 40 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solution was removed under reduced pressure. The solution was separated by column chromatography (petroleum ether: ethyl acetate = 50:1) to obtain a white solid (2.07 g, 70%). 1H NMR (300MHz, DMSO-d6) δ10.41 (s, 1H), 7.80–7.66 (m, 2H), 7.51–7.33 (m, 6H), 7.16 (t, J = 7.5Hz, 1H), 5.26 (s, 2H) ppm.

[0074] Step 3: Dissolve (S)-2,4-diaminobutyric acid (0.2 g, 0.92 mmol) in 5 mL of methanol, add 2-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (0.54 g, 2.29 mmol), stir at room temperature for half an hour, then add sodium cyanoborohydride (0.2 g, 3.21 mmol), stir at room temperature for 12 hours, remove the solvent under reduced pressure, and separate by column chromatography (dichloromethane:methanol = 30:1) to obtain a white solid (0.31 g, 51.35%). HRMS (ESI): m / z [M+H] + .C 41 H 43 The calculated value of N2O6 is 659.3116; the measured value is 659.3127.

[0075] Step 4: Dissolve (S)-4-(bis(2-((((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.24 g, 0.47 mmol) in 10 mL of 1,4-dioxane, then add 8 mL of 4 mol / L hydrochloric acid, heat to 45 °C and react for 3 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.12 g, 60%). 1 H NMR (300MHz, DMSO-d6): δ7.45(t,J=7.6Hz,4H),7.35-7.29(m,10H),7.23(d,J=8.5Hz,2H),7.02(t,J=7.3 Hz,2H),5.02(s,4H),4.30(m,4H),3.70(s,1H),3.26(s,2H),2.39–2.15(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 38 The calculated value of N2O4 is 559.2591; the measured value is 559.2564.

[0076] The compounds described in Examples 2-14 can be prepared by referring to the method and route described in Example 1.

[0077] Example 2: Compound L-2

[0078]

[0079] 1 H NMR (300MHz, DMSO-d6): δ7.49–7.37(m,4H),7.24(dd,J=15.3,7.0Hz,6H),7.19–7.07(m,4H),7.01(t,J=7.3Hz ,2H),5.08(s,4H),4.15(m,4H),3.23(s,1H),3.14(s,2H),2.17(s,6H),1.98(s,1H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 38 Calculated value of N2O4: 587.2904; Measured value: 587.2912.

[0080] Example 3: Compound L-3

[0081]

[0082] 1 H NMR (300MHz, DMSO-d6): δ7.36–7.31(m,4H),7.29–7.23(m,2H),7.19(td,J=7.7,1.5Hz,2H),7.15-7.10(m,4H),6.97–6.89(m, 4H),4.85(s,4H),3.77–3.69(m,4H),3.66(s,1H),3.42-3.38(m,2H),2.21(s,6H),2.01-1.96(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 38 The calculated value of N2O4 is 587.2904; the measured value is 587.2923.

[0083] Example 4: Compound L-4

[0084]

[0085] 1 H NMR (300MHz, DMSO-d6): δ7.49–7.39(m,2H),7.35–7.26(m,10H),7.05–6.97(m,4H),5.01(s,4H),4. 23(m,4H),3.60(s,1H),3.56-3.45(m,2H),2.38(s,6H),1.93–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H38 The calculated value of N2O4 is 587.2904; the measured value is 587.2902.

[0086] Example 5: Compound L-5

[0087]

[0088] 1 H NMR (300MHz, DMSO-d6): δ7.31(t,J=6.9Hz,2H),7.28–7.20(m,4H),7.18(dd,J=7.8,1.6Hz,2H),7.14(d,J=4.1Hz,2H),6.99–6 .89(m,6H),4.85(s,4H),3.82(s,6H),3.76–3.69(m,4H),3.66(s,2H),3.55(s,1H)1.93–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 38 The calculated value of N2O6 is 619.2803; the measured value is 619.2822.

[0089] Example 6: Compound L-6

[0090]

[0091] 1 H NMR (300MHz, DMSO-d6): δ7.53–7.38(m,4H),7.24(t,J=8.1Hz,4H),7.02(t,J=7.4Hz,2H),6.98–6.85(m,6H),5.0 5(s,4H),4.33–4.11(m,4H),3.69(s,6H),3.19(s,2H),3.15(s,1H),2.32–2.10(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 38 The calculated value of N2O6 is 619.2803; the measured value is 619.2809.

[0092] Example 7: Compound L-7

[0093]

[0094] 1H NMR (300MHz, DMSO-d6): δ7.40(d,J=6.7,2H),7.24(m,4H),7.24–7.12(m,6H),6.97–6.89(m,4H), 5.02(s,4H),4.01(s,4H),3.23(s,1H),3.15(s,2H),1.93–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 33 The calculated value of F2N2O4 is 595.2403; the measured value is 595.2408.

[0095] Example 8: Compound L-8

[0096]

[0097] 1 H NMR (300MHz, DMSO-d6): δ7.43–7.35(m,4H),7.01(d,J=2.1Hz,4H),6.97–6.89(m,6H),5.00(s,4H),4.12–4.0 8(m,4H),3.66(d,J=5.0Hz,1H),2.85-2.76(m,2H),2.22(s,12H)1.93–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 40 H 43 The calculated value of N2O4 is 615.3217; the measured value is 615.3225.

[0098] Example 9: Compound L-9

[0099]

[0100] 1 H NMR (300MHz, DMSO-d6): δ7.44–7.33(m,8H),7.29–7.23(m,2H),7.19(td,J=7.7,1.5Hz,2H),6.97–6.89(m,4H),4.9 5(s,4H),3.87–3.80(m,4H),3.70(s,1H),3.21(s,2H)1.93–1.81(m,2H),1.03(s,18H)ppm.HRMS(ESI):m / z[M+H]+.C 44 H 51 The calculated value of N2O4 is 671.3843; the measured value is 671.3859.

[0101] Example 10: Compound L-10

[0102]

[0103] 1 H NMR (300MHz, DMSO-d6): δ7.70–7.64(m,4H),7.63–7.57(m,4H),7.29–7.23(m,2H),7.19(td,J=7.7,1.5Hz,2H),6.97– 6.89(m,4H),4.89(s,4H),3.79(s,4H),3.80(s,1H)3.67-3.60(m,2H),2.01-1.95(m,2H)ppm.HRMS(ESI):m / z[M+H]+.C 38 H 33 The calculated value of F6N2O4 is 695.2339; the measured value is 695.2358.

[0104] Example 11: Compound L-11

[0105]

[0106] 1 H NMR (300MHz, DMSO-d6): δ7.90-7.67(m,6H),7.49(t,J=5.7Hz,2H),7.29–7.19(m,4H),6.97–6.89 (m,4H),5.01(s,4H),3.79(s,1H),3.69-3.58(m,2H),2.05–1.93(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 28 H 33 The calculated value of N4O4 is 609.2496; the measured value is 609.2458.

[0107] Example 12: Compound L-12

[0108]

[0109] 1H NMR (300MHz, DMSO-d6): δ8.62(d,J=4.5Hz,4H),7.91(s,2H),7.58(s,2H),7.47(dd,J=12.4,5.0Hz,4H),7.28(d,J=8.3Hz,2H),7 .05(t,J=7.4Hz,2H),5.13(s,4H),4.46–4.29(m,4H),4.02(s,1H),3.30(d,J=8.0Hz,2H),3.15(s,2H)ppm.HRMS(ESI):m / z[M+H] + .C 34 H 33 The calculated value of N4O4 is 561.2496; the measured value is 561.2485.

