A VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment and a preparation method and application thereof

Abstract

The application provides a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment and a preparation method and application thereof, and relates to the technical field of medicinal chemistry.A VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment has a structural formula as shown in formula A.The VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment with a novel structure is designed and synthesized by using the splicing principle, and the anti-proliferation activity of the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment on cervical cancer cells Siha and chronic myeloid leukemia cells K562 is tested, and the result shows that the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment provided by the application has good inhibitory activity on Siha cells and K562 cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment and a preparation method and application thereof. BACKGROUND

[0002] The vascular endothelial growth factor (VEGF) family tyrosine kinase receptors consists of three protein receptors (VEGFR1, VEGFR2 and VEGFR3). Among the receptors of VEGF, VEGFR2 is the main receptor and plays an important role in regulating endothelial cell proliferation, regulating angiogenesis, etc. Overexpression of VEGFR2 can lead to the occurrence and development of various cancers. Therefore, VEGFR2 has become a hotspot in the current research on anti-tumor angiogenesis. Although many small molecule inhibitors targeting VEGFR2 have been approved for the treatment of tumors, many patients treated with VEGFR2 inhibitors have developed drug resistance and tumor recurrence.

[0003] Histone deacetylase 6 (HDAC6) is a zinc-dependent metalloproteinase, which is highly expressed in various tumor cells and plays an important role in the migration and invasion of tumor cells. There are currently 18 subtypes of HDACs, which can be divided into four families (I, II, III and IV). Among them, HDAC6 is the most widely studied subtype in the IIb subfamily containing two enzyme catalytic regions.

[0004] Many preclinical studies have shown that the combination of HDAC6 inhibitors and VEGFR2 inhibitors has great prospects in overcoming the drug resistance of VEGFR2 inhibitors alone and enhancing the anti-tumor efficacy. Therefore, the development of VEGFR2-HDAC6 dual inhibitors has broad application prospects.

[0005] SUMMARY

[0006] Based on the technical problems existing in the background art, the present application provides a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment and a preparation method and application thereof. The compound containing a chalcone fragment can not only be used for preparing a VEGFR2-HDAC6 dual inhibitor, but also be used for preparing a drug for preventing and / or treating diseases related to abnormal expression of VEGFR2 and HDAC6 activity.

[0007] The technical problems solved by the present application are solved by the following technical solutions:

[0008] One of the purposes of the present application is to provide a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, which comprises a compound of formula A:

[0009]

[0010] Preferably, the VEGFR2-HDAC6 dual inhibitor containing chalcone fragment includes the compound of formula A or pharmaceutically acceptable salt thereof.

[0011] Preferably, the VEGFR2-HDAC6 dual inhibitor containing chalcone fragment includes the compound of formula A, which includes formula A-I or formula A-II:

[0012]

[0013] R is selected from at least one of halogen, substituted or unsubstituted alkyl, alkoxy.

[0014] Preferably, the halogen is selected from one or more of F and Cl.

[0015] Preferably, the alkyl is selected from one or more of methyl and ethyl.

[0016] Preferably, when the substituted or unsubstituted group contains a substituent, the substituent is selected from F or Cl.

[0017] More preferably, the substituted alkyl is selected from one or more of trifluoromethyl and trichloromethyl.

[0018] Preferably, the alkoxy is selected from one or more of methoxy and ethoxy.

[0019] More preferably, R is selected from one of 4-Cl-3-CF3, 4-CH3O, 4-F, 4-CH3-3-CF3, and 4-CH3-3-Cl.

[0020] Preferably, the VEGFR2-HDAC6 dual inhibitor containing chalcone fragment includes any one of the following compounds:

[0021]

[0022] The second object of the present application is to provide a preparation method of the VEGFR2-HDAC6 dual inhibitor containing chalcone fragment, which includes the following steps:

[0023] S1, reacting aminoacetophenone with triphosgene, and then performing urea synthesis reaction with substituted aniline to obtain intermediate MA;

[0024] S2, performing condensation reaction of intermediate MA with 4-formylbenzoic acid to obtain intermediate MB;

[0025] S3, amide condensation reaction of intermediate MB with O-(tetrahydro-2H-pyran)-2-hydroxyamine to obtain intermediate MC;

[0026] S4, mixed intermediate MC, solvent, and deprotection reaction under acidic conditions to obtain compound of formula A.

