A non-covalent crm1 inhibitor and methods of making and using the same

Abstract

The application provides a non-covalent CRM1 inhibitor and a preparation method and use thereof, and belongs to the technical field of biological medicines.The non-covalent CRM1 inhibitor is a compound shown in formula I, a salt thereof, a stereoisomer thereof or a solvate thereof.The application provides a compound which can be used as a non-covalent CRM1 inhibitor, the compound has a good inhibiting effect on CRM1, can be used for inhibiting the growth of various cancer cells, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a non-covalent CRM1 inhibitor, its preparation method, and its uses. Background Technology

[0002] Protein function depends not only on its correct sequence and structure but also on its correct cellular localization. Besides cancer mutations, abnormal protein localization can also lead to the inactivation of tumor suppressor genes and the activation of oncogenes, thus contributing to cancer development. Chromosomal region maintenance 1 (CRM1), also known as exportin 1 (XPO1), is overexpressed and is a recognized pathogenic mechanism in cancer development. CRM1 is a broad-spectrum nuclear export protein that mediates the nuclear export of many factors in tumorigenesis and development, including p53, p21, p27, and nucleophosphoryl 1 (NPM1). CRM1 binds to nuclear export signals (NES) via NES binding grooves and mediates their translocation from the nucleus to the cytoplasm through the nuclear pore complex. Overexpression of CRM1 is highly correlated with tumor grade and prognosis in patients.

[0003] CRM1 inhibitors are broad-spectrum cancer treatments due to their ability to simultaneously inhibit multiple cancer-stimulating pathways. However, the first-generation CRM1 inhibitor, leptomycin B (LMB), failed due to its high toxicity. LMB covalently binds to cysteine ​​residues (C528) in the NES groove and permanently inhibits CRM1, but CRM1-mediated nuclear export is crucial for cell survival; therefore, LMB's high CRM1 inhibitory activity led to safety concerns. Second-generation CRM1 inhibitors, due to their slow and reversible inhibition of CRM1, are less toxic than first-generation CRM1 inhibitors. Among them, celiniso (KPT-330) was approved by the US Food and Drug Administration in 2019 for the treatment of relapsed or refractory multiple myeloma and is currently undergoing extensive clinical trials in hematology and solid tumors. Celiniso binds to XPO1, blocking the nuclear export of tumor suppressor proteins and oncogenic proteins, accumulating tumor suppressor proteins in the nucleus and activating them to exert a chemicalbook effect. Simultaneously, blocking the entry of inhibitory proteins into the cytoplasm prevents oncogenic proteins from functioning properly. Under this dual effect, tumor cells undergo apoptosis.

[0004] However, most existing CRM1 inhibitors are covalent CRM1 inhibitors, and compounds capable of blocking protein-protein interactions between CRM1 and other factors in a non-covalent manner have not been fully developed. Non-covalent CRM1 inhibitors are expected to have a wider therapeutic window, lower toxicity, and insensitivity to C528 mutations because they can reversibly bind to the NES binding groove of CRM1 without relying on covalent binding to cysteine ​​at position 528 (C528). However, developing non-covalent CRM1 inhibitors presents a considerable challenge due to the plasticity of the NES groove and the high binding affinity of NES. Summary of the Invention

[0005] The purpose of this invention is to provide a non-covalent CRM1 inhibitor, its preparation method, and its uses.

[0006] This invention provides compounds of Formula I, their salts, their stereoisomers, or their solvates:

[0007]

[0008] in,

[0009] m is the number of substituents R1 on the benzene ring, selected from 1, 2 or 3;

[0010] Each R1 is independently selected from substituted or unsubstituted C1-C3 alkyl groups and halogens;

[0011] n is the number of substituents R2 on the benzene ring, selected from 1, 2 or 3;

[0012] Each R2 is independently selected from substituted or unsubstituted C1-C3 alkyl groups or halogens, and R2 is not simultaneously selected from two methyl groups;

[0013] The substituents of the alkyl group are selected from halogens.

[0014] Furthermore, the compound is as shown in Formula II:

[0015]

[0016] in,

[0017] R1 is selected from trifluoromethyl or halogen;

[0018] n is the number of substituents R2 on the benzene ring, selected from 1 or 2;

[0019] Each R2 is independently selected from trifluoromethyl, methyl, and halogen, and R2 is not selected from two methyl groups at the same time.

[0020] Furthermore, the compound is of formula IIIa:

[0021]

[0022] in,

[0023] R1 is selected from trifluoromethyl, chlorine, bromine, and fluorine;

[0024] R 21 R 22 Each of the following is independently selected from trifluoromethyl, methyl, chlorine, bromine, and fluorine, and R2 is not simultaneously selected from two methyl groups;

[0025] Alternatively, the compound may be of formula IIIb:

[0026]

[0027] in,

[0028] R1 is selected from trifluoromethyl, chlorine, bromine, and fluorine;

[0029] R 21 R 23 Each of the following is independently selected from trifluoromethyl, methyl, chlorine, bromine, and fluorine.

[0030] Furthermore, the compound is of formula IIIc or IIId:

[0031]

[0032] in,

[0033] R1 is selected from trifluoromethyl, chlorine, bromine, and fluorine;

[0034] R 21 Selected from trifluoromethyl, methyl, chlorine, bromine, and fluorine. Further, the compound is shown in formula V:

[0035]

[0036] in,

[0037] R1 is selected from trifluoromethyl, chlorine, bromine, and fluorine.

[0038] Furthermore, the compound has one of the following structures:

[0039]

[0040]

[0041] The present invention also provides a method for preparing the aforementioned compound, comprising the following steps:

[0042]

[0043] (1) In a solvent, compounds S1 and S2 react with N,N-dimethylethylenediamine to give compound S3;

[0044] (2) In a solvent, compound S3, ammonium formate and zinc powder react to give compound S4;

[0045] (3) In a solvent, compound S4, triethylamine and compound S5 react to give compound S6;

[0046] (4) In a solvent, compound S6 reacts with trifluoroacetic acid to give compound S7;

[0047] (5) In a solvent, compounds S7, S8, BINAP, and Cs2CO3 react under the action of a catalyst to obtain compound S9;

[0048] (6) In a solvent, compound S9 reacts with lithium hydroxide to give compound S10;

[0049] m, R1, n, and R2 are as described above;

[0050] Preferably,

[0051] In step (1), the solvent is ethanol;

[0052] And / or, in step (1), the molar ratio of compound S1, compound S2 and N,N-diisopropylethylamine is 1:1.1 to 1.5:1 to 2;

[0053] And / or, in step (1), the reaction conditions are reflux reaction at 80-100°C for 1-10 hours;

[0054] And / or, in step (2), the solvent is methanol;

[0055] And / or, in step (2), the molar ratio of compound S3, ammonium formate and zinc powder is 1:10-15:10-15;

[0056] And / or, in step (2), the reaction conditions are 20-40°C for 5-10 hours;

[0057] And / or, in step (3), the solvent is dichloromethane;

[0058] And / or, in step (3), the molar ratio of compound S4, triethylamine and compound S5 is 1:1 to 5:1 to 1.5;

[0059] And / or, in step (3), the reaction is carried out at 20-40°C for 4-8 hours;

[0060] And / or, in step (4), the solvent is dichloromethane;

[0061] And / or, in step (4), the molar volume ratio of compound S6 to trifluoroacetic acid is 1-5 mmol: 1 mL;

[0062] And / or, in step (4), the reaction conditions are 20-40°C for 1-10 hours;

[0063] And / or, in step (5), the solvent is toluene;

[0064] And / or, in step (5), the catalyst is Pd2(dba)3;

[0065] And / or, in step (5), the molar ratio of compound S7, compound S8, BINAP, Cs2CO3, and catalyst is 1:1~1.5:0.1~0.5:1~5:0.01~0.05;

[0066] And / or, in step (5), the reaction conditions are 100-120°C for 10-20 hours;

[0067] And / or, in step (6), the solvent is a mixed solution of water and tetrahydrofuran;

[0068] And / or, in step (6), the molar ratio of compound S9 to lithium hydroxide is 1:5 to 10;

[0069] And / or, in step (6), the reaction conditions are 40-60°C for 10-20 hours.

[0070] Furthermore,

[0071] In step (1), after the reaction, the following steps were used to purify compound S3: the solvent was removed by rotary evaporation of the reaction solution, the remaining mixture was diluted with dichloromethane, and extracted with saturated citric acid and saturated brine respectively, dried with anhydrous sodium sulfate, filtered and then the dichloromethane was removed by rotary evaporation, and the residue was purified by silica gel column chromatography.

