Pyridine compounds containing N-hydroxy-benzamide, preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to a pyridine compound containing N-hydroxy-benzamide, a preparation method and an application. The pyridine compound containing N-hydroxy-benzamide of the present invention is a compound shown in Formula I or a pharmaceutically acceptable salt thereof: the compound of the present invention can effectively inhibit HDAC6, and has an inhibitory effect on the proliferation and cloning of tumor cells, and has a promoting effect on the apoptosis of tumor cells. Therefore, it can be used to prepare HDAC6 inhibitors, and can also be used as a new compound for the preparation of anticancer drugs. Therefore, the present invention can provide new research and development directions and structural options for the development of HDAC6 inhibitors or anti-tumor drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a pyridine compound containing N-hydroxy-benzamide, a preparation method and application thereof. Background Art

[0002] Histone acetylation was first reported in 1964. Histone acetylation affects RNA synthesis. Under normal conditions, histones are tightly bound to DNA, but when the lysine residues of histones are acetylated, the positive charge of the lysine residues can be neutralized, thereby weakening their interaction with negatively charged DNA molecules, facilitating the binding of transcription factors and promoting transcription and translation. Acetylation is a reversible mechanism, catalyzed by histone acetyltransferases (HATs) on the one hand and histone deacetylases (HDACs) on the other. The HDAC family has 18 isoforms, which are divided into four major categories, namely class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10) and class IV (HDAC11). Among them, HDAC6 is a structurally and functionally unique deacetylase consisting of 1215 amino acids and containing two independent catalytic domains. It can deacetylate specific cytoplasmic non-histone protein substrates, including heat shock protein (Hsp90), cortactin, peroxiredoxin, α-tubulin, and heat shock transcription factor-1 (HSF-1).

[0003] Studies have shown that HDAC6 is highly expressed in a variety of cancers and plays an important role in the occurrence and development of both hematological tumors and solid tumors. Reducing the expression of HDAC6 or inhibiting its activity can inhibit the growth and metastasis of tumor cells, so HDAC6 has become a molecular target for tumor prevention and treatment. In order to improve the therapeutic effect of tumors, the development and design of efficient HDAC6 inhibitors has become an important strategy for the development of anticancer drugs, which is of great significance for the research and treatment of related diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a pyridine compound containing N-hydroxy-benzamide, which has good HDAC6 inhibitory activity, inhibits the proliferation and cloning of tumor cells, and promotes the apoptosis of tumor cells.

[0005] The present invention also provides a method for preparing a pyridine compound containing N-hydroxy-benzamide. The reaction system is simple and easy to implement, and can realize batch preparation of the pyridine compound containing N-hydroxy-benzamide.

[0006] In addition, the present invention also provides an application of a pyridine compound containing N-hydroxy-benzamide.

[0007] In order to achieve the above object, the pyridine compound containing N-hydroxy-benzamide of the present invention adopts the technical scheme as follows:

[0008] A pyridine compound containing N-hydroxy-benzamide, which is a compound represented by formula I or a pharmaceutically acceptable salt thereof:

[0009]

[0010] The preparation method of the pyridine compound containing N-hydroxy-benzamide of the present invention comprises the following steps:

[0011] (1) Mix 2-aminopyridine, N-bromosuccinimide and a solvent and react at 20 to 35° C. to obtain intermediate A;

[0012] (2) Intermediate A, 4-methoxyphenylboronic acid, anhydrous sodium carbonate, a catalyst, and a solvent are mixed and reacted at 105 to 115° C. for 4 to 6 hours to obtain intermediate B;

[0013] (3) Mixing intermediate B, N-bromosuccinimide and solvent, and reacting at 20-35° C. for 0.5-1 h to obtain intermediate C;

[0014] (4) Intermediate C, 4-isopropylphenylboric acid, anhydrous sodium carbonate, a catalyst, and a solvent are mixed and reacted at 105-115° C. for 4-6 hours to obtain intermediate D;

[0015] (5) Intermediate D, sodium hydroxide, 4-bromobenzoyl bromide and solvent are mixed and reacted at 20-35° C. for 8-12 hours to obtain intermediate E;

[0016] (6) Intermediate E, hydroxylamine, sodium hydroxide and a solvent are mixed and reacted at 20 to 35° C. to obtain a pyridine compound containing N-hydroxy-benzamide.