[0027] The synthetic route is as follows:

[0028]

[0029] Preferably, in S1, the urea synthesis reaction is carried out under the catalysis of an organic base.

[0030] More preferably, the organic base is selected from at least one of triethylamine, pyridine, N,N-diisopropylethylamine.

[0031] Preferably, in S1, the molar ratio of aminoacetophenone to triphosgene is 3:1, and the molar ratio of aminoacetophenone to substituted aniline is 1:(1-1.1).

[0032] Preferably, in S2, the condensation reaction is carried out under basic conditions.

[0033] More preferably, the basic conditions are provided by a basic reagent selected from at least one of sodium hydroxide, potassium hydroxide, lithium hydroxide.

[0034] Preferably, in S2, the reaction temperature is 10-35°C.

[0035] Preferably, in S2, the molar ratio of intermediate MA to 4-formylbenzoic acid is 1:(1-1.1).

[0036] Preferably, in S3, the amide condensation reaction is carried out in the presence of a condensing agent.

[0037] More preferably, the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), carbonyldiimidazole (CDI), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-n-propylphosphonic anhydride (T3P).

[0038] Preferably, in S3, the reaction temperature is 10-35°C.

[0039] Preferably, in S3, the molar ratio of intermediate MB to O-(tetrahydro-2H-pyran)-2-hydroxyamine is 1:(1-1.2).

[0040] Preferably, in the S4, the solvent is selected from one or more of methanol, acetonitrile, acetone.

[0041] Preferably, in the S4, the acidic condition is provided by an acidic reagent selected from one or more of hydrochloric acid, dilute sulfuric acid, trifluoroacetic acid.

[0042] Preferably, in the S4, the volume ratio of the intermediate MC to the solvent is 1:(10-20).

[0043] Preferably, in the S4, the reaction temperature is 10-35℃.

[0044] The third object of the present application is to provide the use of the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment in the preparation of a medicament for preventing and / or treating a disease related to abnormal expression of VEGFR2 and HDAC6 activity. The disease related to abnormal expression of VEGFR2 and HDAC6 activity is a malignant tumor.

[0045] The present application has the following advantages:

[0046] The present application utilizes the splicing principle to design and synthesize a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment with a novel structure, and tests the anti-proliferative activity on cervical cancer cells Siha and chronic myeloid leukemia cells K562. The results show that the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment provided by the present application has good inhibitory activity on Siha cells and K562 cells.

[0047] The inhibitory activity of compounds MD1 and MD5 on Siha cells and K562 cells is stronger than that of the remaining compounds, and the IC 50 values are Siha: 1.04±0.10 μmol / L and 0.91±0.08 μmol / L; K562: 0.67±0.09 μmol / L and 0.84±0.05 μmol / L, respectively, and they have strong affinity with HDAC6 and VEGFR2 proteins, and the IC 50 values are HDAC6: 890±41 nM and 935±57 nM; VEGFR2: 264±28 nmol / L and 197±15 nmol / L, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The H NMR chart of the intermediate MA1 in Example 1. 1

[0049] Figure 2 The H NMR chart of the intermediate MB1 in Example 1. 1

[0050] ​​Figure 3 H NMR chart of the target compound MD1 of Example 1. 1 H NMR chart of the target compound MD1 of Example 1.

[0051] Figure 4 H NMR chart of the target compound MD1 of Example 1. 13 H NMR chart of the target compound MD1 of Example 1.

[0052] Figure 5 H NMR chart of the target compound MD2 of Example 2. 1 H NMR chart of the target compound MD2 of Example 2.

[0053] Figure 6 H NMR chart of the target compound MD2 of Example 2. 13 H NMR chart of the target compound MD2 of Example 2.