[0110] Example 13: Compound L-13

[0111]

[0112] 1 H NMR (300MHz, DMSO-d6): δ8.40(d,J=4.8Hz,2H),7.78(s,2H),7.33–7.23(m,4H),7.19–6.89(m,6H),5.10(s, 4H),3.99(s,6H),3.77–3.70(m,4H),3.68(s,1H),3.50(s,2H),2.30–2.14(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 37 The calculated value of N4O6 is 621.2708; the measured value is 621.2755.

[0113] Example 14: Compound L-14

[0114]

[0115] Step 1: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.15 g, 0.68 mmol) in 5 mL of methanol, add 2-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (0.20 g, 0.83 mmol), stir at room temperature for half an hour, then add sodium cyanoborohydride (65 mg, 1.03 mmol), continue stirring at room temperature for 1.5 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 15:1) to obtain a white solid (0.24 g, 79.63%). HRMS (ESI): m / z [M+H] + .C 25 H30 The calculated value of N2O5 is 438.2155; the measured value is 438.2159.

[0116] Step 2: Dissolve (S)-2-((tert-butoxycarbonyl)amino)-4-((2-(((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)butyric acid (0.29 g, 0.66 mmol) in 8 mL of methanol, add 2-((3-(pyridin-4-yl)prop-2-yn-1-yl)oxy)benzaldehyde (0.18 g, 0.79 mmol), stir at room temperature for half an hour, then add sodium cyanoborohydride (87 mg, 1.37 mmol), stir at room temperature for 12 hours, remove the solvent under reduced pressure, and separate by column chromatography (dichloromethane:methanol = 30:1) to obtain a white solid (0.23 g, 52.71%). HRMS(ESI): m / z [M+H] + .C 41 H 43 The calculated value of N2O6 is 660.3068; the measured value is 660.3054.

[0117] Step 3: Dissolve (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)(2-((3-(pyridin-4-yl)prop-2-en-1-yl)oxy)benzyl)amino)butyric acid (0.22 g, 0.33 mmol) in 3 mL of 1,4-dioxane, then add 5 mL of 4 mol / L hydrochloric acid, react at room temperature for 4 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.12 g, 60%). 1 HNMR (300MHz, DMSO-d6): δ8.61–8.56(m,2H),7.43–7.31(m,7H),7.26-7.10(m,4H),6.97–6.89( m,4H),5.07(s,4H),3.89–3.75(m,5H),3.56(s,2H),2.05-1.98(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 34 The calculated value of N3O4 is 560.2544; the measured value is 560.2564.

[0118] The compounds described in Examples 15 and 16 can be prepared by referring to the method and route described in Example 14.

[0119] Example 15: Compound L-15

[0120]

[0121] 1 H NMR (300MHz, DMSO-d6): δ7.51–7.47(m,5H),7.32–7.27(m,2H),7.19(td,J=7.8,1.6Hz,2H),7.03(d,J=2.1Hz,2H),6.97–6.8 9(m,5H),5.03(s,4H),4.03-3.86(m,4H),3.70(s,1H),3.66(s,2H),2.23(s,6H),2.02–1.95(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 39 Calculated value of N2O4: 587.2904; Measured value: 587.2948.

[0122] Example 16: Compound L-16

[0123]

[0124] 1 H NMR (300MHz, DMSO-d6): δ7.90(s,1H),7.73-7.68(m,1H),7.61–7.37(m,7H),7.26(d,J=8.1,2H),7.19(td,J=7.8,1.6Hz,2H ),6.97–6.89(m,4H),5.01(s,4H),4.20-4.13(m,4H),3.75(s,1H),3.56(s,2H),1.94–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 37 H 34 The calculated value of N3O4 is 584.2544; the measured value is 584.2547.

[0125] Example 17: Compound L-17

[0126]

[0127] Step 1: Dissolve 2-hydroxyacetophenone (2.00 g, 14.69 mmol) in 30 mL of acetone, add 3-bromopropyne (2.62 g, 22.03 mmol) and potassium carbonate (5.08 g, 36.72 mmol), heat to 60 °C and react for 3 hours. After the reaction is complete, cool to room temperature, remove potassium carbonate by filtration, remove acetone under reduced pressure, and perform column chromatography (petroleum ether: ethyl acetate = 20:1) to give a white solid (2.03 g, 79.33%). 1H NMR(300MHz,Chloroform-d)δ7.76(s,1H),7.41(td,J=7.8,1.5Hz,1H),7.15(dd,J=8.0 ,1.2Hz,1H),7.00(s,1H),4.84(d,J=3.0Hz,2H),2.59(s,3H),2.55(t,J=3.0Hz,1H)ppm.

[0128] Step 2: Dissolve 2-((propyl-2-yn-1-oxy)acetophenone (2.00 g, 11.48 mmol) in 30 mL of tetrahydrofuran, add iodobenzene (2.13 g, 10.44 mmol), triethylamine (5.28 g, 52.19 mmol), cuprous iodide (0.20 g, 1.04 mmol), and Pd(PPh3)4 (0.60 g, 0.52 mmol). Under nitrogen protection, react at 60 °C for 3 hours. Cool to room temperature, pour the reaction solution into 40 mL of water, extract with ethyl acetate (50 mL × 3), dry with anhydrous sodium sulfate, remove the solution under reduced pressure, and separate by column chromatography (petroleum ether:ethyl acetate = 50:1) to obtain a white solid (1.70 g, 65.07%). 1 H NMR(300MHz,Chloroform-d)δ7.76(dd,J=7.9,1.6Hz,1H),7.47–7.33(m,6H),7.15 (dd,J=8.0,1.2Hz,1H),7.00(td,J=7.8,1.2Hz,1H),4.87(s,2H),2.59(s,3H)ppm.

[0129] Step 3: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.92 mmol) in 5 mL of methanol, add 2-((3-phenylprop-2-yn-1-yl)oxy)acetophenone (0.57 g, 2.29 mmol), stir at room temperature for one hour, then add sodium cyanoborohydride (0.2 g, 3.21 mmol), continue stirring at room temperature for 8 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 20:1) to obtain a white solid (0.31 g, 49.18%). HRMS (ESI): m / z [M+H] + .C 43 H 47 The calculated value of N2O6 is 687.3429; the measured value is 687.3450.

[0130] Step 4: Dissolve (S)-4-(bis(2-((((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.20 g, 0.30 mmol) in 1,4-dioxane (5 mL), then add 5 mL of 4 mol / L hydrochloric acid, stir at room temperature for 6 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.12 g, 70.24%). 1 H NMR (300MHz, DMSO-d6): δ7.43–7.34(m,10H),7.27–7.19(m,4H),7.04–6.94(m,4H),5.16(s,4H),3.7 7-3.66(m,3H),2.87-2.76(m,2H),2.00-1.92(m,2H),1.28(d,J=6.6Hz,6H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 39 Calculated value of N2O4: 587.2904; Measured value: 587.2952.

[0131] Example 18: Compound L-18

[0132]

[0133] Step 1: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.15 g, 0.69 mmol) in 5 mL of methanol, add 2-((3-phenylprop-2-yn-1-yl)oxy)acetophenone (0.20 g, 0.82 mmol), stir at room temperature for one hour, then add sodium cyanoborohydride (64 mg, 1.03 mmol), continue stirring at room temperature for 8 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 15:1) to obtain a white solid (0.24 g, 79.63%). HRMS (ESI): m / z [M+H] + .C 26 H 33 The calculated value of N2O5 is 453.2384; the measured value is 453.2376.