[0072] And / or, in step (2), after the reaction, the following steps are used to purify the compound S4: the reaction solution is filtered with diatomaceous earth, the solvent is removed by rotary evaporation of the filtrate, the residue is dissolved in dichloromethane, the insoluble matter is removed by diatomaceous earth filtration, the solvent is removed by rotary evaporation, and the residue is purified by silica gel column chromatography.

[0073] And / or, in step (3), after the reaction, the following steps are used to purify compound S6: the reaction solution is desolventized under vacuum, the residue is diluted with water and extracted with ethyl acetate, the organic phase is back-extracted with saturated brine and saturated citric acid aqueous solution, dried with anhydrous sodium sulfate, filtered and then the solvent is removed by rotary evaporation, and the residue is purified by silica gel column chromatography.

[0074] And / or, in step (4), after the reaction, the following steps are used to purify the compound S7: the pH of the reaction solution is adjusted to 9-10, extracted with dichloromethane, the organic phase is back-extracted with saturated brine, the organic phase is dried with anhydrous sodium sulfate, and the solvent is removed under vacuum after filtration.

[0075] And / or, in step (5), after the reaction, the following steps are used to purify the compound S9: the reaction solution is filtered with diatomaceous earth, the solvent is removed from the filtrate under vacuum, the residue is dissolved with ethyl acetate and extracted with semi-saturated brine, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed under vacuum after filtration, and the residue is purified by passing it through a silica gel column.

[0076] And / or, in step (6), after the reaction, the compound S10 is purified by the following steps: the pH of the reaction solution is adjusted to 5.0.

[0077] The present invention also provides the use of the aforementioned compounds, their salts, their stereoisomers or solvates thereof in the preparation of medicaments for the prevention and / or treatment of CRM1-related diseases;

[0078] Preferably, the CRM1 inhibitor is a non-covalent CRM1 inhibitor;

[0079] And / or, the diseases associated with CRM1 are pancreatic cancer, endometrial cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, and cervical cancer.

[0080] The present invention also provides a drug preparation which is a formulation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients to the aforementioned compound, its salt, its stereoisomer or its solvate as the active ingredient.

[0081] The compounds and derivatives provided in this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0082] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0083] "Substitution" refers to the replacement of hydrogen atoms in a molecule by other different atoms or molecules.

[0084] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a ~C bAlkyl indicates any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, "C1 to C6 alkyl" refers to an alkyl group containing 1 to 6 carbon atoms.

[0085] "Alkyl" refers to a saturated hydrocarbon chain with a specified number of carbon atoms. For example, C1-C6 alkyl refers to an alkyl group with 1 to 6 carbon atoms, that is, alkyl groups with 1, 2, 3, 4, 5, or 6 carbon atoms. Alkyl groups can be straight-chain or branched. Representative branched alkyl groups have one, two, or three branches. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and neopentyl), and hexyl, etc.

[0086] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0087] This invention provides a class of compounds that can be used as non-covalent CRM1 inhibitors. These compounds have a good inhibitory effect on CRM1 and can be used to inhibit the growth of various cancer cells, showing good application prospects.

[0088] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0089] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0090] Figure 1The following graphs show the inhibitory activity results of the compounds of this invention against CRM1: A shows the inhibitory activity results of compounds G1-G12 and B28 against CRM1, with a compound concentration of 20 μM. The bottom number in graph A represents the ratio of CRM1 intensity to NES intensity, with DMSO sample set to 1; B shows the inhibitory activity results of compounds G1M-G12M and B28M against CRM1, with a compound concentration of 100 μM; C shows the binding affinity of G1 and human CRM1 determined by MST competitive assay in the presence of RanGTP. The blue and red lines represent the fitting of CRM1 titration and G1 titration, respectively. Each titration was repeated three times, and the graph shows the average value of the three titrations. Kd(AB*) is the affinity constant (equilibrium dissociation constant) of CRM1 with PKI-IAF; Kd(AC) represents the affinity constant at 50 nM. The competitive affinity constant between compound G1 and CRM1 in the presence of PKI-IAF; [FLuo]: 50 nM means the amount of PKI-IAF used is 50 nM; [A]: 350 nM means the concentration of CRM1 used in the differential competition experiment is 350 nM; Norm.Rel.Fluo.X1000 is the normalized fluorescence value shown in the isotherm plot; Residuals is the data deviation value; [titrant] at the bottom is translated as titration concentration (nM).

[0091] Figure 2 The graph shows the results of determining the affinity of G8 and G9 for CRM1 in the presence of RanGTP using the micro-thermal surge competitive difference method: A is the result graph of G8; B is the result graph of G9; the blue and red lines represent the fitting of CRM1 titration and G8 or G9 titration, respectively. Each titration was repeated three times, and the graph shows the average value of the three titrations.

[0092] Figure 3The following graphs show the inhibitory effects of the compounds of this invention on tumor cells: A shows the inhibitory effect of the compounds of this invention on HeLa cell growth (20 μM, 72 h), with error bars representing the standard deviation of 6 biological replicates; B shows the concentration-dependent inhibitory effect of compounds G1, G8, and G9 on HeLa cell growth, where cells were treated with different concentrations of the compounds for 72 hours before cell density analysis, with error bars representing the standard deviation of 6 biological replicates; C shows the inhibitory effect of compounds G1, G8, and G9 (20 μM, 72 h) on the growth of various cancer cells, with error bars representing the standard deviation of four biological replicates. Figure D shows the results of CESTA analysis of the thermostability of intracellular CRM1 in the presence of DMSO or 20 μM compounds (G1, G8, and G9). The numbers below represent the grayscale analysis of the bands, with each sample treated at 4 °C as 1. Figure E shows the results of the inhibitory effects of compounds G1, G8, and G9 (50 μM) on nuclear output in A549 cells. Endogenous NFκB was stained with NFκB antibody. The scale bar is 20 μm. The right figure is a statistical analysis of the nuclear localization ratio (nucleus / (nucleus + cytoplasm). Each point represents the nuclear localization ratio of one cell, and the error bars represent the standard deviation of at least 20 cells.

[0093] Figure 4 The figure shows the inhibitory effects of compounds G1, G8, and G9 (50 μM) on nuclear export from HeLa cells permeable to digitalis saponins. Detailed Implementation

[0094] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0095] The silica gel (300-400 mesh) used in the chromatographic column was purchased from Qingdao Ocean Chemical Co., Ltd. Pd2(dba)3, BINAP, ultra-dry THF, and other ultra-dry solvents were purchased directly from Anhui Zesheng Technology Co., Ltd. (Annegi Chemical) or Beijing Innocare Technology Co., Ltd. Ultra-dry toluene was refluxed under nitrogen using sodium / benzophenone. Other starting materials were purchased directly from Anhui Zesheng Technology Co., Ltd. (Annegi Chemical), TCI (Shanghai) Chemical Industry Development Co., Ltd., or Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0096] Unless otherwise specified, all reported reaction yields are isolated yields of the purified product. All new compounds were characterized by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). NMR spectra of all new compounds were recorded on a Bruker AMX 400 spectrometer with tetramethylsilane (TMS) (0.00 ppm) or deuterated solvent as internal standards (deuterated chloroform (CDCl3)). 1 In the 1H NMR, 7.26 ppm was used as an internal standard. 13The internal standard in the C NMR was 77.16 ppm; deuterated tetrahydrofuran (THF-d8): 1 In the 1H NMR, 1.72 ppm and 3.58 ppm were used as internal standards. 13 The internal standards for C NMR were 67.21 ppm and 25.31 ppm; deuterated dimethyl sulfoxide ((CD3)2SO4): 1 In H NMR, 2.50 ppm was used as an internal standard. 13 The calibration was performed using 39.52 ppm as an internal standard in the C NMR, and the data in the table are reported in ppm for TMS (0.00 ppm). High-resolution mass spectra were recorded on a Waters Q-TOF Premier.

[0097] The general synthetic steps of the compounds of this invention are as follows:

[0098]

[0099] Take a 100 mL round-bottom flask and add methyl 4-chloro-3-nitrobenzoate S2 (2.37 g, 11.00 mmol, 1.1 equiv.), N-tert-butoxycarbonylpiperazine S1 (1.86 g, 10.00 mmol, 1.0 equiv.), N,N-dimethylethylenediamine (1.66 mL, 10.00 mmol, 1.0 equiv.), and ethanol (30 mL) as solvent. Reflux at 80 °C for 4 h. After the reaction is complete, cool to room temperature. Remove the ethanol from the reaction solution by rotary evaporation. Dilute the remaining mixture with dichloromethane and extract with saturated citric acid and saturated brine, respectively. Dry with anhydrous sodium sulfate, filter, and remove the dichloromethane by rotary evaporation. Purify the residue by silica gel column chromatography (eluent: petroleum ether:ethyl acetate = 5:1, v:v) to obtain S3 (pale yellow solid).