[0054] Figure 7 H NMR chart of the target compound MD3 of Example 3. 1 H NMR chart of the target compound MD3 of Example 3.

[0055] Figure 8 H NMR chart of the target compound MD3 of Example 3. 13 H NMR chart of the target compound MD3 of Example 3.

[0056] Figure 9 H NMR chart of the target compound MD4 of Example 4. 1 H NMR chart of the target compound MD4 of Example 4.

[0057] Figure 10 H NMR chart of the target compound MD4 of Example 4. 13 H NMR chart of the target compound MD4 of Example 4.

[0058] Figure 11 H NMR chart of the target compound MA5 of Example 5. 1 H NMR chart of the target compound MD5 of Example 5.

[0059] Figure 12 H NMR chart of the target compound MD5 of Example 5. 1 H NMR chart of the target compound MD5 of Example 5.

[0060] Figure 13 H NMR chart of the target compound MD5 of Example 5. 13 H NMR chart of the target compound MD5 of Example 5. DETAILED DESCRIPTION

[0061] The technical solutions of the present application are described in detail through specific embodiments.

[0062] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0063] Example 1

[0064] A method for preparing a VEGFR2-HDAC6 dual inhibitor (MD1) containing a chalcone fragment, comprising the following steps:

[0065] S1, synthesis of intermediate MA1

[0066]

[0067] Into a 100 mL round bottom flask, 4-aminoacetophenone (0.405 g, 3.0 mmol), triethylamine (6 mL, 6.0 mmol) and 20 mL of dichloromethane were cooled to 0 °C, then triphosgene (0.3 g, 1.0 mmol) was added, and the reaction was carried out at this temperature for 4 h. To the reaction solution, 4-chloro-3-trifluoromethylaniline (0.615 g, 3.1 mmol) was added, and the reaction was carried out at 0 °C for 1 h, then the temperature was raised to 22 °C, and the reaction was carried out at this temperature for 2 h. A large amount of solid was generated in the reaction solution, and the excess was washed with a small amount of dichloromethane to obtain 0.73 g of a gray solid, with a yield of 68.4%. As shown in Figure 1 , 1 H NMR (600 MHz, DMSO-d6) δ 9.28 (d, J = 4.6 Hz, 2H), 8.11 (d, J = 2.3 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.64 (dt, J = 16.6, 5.6 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 2.52 (s, 3H).

[0068] S2, synthesis of intermediate MB1

[0069] Into a 100 mL round bottom flask, intermediate MA1 (0.71 g, 2.0 mmol), 4-formylbenzoic acid (0.33 g, 2.2 mmol), sodium hydroxide (0.8 g, 20 mmol) and methanol (25 mL) were added, and the reaction was carried out at 25 °C for 8 h. Then the pH was adjusted to 4 with 5% dilute hydrochloric acid, and the filtrate was washed with a mixed solvent of methanol and water (1:1 by volume) and dried to obtain a white solid 0.79 g, with a yield of 80.9%. As shown in Figure 2 , 1 H NMR (600 MHz, DMSO-d6) δ 9.28 (d, J = 4.6 Hz, 2H), 8.11 (d, J = 2.3 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.64 (dt, J = 16.6, 5.6 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H), 2.52 (s, 3H).

[0070] S3, synthesis of intermediate MC1

[0071] MB1 (0.49 g, 1.0 mmol), EDCI (0.22 g, 1.1 mmol), O-(tetrahydro-2H-pyran)-2- hydroxylamine (0.14 g, 1.2 mmol), HOBt (20 mg), triethylamine (0.5 mL, 3.6 mmol) and tetrahydrofuran (25 mL) were added into a 100 mL round bottom flask and reacted at 25 °C for 8 h. Ethyl acetate (20 mL) was added to the reaction solution, which was then washed twice with water, and the organic layer was concentrated to remove the solvent to obtain a crude product. The crude product was purified by column chromatography (eluent: dichloromethane / methanol) to obtain the product 0.39 g, with a yield of 66.3%.