[0134] Step 2: (S)-2-((tert-Butoxycarbonyl)amino)-4-((1-(2-(((3-phenylprop-2-yn-1-yl)oxy)phenyl)ethylamino)butyric acid (0.20 g, 0.44 mmol) was dissolved in 8 mL of methanol. 2-((3-phenylprop-2-yn-1-yl)oxy)acetophenone (0.125 g, 0.53 mmol) was added, and the mixture was stirred at room temperature for half an hour. Then, sodium cyanoborohydride (42 mg, 0.66 mmol) was added, and the mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the solid was separated by column chromatography (dichloromethane:methanol = 30:1) to give a white solid (0.21 g, 70.62%). HRMS (ESI): m / z [M+H] + .C 42 H 45 The calculated value of N2O6 is 673.3272; the measured value is 673.3258.

[0135] Step 3: Dissolve (2S)-4-(bis(1-(2-((3-phenylprop-2-yn-1-yl)oxy)phenyl)ethyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.20 g, 0.29 mmol) in 5 mL of 1,4-dioxane, then add 5 mL of 4 mol / L hydrochloric acid, react at room temperature for 6 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.12 g, 70.24%). 1 H NMR (300MHz, DMSO-d6): δ7.43–7.34(m,10H),7.27–7.16(m,4H),7.04–6.89(m,4H),5.13(s,4H),3.89–3 .75(m,3H),3.70(s,1H),2.81(s,2H),2.15-1.97(m,2H),1.41(d,J=6.8Hz,3H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 39 The calculated value of N2O4 is 587.2904; the measured value is 587.2911.

[0136] Example 19: Compound L-19

[0137]

[0138] Step 1: CuI (15 mg, 81 μmol), triethylamine (3 mL), 2-chlorobenzo[d]oxazole (250 mg, 1.63 mmol), Pd(PPh3)2Cl2 (57 mg, 81 μmol), triphenylphosphine (98 mg, 293 μmol), and 2-((propyl-2-ynyl-1-oxy)benzaldehyde (250 mg, 1.92 mmol) were added to 5 mL of N,N-dimethylformamide. The mixture was heated to 120°C and reacted for 1 hour. The reaction was confirmed to be complete by TLC. After cooling to room temperature, the mixture was extracted with ethyl acetate and water. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude residue was purified by silica gel column chromatography (cyclohexane:ethyl acetate = 60:1) to obtain an orange oil (163 mg, 40%). 1 H NMR(300MHz,Chloroform-d)δ7.88–7.79(m,1H),7.76–7.53(m,2H),7.48–7.31( m,3H),7.17(dd,J=8.1,1.2Hz,1H),7.09(td,J=7.7,1.2Hz,1H),4.88(s,2H)ppm.

[0139] Step 2: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.92 mmol) in 5 mL of methanol, add 2-((3-(benzo[d]oxazol-2-yl)prop-2-yn-1-yl)oxy)benzaldehyde (0.635 g, 2.29 mmol), stir at room temperature for one hour, then add sodium cyanoborohydride (0.2 g, 3.21 mmol), continue stirring at room temperature for 8 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 20:1) to obtain a yellow solid (0.25 g, 36.83%). HRMS (ESI): m / z [M+H] + .C 43 H 41 The calculated value of N4O8 is 741.2919, and the measured value is 741.2978.

[0140] Step 3: Dissolve (S)-4-(bis(2-((3-(benzo[d]oxazol-2-yl)prop-2-yn-1-yl)oxy)benzylamino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.27 mmol) in 5 mL of dioxane, add 5 mL of 4 mol / L hydrochloric acid, react at room temperature for 6 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a yellow solid (0.11 g, 63.59%).

[0141] 1H NMR (300MHz, DMSO-d6): δ7.72–7.61(m,4H),7.44–7.34(m,4H),7.29–7.23(m,2H),7.19(td,J=7.7,1.5Hz,2H),6.97–6. 89(m,4H),4.99(s,4H),4.01-3.95(m,4H),3.77(s,1H),3.69–3.63(m,2H),1.93–1.81(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 33 The calculated value of N4O6 is 641.2395; the measured value is 641.2376.

[0142] Example 20: Compound L-20

[0143]

[0144] Step 1: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.92 mmol) in 5 mL of methanol, add 2-((3-(benzo[d]oxazol-2-yl)prop-2-yn-1-yl)oxy)benzaldehyde (0.304 g, 1.10 mmol), stir at room temperature for one hour, then add sodium cyanoborohydride (86 mg, 1.37 mmol), continue stirring at room temperature for 8 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 10:1) to obtain a yellow solid (0.38 g, 86.48%). HRMS (ESI): m / z [M+H] + .C 26 H 30 The calculated value of N3O6 is 480.2129; the measured value is 480.2146.

[0145] Step 2: (S)-4-((2-((3-(benzo[d]oxazol-2-yl)prop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.20 g, 0.54 mmol) was dissolved in 5 mL of methanol. 2-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (0.13 g, 0.53 mmol) was added, and the mixture was stirred at room temperature for half an hour. Then, sodium cyanoborohydride (52 mg, 0.83 mmol) was added, and the mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure, and the solid was separated by column chromatography (dichloromethane:methanol = 30:1) to give a white solid (0.12 g, 41.06%). HRMS (ESI): m / z [M+H] + .C 42 H 42Calculated value of N3O7: 700.3017; Measured value: 700.3023.

[0146] Step 3: Dissolve (S)-4-((2-((3-(benzo[d]oxazol-2-yl)prop-2-yn-1-yl)oxy)benzyl)(2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.1 g, 0.14 mmol) in 5 mL of 1,4-dioxane, then add 5 mL of 4 mol / L hydrochloric acid, stir at room temperature for 6 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a yellow solid (50 mg, 58.35%). 1 H NMR (300MHz, DMSO-d6): δ7.72–7.39(m,9H),7.29–7.19(m,4H),6.97–6.89(m,4H),5.16(s,4 H),3.95-3.89(m,4H),3.70(s,1H),3.66(s,2H),2.03-1.90(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 42 H 42 The calculated value of N3O7 is 600.2493; the measured value is 600.2487.

[0147] The compounds described in Examples 21-23 can be prepared by referring to the method and route described in Example 14.

[0148] Example 21: Compound L-21

[0149]

[0150] 1 H NMR (300MHz, DMSO-d6): δ7.82–7.42(m,9H),7.24–7.13(m,4H),6.97–6.89(m,4H),5.11(s,4H),4.0 2-3.95(m,4H),3.79(s,1H),3.66(s,2H),2.00-1.79(m,2H),1.35(s,3H)ppm.HRMS(ESI):m / z[M+H] + .C 28 H 36 The calculated value of N3O5 is 614.2649; the measured value is 614.2623.

[0151] Example 22: Compound L-22

[0152]

[0153] 1 H NMR (300MHz, DMSO-d6): δ7.72–7.56(m,2H),7.44–7.34(m,7H),7.29–7.16(m,3H),7.02(s,1H),6.97–6.85(m,3H),5.0 5(s,4H),4.32–4.13(m,4H),3.81(s,1H),3.63–3.58(m,2H),2.27(s,3H),1.94–1.73(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 28 H 36 The calculated value of N3O5 is 614.2649; the measured value is 614.2627.

[0154] Example 23: Compound L-23

[0155]

[0156] 1 H NMR (300MHz, DMSO-d6): δ7.86–7.79(m,2H),7.44–7.23(m,9H),7.23–6.92(m,4H),6.81(d,J=7.6Hz,1H) ,5.08(s,4H),3.89–3.72(m,5H),3.54(s,4H),2.11-1.84(m,2H),1.31(s,9H)ppm.HRMS(ESI):m / z[M+H] + .C 41 H 42 The calculated value of N3O5 is 656.3119; the measured value is 656.3137.