[0100] The S3 obtained in the previous step (3.0 g, 8.54 mmol, 1.0 equiv.) was placed in a 100 mL round-bottom flask, and ammonium formate (5.38 g, 85.4 mmol, 10.0 equiv.) and zinc powder (5.55 g, 85.4 mmol, 10.0 equiv.) were added. The mixture was reacted with methanol (40 mL) at room temperature for 6 h. After the reaction was complete, the remaining zinc powder was removed by vacuum filtration with diatomaceous earth, and the methanol was removed by rotary evaporation. The residue was dissolved in dichloromethane, and the insoluble matter was removed by vacuum filtration with diatomaceous earth. Excess volume was removed by rotary evaporation. The residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1, v:v) to obtain S4 (pale yellow solid).

[0101] The S4 obtained in the previous step was placed in a 100 mL (2.23 g, 6.66 mmol, 1.0 equiv.) round-bottom flask and dissolved in dichloromethane (20 mL). Triethylamine (2.78 mL, 19.98 mmol, 3.0 equiv.) and substituted benzoyl chloride S5 (1.02 mL, 8.00 mmol, 1.2 equiv.) were added at 0 °C, and the mixture was stirred at room temperature for 4–8 h. After the reaction was complete, excess solvent was removed under vacuum. The residue was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined and back-extracted with saturated brine (40 mL) and saturated citric acid aqueous solution (40 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and the excess solvent was removed by rotary evaporation. The residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1, v:v) to obtain S6 (white solid).

[0102] S6 (6.12 mmol, 1.0 equiv.) was placed in a 50 mL round-bottom flask, dissolved in a small amount of dichloromethane, and trifluoroacetic acid (6 mL) was slowly added dropwise at 0 °C. The mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was neutralized to pH 9-10 with a semi-saturated sodium bicarbonate aqueous solution. The organic phase was then extracted with dichloromethane, back-extracted with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered under vacuum to remove excess solvent, yielding S7 as a white solid.

[0103] S7 (0.90 mmol, 1.0 equiv.) and S8 (1.08 mmol, 1.2 equiv.) were placed in a thick-walled, pressure-resistant flask equipped with a stir bar. The flask was then transferred to a glove box, and Pd2(dba)3 (0.045 mmol, 0.05 equiv.), BINAP (0.09 mmol, 0.10 equiv.), Cs2CO3 (1.8 mmol, 2.0 equiv.), and dried toluene (50 mL) were added sequentially. The mixture was removed from the glove box and refluxed at 110 °C for 12 h. After the reaction was complete, the mixture was cooled to room temperature. Insoluble matter was removed by diatomaceous earth filtration, and toluene was removed under vacuum. The residue was dissolved in ethyl acetate and extracted with semi-saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (using a mixture of petroleum ether and ethyl acetate as the eluent, 4:1 or 5:1, v:v) to obtain S9 as a white solid.

[0104] S9 (0.2 mmol, 1.0 equiv.) was added to a 50 mL round-bottom flask and dissolved in water (5 mL) and tetrahydrofuran (5 mL). Then, LiOH·H₂O (2.0 mmol, 10.0 equiv.) was slowly added dropwise with stirring to dissolve the dissolved substance. The reaction was carried out overnight at 50 °C with stirring. After the reaction was complete, the solution was neutralized to pH 5.0 with 2N HCl. The residue was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v) to obtain S10 as a white solid.

[0105] Example 1: Synthesis of methyl 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G1M)

[0106]

[0107] The compound was synthesized following the general synthetic steps described above, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (299.1 mg, 0.8 mmol) and 2-bromo-1,4-bis(trifluoromethyl)benzene (S8) (208 μL, 1.2 mmol) as starting materials. Compound G1M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v), yielding a white solid (173.4 mg, overall yield of 37% across five steps).

[0108] 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.16 (s, 1H), 9.14 (d, J = 2.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.87 (dd, J = 8.4, 2.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.55–7.52 (m, 3H), 7.34 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.18 (d, J = 5.2 Hz, 4H), 3.13 (d, J = 5.2 Hz, 4H). 13 C NMR (101MHz, deuterated chloroform) δ 166.7, 163.9, 152.5, 145.6, 138.6, 135.1 (q, J = 32.7 Hz), 133.3, 133.0, 130.4 (d, J = 30.5 Hz), 130.0, 129.4, 128.7 (q, J = 5.3 Hz), 128.5, 127.6, 126.2, 124.7 (d, J = 5.0 Hz), 122.0 (d, J = 3.9 Hz), 121.2, 120.7 (d, J = 3.6 Hz). 120.6, 54.2, 52.3, 52.3.19 FNMR (376MHz, deuterated chloroform) δ -60.70, -63.12. HRMS (DART-TOF) calculated value is C 27 H 23 ClF6N3O3 + [M+H] + m / z 586.1327, detected value 586.1333.

[0109] Example 2: Synthesis of methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-{4-[2-methyl-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G2M)

[0110]

[0111] The compound was synthesized following the general synthetic steps described above, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (299.1 mg, 0.8 mmol) and 2-bromo-1-methyl-4-(trifluoromethyl)benzene (S8) (187 μL, 1.2 mmol) as starting materials. Compound G2M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v), yielding a white solid (89.4 mg, overall yield of 21% across five steps).

[0112] 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.15 (s, 2H), 7.91–7.86 (m, 3H), 7.52 (d, J = 8.0 Hz, 2H), 7.38–7.22 (m, 3H), 3.93 (s, 3H), 3.14 (s, 8H), 2.39 (s, 3H). 13 CNMR (101MHz, deuterated chloroform) δ 166.8, 163.9, 151.3, 145.7, 138.7, 136.9, 133.1, 133.1, 131.8, 129.5, 129.2 (d, J = 32.2Hz), 128.4, 127.6, 126.2, 124.4 (d, J = 272.1Hz), 121.2, 120.5, 120.4, 115.9 (q, J = 3.7Hz), 52.5, 52.5, 52.3, 18.2. 19 FNMR (376MHz, deuterated chloroform) δ -62.16. HRMS (DART-TOF) calculated value is C 27 H 26 ClF3N3O3 + [M+H] + m / z 532.1609, detected value 532.1618.

[0113] Example 3: Synthesis of methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dichlorophenyl)piperazin-1-yl]benzoate (G3M)

[0114]

[0115] The compound was synthesized following the general synthetic steps described above, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (299.1 mg, 0.8 mmol) and 2-bromo-1,4-dichlorobenzene (S8) (144 μL, 1.04 mmol) as starting materials. Compound G3M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v), yielding a white solid (212.7 mg, overall yield of 41% across five steps).

[0116] 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.16–9.14 (m, 2H), 7.90–7.86 (m, 3H), 7.53 (d, J = 8.4 Hz, 2H), 7.33 (t, J = 8.0 Hz, 2H), 7.06 (d, J = 2.4 Hz, 1H), 7.01 (dd, J = 8.4, 2.4 Hz, 1H), 3.93 (s, 3H), 3.25 (s, 4H), 3.18–3.06 (m, 4H). 13 CNMR (101MHz, deuterated chloroform) δ 166.8, 163.9, 149.7, 145.6, 138.6, 133.3, 133.2, 133.1, 131.7, 129.5, 128.4, 127.6, 127.2, 126.2, 124.2, 121.1, 121.0, 120.6, 52.3, 52.2, 52.0. HRMS (DART-TOF) calculated value C 25 H 23 Cl3N3O3 + [M+H] + m / z 518.0800, 520.0770, detected values ​​518.0804, 520.0777.

[0117] Example 4: Synthesis of methyl 4-[4-(5-chloro-2-methoxyphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G4M)

[0118]

[0119] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (299.1 mg, 0.8 mmol) and 2-bromo-4-chloro-1-methoxybenzene (S8) (164 μL, 1.5 mmol) as starting materials. Compound G4M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v) to obtain a white solid (61.7 mg, overall yield of 15% across five steps).

[0120] 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.20 (s, 1H), 9.14 (d, J = 1.2 Hz, 1H), 7.89–7.84 (m, 3H), 7.51 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.00 (dd, J = 8.4, 2.0 Hz, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.87 (s, 3H), 3.25 (s, 4H), 3.14 (s, 4H). 13 C10 NMR (101 MHz, deuterated chloroform) δ 166.8, 163.8, 151.0, 145.7, 141.8, 138.5, 133.1, 133.0, 129.4, 128.4, 127.4, 126.1, 126.0, 122.8, 121.0, 120.4, 118.8, 112.4, 55.9, 52.3, 52.2, 51.3. HRMS (DART-TOF) calculated value is C10. 26 H 26 Cl2N3O4+[M+H]+ m / z 514.1295, detected value 514.1299.

