VEGFR2-HDAC6 dual inhibitor containing chalcone fragment as well as preparation method and application of VEGFR2-HDAC6 dual inhibitor

Abstract

The invention provides a VEGFR2-HDAC6 dual inhibitor containing chalcone fragments as well as a preparation method and application of the VEGFR2-HDAC6 dual inhibitor, and relates to the technical field of medicinal chemistry. The invention discloses a VEGFR2-HDAC6 dual inhibitor containing chalcone fragments. The structural formula of the VEGFR2-HDAC6 dual inhibitor is shown as a formula A, according to the invention, the VEGFR2-HDAC6 dual inhibitor containing the chalcone fragment and having a novel structure is designed and synthesized by utilizing a splicing principle, the anti-proliferative activity test is carried out on cervical cancer cells Siha and chronic granulocytic leukemia cells K562, and the result shows that the VEGFR2-HDAC6 dual inhibitor containing the chalcone fragment provided by the invention has relatively good inhibitory activity on the Siha cells and the K562 cells.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, and a preparation method and application thereof. Background Art

[0002] The vascular endothelial growth factor (VEGF) family of 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 and angiogenesis. Overexpression of VEGFR2 can lead to the occurrence and development of a variety of cancers. Therefore, VEGFR2 has become a hot topic in current anti-tumor angiogenesis research. 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 that is highly expressed in a variety of tumor cells and plays an important role in the migration and invasion of tumor cells. Currently, there are 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 combined use of HDAC6 inhibitors and VEGFR2 inhibitors has great promise 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 of the invention

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

[0007] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

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

[0009]

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

[0011] Preferably, the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, the compound of formula A includes the structures shown in formula AI and formula A-II:

[0012]

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

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

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

[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 group is selected from one or more of trifluoromethyl and trichloromethyl.

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

[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 a chalcone fragment comprises any one of the following compounds:

[0021]

[0022] The second object of the present invention is to provide a method for preparing a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, comprising the following steps:

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

[0024] S2, intermediate MA and 4-formylbenzoic acid undergo condensation reaction to obtain intermediate MB;

[0025] S3, intermediate MB and O-(tetrahydro-2H-pyran)-2-hydroxylamine undergo amide condensation reaction to obtain intermediate MC;

[0026] S4. The intermediate MC and the solvent are mixed, and a deprotection reaction is carried out under acidic conditions to obtain a 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 and 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 said S2, the condensation reaction is carried out under alkaline conditions.

[0033] More preferably, the alkaline condition is provided by an alkaline reagent, and the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide and lithium hydroxide.

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

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

[0036] Preferably, in said S3, the amide condensation reaction is carried out in the presence of a condensation 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-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), and 1-n-propyl phosphoric anhydride (T3P).

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

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

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

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

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

[0043] Preferably, in said S4, the reaction temperature is 10-35°C.

[0044] The third object of the present invention is to provide the use of the above-mentioned VEGFR2-HDAC6 dual inhibitor containing chalcone fragment in the preparation of drugs for preventing and / or treating diseases 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 beneficial effects of the present invention are:

[0046] The present invention designs and synthesizes a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment with a novel structure by utilizing the splicing principle, and tests the anti-proliferation activity on cervical cancer cell Siha and chronic myeloid leukemia cell K562. The results show that the VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment provided by the present invention has good inhibitory activity on Siha cells and K562 cells.

[0047] Compounds MD1 and MD5 had stronger inhibitory activities against Siha cells and K562 cells than the other compounds. Their IC 50 The values ​​were Siha: 1.04±0.10μmol / L and 0.91±0.08μmol / L; K562: 0.67±0.09μmol / L and 0.84±0.05μmol / L, respectively. It has a strong affinity with HDAC6 and VEGFR2 proteins, and its IC 50 The values ​​were: HDAC6: 890±41nM, 935±57nM; VEGFR2: 264±28nmol / L and 197±15nmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 For the intermediate MA1 of Example 1 1 H NMR spectra.

[0049] Figure 2 For the intermediate MB1 of Example 1 1 H NMR spectra.

[0050] Figure 3 The target compound MD1 of Example 1 1 H NMR spectra.

[0051] Figure 4 The target compound MD1 of Example 1 13 C NMR spectrum.

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

[0053] Figure 6 For the target compound MD2 of Example 2 13 C NMR spectrum.

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

[0055] Figure 8 For the target compound MD3 of Example 3 13 C NMR spectrum.

[0056] Fig. 9 For the target compound MD4 of Example 4 1 H NMR spectra.

[0057] Fig.10 For the target compound MD4 of Example 4 13 C NMR spectrum.

[0058] Fig.11 For Example 5 target compound MA5 1 H NMR spectra.

[0059] Fig.12 For the target compound MD5 of Example 5 1 H NMR spectra.