[0157] Example 24: Compound L-24

[0158]

[0159] Step 1: Dissolve 5-methylsalicylaldehyde (2.0 g, 14.64 mmol) in 30 mL of acetone, then add potassium carbonate (4.06 g, 29.38 mmol) and bromopropyne (2.62 g, 22.03 mmol) sequentially. Stir and heat to reflux for 2 h. Confirm the reaction is complete by TLC. Cool to room temperature, filter, wash the filter cake with acetone, and evaporate the filtrate to dryness. After evaporation, slurry the solid with an appropriate amount of isopropyl ether to give a pale yellow solid (2.12 g, 82.85%). 1H NMR (300MHz, Chloroform-d) δ10.07(s,1H),7.77–7.70(m,1H),7.24–7.08(m,2H),5.06(s,2H),2.55(s,1H),2.39(s,3H)ppm.

[0160] Step 2: Iodobenzene (1.7 g, 8.61 mmol), triethylamine (4.36 g, 43.05 mmol), cuprous iodide (164 mg, 0.86 mmol), and Pd(PPh3)4 (498 mg, 0.43 mmol) were dissolved in 15 mL of tetrahydrofuran under nitrogen protection and heated to 60 °C. 5-Methyl-2-((propyl-2-yn-1-oxy)benzaldehyde (1.5 g, 8.61 mmol) was dissolved in 5 mL of tetrahydrofuran and added dropwise to the reaction mixture. After complete addition, the reaction was carried out for 3 hours. The reaction was confirmed to be complete by TLC. The mixture was cooled to room temperature, poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the solution was removed under reduced pressure. The solution was separated by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1) to obtain a pale yellow solid (1.73 g, 80.27%). 1 H NMR (300MHz, Chloroform-d) δ 10.10 (s, 1H), 7.74 (d, J = 2.5Hz, 1H), 7.46–7.33 (m, 5H), 7.24–7.08 (m, 2H), 5.09 (s, 2H), 2.45 (s, 3H) ppm.

[0161] Step 3: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.92 mmol) in 5 mL of methanol, add 5-methyl-2-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (0.57 g, 2.29 mmol), and stir at room temperature for 30 minutes. Slowly add NaBH3CN (0.17 g, 2.75 mmol), and stir at room temperature for 12 hours. Confirm the reaction is complete by TLC. The solvent is evaporated to dryness, water is added, and the mixture is extracted with ethyl acetate, washed with saturated saline solution, dried over anhydrous sodium sulfate, and the organic layer is evaporated to dryness. Purify by silica gel column chromatography (dichloromethane:methanol = 20:1) to give a white solid (0.2 g, 33.68%). HRMS (ESI): m / z [M+H] + .C 43 H 47 The calculated value of N2O4 is 687.3429; the measured value is 587.3456.

[0162] Step 4: Dissolve (S)-4-(bis(5-methyl-2-((3-phenylprop-2-yn-1-yl)oxy)benzylamino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.29 mmol) in 5 mL of 1,4-dioxane, then add 5 mL of 4 mol / L hydrochloric acid, react at room temperature for 3 hours, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.12 g, 70.24%). 1 H NMR (300MHz, DMSO-d6) δ7.36 (s, 10H), 7.29–7.20 (m, 4H), 7.14 (d, J = 8.2Hz, 2H), 5.0 5(s,4H),4.17(q,J=13.5Hz,4H),3.21(s,1H),2.23(s,8H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 39 The calculated value of N2O4 is 587.2904; the measured value is 587.2917.

[0163] The compounds described in Examples 25-31 can be prepared by referring to the method and route described in Example 14.

[0164] Example 25: Compound L-25

[0165]

[0166] 1 H NMR (300MHz, DMSO-d6) δ7.34 (s, 10H), 7.20 (d, J = 9.0Hz, 2H), 7.07 (d, J = 16.6Hz, 4H) ,4.97(s,4H),3.64(s,4H),2.64(s,2H),2.11–1.77(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 33 The calculated value of Br2N2O4 is 715.0802; the measured value is 715.0815.

[0167] Example 26: Compound L-26

[0168]

[0169] 1H NMR(300MHz,DMSO-d6)δ7.62(s,2H),7.55(dd,J=8.8,2.5Hz,2H),7.34(s,10H),7.17(d,J=8.9Hz ,2H),5.06(s,4H),4.13(s,4H),3.88(s,1H),3.14(s,2H),2.28(s,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 33 The calculated value of F2N2O4 is 595.2403; the measured value is 595.2426.

[0170] Example 27: Compound L-27

[0171]

[0172] 1 H NMR (300MHz, DMSO-d6) δ7.43–7.34(m,10H),7.26(s,2H),7.15(dd,J=7.6,1.9Hz,2H),6.81(d,J=7.6Hz,2H), 5.04(s,4H),3.79–3.67(m,5H),3.16-3.02(m,2H),1.93–1.81(m,2H),1.31(s,18H)ppm.HRMS(ESI):m / z[M+H] + .C 44 H 51 The calculated value of N2O4 is 671.3843; the measured value is 671.3841.

[0173] Example 28: Compound L-28

[0174]

[0175] 1 H NMR(300MHz,DMSO-d6)δ7.45–7.37(m,10H),6.86(d,J=9.0Hz,2H),6.81–6.73(m,4H),5.01 (s,4H),3.83–3.65(m,11H),3.13-3.03(s,2H),2.13–1.87(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 39 The calculated value of N2O6 is 619.2803; the measured value is 619.2827.

[0176] Example 29: Compound L-29

[0177]

[0178] 1 H NMR (300MHz, DMSO-d6) δ7.57–7.41(m,10H),7.32–7.16(m,2H),7.07(d,J=1.8,2H),6.88(d,J=7.9Hz,2H),4.98(s,4H),3.91-3.72(m ,5H),3.55-3.43(m,2H),2.93–2.83(m,2H),2.13–1.82(m,2H),1.29(d,J=6.6Hz,6H),1.24(d,J=6.6Hz,6H)ppm.HRMS(ESI):m / z[M+H] + .C 42 H 47 Calculated value of N2O4: 643.3530; Measured value: 643.3503.

[0179] Example 30: Compound L-30

[0180]

[0181] 1 H NMR(300MHz,DMSO-d6)δ7.72–7.59(m,4H),7.43–7.34(m,10H),7.00(d,J=7.3Hz,2H),5.09 (s,4H),4.03–3.87(m,5H),3.41-3.37(m,2H),2.10–1.84(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 33 The calculated value of F6N2O4 is 695.2339; the measured value is 695.2348.

[0182] Example 31: Compound L-31

[0183]

[0184] 1 H NMR(300MHz,DMSO-d6)δ7.40-7.37(m,14H),7.12(d,J=8.0Hz,2H),5.10(s,4H),3 .80(s,4H),3.62(s,1H),2.87(s,2H),2.05–1.90(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 33The calculated value of Br2N2O4 is 715.0802; the measured value is 715.0817.

[0185] Example 32: Compound L-32

[0186]

[0187] Step 1: Dissolve (S)-4-amino-2-((tert-butoxycarbonyl)amino)butyric acid (0.15 g, 0.68 mmol) in 5 mL of methanol, add 5-methyl-2-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (0.21 g, 0.83 mmol), stir at room temperature for half an hour, then add sodium cyanoborohydride (65 mg, 1.03 mmol), continue stirring at room temperature for 6 hours, and remove the solvent under reduced pressure. Separate by column chromatography (dichloromethane:methanol = 20:1) to obtain a white solid (0.24 g, 77.02%). HRMS (ESI): m / z [M+H] + .C 26 H 33 The calculated value of N2O5 is 453.2384; the measured value is 453.2372.