[0017] Preferably, the solvent used in step (1), step (3) and step (5) is tetrahydrofuran; the solvent used in step (2) and step (4) is a mixture of dioxane and water in a volume ratio of 5:1; and the solvent used in step (6) is a mixture of dichloromethane and methanol in a volume ratio of 1:2.

[0018] To improve the reaction efficiency and product selectivity, preferably, the catalyst used in step (2) is bis(triphenylphosphine)palladium dichloride; and the catalyst used in step (4) is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex.

[0019] The application of the pyridine compound containing N-hydroxy-benzamide provided by the present invention is specifically the application of the pyridine compound containing N-hydroxy-benzamide in the preparation of HDAC6 inhibitors or anti-tumor drugs.

[0020] As a further preferred embodiment, the anti-tumor drug is a drug that inhibits tumor cell proliferation and cloning and promotes tumor cell apoptosis.

[0021] More preferably, the tumor cells are ovarian cancer cells.

[0022] To further optimize the anti-tumor effect, preferably, the ovarian cancer cells are one or both of A2780 ovarian cancer cells and SKOV3 ovarian cancer cells.

[0023] The beneficial effects of the present invention are:

[0024] The pyridine compound containing N-hydroxy-benzamide of the present invention is modified with a characteristic N-hydroxy-benzamide group. The present invention unexpectedly found through experimental exploration that the modification with the above group can not only enhance the proliferation inhibition activity of the compound on ovarian cancer cells and induce apoptosis thereof, but also effectively inhibit the HDAC6 in ovarian cancer cells, so it can be used to prepare HDAC6 inhibitors, and can also be used as a new compound for the preparation of anticancer drugs. Therefore, the present invention can provide a new research and development direction and material selection for the development of HDAC6 inhibitors or anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The structural characterization of the compound of Example 1 of the present invention 1 H NMR spectrum;

[0026] Figure 2 The structural characterization of the compound of Example 1 of the present invention 13 C NMR spectrum;

[0027] Figure 3 This is a graph showing the inhibitory effect on the proliferation of ovarian cancer cells SKOV3 and A2780 after the compound of Example 1 of the present invention is treated;

[0028] Figure 4 The clone formation results obtained after the ovarian cancer A2780 cells were treated with the compound of Example 1 of the present invention;

[0029] Figure 5 This is a diagram showing the apoptosis results of ovarian cancer cells SKOV3 and A2780 after being treated with the compound of Example 1 of the present invention;

[0030] Figure 6This is a graph showing the results of WB detection of the expression level of HDAC6 in cells and the acetylation of H3 and α-tubulin after the ovarian cancer cells SKOV3 and A2780 were treated with the compound of Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the following embodiments are only used to exemplarily illustrate and explain the present invention and should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] The pyridine compound containing N-hydroxy-benzamide in this embodiment is a compound represented by Formula I or a pharmaceutically acceptable salt thereof:

[0034]

[0035] Example 2

[0036] This example is to prepare the pyridine compound containing N-hydroxy-benzamide of Example 1, and the specific steps are as follows:

[0037]

[0038] (1) 1 mM 2-aminopyridine was weighed in a 250 mL round-bottom flask, and tetrahydrofuran (150 mL) was added to slowly dissolve. Within half an hour, (2×0.5 mM) N-bromosuccinimide (NBS) was added in portions under stirring at room temperature, and the reaction was monitored by thin layer chromatography (petroleum ether: ethyl acetate = 5:1). After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate 3 times, combined with organic phase, washed with saturated brine 2 times (50 mL×2), and the organic phase was removed by vacuum distillation to obtain intermediate A. Since the reaction system is relatively simple, intermediate A can be directly used in the next step.