[0121] Example 5: Synthesis of methyl 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G5M)

[0122]

[0123] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (336.4 mg, 0.9 mmol) and 2-bromo-4-chloro-1-methylbenzene (S8) (144 μL, 1.08 mmol) as starting materials. Compound G5M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v) to obtain a white solid (129 mg, overall yield of 29% across five steps).

[0124] 1 ¹H NMR (400MHz, deuterated chloroform) δ 9.16 (s, 1H), 9.14 (d, J = 1.6 Hz, 1H), 7.90–7.85 (m, 3H), 7.52 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 7.01 (dd, J = 10.4, 2.4 Hz, 2H), 3.92 (s, 3H), 3.10 (s, 8H), 2.29 (s, 3H). 13 C10 NMR (101 MHz, deuterated chloroform) δ 166.8, 163.9, 152.0, 145.7, 138.6, 133.1, 133.0, 132.3, 132.0, 131.0, 129.4, 128.4, 127.5, 126.1, 123.7, 121.1, 120.4, 119.6, 52.5, 52.4, 52.3, 17.7. HRMS (DART-TOF) calculated value is C10. 26 H 26 Cl2N3O3 + [M+H] + m / z 498.1346, detected value 498.1348.

[0125] Example 6: Synthesis of methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]benzoate (G6M)

[0126]

[0127] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (606.4 mg, 1.5 mmol) and 2-bromo-4-chlorotoluene (S8) (200 μL, 1.5 mmol) as starting materials. Compound G6M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v), yielding a white solid (63.5 mg, overall yield of 8% across five steps).

[0128] 1¹H NMR (400MHz, deuterated chloroform) δ 9.15 (d, J = 8.0 Hz, 2H), 7.86 (dd, J = 8.4, 1.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 10.4 Hz, 2H), 3.92 (s, 3H), 3.10 (s, 8H), 2.29 (s, 3H). 13 C10 NMR (101 MHz, deuterated chloroform) δ 166.8, 164.0, 152.0, 145.7, 133.6, 133.0, 132.4, 132.3, 132.0, 131.0, 128.6, 127.5, 127.1, 126.2, 123.7, 121.1, 120.4, 119.6, 52.5, 52.5, 52.3, 17.7. HRMS (DART-TOF) calculated value is C10. 26 H 26 BrClN3O3 + [M+H] + m / z 542.0841, 544.0820, detected values ​​542.0834, 544.0812.

[0129] Example 7: Synthesis of methyl 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-({[4-(trifluoromethyl)phenyl]carbonyl}amino)benzoate (G7M)

[0130]

[0131] The compound was synthesized following standard synthetic procedures, using methyl 4-(piperazin-1-yl)-3-({[4-(trifluoromethyl)phenyl]carbonyl}amino)benzoate (S7) (590 mg, 1.5 mmol) and 2-bromo-4-chlorotoluene (S8) (200 μL, 1.5 mmol) as starting materials. Compound G7M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v), yielding a white solid (233.1 mg, overall yield of 30% across five steps).

[0132] 1¹H NMR (400MHz, deuterated chloroform) δ 9.26 (s, 1H), 9.17 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.89 (dd, J = 8.4, 2.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.03–7.00 (m, 2H), 3.94 (s, 3H), 3.12 (d, J = 3.2 Hz, 8H), 2.30 (s, 3H). 13 C NMR (101MHz, deuterated chloroform) δ 166.8, 163.6, 152.0, 145.8, 138.0, 134.9 (d, J = 30.5Hz), 132.9, 132.4, 132.0, 131.0, 127.6, 127.5, 126.4, 126.3 (q, J = 2.3Hz), 123.7, 122.4, 121.2, 120.6, 119.6, 52.5, 52.4, 52.4, 17.7. 19 F NMR (376MHz, deuterated chloroform) δ -62.94. HRMS (DART-TOF) calculated value is C 27 H 26 ClF3N3O3 + [M+H] + m / z 532.1609, detected value 532.1614.

[0133] Example 8: Synthesis of methyl 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoate (G8M)

[0134]

[0135] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (209.1 mg, 0.5 mmol) and 2,5-bis(trifluoromethyl)bromobenzene (S8) (130 μL, 0.75 mmol) as starting materials. Compound G8M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v:v) to obtain a white solid (72.3 mg, overall yield of 23% across five steps).

[0136] 1¹H NMR (400MHz, deuterated chloroform) δ 9.16 (s, 1H), 9.12 (d, J = 1.2 Hz, 1H), 7.90–7.77 (m, 4H), 7.68 (d, J = 8.4 Hz, 2H), 7.63 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.18 (d, J = 4.0 Hz, 4H), 3.13 (d, J = 4.0 Hz, 4H). 13 C NMR (101MHz, deuterated chloroform) δ 166.7, 164.0, 152.5, 145.6, 135.1 (q, J = 32.7 Hz), 133.7, 133.0, 132.3, 130.4 (q, J = 28.9 Hz), 128.7, 128.7, 128.6, 127.5, 127.0, 126.2, 124.7 (d, J = 5.4 Hz), 122.0 (d, J = 3.2 Hz), 121.2, 120.7 (d, J = 3.6 Hz), 120.6, 54.2, 52.3, 52.2. 19 F NMR (376MHz, deuterated chloroform) δ -60.68, -63.09. HRMS (DART-TOF) calculated value is C 27 H 23 BrF6N3O3 + [M+H] + The m / z values ​​are 630.0821 and 632.0801, respectively, and the detected values ​​are 630.0821 and 632.0805.

[0137] Example 9: Synthesis of methyl 4-{4-[3,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoate (G9M)

[0138]

[0139] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (167.3 mg, 0.4 mmol) and 3,5-bis(trifluoromethyl)bromobenzene (S8) (103 μL, 0.6 mmol) as starting materials. Compound G9M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v:v) to obtain a white solid (82.0 mg, overall yield of 21% across five steps).

[0140] 1¹H NMR (400MHz, deuterated chloroform) δ 9.13 (d, J = 2.0 Hz, 1H), 9.10 (s, 1H), 7.86 (dd, J = 8.4, 2.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.36 (s, 1H), 7.32 (s, 2H), 7.29 (d, J = 8.4 Hz, 1H), 3.92 (s, 3H), 3.51–3.46 (m, 4H), 3.18–3.09 (m, 4H). 13 C NMR (101MHz, deuterated chloroform) δ 166.7, 164.0, 151.4, 145.2, 133.5, 133.0, 132.8 (q, J = 32.6 Hz), 132.4, 128.5, 127.8, 127.6, 127.1, 126.2, 123.6 (d, J = 813.5 Hz), 123.6 (d, J = 271.2 Hz), 120.8 (d, J = 104.2 Hz), 115.2 (d, J = 3.1 Hz), 113.0, 113.0 (d, J = 7.5 Hz), 52.3, 52.3, 51.8, 49.2. 19 FNMR (376MHz, deuterated chloroform) δ -62.99. HRMS (DART-TOF) calculated value is C 27 H 23 BrF6N3O3 + [M+H] + m / z 630.0821, 632.0801, detected values ​​630.0828, 632.0812.

[0141] Example 10: Synthesis of methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-chloro-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G10M)

[0142]

[0143] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (167.3 mg, 0.4 mmol) and 3-bromo-4-chlorotrifluorotoluene (S8) (90 μL, 0.6 mmol) as starting materials. Compound G10M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v:v), yielding a white solid (85.0 mg, overall yield of 21% across five steps).

[0144] 1¹H NMR (400MHz, deuterated chloroform) δ 9.18–9.10 (m, 2H), 7.87 (dd, J = 8.4, 2.0Hz, 1H), 7.81 (d, J = 8.4Hz, 2H), 7.67 (d, J = 8.4Hz, 2H), 7.51 (d, J = 8.0Hz, 1H), 7.34 (d, J = 8.4Hz, 1H), 7.30–7.27 (m, 2H), 3.92 (s, 3H), 3.30 (s, 4H), 3.22–3.07 (m, 4H). 13 C NMR (101MHz, deuterated chloroform) δ 166.8, 164.0, 149.2, 145.5, 133.6, 133.0, 132.6, 132.4, 131.5, 130.2 (d, J = 64.8 Hz), 128.6, 127.6, 127.1, 126.2, 123.8 (d, J = 270.6 Hz), 120.9 (d, J = 61.9 Hz), 120.9 (q, J = 3.1 Hz), 117.4 (d, J = 3.6 Hz), 52.3, 52.3, 52.2, 51.9. 19 F NMR (376MHz, deuterated chloroform) δ -62.43. HRMS (DART-TOF) calculated value is C 26 H 23 BrClF3N3O3 + [M+H] + m / z 596.0558, 598.0537 Detected values ​​are 596.0558, 598.0536.