[0188] Step 2: Dissolve (S)-2-((tert-butoxycarbonyl)amino)-4-((5-methyl-2-((3-phenylprop-2-yn-1-yl)oxy)benzylamino)butyric acid (0.20 g, 0.44 mmol) in 8 mL of methanol, add 3-(3-(2-formylphenoxy)prop-1-yn-1-yl)benzonitrile (0.14 g, 0.53 mmol), stir at room temperature for half an hour, then add sodium cyanoborohydride (42 mg, 0.66 mmol), stir at room temperature for 12 hours, remove the solvent under reduced pressure, and separate by column chromatography (dichloromethane:methanol = 30:1) to obtain a white solid (0.15 g, 48.64%). HRMS (ESI): m / z [M+H] + .C 43 H 44 The calculated value of N3O6 is 698.3325; the measured value is 698.3346.

[0189] Step 3: Dissolve (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-(3-cyanophenyl)prop-2-yn-1-yl)oxy)benzyl)(5-methyl-2-((3-phenylprop-2-yn-1)oxy)benzyl)amino)butyric acid (0.12 g, 0.17 mmol) in 3 mL of 1,4-dioxane, then add 5 mL of 4 mol / L hydrochloric acid, react at room temperature for 4 h, remove the solvent under reduced pressure, and purify by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (65 mg, 63.24%). 1H NMR(300MHz, DMSO-d6)δ7.90–7.73(m,2H),7.61-7.56(m,1H),7.49(t,J=5.7Hz,1H),7.43–7.34(m,5H),7.26(s,1H),7.23–6.89(m,5H ),6.75(d,J=8.8Hz,1H),5.01(s,4H),3.86–3.73(m,5H),3.16–3.07(m,2H),2.27(s,3H),2.12-1.93(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 36 The calculated value of N3O4 is 598.2700; the measured value is 598.2734.

[0190] The compound described in Example 33 can be prepared by referring to the method and route described in Example 12.

[0191] Example 33: Compound L-33

[0192]

[0193] 1 H NMR (300MHz, DMSO-d6) δ7.43–7.28(m,8H),7.23–7.16(m,2H),7.02(s,1H),6.97–6.89(m,2H),6.85(d,J=8.8Hz,1H),6.81 –6.75(m,2H),4.98(s,4H),3.89(s,4H),3.76–3.63(m,1H),3.13-3.07(m,2H),2.29–1.83(m,5H)ppm.HRMS(ESI):m / z[M+H] + .C 37 H 37 Calculated N2O5 value: 589.2697; Measured value: 589.2697.

[0194] Example 34: Compound L-34

[0195]

[0196] Step 1: [1,1'-Biphenyl]-3-carboxaldehyde (0.19 g, 1.03 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)butyric acid (0.3 g, 0.68 mmol) were dissolved in 5 mL of methanol and stirred at room temperature for 1 hour. Sodium cyanoborohydride (0.11 g, 1.71 mmol) was added, and the mixture was stirred at room temperature for 8 hours. The reaction was confirmed to be complete by TLC, and the solvent was removed under reduced pressure. Separation by column chromatography (dichloromethane:methanol = 20:1) yielded a white solid (0.21 g, 50.76%). HRMS (ESI): m / z [M+H] + .C 38 H 41 Calculated N2O5 value: 605.3010; Measured value: 605.3015.

[0197] Step 2: (S)-4-(([1,1'-biphenyl]-3-ylmethyl)(2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.33 mmol) was dissolved in 5 mL of dioxane, and 5 mL of 4N HCl solution was added. The mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid (130 mg, 77.90%). 1 H NMR (300MHz, DMSO-d6) δ7.61–7.55(m,3H),7.50(s,1H),7.47–7.41(m,3H),7.41–7.35(m,6H),7.32–7.26(m,2H),7.19(td,J=7.7,1.5H z,1H),6.97–6.89(m,2H),4.97(s,2H),4.10–4.03(m,4H),3.51(s,1H),2.79-2.69(m,2H),2.01-1.87(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 33 H 33 Calculated value of N2O3: 505.2486; Measured value: 505.2465.

[0198] Example 35: Compound L-35

[0199]

[0200] Step 1: 2-Chlorobenzo[d]oxazole (1 g, 6.51 mmol), potassium carbonate (1.8 g, 13.02 mmol), 3-formylphenylboronic acid (1.17 g, 7.81 mmol), and Pd(PPh3)2Cl2 (200 mg, 0.65 mmol) were added to 5 mL of dioxane and 0.5 mL of water. The mixture was heated to 100 °C and reacted for 3 hours. The reaction was confirmed to be complete by TLC. After cooling to room temperature, the mixture was filtered, the filtrate was evaporated to dryness, and water was added. The filtrate was extracted with ethyl acetate, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the organic layer was evaporated to dryness. The crude residue was purified by silica gel column chromatography (cyclohexane:ethyl acetate = 50:1) to give a white solid (163 mg, 40%).

[0201] Step 2: 3-(benzo[d]oxazol-2-yl)benzaldehyde (0.23 g, 1.03 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)butyric acid (0.3 g, 0.68 mmol) were dissolved in 5 mL of methanol and stirred at room temperature for 1 hour. Sodium cyanoborohydride (0.11 g, 1.71 mmol) was added, and the mixture was stirred at room temperature for 8 hours. The reaction was confirmed to be complete by TLC, and the solvent was removed under reduced pressure. Separation by column chromatography (dichloromethane:methanol = 20:1) yielded a white solid (0.21 g, 47.54%). HRMS (ESI): m / z [M+H] + .C 39 H 40 The calculated value of N3O6 is 646.2912; the measured value is 646.2937.

[0202] Step 3: (S)-4-((3-(benzo[d]oxazol-2-yl)benzyl)(2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.2 g, 0.31 mmol) was dissolved in 5 mL of dioxane, and 5 mL of 4N HCl solution was added. The mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography (dichloromethane:methanol = 10:1) to give a white solid (130 mg, 76.93%). 1 H NMR(300MHz, DMSO-d6)δ8.45(s,1H),8.28(d,J=7.8Hz,1H),7.89-7.69(m,4H),7.51(q,J=6.7,5.3Hz,4H),7.33–7.18(m, 6H),5.04(s,2H),4.67–4.49(m,2H),4.46–4.31(m,2H),3.98(s,1H),3.40(s,2H),2.45(s,2H)ppm.HRMS(ESI):m / z[M+H]+ .C 34 H 32 The calculated value of N3O4 is 546.2387; the measured value is 546.2381.

[0203] The compound described in Example 36 can be prepared by referring to the method and route described in Example 14.

[0204] Example 36: Compound L-36

[0205]

[0206] 1 H NMR(300MHz, DMSO-d6)δ8.57(s,1H),8.23(d,J=7.7Hz,1H),7.91-7.69(m,4H),7.64-7.51(m,4H),7.33–7.18(m,6H),5. 07(s,2H),4.63–4.44(m,2H),4.46–4.31(m,2H),3.89(s,1H),3.40(s,2H),2.43-2.24(m,5H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 34 The calculated value of N3O4 is 560.2544; the measured value is 560.2529.

[0207] Example 37: Compound L-37

[0208]

[0209] Step 1: Methyl salicylate (1 g, 6.57 mmol) was dissolved in 30 mL of tetrahydrofuran, and potassium carbonate (1.82 g, 13.15 mmol) and bromopropyne (0.94 g, 7.89 mmol) were added. The mixture was heated to 60 °C and stirred for 8 hours. The reaction was confirmed to be complete by TLC. After cooling to room temperature, the mixture was filtered, and the filtrate was evaporated to dryness. The residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) to obtain a colorless oil (1 g, 80%). HRMS (ESI): m / z [M+Na] + .C 11 H 10 Calculated value of NaO3: 213.0522; measured value: 213.0546.