[0072] S4, synthesis of target product MD1

[0073] MC1 (0.29 g, 0.49 mmol), 5% dilute hydrochloric acid (2 mL) and methanol (4 mL) were added into a 50 mL round bottom flask and reacted at 25 °C for 4 h, to obtain a light yellow solid after filtration. The crude product was then added into ethyl acetate (2 mL) and stirred at room temperature for 2 h, filtered and dried to obtain a light yellow solid 0.15 g, with a yield of 60.7%. Figures 3-4 1 H NMR (600 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.41 (s, 1H), 9.40 (s, 1H), 9.12 (s, 1H), 8.17 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 15.6 Hz, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 15.6 Hz, 1H), 7.70 - 7.60 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 187.27, 163.52, 152.14, 144.16, 141.99, 138.99, 137.42, 133.99, 132.08, 131.25, 130.18, 128.73, 127.36, 126.76 (q, J = 30.7 Hz), 123.48, 123.35, 122.80 (q, J = 273.5 Hz), 122.77, 117.68, 117.01 (q, J = 6.1 Hz).

[0074] Example 2

[0075] A method for preparing a VEGFR2-HDAC6 dual inhibitor (MD2) containing a chalcone fragment, comprising the following steps:

[0076] S1, synthesis of intermediate MA2 ​

[0077]

[0078] The synthesis of intermediate MA2 is the same as MA1, except that 4-chloro-3- trifluoromethylaniline is replaced by 4-methoxyaniline, to give purple-black solid 0.58 g, yield 68.1%. 1 H NMR (600 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.61 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.72 (s, 3H), 2.51 (s, 3H).

[0079] S2, synthesis of intermediate MB2

[0080] The synthesis of intermediate MB2 is the same as MB1, except that intermediate MA1 is replaced by MA2, to give off-white solid 0.63 g, yield 75.7%.

[0081] S3, synthesis of intermediate MC2

[0082] The synthesis of intermediate MC2 is the same as MC1, except that intermediate MB1 is replaced by MB2, to give off-white solid 0.31 g, yield 60.1%.

[0083] S4, synthesis of target product MD2

[0084] The synthesis of target product MD2 is the same as MD1, except that intermediate MC1 is replaced by MC2, to give yellowish solid 0.11 g, yield 52.1%. As shown in Figures 5-6 1 H NMR (600 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.61 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 3.72 (s, 3H), 2.51 (s, 3H). 13 ​C NMR (151 MHz, DMSO-d6) δ 187.17, 163.56, 154.80, 152.37, 144.93, 141.87, 137.51, 133.97, 132.25, 130.68, 130.29, 128.77, 127.40, 123.53, 120.39, 117.21, 114.06, 55.22.

[0085] Example 3

[0086] A method for preparing a VEGFR2-HDAC6 dual inhibitor (MD3) containing a chalcone fragment, comprising the following steps:

[0087] S1, synthesis of intermediate MA3

[0088]

[0089] The synthesis method of intermediate MA3 is the same as that of MA1, except that 4-chloro-3-trifluoromethylaniline is replaced by 3-chloro-4-methyl aniline to obtain 0.66 g of gray solid with a yield of 72.8%.

[0090] S2, synthesis of intermediate MB3

[0091] The synthesis method of intermediate MB3 is the same as that of MB1, except that intermediate MA1 is replaced by MA3 to obtain 0.74 g of white-like solid with a yield of 85.1%.

[0092] S3, synthesis of intermediate MC3

[0093] The synthesis method of intermediate MC3 is the same as that of MC1, except that intermediate MB1 is replaced by MB3 to obtain 0.38 g of white-like solid with a yield of 71.2%.