[0145] Example 11: Synthesis of methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G11M)

[0146]

[0147] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (167.3 mg, 0.4 mmol) and o-bromotrifluorotoluene (S8) (82 μL, 0.6 mmol) as starting materials. Compound G11M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v:v), yielding a white solid (20.2 mg, overall yield of 9% across five steps).

[0148] 1¹H NMR (400MHz, deuterated chloroform) δ 9.23 (s, 1H), 9.14 (s, 1H), 7.90–7.80 (m, 3H), 7.68 (t, J = 8.4 Hz, 3H), 7.59 (t, J = 7.2 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 3.92 (s, 3H), 3.11 (d, J = 5.6 Hz, 8H). 13 C NMR (101MHz, deuterated chloroform) δ 166.8, 164.0, 151.8, 145.9, 133.8, 133.0, 132.3, 128.6, 127.7, 127.6 (q, J = 5.3Hz), 127.4, 127.1, 126.9, 126.2, 125.5, 125.4, 123.9, 120.9, 120.6, 54.3, 52.5, 52.3. 19 F NMR (376MHz, deuterated chloroform) δ-60.26. HRMS (DART-TOF) calculated value is C 26 H 23 BrF3N3NaO3 + [M+Na] + m / z 584.0767, 586.0747, detected values ​​584.0765, 586.0748.

[0149] Example 12: Synthesis of methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G12M)

[0150]

[0151] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (167.3 mg, 0.4 mmol) and m-bromotrifluorotoluene (S8) (84 μL, 0.6 mmol) as starting materials. Compound G12M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1, v:v) to obtain a white solid (114.5 mg, overall yield of 51% across five steps).

[0152] 1¹H NMR (400MHz, deuterated chloroform) δ 9.15 (s, 1H), 9.14 (s, 1H), 7.86 (dd, J = 8.0, 2.0Hz, 1H), 7.79 (d, J = 8.4Hz, 2H), 7.65 (d, J = 8.4Hz, 2H), 7.40 (t, J = 8.0Hz, 1H), 7.33–7.23 (m, 1H), 7.21–7.05 (m, 3H), 3.92 (s, 3H), 3.42 (t, J = 5.2Hz, 4H), 3.13 (t, J = 5.2Hz, 4H). 13 C NMR (101MHz, deuterated chloroform) δ 166.8, 164.0, 151.1, 145.4, 133.6, 133.0, 132.4, 131.8 (q, J = 31.8 Hz), 129.9, 128.5, 127.7, 127.1, 126.2, 124.3 (d, J = 271.0 Hz), 121.2, 120.3, 119.2, 116.8 (d, J = 3.8 Hz), 112.6 (d, J = 3.9 Hz), 52.3, 52.0, 49.8. 19 F NMR (376MHz, deuterated chloroform) δ -62.71. HRMS (DART-TOF) calculated value is C 26 H 23 BrF3N3NaO3 + [M+Na] + m / z 584.0767, 586.0747, detected values ​​are 584.0760, 586.0743.

[0153] Example 13: Synthesis of methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dimethylphenyl)cyclohexyl]benzoate (B28M)

[0154]

[0155] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-(piperazin-1-yl)benzoate (S7) (299.1 mg, 0.8 mmol) and 2,5-dimethylbromobenzene (S8) (144 μL, 1.04 mmol) as starting materials. Compound B28M was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v:v) to obtain a white solid (71.7 mg, overall yield of 19% across five steps).

[0156] 1¹H NMR (400MHz, deuterated chloroform) δ 9.22 (s, 1H), 9.15 (d, J = 2.0 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.87 (dd, J = 8.4, 2.0 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.87–6.85 (m, 2H), 3.92 (s, 3H), 3.11 (s, 8H), 2.35 (s, 3H), 2.30 (s, 3H). 13 C NMR (101MHz, deuterated chloroform) δ 166.9, 163.9, 150.8, 145.9, 138.5, 136.4, 133.3, 133.1, 131.3, 129.4, 129.4, 128.5, 127.4, 126.2, 124.4, 121.0, 120.5, 119.7, 52.7, 52.6, 52.3, 21.4, 17.6. HRMS (DART-TOF) calculated value C 27 H 29 ClN3O3 + [M+H] + m / z 478.1892, detected value 478.1893.

[0157] Example 14: Synthesis of 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-chlorophenyl)carbonyl]amino}benzoic acid (G1)

[0158]

[0159] The compound was synthesized following standard synthetic procedures, starting with methyl 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G1M) (117.2 mg, 0.2 mmol). Compound G1 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (112.1 mg, 98% yield).

[0160] 1¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.25 (s, 1H), 9.07 (d, J = 1.6 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.0 Hz, 1H), 7.85–7.78 (m, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 1H), 3.25 (d, J = 4.0 Hz, 4H), 3.18 (d, J = 4.0 Hz, 4H). 13 C NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.8, 153.8, 147.0, 138.3, 135.5 (q, J = 33.1 Hz), 134.7, 133.8, 130.6 (d, J = 29.9 Hz), 129.5, 129.4, 129.4, 129.3, 128.3, 126.6, 125.73 (d, J = 6.5 Hz), 123.0, 122.3 (d, J = 3.7 Hz), 121.5 (d, J = 3.7 Hz), 120.6, 54.6, 52.7. 19 F NMR (376 MHz, deuterated tetrahydrofuran) δ -61.13, -63.90. HRMS (DART-TOF) calculated value is C 26 H 21 ClF6N3O3 + [M+H] + m / z 572.1170, detected value 572.1176.

[0161] Example 15: Synthesis of 3-{[(4-chlorophenyl)carbonyl]amino}-4-{4-[2-methyl-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoic acid (G2)

[0162]

[0163] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-{4-[2-methyl-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G2M) (106.4 mg, 0.2 mmol) as the starting material. Compound G2 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (99.4 mg, 96% yield).

[0164] 1¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.27 (s, 1H), 9.08 (d, J = 1.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.81 (dd, J = 8.4, 1.6 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.40–7.32 (m, 3H), 7.28 (d, J = 8.0 Hz, 1H), 3.19 (s, 8H), 2.41 (s, 3H). 13 C NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.7, 152.6, 147.1, 138.3, 137.7, 134.5, 133.8, 132.3, 129.6, 129.4 (q, J = 31.6 Hz), 129.4, 128.2, 126.6, 125.4 (d, J = 270.1 Hz), 123.0, 120.5, 120.3 (d, J = 3.9 Hz), 116.1 (d, J = 3.7 Hz), 52.8, 52.8, 18.1. 19 F NMR (376 MHz, deuterated tetrahydrofuran) δ -62.88. HRMS (DART-TOF) calculated value is C 26 H 24 ClF3N3O3 + [M+H] + m / z 518.1453, detected value 518.1461.

[0165] Example 16: Synthesis of 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dichlorophenyl)piperazin-1-yl]benzoic acid (G3)

[0166]

[0167] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dichlorophenyl)piperazin-1-yl]benzoate (G3M) (103.8 mg, 0.2 mmol) as the starting material. Compound G3 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (92.9 mg, yield 92%).

[0168] 1¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.30 (s, 1H), 9.09 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 6.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 1.6 Hz, 1H), 7.04 (dd, J = 8.4, 2.0 Hz, 1H), 3.29 (s, 4H), 3.16 (s, 4H). 13 C10 NMR (101 MHz, deuterated tetrahydrofuran) δ 163.7, 151.1, 146.8, 138.2, 134.6, 133.8, 132.2, 129.6, 129.4, 127.6, 126.6, 124.3, 122.9, 121.7, 120.6, 52.60, 52.4. HRMS (DART-TOF) calculated value is C10. 24 H 21 Cl3N3O3 + [M+H] + m / z 504.0643, detected value 504.0651.

[0169] Example 17: Synthesis of 4-[4-(5-chloro-2-methoxyphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoic acid (G4)

[0170]

[0171] The compound was synthesized following standard synthetic procedures, using methyl 4-[4-(5-chloro-2-methoxyphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G4M) (102.9 mg, 0.2 mmol) as the starting material. Compound G4 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (90.1 mg, 90% yield).

[0172] 1 ¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.32 (s, 1H), 9.09 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.80 (dd, J = 8.4, 2.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 6.94–6.88 (m, 3H), 3.82 (s, 3H), 3.28 (s, 4H), 3.16–3.10 (m, 4H). 13C10 NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.6, 152.2, 147.1, 143.3, 138.2, 134.5, 133.9, 129.6, 129.4, 128.2, 126.5, 126.3, 122.8, 122.5, 120.5, 119.0, 113.5, 55.9, 52.7, 51.6. HRMS (DART-TOF) calculated value C10. 25 H 24 Cl2N3O4 + [M+H] + m / z 500.1138, detected value 500.1140.