[0210] Step 2: Methyl 2-(propane-2-yne-1-oxy)benzoate (1 g, 5.26 mmol), iodobenzene (0.98 g, 4.78 mmol), triethylamine (2.42 g, 23.90 mmol), cuprous iodide (91 mg, 0.48 mmol), and Pd(PPh3)4 (276 mg, 0.24 mmol) were dissolved in 15 mL of tetrahydrofuran. Under nitrogen protection, the mixture was heated to 60 °C and reacted for 3 hours. The reaction was confirmed to be complete by TLC. After cooling to room temperature, the reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and the solution was removed under reduced pressure. The solution was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) to obtain a pale yellow solid (0.81 g, 63.64%).

[0211] Step 3: Methyl 2-((3-phenylprop-2-yn-1-yl)oxy)benzoate (0.8 g, 3.00 mmol) was dissolved in tetrahydrofuran, and 1N lithium hydroxide solution was added dropwise. The reaction was carried out at room temperature for 8 hours. The reaction was confirmed to be complete by TLC. The reaction solution was concentrated under vacuum, water was added to the residue, and the pH was adjusted to 5 using 1N HCl solution. The residue was filtered to obtain a white solid (0.7 g, 92.37%).

[0212] Step 4: Dissolve 2-((3-phenylprop-2-yn-1-yl)oxy)benzoic acid (0.17 g, 0.68 mmol) in 8 mL of LDM, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.39 g, 1.03 mmol) and diisopropylethylamine (0.27 g, 2.05 mmol), and stir at room temperature for half an hour. Then add (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)butyric acid (0.3 g, 0.68 mmol), stir at room temperature for 12 hours, add water, extract with EA, wash with saturated brine, dry with anhydrous sodium sulfate, and separate by column chromatography (dichloromethane:methanol = 30:1) to obtain a white solid (0.31 g, 67.37%). HRMS(ESI): m / z[M+H] + .C 41 H 40 The calculated value of N2O7 is 673.2908; the measured value is 673.2947.

[0213] Step 5: Dissolve (S)-2-((tert-butoxycarbonyl)amino)-4-(2-((3-phenylprop-2-yn-1-yl)oxy)-N-(2-((3-phenylprop-2-yn-1-yl)oxy)benzamide)butyric acid (0.3 g, 0.45 mmol) in 5 mL of dioxane, add 5 mL of 4N HCl solution, stir at room temperature for 6 hours, confirm the reaction is complete by TLC, remove the solvent under reduced pressure, and separate by column chromatography (dichloromethane:methanol = 10:1) to give a white solid (0.2 g, 78.32%). 1 H NMR(300MHz,DMSO-d6)δ7.75(s,1H),7.48–7.41(m,1H),7.41–7.34(m,10H),7.30-7.19(m,2H),7.14–6.97(m,4H ),5.23(s,4H),4.61-4.55(m,2H),3.73(t,J=6.6,1H),3.48–3.38(m,2H),2.33(s,2H)ppm.HRMS(ESI):m / z[M+H] + .C 41 H 40 The calculated value of N2O7 is 573.2384; the measured value is 573.2326.

[0214] The compounds described in Examples 38-40 can be prepared by referring to the method and route described in Example 14.

[0215] Example 38: Compound L-38

[0216]

[0217] 1 H NMR (300MHz, DMSO-d6) δ8.01(t,J=2.0Hz,1H),7.91(s,1H),7.88(d,J=7.1Hz,1H),7.87–7.24(m,12H),7.14–7.07(m,2H),7. 07–6.94(m,2H),5.15(s,4H),4.56-4.48(m,2H),3.89(s,1H),3.58–3.48(m,2H),2.10–1.85(m,5H)ppm.HRMS(ESI):m / z[M+H] + .C 24 H 27 The calculated value of N2O4 is 407.1965; the measured value is 407.1954.

[0218] Example 39: Compound L-39

[0219]

[0220] 1 H NMR(300MHz,DMSO-d6)δ8.10(d,J=7.9Hz,1H),7.83-7.73(m,3H),7.55–7.39(m,9H),7.21(s,1H),7.19–7.15(m,1H),7.08–7.01(m,2H),6 .87(d,J=8.9Hz,1H),5.22(s,2H),4.85(s,2H),3.73(s,1H),3.48–3.38(m,2H),2.45–2.24(m,5H),2.17(s,3H)ppm.HRMS(ESI):m / z[M+H] + .C 31 H 29 The calculated value of N2O4 is 493.2122; the measured value is 493.2153.

[0221] Example 40: Compound L-40

[0222]

[0223] 1 H NMR (300MHz, DMSO-d6) δ7.59–7.44(m,12H),7.34–7.27(m,2H),7.17(td,J=7.7,1.1Hz,2H),7.04–6.94(m,2H),4.96(s ,1H),5.03(s,4H),3.77(s,1H),3.50–3.41(m,2H),2.42–2.31(m,2H),1.48(d,J=6.5Hz,3H)ppm.HRMS(ESI):m / z[M+H] + .C 37 H 35 Calculated N2O5 value: 587.2540; Measured value: 587.2529.

[0224] Example 41: Compound L-41

[0225]

[0226] Step 1: Dissolve 2-aminobenzaldehyde (1.0 g, 8.25 mmol) in tetrahydrofuran, add triethylamine (1.67 g, 16.51 mmol), cool to 0 °C, and add benzoyl chloride (1.16 g, 8.25 mmol) dropwise. After the addition is complete, move the mixture to room temperature and react for 3 hours. Confirm the reaction is complete by TLC. Filter the mixture, evaporate the filtrate to dryness, wash the residue with n-hexane, and filter to give a white solid (1.3 g, 69.91%). HRMS (ESI): m / z [M+H]+ .C 14 H 12 NO2 calculated value: 226.0863; measured value: 226.0837.

[0227] Step 2: N-(2-formylphenyl)benzamide (0.31 g, 1.37 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzylamino)butyric acid (0.3 g, 0.68 mmol) were dissolved in methanol and stirred at room temperature for 1 hour. Sodium cyanoborohydride (0.13 g, 2.05 mmol) was added, and the reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 20:1) to give a white solid (0.2 g, 45.13%). HRMS (ESI): m / z [M+H] + .C 39 H 42 The calculated value of N3O6 is 648.3068; the measured value is 648.3042.

[0228] Step 3: Dissolve (S)-4-((2-benzamido)(2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)amino)-2-((tert-butoxycarbonyl)amino)butyric acid (0.15 g, 0.23 mmol) in 3 mL of dioxane, add 3 mL of 4N HCl solution, stir at room temperature for 3 hours, confirm the reaction is complete by TLC, remove the solvent under reduced pressure, and separate by column chromatography (dichloromethane:methanol = 10:1) to obtain a white solid (0.2 g, 78.32%). 1 H NMR(300MHz, DMSO-d6)δ9.40(s,1H),7.95(dt,J=8.0,1.2Hz,2H),7.67–7.60(m,2H),7.50–7.43(m,2H),7.43–7.18(m,9H),7. 11-6.98(m,3H),5.10(s,2H),4.35-4.29(m,4H),3.71–3.68(m,1H),3.54(s,2H),2.21-2.04(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 34 H 34 The calculated value of N3O4 is 548.2544; the measured value is 548.2561.

[0229] The compounds described in Examples 42-48 can be prepared by referring to the method and route described in Example 14.

[0230] Example 42: Compound L-42

[0231]

[0232] 1 H NMR(300MHz, DMSO-d6)δ9.45(s,1H),8.24–8.16(m,2H),7.73–7.65(m,2H),7.55(d,J=8.3,1H),7.43–7.34(m,5H),7.29–7.16(m ,4H),7.11-6.95(m,3H),5.10(s,2H),4.35-4.28(m,4H),3.75(s,1H),3.56(s,2H),2.20-2.08(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 33 The calculated value of F3N3O4 is 616.2418; the measured value is 616.2435.