[0060] Fig.13 For the target compound MD5 of Example 5 13 C NMR spectrum. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is described in detail through specific embodiments.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] Example 1

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

[0065] S1. Synthesis of intermediate MA1

[0066]

[0067] Add 4-aminoacetophenone (0.405g, 3.0mmol), triethylamine (6mL, 6.0mmol) and 20mL of dichloromethane to a 100mL round-bottom flask, cool to 0℃, then add triphosgene (0.3g, 1.0mmol) and react at this temperature for 4h. Add 4-chloro-3-trifluoromethylaniline (0.615g, 3.1mmol) to the reaction solution, react at 0℃ for 1h, then heat to 22℃ and react at this temperature for 2h. A large amount of solid is generated in the reaction solution, which is excessive. Wash the filter cake with a small amount of dichloromethane to obtain 0.73g of gray solid with a yield of 68.4%. Figure 1 As shown, 1 H NMR (600MHz, DMSO-d6) δ9.28(d,J=4.6Hz,2H),8.11(d,J=2.3Hz,1H),7.91(d,J =8.7Hz, 2H), 7.64 (dt, J = 16.6, 5.6Hz, 2H), 7.60 (d, J = 8.7Hz, 2H), 2.52 (s, 3H).

[0068] S2. Synthesis of intermediate MB1

[0069] The 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 to a 100 mL round-bottom flask and reacted at 25 °C for 8 h. Then the pH was adjusted to 4 with 5% dilute hydrochloric acid, filtered, and the filter cake was washed with a mixed solvent of methanol and water in a volume ratio of 1:1, and dried to obtain 0.79 g of an off-white solid with a yield of 80.9%. Figure 2 As shown, 1 H NMR (600MHz, DMSO-d6) δ9.28(d,J=4.6Hz,2H),8.11(d,J=2.3Hz,1H),7.91(d,J =8.7Hz, 2H), 7.64 (dt, J = 16.6, 5.6Hz, 2H), 7.60 (d, J = 8.7Hz, 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 to a 100 ml round-bottom flask and reacted at 25 ° C for 8 h. Ethyl acetate (20 mL) was added to the reaction solution, and 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 0.39 g of the product, 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 to a 50 mL round-bottom flask, reacted at 25 ° C for 4 h, filtered to obtain a light yellow solid, and then the crude product was added to ethyl acetate (2 mL), stirred at room temperature for 2 h, filtered, and dried to obtain 0.15 g of a light yellow solid, with a yield of 60.7%. Figures 3-4 As shown, 1 H NMR (600MHz, DMSO-d6) δ11.34(s,1H),9.41(s,1H),9.40(s,1H),9.12(s,1H),8.17(d,J=8.8Hz,2H),8.12(d,J=2.4Hz ,1H),8.03(d,J=15.6Hz,1H),7.97(d,J=8.3Hz,2H),7.83(d,J=8.3Hz,2H),7.74(d,J=15.6Hz,1H),7.70–7.60(m,4H). 13 C NMR(151MHz,DMSO-d6)δ187.27,163.52,152.14,144.16,141.99,138.99,137.42,133.99,132.08,131.25,130.18,1 28.73, 127.36, 126.76 (q, J = 30.7Hz), 123.48, 123.35, 122.80 (q, J = 273.5Hz), 122.77, 117.68, 117.01 (q, J = 6.1Hz).

[0074] Example 2

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

[0076] S1. Synthesis of intermediate MA2

[0077]

[0078] The synthesis method of intermediate MA2 is the same as that of MA1, except that 4-chloro-3-trifluoromethylaniline is replaced by 4-methoxyaniline, and 0.58 g of purple-black solid is obtained with a yield of 68.1%. 1 H NMR(600MHz,DMSO-d6)δ9.02(s,1H),8.61(s,1H),7.89(d,J=8.7Hz,2H),7.57(d,J= 8.7Hz, 2H), 7.37 (d, J = 8.9Hz, 2H), 6.88 (d, J = 8.9Hz, 2H), 3.72 (s, 3H), 2.51 (s, 3H).

[0079] S2. Synthesis of intermediate MB2

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

[0081] S3. Synthesis of intermediate MC2

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

[0083] S4. Synthesis of target product MD2

[0084] The synthesis method of the target product MD2 is the same as that of MD1, except that the intermediate MC1 is replaced by MC2, and 0.11 g of a light yellow solid is obtained with a yield of 52.1%. Figures 5-6 As shown, 1 H NMR (600MHz, DMSO-d6) δ11.36(s,1H),9.15(d,J=1.2Hz,1H),9.12(s,1H),8.66(s,1H),8.16(d,J=8.9Hz,2H),8.04(d,J=15.6Hz,1H),7.97 (d,J=8.4Hz,2H),7.83(d,J=8.3Hz,2H),7.73(d,J=15.5Hz,1H),7.63(d,J=8.8Hz,2H),7.41–7.36(m,2H),6.91–6.86(m,2H),3.72(s,3H). 13C NMR(151MHz,DMSO-d6)δ187.17,163.56,154.80,152.37,144.93,141.87,137.51,133 .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 comprises 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-methylaniline, 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, and 0.74 g of off-white solid is obtained with a yield of 85.1%.