[0094] S4, synthesis of target product MD3

[0095] The synthesis method of target product MD3 is the same as that of MD1, except that intermediate MC1 is replaced by MC3 to obtain 0.12 g of yellowish solid with a yield of 54.4%. As shown in Figures 7-8 1 H NMR (600 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.17 (dd, J = 61.8, 4.9 Hz, 2H), 8.94 (d, J = 4.8 Hz, 1H), 8.16 (t, J = 6.7 Hz, 2H), 8.07 - 7.88 (m, 3H), 7.84 (d, J = 6.6 Hz, 2H), 7.76 - 7.67 (m, 2H), 7.64 (d, J = 6.5 Hz, 2H), 7.24 (dt, J = 23.4, 7.8 Hz, 2H), 2.26 (s, 3H).​13 C NMR(151MHz,DMSO-d6)δ187.23,163.54,152.22,144.48,141.91,138.47,137.46,133.97,133 .16,131.23,131.00,130.21,128.75,128.72,127.37,123.51,118.41,117.44,117.26,18.85.

[0096] Example 4

[0097] A method for preparing a VEGFR2-HDAC6 dual inhibitor (MD4) containing a chalcone fragment comprises the following steps:

[0098] S1. Synthesis of intermediate MA4

[0099]

[0100] The synthesis method of intermediate MA4 is the same as that of MA1, except that 4-chloro-3-trifluoromethylaniline is replaced by 4-fluoroaniline, to obtain 0.51 g of gray solid with a yield of 62.8%.

[0101] S2. Synthesis of intermediate MB4

[0102] The synthesis method of intermediate MB4 was the same as that of MB1, except that intermediate MA1 was replaced by MA4, to obtain 0.66 g of off-white solid with a yield of 81.6%.

[0103] S3. Synthesis of intermediate MC4

[0104] The synthesis method of intermediate MC4 is the same as that of MC1, except that intermediate MB1 is replaced by MB4, to obtain 0.32 g of off-white solid with a yield of 63.5%.

[0105] S4. Synthesis of target product MD4

[0106] The target product MD4 was synthesized in the same manner as MD1, except that the intermediate MC1 was replaced by MC4, yielding 92 mg of a pale yellow solid with a yield of 44.8%. Figures 9-10 As shown, 1H NMR (600 MHz, DMSO-d6) δ 11.36 (s, 1H), 9.20 (s, 1H), 9.13 (s, 1H), 8.90 (s, 1H), 8.16 (d, J = 8.7 Hz, 2H), 8.04 (d, J = 15.6 Hz, 1H), 7.97 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 15.5 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.53 - 7.46 (m, 2H), 7.15 (t, J = 8.8 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 187.21, 163.55, 157.10 (d, J = 234.9 Hz), 152.31, 144.70, 141.93, 137.49, 135.63, 133.98, 130.88, 130.28, 128.78, 127.40, 123.51, 120.36 (d, J = 7.8 Hz), 117.36, 115.43 (d, J = 22.2 Hz).

[0107] Example 5

[0108] A method for preparing a VEGFR2-HDAC6 dual inhibitor (MD5) containing a chalcone fragment, comprising the following steps:

[0109] S1, synthesis of intermediate MA5

[0110]

[0111] The synthesis method of intermediate MA5 is the same as MA1, except that 4- aminophenylacetone is replaced by 3-aminophenylacetone to obtain 0.56 g of gray solid with a yield of 62.8%. As shown in Figure 11 , 1 H NMR (600 MHz, DMSO-d6) δ 9.23 (s, 1H), 9.10 (s, 1H), 8.11 (d, J = 2.5 Hz, 1H), 8.09 - 8.05 (m, 1H), 7.68 (ddd, J = 16.6, 8.4, 2.4 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.45 (t, J = 7.8 Hz, 1H), 2.57 (s, 3H).

[0112] S2, synthesis of intermediate MB5

[0113] The synthesis method of intermediate MB5 is the same as MB1, except that intermediate MA1 is replaced by MA5 to obtain 0.55 g of white-like solid with a yield of 56.4%.

[0114] S3, synthesis of intermediate MC5

[0115] The synthesis method of intermediate MC5 is the same as that of MC1, except that intermediate MB1 is replaced by MB5, to obtain a off-white solid 0.35 g, yield 59.5%.