[0233] Example 43: Compound L-43

[0234]

[0235] 1 H NMR(300MHz,DMSO-d6)δ9.37(s,1H),7.98–7.92(m,2H),7.55(s,1H),7.43–7.34(m,5H),7.29–7.16(m,4H),7.11(dd,J=8.3,7.3,1H),7.0 1–6.97(m,2H),6.97–6.89(m,2H),5.09(s,2H),4.01-3.96(m,4H),3.83(s,4H),3.49(s,2H),2.19-2.03(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 36 The calculated value of N3O5 is 578.2649; the measured value is 578.2638.

[0236] Example 44: Compound L-44

[0237]

[0238] 1H NMR(300MHz, DMSO-d6)δ9.46(s,1H),8.35(t,J=1.3Hz,1H),8.05–7.96(m,2H),7.94–7.86(m,2H),7.61–7.51(m,3H),7.43–7.34(m,5H),7.29 –7.16(m,4H),7.05–6.89(m,2H),5.09(s,3H),4.38-4.29(m,4H),3.87(s,1H),3.68–3.57(m,2H),2.13-2.01(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 36 The calculated value of N3O4 is 598.2700; the measured value is 598.2741.

[0239] Example 45: Compound L-45

[0240]

[0241] 1 H NMR (300MHz, DMSO-d6) δ9.40(s,1H),7.95(dt,J=8.0,1.2Hz,2H),7.67–7.46(m,4H),7.29–7.16(m,4H),7.11(s,1H),7.01(d,J=2. 1Hz,2H),6.97–6.89(m,3H),5.19(s,2H),4.10-3.98(m,4H),3.87(s,1H),3.56(s,2H),2.38–2.12(m,8H)ppm.HRMS(ESI):m / z[M+H] + .C 36 H 38 The calculated value of N3O4 is 576.2857; the measured value is 576.2841.

[0242] Example 46: Compound L-46

[0243]

[0244] 1H NMR(300MHz, DMSO-d6)δ9.39(s,1H),7.95(dt,J=8.0,1.2Hz,2H),7.57–7.31(m,9H),7.27–7.21(m,2H),7.18(dt,J=1.9,1.0Hz,1H ),7.11(s,1H),7.05–6.99(m,2H),5.09(s,2H),3.79–3.65(m,5H),3.15–3.07(m,2H),2.29–1.97(m,5H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 36 Calculated value of N3O4: 562.2700; Measured value: 562.2754.

[0245] Example 47: Compound L-47

[0246]

[0247] 1 H NMR(300MHz, DMSO-d6)δ9.45(s,1H),7.95(dt,J=8.0,1.2Hz,2H),7.57–7.47(m,4H),7.43–7.34(m,5H),7.28–7.14(m,4H),7.07(s,1H),6 .81(d,J=7.6Hz,1H),5.17(s,2H),4.32-4.12(m,4H),3.84(s,1H),3.19(s,1H),2.27–2.14(m,2H),1.31(s,9H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 42 The calculated value of N3O4 is 604.3170; the measured value is 604.3175.

[0248] Example 48: Example L-48

[0249]

[0250] 1H NMR(300MHz,DMSO-d6)δ9.35(s,1H),7.88–7.82(m,2H),7.45–7.38(m,2H) ,7.30(s,3H),7.18(dt,J=2.0,0.9Hz,1H),7.10–6.99(m,3H),6.92–6.87(m ,2H),6.85(d,J=8.8Hz,1H),5.13(s,2H),4.10-4.03(m,4H),3.83(s,3H),3 .76(s,1H),3.45–3.41(m,2H),2.32–1.89(m,8H)ppm.HRMS(ESI):m / z[M+H] + .C 38 H 42 The calculated value of N3O5 is 620.3119; the measured value is 620.3146.

[0251] Example 49: Example L-49

[0252]

[0253] Step 1: Dissolve 2-aminobenzaldehyde (1.0 g, 8.25 mmol) in tetrahydrofuran, add triethylamine (1.67 g, 16.51 mmol), cool to 0 °C, and add benzenesulfonyl chloride (1.46 g, 8.25 mmol) dropwise. After the addition is complete, move the mixture to room temperature and react for 3 hours. Confirm the reaction is complete by TLC. Filter the mixture, evaporate the filtrate to dryness, wash the residue with n-hexane, and filter to give a white solid (1.5 g, 69.54%). HRMS (ESI): m / z [M+H] + .C 13 H 12 NO3S calculated value: 262.0532; measured value: 262.0516.

[0254] Step 2: N-(2-formylphenyl)benzamide (0.36 g, 1.37 mmol) and (S)-2-((tert-butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzylamino)butyric acid (0.3 g, 0.68 mmol) were dissolved in methanol and stirred at room temperature for 1 hour. Sodium cyanoborohydride (0.13 g, 2.05 mmol) was added, and the reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 20:1) to give a white solid (0.2 g, 42.75%). HRMS (ESI): m / z [M+H] + .C 38 H 42 The calculated value of N3O7S is 684.2738; the measured value is 684.2761.

[0255] Step 3: (S)-2-((tert-Butoxycarbonyl)amino)-4-((2-((3-phenylprop-2-yn-1-yl)oxy)benzyl)(2-(benzenesulfonamide)benzyl)amino)butyric acid (0.13 g, 0.19 mmol) was dissolved in 3 mL of dioxane, and 3 mL of 4N HCl solution was added. The mixture was stirred at room temperature for 3 hours. The reaction was confirmed to be complete by TLC. The solvent was removed under reduced pressure, and the mixture was separated by column chromatography (dichloromethane:methanol = 10:1) to give a white solid (60 mg, 46.15%). 1 H NMR(300MHz, DMSO-d6)δ8.90(s,1H),7.81–7.73(m,3H),7.48–7.34(m,7H),7.32(d,J=7.9,1H),7.29–7.14(m,4H),7.00(s,1H), 6.97–6.89(m,2H),5.12(s,2H),4.01-3.96(m,4H),3.78–3.73(m,1H),3.68(s,2H),2.31-2.07(m,2H)ppm.HRMS(ESI):m / z[M+H] + .C 33 H 34 The calculated value of N3O5S is 584.2214; the measured value is 584.2231.

[0256] The compounds described in Examples 50-52 were prepared by referring to the method and route described in Example 14.

[0257] Example 50: Example L-50

[0258]

[0259] 1 H NMR(300MHz,DMSO-d6)δ8.76(s,1H),7.90–7.81(m,3H),7.58–7.41(m,7H),7.19–7.04(m,4H),6.98(d,J=7.6Hz,1H),6.85 (s,1H),5.09(s,2H),4.01-3.96(s,2H),3.76–3.71(m,1H),3.54–3.47(m,2H),2.32–2.11(m,8H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 38 The calculated value of N3O5S is 612.2527; the measured value is 612.2546.

[0260] Example 51: Example L-51

[0261]

[0262] 1 H NMR(300MHz, DMSO-d6)δ9.50(s,1H),7.49(dd,J=8.2,1.9Hz,2H),7.43–7.34(m,5H),7.18(dd,J=2.0,0.9Hz,1H),7.10–6.95(m,5H) ,6.85(d,J=8.8Hz,1H),5.16(s,2H),3.89–3.77(m,4H),3.71(s,1H),3.23–3.19(m,2H),2.27-1.96(m,8H)ppm.HRMS(ESI):m / z[M+H] + .C 35 H 36 The calculated value of F2N3O5S is 648.2338; the measured value is 618.2353.

[0263] Example 52: Example L-52

[0264]

[0265] 1 H NMR(300MHz,DMSO-d6)δ9.14(s,1H),8.43(t,J=1.9Hz,1H),8.09(d,J=8.9Hz,1H),8.01 (dt,J=8.3,1.8Hz,1H),7.96–7.85(m,2H),7.57–7.48(m,2H),7.43–7.34(m,5H),7.18(d ,J=1.9Hz,1H),7.06–7.00(m,3H),6.98(d,J=7.6Hz,1H),6.85(d,J=8.8Hz,1H),5.09(s ,2H),3.98–3.68(m,5H),3.23–3.12(s,2H),2.32–2.02(m,8H)ppm.HRMS(ESI):m / z[M+H] + .C 39 H 40 The calculated value of N3O5S is 662.2683; the measured value is 662.2647.