[0092] Synthesis of S3 and intermediate MC3

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

[0094] S4. Synthesis of target product MD3

[0095] The synthesis method of the target product MD3 is the same as that of MD1, except that the intermediate MC1 is replaced by MC3, and 0.12 g of light yellow solid is obtained with a yield of 54.4%. Figures 7-8 As shown, 1 H NMR (600MHz, DMSO-d6) δ11.34(s,1H),9.17(dd,J=61.8,4.9Hz,2H),8.94(d,J=4.8Hz,1H),8.16(t,J=6.7Hz,2H),8.07– 7.88(m,3H),7.84(d,J=6.6Hz,2H),7.76–7.67(m,2H),7.64(d,J=6.5Hz,2H),7.24(dt,J=23.4,7.8Hz,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, and 0.51 g of gray solid is obtained with a yield of 62.8%.

[0101] S2. Synthesis of intermediate MB4

[0102] The synthesis method of intermediate MB4 is the same as that of MB1, except that intermediate MA1 is replaced by MA4, and 0.66 g of off-white solid is obtained 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, and 0.32 g of off-white solid is obtained with a yield of 63.5%.

[0105] S4. Synthesis of target product MD4

[0106] The synthesis method of the target product MD4 is the same as that of MD1, except that the intermediate MC1 is replaced by MC4, and 92 mg of light yellow solid is obtained with a yield of 44.8%. Figures 9-10 As shown, 1H NMR (600MHz, DMSO-d6) δ11.36(s,1H),9.20(s,1H),9.13(s,1H),8.90(s,1H),8.16(d,J=8.7Hz,2H),8.04(d,J=15.6Hz,1H),7.97 (d,J=8.2Hz,2H),7.83(d,J=8.2Hz,2H),7.73(d,J=15.5Hz,1H),7.64(d,J=8.7Hz,2H),7.53–7.46(m,2H),7.15(t,J=8.8Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ187.21, 163.55, 157.10 (d, J = 234.9Hz), 152.31, 144.70, 141.93, 137.49, 135.6 3,133.98,130.88,130.28,128.78,127.40,123.51,120.36(d,J=7.8Hz),117.36,115.43(d,J=22.2Hz).

[0107] Example 5

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

[0109] S1. Synthesis of intermediate MA5

[0110]

[0111] The synthesis method of intermediate MA5 is the same as that of MA1, except that 4-aminoacetophenone is replaced by 3-aminoacetophenone to obtain 0.56 g of gray solid with a yield of 62.8%. Fig.11 As shown, 1 H NMR(600MHz,DMSO-d6)δ9.23(s,1H),9.10(s,1H),8.11(d,J=2.5Hz,1H),8.09–8.05(m,1H),7 .68(ddd,J=16.6,8.4,2.4Hz,2H),7.62(d,J=8.5Hz,2H),7.45(t,J=7.8Hz,1H),2.57(s,3H).

[0112] S2. Synthesis of intermediate MB5

[0113] The synthesis method of intermediate MB5 is the same as that of MB1, except that intermediate MA1 is replaced by MA5, and 0.55 g of off-white solid is obtained 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, and 0.35 g of off-white solid is obtained with a yield of 59.5%.

[0116] S4. Synthesis of target product MD5

[0117] The synthesis method of the target product MD5 is the same as that of MD1, except that the intermediate MC1 is replaced by MC5, and 0.12 g of light yellow solid is obtained with a yield of 48.6%. Figures 12-13 As shown, 1 H NMR (600MHz, DMSO-d6) δ11.34(s,1H),9.26(d,J=4.7Hz,1H),9.12(d,J=4.8Hz,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(151MHz,DMSO-d6)δ189.11,163.50,152.51,142.87,139.86,139.25,138.07,137.23,134.20,132.02,129.32,128.80,1 27.41, 126.73 (q, J = 31.4Hz), 123.65, 123.54, 123.27, 122.94, 122.83 (q, J = 272.7Hz), 122.50, 118.22, 116.96 (q, J = 6.1Hz).

[0118] The cytotoxicity of the MD1-MD5 compounds obtained above on cervical cancer cell Siha and chronic myeloid leukemia cell K562 was determined by CCK8 method.