[0173] Example 18: Synthesis of 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoic acid (G5)

[0174]

[0175] The compound was synthesized following standard synthetic procedures, using methyl 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-{[(4-chlorophenyl)carbonyl]amino}benzoate (G5M) (99.7 mg, 0.2 mmol) as the starting material. Compound G5 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (92.0 mg, 95% yield).

[0176] 1 ¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.28 (s, 1H), 9.09 (d, J = 1.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.80 (dd, J = 8.4, 1.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.0, 2.0 Hz, 1H), 3.14 (s, 8H), 2.30 (s, 3H). 13 C10 NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.6, 153.3, 147.1, 138.3, 134.5, 133.8, 132.7, 132.3, 131.7, 129.6, 129.4, 128.2, 126.5, 123.7, 122.9, 120.5, 120.0, 52.9, 52.8, 17.5. HRMS (DART-TOF) calculated value C10. 25 H24 Cl2N3O3 + [M+H] + m / z 484.1189, detected value 484.1188.

[0177] Example 19: Synthesis of 3-{[(4-bromophenyl)carbonyl]amino}-4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]benzoic acid (G6)

[0178]

[0179] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]benzoate (G6M) (108.6 mg, 0.2 mmol) as the starting material. Compound G6 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (89.4 mg, yield 93%).

[0180] 1 ¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.29 (s, 1H), 9.08 (d, J = 1.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.81 (dd, J = 8.4, 1.6 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.97 (dd, J = 8.0, 2.0 Hz, 1H), 3.14 (s, 8H), 2.30 (s, 3H). 13 C10 NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.8, 153.3, 147.1, 134.9, 133.7, 132.7, 132.6, 132.3, 131.7, 129.5, 128.2, 126.8, 126.6, 123.7, 122.9, 120.5, 120.0, 52.9, 52.8, 17.5. HRMS (DART-TOF) calculated value C10. 25 H 24 BrClN3O3 + [M+H] + m / z 528.0684, 530.0664, detected values ​​528.0688, 530.0670.

[0181] Example 20: Synthesis of 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-({[4-(trifluoromethyl)phenyl]carbonyl}amino)benzoic acid (G7)

[0182]

[0183] The compound was synthesized following standard synthetic procedures, using methyl 4-[4-(5-chloro-2-methylphenyl)piperazin-1-yl]-3-({[4-(trifluoromethyl)phenyl]carbonyl}amino)benzoate (G7M) (106.4 mg, 0.2 mmol) as the starting material. Compound G7 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (91.2 mg, yield 88%).

[0184] 1 ¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.39 (s, 1H), 9.11 (d, J = 1.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.81 (dd, J = 8.4, 1.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.96 (dd, J = 8.0, 2.0 Hz, 1H), 3.14 (s, 8H), 2.30 (s, 3H). 13 C NMR (101 MHz, deuterated tetrahydrofuran) δ 167.3, 163.6, 153.3, 147.3, 139.3, 133.6, 133.5 (d, J = 32.1 Hz), 132.7, 132.3, 131.6, 131.3 (d, J = 94.3 Hz), 128.5, 128.1, 126.8, 126.43 (q, J = 3.6 Hz), 124.8 (d, J = 270.7 Hz), 123.6, 123.0, 120.6, 120.0, 119.5 (d, J = 127.5 Hz), 52.8, 17.6. 19 F NMR (376 MHz, deuterated tetrahydrofuran) δ -63.49. HRMS (DART-TOF) calculated value is C 26 H 24 ClF3N3O3 + [M+H] + m / z 518.1453, detected value 518.1460.

[0185] Example 21: Synthesis of 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoic acid (G8)

[0186]

[0187] The compound was synthesized following standard synthetic procedures, starting with methyl 4-{4-[2,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoate (G8M) (126.1 mg, 0.2 mmol). Compound G8 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (117.1 mg, 95% yield).

[0188] 1 H NMR(400MHz,DMSO)δ9.64(s,1H),8.47(s,1H),8.00–7.91(m,3H),7.86(s,1H),7 .77(d,J=8.0Hz,3H),7.71(d,J=8.0Hz,1H),7.35(d,J=8.4Hz,1H),3.13(s,8H). 13 C NMR (101MHz, DMSO) δ166.9,164.0,152.8,148.6,133.8(d,J=32.2Hz),133.4,131.7,131.2,129.5,128.8(d,J=14.0H z), 127.0, 125.6 (d, J = 5.2Hz), 125.1, 124.7 (d, J = 6.0Hz), 121.9 (d, J = 3.8Hz), 121.2 (d, J = 1.8Hz), 120.0, 53.0, 51.1. 19 F NMR (376MHz, DMSO) δ -59.31, -61.64. HRMS (DART-TOF) calculated value is C 26 H 21 BrF6N3O3 + [M+H] + m / z 616.0665, 618.0645, detected values ​​616.0663, 618.0647.

[0189] Example 22: Synthesis of 4-{4-[3,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoic acid (G9)

[0190]

[0191] The compound was synthesized following standard synthetic procedures, using methyl 4-{4-[3,5-bis(trifluoromethyl)phenyl]piperazin-1-yl}-3-{[(4-bromophenyl)carbonyl]amino}benzoate (G9M) (126.1 mg, 0.2 mmol) as the starting material. Compound G9 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (114.6 mg, 93% yield).

[0192] 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 12.82 (s, 1H), 9.74 (s, 1H), 8.45 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.80–7.76 (m, 3H), 7.53 (s, 2H), 7.32 (d, J = 8.0 Hz, 2H), 3.54 (s, 4H), 3.11 (s, 4H). 13 C NMR (101 MHz, deuterated dimethyl sulfoxide) δ 166.9, 164.0, 151.5, 148.6, 133.3, 131.8, 131.3, 131.2, 131.1 (d, J = 95.9 Hz), 131.0, 129.5, 127.0, 125.7 (d, J = 2.9 Hz), 125.3, 125.0, 123.6 (d, J = 815.7 Hz), 122.2, 119.9, 114.4 (d, J = 2.4 Hz), 110.2 (d, J = 4.1 Hz), 50.4, 47.3. 19 FNMR (376 MHz, deuterated dimethyl sulfoxide) δ -61.40. HRMS (DART-TOF) calculated value: C 26 H 21 BrF6N3O3 + [M+H] + m / z 616.0665, 618.0645, detected values ​​616.0658, 618.0641.

[0193] Example 23: Synthesis of 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-chloro-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoic acid (G10)

[0194]

[0195] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-chloro-5-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G10M) (119.4 mg, 0.2 mmol) as the starting material. Compound G10 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (110.7 mg, 95% yield).

[0196] 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 12.79 (s, 1H), 9.68 (s, 1H), 8.46 (s, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.3 Hz, 3H), 7.67 (d, J = 8.0 Hz, 1H), 7.43–7.40 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H), 3.25 (s, 4H), 3.16 (s, 4H). 13 C NMR (101 MHz, deuterated dimethyl sulfoxide) δ 166.9, 164.1, 149.4, 148.6, 133.4, 131.8, 131.7, 131.5, 131.3, 129.5, 128.8 (d, J = 31.9 Hz), 127.0, 125.6, 125.2, 122.4, 120.4 (d, J = 2.9 Hz), 120.0, 117.4 (d, J = 3.2 Hz), 50.8, 50.7. 19 F NMR (376 MHz, deuterated dimethyl sulfoxide) δ -60.91. HRMS (DART-TOF) calculated value is C 25 H 21 BrClF3N3O3 + [M+H] + m / z 582.0401, 584.0381, detected values ​​582.0406, 584.0386.

[0197] Example 24: Synthesis of 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-(trifluoromethyl)phenyl]piperazin-1-yl}benzoic acid (G11)

[0198]

[0199] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[2-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G11M) (112.5 mg, 0.2 mmol) as the starting material. Compound G11 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (103.1 mg, 94% yield).

[0200] 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 9.68 (s, 1H), 8.49 (d, J = 1.6 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.82–7.75 (m, 3H), 7.69 (t, J = 7.6 Hz, 2H), 7.60 (d, J = 8.0 Hz, 1H), 7.43–7.26 (m, 2H), 3.08 (s, 4H), 3.05 (s, 4H). 13 C NMR (101 MHz, deuterated dimethyl sulfoxide) δ 167.0, 164.0, 152.0, 148.7, 133.7, 133.5, 131.8, 131.3, 129.5, 127.1, 127.0, 125.7, 125.6, 125.5 (d, J = 2.6 Hz), 125.4, 125.0, 124.6, 122.8, 120.0, 53.4, 51.3. 19 F NMR (376 MHz, deuterated dimethyl sulfoxide) δ -58.69. HRMS (DART-TOF) calculated value is C 25 H 22 BrF3N3O3 + [M+H] + m / z 548.0791, 550.0771, detected values ​​548.0787, 550.0770.