[0116] S4, synthesis of target product MD5

[0117] The synthesis method of target product MD5 is the same as that of MD1, except that intermediate MC1 is replaced by MC5, to obtain a light yellow solid 0.12 g, yield 48.6%. As shown in Figures 12-13 1 H NMR (600 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.26 (d, J = 4.7 Hz, 1H), 9.12 (d, J = 4.8 Hz, 2H), 8.18 - 8.09 (m, 2H), 8.00 - 7.91 (m, 3H), 7.86 (dt, J = 26.5, 6.5 Hz, 3H), 7.77 (d, J = 11.7 Hz, 2H), 7.67 (d, J = 6.0 Hz, 1H), 7.63 (d, J = 5.6 Hz, 1H), 7.51 (q, J = 7.2 Hz, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 189.11, 163.50, 152.51, 142.87, 139.86, 139.25, 138.07, 137.23, 134.20, 132.02, 129.32, 128.80, 127.41, 126.73 (q, J = 31.4 Hz), 123.65, 123.54, 123.27, 122.94, 122.83 (q, J = 272.7 Hz), 122.50, 118.22, 116.96 (q, J = 6.1 Hz).

[0118] The above obtained MD1- MD5 compounds were determined for their cytotoxicity on cervical cancer cells Siha and chronic myeloid leukemia cells K562 by CCK8 method.

[0119] Siha cells in a special medium (CM-0210, Pnnsay), K562 in a special cell culture medium (CM-0130, Pnnsay) were cultured in a cell incubator at 37°C containing 5% CO2 air. The logarithmic growth phase Siha cells and K562 were taken, about 2 x 10 3 ​The cells were incubated in an incubator for 12 h, then different concentrations of compounds MD1- MD5 were added, and incubation was continued for 48 h, then 10 μL of CCK8 solution (10 mg / mL, Adamas life) was added, and incubation was continued for 2 h. The absorbance of each well was determined at 450 nm by an enzyme marker, and the IC 50 values were calculated by Prism 8.0 software, and the results are shown in Table 1.

[0120] Table 1 Inhibitory activity of compounds MD1- MD5 on Siha cells and K562 cells in vitro

[0121]

[0122] As shown in Table 1, compounds MD1- MD5 have strong inhibitory effects on cervical cancer cells Siha and chronic myeloid leukemia cells K562, and the Siha IC 50 values are 0.91-5.24 μmol / L, and the K562 IC 50 values are 0.67-7.35 μmol / L, and compounds MD1 and MD5 have stronger inhibitory activity on Siha cells and K562 cells than the control Sorafenib and SAHA.

[0123] Example 6

[0124] Test of affinity of compounds MD1 and MD5 to HDAC6 protein

[0125] The affinity of compounds MD1 and MD5 to HDAC6 protein was detected using an HDAC6 Fluorogenic assay kit (BPS bioscience, USA), and the steps were performed according to the instructions. In a black 96-well plate, 5 μL of Fluorogenic HDAC substrate (200 μM), 5 μL of BSA solution (0.1%), and 30 μL of HDAC detection buffer solution were added, then 5 μL of an aqueous solution containing 10% DMSO was added in the control group and the blank group, 5 μL of different concentrations of test samples was added in the test group, 5 μL of a DMSO solution of Trichostatin A (20 μM) was added in the positive control group, 5 μL of HDAC detection buffer solution was added in the blank group, and the rest of each well was added with HDAC6 human recombinant enzyme (7 ng / μL). Incubate at 37°C for 30 min, then add HDAC Developer solution (50 μL) in the kit, and incubate at room temperature for 15 min. The absorbance (OD) was measured in a fluorescence detector at 380 nm excitation and 450 nm emission. Three replicate wells were set for each group, and the inhibition rate was calculated according to the formula shown in 1-1:

[0126]

[0127] IC 50 The affinity of MD1 and MD5 to the HDAC6 protein is represented by IC, and the results are shown in Table 2.