[0119] Siha cells were cultured in a dedicated culture medium (CM-0210, Pronocell) and K562 cells were cultured in a dedicated cell culture medium (CM-0130, Pronocell) in a cell culture incubator at 37°C with 5% CO2 in air. Siha cells and K562 cells in logarithmic growth phase were taken and approximately 2×10 3Cells were incubated in an incubator for 12 h, and then different concentrations of compounds MD1 to MD5 were added and incubated for 48 h. Then 10 μL of CCK8 solution (10 mg / mL, Adamas life) was added and incubated for 2 h. The absorbance of each well was measured at 450 nm using an ELISA reader, and the IC was calculated using Prism 8.0 software. 50 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 to MD5 have strong inhibitory effects on cervical cancer cell Siha and chronic myeloid leukemia cell K562, among which Siha IC 50 The value is between 0.91 and 5.24 μmol / L, K562 IC 50 The values ​​were between 0.67 and 7.35 μmol / L, among which compounds MD1 and MD5 had stronger inhibitory activities on Siha cells and K562 cells than the controls Sorafenib and SAHA.

[0123] Example 6

[0124] Affinity test of compounds MD1 and MD5 with HDAC6 protein

[0125] The affinity of compounds MD1 and MD5 to HDAC6 protein was detected using the HDAC6 Fluorogenic assay kit (BPS bioscience, USA), and the operation was performed according to the steps in the instructions. 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 to a black 96-well plate, and then 5 μL of an aqueous solution containing 10% DMSO was added to the control group and the blank group, 5 μL of test samples of different concentrations were added to the test group, 5 μL of Trichostatin A DMSO solution (20 μM) was added to the positive control group, 5 μL of HDAC detection buffer solution was added to the blank group, and HDAC6 human recombinant enzyme (7 ng / μL) was added to each well. Incubate at 37°C for 30 min, then add the 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 that excites at a wavelength of 380 nm and detects the emitted light at 450 nm. Three replicate wells were set for each group, and the calculation formula of inhibition rate is shown in 1-1:

[0126]

[0127] IC 50 The values ​​represent the affinity of MD1 and MD5 to HDAC6 protein, and the results are shown in Table 2.

[0128] Example 7

[0129] Affinity test of compounds MD1 and MD5 with VEGFR2 protein

[0130] The affinity of compounds MD1 and MD5 to VEGFR2 protein was detected using the VEGFR2 (KDR) Kinase Assay Kit (BPS bioscience, USA) and the operation was performed according to 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 the 96-well plate, 5 μL of distilled water was added to the control group and the blank group, 5 μL of the test solution of different concentrations was added to the test group, and then 20 μL of 1x kinase buffer was added to the blank group, and 20 μL of 1ng / μL protein kinase solution was added to the control group and the test group. Incubate at 30°C for 45 minutes, and then add 50 μl Kinase Glo to each well. TM MAX reagent was wrapped with aluminum foil and incubated at room temperature for 15 min. The fluorescence data was measured in an ELISA reader. Three replicate wells were set up for each well. The inhibition rate was calculated according to formula 1-1. The results are shown in Table 2.

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

[0132]

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

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

[0135] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment, characterized in that: Including compounds of formula A: R is selected from at least one of halogen, substituted or unsubstituted alkyl, and alkoxy; 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 compounds of formula A include the structures shown in formula AI and formula A-II:

3. The VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment according to any one of claims 1 to 2, characterized in that: The VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment includes any one of the following compounds:

4. A method for preparing a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, reacting aminoacetophenone with triphosgene, and then reacting with substituted aniline to synthesize urea to obtain intermediate MA; S2, intermediate MA and 4-formylbenzoic acid undergo condensation reaction to obtain intermediate MB; S3, intermediate MB and O-(tetrahydro-2H-pyran)-2-hydroxylamine undergo amide condensation reaction to obtain intermediate MC; S4. The intermediate MC and the solvent are mixed, and a deprotection reaction is carried out under acidic conditions to obtain a compound of formula A.

5. The preparation method according to claim 4, characterized in that: In the above S1, the urea synthesis reaction is carried out under the catalysis of an organic base; the organic base is selected from at least one of triethylamine, pyridine, and N,N-diisopropylethylamine.

6. The preparation method according to claim 4, characterized in that: In the 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).

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

8. The preparation method according to claim 4, characterized in that: In S3, the amide condensation reaction is carried out in the presence of a condensing agent, and the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, carbonyldiimidazole, O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate, and 1-n-propyl phosphoric anhydride.

9. The preparation method according to claim 4, characterized in that: In the step S4, the acidic condition is provided by an acidic reagent, and the acidic reagent is selected from one or more of hydrochloric acid, dilute sulfuric acid, and trifluoroacetic acid.

10. Use of a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment as claimed in any one of claims 1 to 3 or a VEGFR2-HDAC6 dual inhibitor containing a chalcone fragment obtained by the preparation method as claimed in any one of claims 4 to 9 in the preparation of a drug for preventing and / or treating diseases associated with abnormal expression of VEGFR2 and HDAC6 activity.

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