[0201] Example 25: Synthesis of 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}benzoic acid (G12)

[0202]

[0203] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-bromophenyl)carbonyl]amino}-4-{4-[3-(trifluoromethyl)phenyl]piperazin-1-yl}benzoate (G12M) (112.5 mg, 0.2 mmol) as the starting material. Compound G12 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (92.1 mg, yield 84%).

[0204] 1 ¹H NMR (400 MHz, deuterated dimethyl sulfoxide) δ 9.70 (s, 1H), 8.50 (s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27–7.18 (m, 2H), 7.08 (d, J = 7.6 Hz, 1H), 3.41 (s, 4H), 3.10 (s, 4H). 13 C NMR (101 MHz, deuterated dimethyl sulfoxide) δ 167.0, 164.0, 151.1, 148.6, 141.9, 133.4, 131.8, 131.3, 130.1, 130.0 (d, J = 30.7 Hz), 129.4, 127.0, 125.7, 125.2, 124.5 (d, J = 270.8 Hz), 119.8, 118.9, 114.9, 111.1, 50.6, 48.0. 19 F NMR (376 MHz, deuterated dimethyl sulfoxide) δ -61.14. HRMS (DART-TOF) calculated value is C 25 H 22 BrF3N3O3 + [M+H] + m / z 548.0791, 550.0771, detected values ​​548.0794, 550.0777.

[0205] Example 26: Synthesis of 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dimethylphenyl)piperazin-1-yl]benzoic acid (B28)

[0206]

[0207] The compound was synthesized following standard synthetic procedures, using methyl 3-{[(4-chlorophenyl)carbonyl]amino}-4-[4-(2,5-dimethylphenyl)cyclohexyl]benzoate (B28M) (95.6 mg, 0.2 mmol) as the starting material. Compound B28 was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v), yielding a white solid (85.4 mg, yield 92%).

[0208] 1¹H NMR (400 MHz, deuterated tetrahydrofuran) δ 9.30 (s, 1H), 9.10 (d, J = 1.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.81 (dd, J = 8.4, 2.0 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H), 6.91 (s, 1H), 6.77 (d, J = 7.6 Hz, 1H), 3.13 (d, J = 4.0 Hz, 8H), 2.29 (s, 3H), 2.28 (s, 3H). 13 C10 NMR (101 MHz, deuterated tetrahydrofuran) δ 167.2, 163.6, 151.9, 147.2, 138.2, 136.5, 134.6, 133.8, 131.4, 129.8, 129.5, 129.4, 128.1, 126.5, 124.5, 122.8, 120.5, 120.2, 53.2, 53.0, 21.1, 17.5. HRMS (DART-TOF) calculated value C10. 26 H 27 ClN3O3 + [M+H] + m / z 464.1735, detected value 464.1737.

[0209] The following specific experimental examples demonstrate the beneficial effects of the present invention.

[0210] Experimental Example 1: Inhibitory effect of the compound of the present invention on CRM1

[0211] 1. Experimental Methods

[0212] 1.1 Pull-down experiment:

[0213] To test the CRM1 inhibitory activity of different compounds of this invention, NES sequence protein (MVM) with a 0.5 μM GST tag was used. MVM Immobilized on glutathione agarose beads, 1 μM CRM1 and different concentrations of compounds were combined with immobilized GST-NES. MVMIncubate at 4°C with a total volume of 500 μL for 1.5 hours using pull-down buffer. Centrifuge at 3800 rpm for 1 min at 4°C, discard the supernatant, add pull-down buffer, wash thoroughly by inverting the EP tube, and centrifuge again. Repeat this washing step three times. Separate the bound proteins by SDS / PAGE gel electrophoresis and visualize by Coomassie brilliant blue staining. The pull-down buffer contains 20 mM tris(hydroxymethyl)aminomethane (pH 8.0), 200 mM sodium chloride, 10% (v / v) glycerol, 2 mM magnesium chloride, 0.005% Triton X-100, and 1 mM dithiothreitol.

[0214] 1.2 Micro-heat surge competition difference test (MST):

[0215] MBP-PKI NES (SEQ ID NO.1: NSNELALKLAGLDI) was mutated to CSNELALKLAGLDI (SEQ ID NO.2) to specifically bind to the fluorescent molecule 5-iodoacetamide fluorescein (IAF). The MBP tag was removed by TEV restriction enzyme digestion. To determine the affinity of PKI, different concentrations of CRM1 were co-incubated with 50 nM IAF-PKI (PKI*) and 10 μM Ran-GTP at 25 °C for 1 h. In the competition experiment, different concentrations of the compound were incubated with 350 nM CRM1, 50 nM PKI*, and 10 μM Ran-GTP. After incubation, the samples were measured using a Nano Temper NT.115 instrument at 20% LED power and 60% MST power. The binding affinity of CRM1 was determined using PALMIST software.

[0216] 2. Experimental Results

[0217] The inhibitory activity of the compound of this invention against CRM1 was tested using a pull-down experiment, and the results are as follows: Figure 1 As shown in A and 1B. Experimental results ( Figure 1 A) It was found that, except for compound G4, which showed weaker activity against CRM1 than compound B28, all other compounds exhibited better activity against CRM1 than compound B28. This indicates that, compared to compound B28, the larger substituents on the benzene ring of the piperazine linked to the compound effectively inhibit CRM1. Furthermore, comparing the inhibitory activities of compounds G1 and G3 shows that substitution of the CF3 group is more effective than that of the Cl atom; comparing the inhibitory activities of G3 and B28 shows that substitution of the Cl atom is more effective than that of the CH3 group; comparing G8 with G11 or G12 shows that the disubstituted compounds are more effective at inhibiting CRM1 than the monosubstituted compounds, and that di-meta substitution is also possible. G4 almost completely lost its activity against CRM1, indicating that OMe substitution is not allowed at the ortho position on the benzene ring linked to the piperazine. Notably, methylation of the carboxyl group completely eliminated the compound's inhibitory effect on CRM1. Figure 1 B, G1M-G12M).

[0218] The binding affinity of G1, G8, and G9 to CRM1 was determined using the micro-thermal surge (MST) differential competitive method, and the affinity constants KD were 2.3 μM, 3.2 μM, and 4.7 μM, respectively. Figure 1 C Figure 2 A and Figure 2 B).

[0219] Experimental Example 2: Inhibitory effect of the compound of the present invention on tumor cells

[0220] 1. Experimental Methods

[0221] Nuclear export inhibition assay: A549 cells were seeded into 24-well cell culture plates and treated with a specified concentration of compound or DMSO for 4 h. Cells were fixed and permeabilized, blocked, and then incubated with primary antibody, secondary antibody, and Dapi before mounting. The cells were then observed and photographed using an Olympus FV1000 confocal microscope and analyzed using NIH ImageJ software. Anti-NFkB (Proteintech, rabbit) and anti-rabbit (JacksonLab, FITC) antibodies were used at 1:200 and 1:1000 dilutions, respectively.

[0222] Cell heat transfer assay (CESTA): HeLa cells were cultured to 80% confluence in 10 cm culture dishes and collected in PBS containing 1 mm PMSF. Cells were lysed and centrifuged to remove cell debris. The supernatant was incubated with 20 μM compound or DMSO and then heated at different temperatures for 3 min. The heated sample was centrifuged at 10,000 rpm for 10 min. The supernatant was used for sample preparation and Western blot analysis.

[0223] Cell viability assay: In this invention, cancer cells including HeLa, HepG2, Panc-1, A2780, MDA-MB-453, SW480, A549, HUCC-T1, OVCAR-8, and Ishikawa were seeded in 96-well plates (500 cells / well) and cultured overnight at 37°C. The following morning, after cell attachment, 20 μM of the test compound was added to the cells for 72 h. Cells were fixed with 10% (w / v) trichloroacetic acid, stained with sulfanilamide (SRB) for 30 min, and repeatedly washed with 1% (v / v) acetic acid to remove excess dye. 10 mM Tris base solution was added to each well to dissolve the dye. OD values ​​were measured at 570 nm using a microplate reader.