[0266] Example 53: In vitro activity test of the compound

[0267] 1. Anti-tumor cell proliferation experiment

[0268] A549 cells (3000 / well) were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 12 h. The cells were then given different concentrations of the compound and cultured for another 72 h. 20 μL of MTT was added to each well, and the cells were incubated at 37°C with 5% CO2 for 4 h. The culture medium was discarded, and 150 μL of formazan crystals were added to dissolve them. The absorbance was measured at 490 nm. / 2. Glutamine transport assay

[0269] (1) Cell Culture

[0270] (2) Plate preparation: 24-well plate, 50,000 / well, 37℃, 5% CO2, culture for 24h;

[0271] (3) Washing the cell plate: After the cells have grown to a high level, remove the 24-well plate from the incubator, discard the culture medium in the plate, and wash it 3 times with Assay buffer preheated at 37°C.

[0272] (4) Add 250 μL of inhibitor solutions of different concentrations (10 mM compound stock solution diluted with Assay buffer to 100 μM, 50 μM, 20 μM, 10 μM, 5 μM, 2 μM, and 1 μM), preheat at 37°C for 15 minutes, discard the compound solution, and add another 250 μL (using 5 μM... 13 Incubate C5-Glutamine (diluted with Assay buffer) at 37°C for 15 minutes. After incubation, aspirate the liquid and wash cells three times with Assay buffer. Add 250 μL of ultrapure water. Perform three freeze-thaw cycles at -80°C to scrape cells into a 1.5 mL EP tube and sonicate for 15 minutes. Take 30 μL of the sonicated cell sample and add it to a 1.5 mL EP tube, along with 30 μL of diluent and 60 μL of internal standard solution. Vortex for 3 minutes; centrifuge at 12000 rpm for 10 minutes at 4°C. Transfer 60 μL of the supernatant to a vial for LC-MS analysis. 13 C5-Glutamine concentration.

[0273] 3. Data Analysis

[0274] (1) Calculation formula for anti-tumor cell proliferation experiment

[0275] %Inhibition=[1-(OD_sample-OD_min)] / (OD_max-OD_min)

[0276] Where: OD_sample represents the absorbance of the drug delivery well, OD_min represents the absorbance of the blank well, and OD_max represents the absorbance of the negative well.

[0277] (2) Calculation formula for glutamine transport experiment

[0278] %Inhibition=[1-(A_sample / A_max)]

[0279] Where: A_sample represents the sample 13 C5-Glutamine content, A_max represents airborne .... 13 C5-Glutamine content.

[0280] (3) The dose-response curves were fitted using the log value of concentration as the X-axis and the percentage inhibition rate as the Y-axis. The log(inhibitor) vs. response-variable slope function of the analysis software GraphPad Prism5 was used to fit the dose-response curves, thereby obtaining the IC50 of each compound on the enzyme activity. 50 value.

[0281] Calculation formula:

[0282] Y=Bottom+(Top-Bottom) / (1+10^((Log IC 50 -X)×Hill Slope)).

[0283] IC 50 The specific data is shown in Table 1.

[0284] Table 1. In vitro activity of compounds (IC50) 50 )

[0285]

[0286]

[0287] Note: A: 1-10μM, B: 10-50μM, C: >50μM.

[0288] As shown in Table 1, all tested compounds inhibited the proliferation of A549 tumor cells and glutamine transporter enzyme activity. Among them, compounds L-1, L-3, L-6, L-17–L-18, and L-25 showed the highest IC50 values ​​for the transporter enzyme. 50 The values ​​were all less than 10 μM, and the IC50 values ​​for the antitumor activity of compounds L-25~L-26 and L-30 were all less than 10 μM. 50 The values ​​were all less than 10 μM, and the IC50 values ​​of the other compounds were... 50 The value is also at the micromolar concentration level.

Claims

1. A glutamine transport inhibitor, characterized in that, Having the structure of Formula I, the compound further comprises a pharmaceutically acceptable salt thereof: ， in: X is or Q is ; R a It can be hydrogen or methyl; R 1 It is a halogen, a C1-C4 alkyl, a C1-C4 haloalkyl, a C1-C4 alkoxy, or a C1-C4 haloalkoxy; R 2 For 1-4 hydrogens R 2a Substituted 6-membered aryl; R 2a It can be hydrogen, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy; R 3 For R 3a -CH2O-、R 3a -C≡C-CH2O-; R 3a For 1-4 hydrogens R 3c Substituted 6-10 aryl or benzoxazolyl groups; R 3c It can be hydrogen, halogen, cyano, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy or C1-C4 haloalkoxy; m is 0 or 1; n is 1; z is 2.

2. The inhibitor according to claim 1, characterized in that, In the structure: X and Q are ; R a It can be hydrogen or methyl; R 1 It can be fluorine, bromine, iodine, methyl, isopropyl, tert-butyl, trifluoromethyl, or methoxy; R 2 For 1-4 hydrogens R 2a Substituted phenyl; R 2a It can be hydrogen, fluorine, methyl, isopropyl, tert-butyl, trifluoromethyl, methoxy, ethoxy, or isopropoxy; R 3 For R 3a -CH2O-、R 3a -C≡C-CH2O-; R 3a For 1-4 hydrogens R 3c Substituted phenyl, naphthyl, or benzoxazolyl groups; R 3c It can be hydrogen, fluorine, chlorine, cyano, methyl, ethyl, isopropyl, trifluoromethyl, methoxy, ethoxy, or isopropoxy; z is 2.

3. The inhibitor according to claim 1, characterized in that, In the structure: X is -C(O)-; Q is R a It can be hydrogen or methyl; R 1 It is methyl; R 2 It is phenyl; R 3 For R 3a -CH2O-; R 3a It is phenyl or naphthyl; z is 2.

4. The inhibitor according to claim 1, characterized in that, In the structure: X and Q are ; R a It can be hydrogen or methyl; R 1 It can be bromine, iodine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl or methoxy; R 2 For 1-4 hydrogens R 2a Substituted phenyl; R 2a It can be hydrogen, fluorine, methyl, isopropyl, tert-butyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy; R 3 For R 3a -C≡C-CH2O-; R 3a For 1-4 hydrogens R 3c Substituted phenyl or benzoxazole group; R 3c It can be hydrogen, fluorine, chlorine, cyano, methyl, ethyl, isopropyl, C1-C4 fluoroalkyl, methoxy, ethoxy, or isopropoxy.

5. A glutamine transport inhibitor, characterized in that, It has any of the following general formula structures: ， Where y is 0, 1, or 2, and n is 0; R 1 R 2a R 3 R 3a The definitions of m are as described in any one of claims 1-4.

6. A glutamine transport inhibitor, characterized in that, Selected from any of the following compounds: 。 7. The inhibitor according to any one of claims 1-4, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound with an acid or base, wherein the acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, or mandelic acid, and the base is an inorganic base containing a basic metal cation, an alkaline earth metal cation, or an ammonium cation.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the inhibitor of any one of claims 1-7 and a pharmaceutically acceptable carrier.

9. An inhibitor according to any one of claims 1-7 or a pharmaceutical composition according to claim 8, in the preparation of a treatment for sodium-related diseases. + Application in drugs for diseases related to glutamine carrier 2 dependence.

10. The application according to claim 9, characterized in that, The relationship with Na + Diseases associated with glutamine carrier 2 dependence include cancer or viral infections.

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