[0128] Example 7

[0129] Test of the affinity of compounds MD1 and MD5 to VEGFR2 protein

[0130] The affinity of compounds MD1 and MD5 to VEGFR2 protein was detected using a VEGFR2 (KDR) Kinase Assay Kit (BPS bioscience, USA) according to the steps of the instructions. A mixture containing 5x kinase buffer 1, 500 μM ATP and PTK substrate (Poly-Glu, Tyr 4:1) was prepared according to the instructions. 25 μL of the mixture was added to each well of a 96-well plate, 5 μL of distilled water was added to the control and blank groups, 5 μL of different concentrations of the test solution was added to the test group, then 20 μL of 1x kinase buffer was added to the blank group, 20 μL of 1 ng / μL protein kinase solution was added to the control and test groups. Incubate at 30°C for 45 min, then add 50 μl Kinase Glo TM MAX reagent, wrap with aluminum foil, and incubate at room temperature for 15 min. Measure the fluorescence emission data in a microplate reader. Set 3 replicates per well, calculate the inhibition rate according to formula 1-1, and the results are shown in Table 2.

[0131] Table 2 Affinity of MD1 and MD5 to HDAC6 and VEGFR2 proteins in vitro

[0132]

[0133] As can be seen from Table 2, the affinity of target compounds MD1 and MD2 to HDAC6 protein in vitro is lower than that of positive control SAHA, and the affinity to VEGFR2 protein is lower than that of control Sorafenib, but they both have good affinity to HDAC6 and VEGFR2 proteins, indicating that the target compounds are HDAC6 and VEGFR2 dual inhibitors.

[0134] In summary, the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment provided by the present application has good inhibitory activity on Siha cells and K562 cells, and the dual inhibitor has good affinity to HDAC6 and VEGFR2 proteins.

[0135] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, characterized in that, A compound of formula A: ； Formula A The compound of formula A is shown in formula A-I or formula A-II: ； Formula A-I Formula A-II R is selected from at least one of halogen, substituted or unsubstituted methyl, methoxy; When the substituted or unsubstituted group contains a substituent, the substituent is selected from F or Cl.

2. The VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment according to claim 1, characterized in that, The VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment is any one of the following compounds: 、 、 、 、 。 3. A process for the preparation of a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: S1, reacting aminoacetophenone with triphosgene, and then with substituted aniline to obtain an intermediate MA through a urea synthesis reaction; S2, condensing the intermediate MA with 4-formylbenzoic acid to obtain an intermediate MB; S3, performing an amide condensation reaction between the intermediate MB and O-(tetrahydro-2H-pyran)-2-hydroxylamine to obtain an intermediate MC; S4, mixing the intermediate MC with a solvent, and performing a deprotection reaction under acidic conditions to obtain a compound of formula A.

4. The production method according to claim 3, characterized by, In S1, the urea synthesis reaction is performed under catalysis of an organic base selected from at least one of triethylamine, pyridine, and N,N-diisopropylethylamine.

5. The preparation method according to claim 3, characterized in that In S1, the molar ratio of aminoacetophenone to triphosgene is 3:1, and the molar ratio of aminoacetophenone to substituted aniline is 1:(1-1.1).

6. The preparation method according to claim 3, characterized in that In S2, the condensation reaction is performed under alkaline conditions provided by an alkaline reagent selected from at least one of sodium hydroxide, potassium hydroxide, and lithium hydroxide; and the molar ratio of the intermediate MA to 4-formylbenzoic acid is 1:(1-1.1).

7. The preparation method according to claim 3, characterized in that In S3, the amide condensation reaction is performed in the presence of a condensing agent selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, carbonyldiimidazole, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 1-n-propylphosphonic anhydride.

8. The preparation method according to claim 3, characterized in that In S4, the acidic conditions are provided by an acidic reagent selected from one or more of hydrochloric acid, dilute sulfuric acid, and trifluoroacetic acid.

9. Use of the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment according to any one of claims 1-2 or prepared by the method according to any one of claims 3-8 in the preparation of a drug for preventing and / or treating a disease associated with abnormal expression of VEGFR2 and HDAC6 activity.

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