[0224] 2. Experimental Results

[0225] B28 and its compounds G1-G12 exhibited different growth-inhibiting effects on HeLa cells. Their cell growth-inhibiting effects were largely consistent with the inhibitory activities observed in the pull-down assay. Figure 3 A). Among them, compounds G1, G8, and G9 exhibited the strongest inhibitory activity against cell growth. The IC50 values ​​of G1, G8, and G9 in HeLa cells were 14.0 μM, 17.0 μM, and 8.5 μM, respectively. Figure 3 B). Similar to known CRM1 inhibitors, these three compounds can broadly inhibit the growth of cancer cells, such as pancreatic cancer, endometrial cancer, colorectal cancer, and breast cancer. Figure 3 C). Furthermore, the cell heat transfer experiment (CESTA) conducted in this invention showed that these three compounds can improve the thermal stability of CRM1 in HeLa cells. Figure 3 D). Furthermore, compounds G1, G8, and G9 all inhibited nuclear export of CRM1 cargoes from A549 cells or semi-permeable HeLa cells. Figure 3 E and Figure 4 ).

[0226] The cysteine ​​residue at position 528 in the CRM1 NES binding groove (hereinafter referred to as C528) can undergo a Michael addition reaction with certain compounds containing unsaturated bonds, i.e., covalent binding. These small molecules are called CRM1 covalent inhibitors. Non-covalent inhibitors generally occupy the CRM1 NES binding groove through non-covalent interactions such as hydrophobic interactions, electrostatic interactions, and hydrogen bonds, and are therefore called CRM1 non-covalent inhibitors. DTT (dithiothreitol) is a strong reducing agent that can promote the reduction reaction of the unsaturated bonds in covalent inhibitors. By co-incubating DTT, the inhibitor, CRM1, and the NES sequence protein, and verifying the effect through a pull-down assay, if the inhibitor can inhibit the binding of CRM1 to NES without DTT incubation, but loses its inhibitory effect on CRM1 upon the addition of DTT, then the compound is a covalent inhibitor. Conversely, if the inhibitor's inhibitory effect on CRM1 is unaffected in the presence of DTT, then the inhibitor is a non-covalent inhibitor. Therefore, the control experiment with added DTT described above proves that the compound of the present invention inhibits CRM1 as a non-covalent inhibitor.

[0227] In summary, this invention provides a class of compounds that can be used as non-covalent CRM1 inhibitors. These compounds exhibit good inhibitory effects on CRM1 and can be used to inhibit the growth of various cancer cells, showing promising application prospects.

Claims

1. A compound or a salt thereof, characterized in that: The compound is shown in Formula II: Formula II in, R1 is selected from trifluoromethyl; n is the number of substituents R2 on the benzene ring, selected from 1 or 2; Each R2 is independently selected from trifluoromethyl, methyl, and halogen, and R2 is not selected from two methyl groups at the same time.

2. A compound or a salt thereof, characterized in that: The compound is shown in formula IIIa: Formula IIIa in, R1 is selected from trifluoromethyl, chlorine, and bromine; R 21 R 22 Each of the following is independently selected from trifluoromethyl, chlorine, bromine, and fluorine; Alternatively, the compound may be of formula IIIb: Formula IIIb in, R1 is selected from trifluoromethyl, chlorine, and bromine; R 21 R 23 Each of the following is independently selected from trifluoromethyl, methyl, chlorine, bromine, and fluorine.

3. A compound or a salt thereof, characterized in that: The compound is shown in formula IIId: Formula IIId in, R1 is selected from trifluoromethyl, chlorine, and bromine; R 21 Selected from trifluoromethyl, methyl, chlorine, bromine, and fluorine.

4. A compound or a salt thereof, characterized in that: The compound is shown in formula V: Formula V in, R1 is selected from trifluoromethyl, chlorine, bromine, and fluorine.

5. A compound or a salt thereof, characterized in that: The compound has one of the following structures: 。 6. A method for preparing the compound according to any one of claims 1 to 5, characterized in that: Includes the following steps: (1) In a solvent, compounds S1 and S2 react with N,N-dimethylethylenediamine to give compound S3; (2) In a solvent, compound S3, ammonium formate and zinc powder react to give compound S4; (3) In a solvent, compound S4, triethylamine and compound S5 react to give compound S6; (4) In a solvent, compound S6 reacts with trifluoroacetic acid to give compound S7; (5) In a solvent, compounds S7, S8, BINAP, and Cs2CO3 react under the action of a catalyst to obtain compound S9; (6) In a solvent, compound S9 reacts with lithium hydroxide to give compound S10; m, R1, n, and R2 are as described in any one of claims 1 to 5.

7. The method according to claim 6, characterized in that: In step (1), the solvent is ethanol; And / or, in step (1), the molar ratio of compound S1, compound S2 and N,N-diisopropylethylamine is 1:1.1~1.5:1~2; And / or, in step (1), the reaction conditions are reflux reaction at 80~100℃ for 1~10 hours; And / or, in step (2), the solvent is methanol; And / or, in step (2), the molar ratio of compound S3, ammonium formate and zinc powder is 1:10~15:10~15; And / or, in step (2), the reaction conditions are 20~40℃ for 5~10 hours; And / or, in step (3), the solvent is dichloromethane; And / or, in step (3), the molar ratio of compound S4, triethylamine and compound S5 is 1:1~5:1~1.5; And / or, in step (3), the reaction is carried out at 20~40℃ for 4-8 hours; And / or, in step (4), the solvent is dichloromethane; And / or, in step (4), the molar volume ratio of compound S6 to trifluoroacetic acid is 1~5 mmol: 1 mL; And / or, in step (4), the reaction conditions are 20~40℃ for 1~10 hours; And / or, in step (5), the solvent is toluene; And / or, in step (5), the catalyst is Pd2(dba)3; And / or, in step (5), the molar ratio of compound S7, compound S8, BINAP, Cs2CO3, and catalyst is 1:1~1.5:0.1~0.5:1~5:0.01~0.05; And / or, in step (5), the reaction conditions are 100~120℃ for 10~20 hours; And / or, in step (6), the solvent is a mixed solution of water and tetrahydrofuran; And / or, in step (6), the molar ratio of compound S9 to lithium hydroxide is 1:5~10; And / or, in step (6), the reaction conditions are 40~60℃ for 10~20 hours.

8. The method according to claim 6, characterized in that: In step (1), after the reaction, the following steps were used to purify compound S3: the solvent was removed by rotary evaporation of the reaction solution, the remaining mixture was diluted with dichloromethane and extracted with saturated citric acid and saturated brine respectively, dried with anhydrous sodium sulfate, filtered and then the dichloromethane was removed by rotary evaporation, and the residue was purified by silica gel column chromatography. And / or, in step (2), after the reaction, the following steps are used to purify the compound S4: the reaction solution is filtered with diatomaceous earth, the solvent is removed by rotary evaporation of the filtrate, the residue is dissolved in dichloromethane, the insoluble matter is removed by diatomaceous earth filtration, the solvent is removed by rotary evaporation, and the residue is purified by silica gel column chromatography. And / or, in step (3), after the reaction, the following steps are used to purify compound S6: the reaction solution is desolventized under vacuum, the residue is diluted with water and extracted with ethyl acetate, the organic phase is back-extracted with saturated brine and saturated citric acid aqueous solution, dried with anhydrous sodium sulfate, filtered and then the solvent is removed by rotary evaporation, and the residue is purified by silica gel column chromatography. And / or, in step (4), after the reaction, the following steps are used to purify the compound S7: the pH of the reaction solution is adjusted to 9-10, extracted with dichloromethane, the organic phase is back-extracted with saturated brine, the organic phase is dried with anhydrous sodium sulfate, and the solvent is removed under vacuum after filtration. And / or, in step (5), after the reaction, the following steps are used to purify the compound S9: the reaction solution is filtered with diatomaceous earth, the solvent is removed from the filtrate under vacuum, the residue is dissolved with ethyl acetate and extracted with semi-saturated brine, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed under vacuum after filtration, and the residue is purified by silica gel column chromatography. And / or, in step (6), after the reaction, the compound S10 is purified by the following steps: pH of the reaction solution is adjusted to 5.

0.

9. Use of the compound or salt thereof according to any one of claims 1 to 5 in the preparation of CRM1 inhibitors.

10. The use according to claim 9, characterized in that: The CRM1 inhibitor is a non-covalent CRM1 inhibitor.

11. Use of the compound or salt thereof according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of diseases associated with CRM1; wherein the diseases associated with CRM1 are pancreatic cancer, endometrial cancer, colorectal cancer, breast cancer, ovarian cancer, lung cancer, and cervical cancer.

12. A drug, characterized in that: It is a formulation prepared by adding pharmaceutically acceptable excipients to the compound or its salt as the active ingredient according to any one of claims 1 to 5.

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