[0039] (2) Place intermediate A (1 mM) in a 100 mL round-bottom flask and add a dioxane-water (volume ratio 5:1) solvent mixture to dissolve. Under room temperature stirring, add 4-methoxyphenylboronic acid (1.2 mM), anhydrous sodium carbonate (2 mM), and di(triphenylphosphine)palladium dichloride (0.2 mM) to the reaction system in sequence. Then transfer to a 110°C oil bath, heat under reflux for 5 h under nitrogen protection, and monitor with thin layer chromatography (petroleum ether-ethyl acetate = 5:1). After the reaction of intermediate A is completed, stop the reaction, cool to room temperature, quench with water, extract with ethyl acetate three times, combine the organic phase, wash with saturated brine, and remove the organic phase by reduced pressure distillation. Finally, column chromatography (petroleum ether: ethyl acetate = 3:1) is performed to obtain intermediate B as a light yellow solid.

[0040] (3) Add intermediate B (1 mM) to a 250 mL round-bottom flask and add tetrahydrofuran solvent to dissolve. Add NBS (2×0.5 mM) and react at room temperature for 0.5 h. After the reaction is completed, extract the organic phase three times with water and ethyl acetate, then combine with the organic phase, wash with saturated brine, and remove under reduced pressure to generate intermediate C. Since the reaction system is simple, intermediate C can be directly put into the next step.

[0041] (4) Intermediate C was dissolved in a 100 mL round-bottom flask, and a mixture of dioxane and water (volume ratio 5:1) was added, followed by 4-isopropylphenylboronic acid (1.2 mM) and anhydrous sodium carbonate (2 mM), and then [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloromethane complex (0.2 mM). The reaction system was then transferred to an oil bath at 110 °C and heated under reflux for 5 h under nitrogen protection. The reaction was stopped when intermediate C disappeared by thin layer chromatography. The mixture was then cooled to room temperature, quenched with water, extracted three times with ethyl acetate, combined with an organic phase, washed with saturated brine, and the organic phase was removed by reduced pressure distillation. Finally, intermediate D was obtained by column chromatography (petroleum ether: ethyl acetate = 2:1).

[0042] (5) Place intermediate D (1 mM) in a 25 mL round-bottom flask and add ultra-dry tetrahydrofuran to dissolve. Add sodium hydroxide (2 mM) while stirring at room temperature. After stirring for 0.5 hours, add 4-bromobenzoyl bromide (1.1 mM). Finally, react overnight at room temperature. After the system reaction is completed, extract with water and ethyl acetate three times, wash with saturated brine, and remove the organic phase by vacuum distillation. Finally, intermediate E is obtained by column chromatography (petroleum ether: ethyl acetate = 5:1).

[0043] (6) Intermediate E was placed in a 25 mL round-bottom flask and dissolved in a DCM-MEOH (1:2) solvent. While stirring, a 50% mass percent hydroxylamine aqueous solution was added, and after stirring at room temperature for 0.5 h, 10 eq of sodium hydroxide was added. After 10 min, the reaction of intermediate E was completely detected by thin layer chromatography. Then, the organic solvent was removed by vacuum distillation. Then, water was added to dissolve the residue, and dilute hydrochloric acid was added in an ice bath to adjust the pH value to 5-6. The filtered solid was washed twice with a mixture of petroleum ether-ethyl acetate (1:1) and finally dried to obtain a white solid final product, which is the compound shown in Formula I, and its Chinese name is: N-hydroxy-4-(((5-(4-isopropylphenyl)-3-(4-methoxyphenyl)pyridin-2-yl)amino)methyl)benzamide.

[0044] The compound shown in formula I is a white powder with a yield of 70% and a melting point of 237.2-238.8°C. Figure 1 and Figure 2 The characterization results are as follows: 1H NMR(400MHz,DMSO-d6)δ11.13(s,1H,OH),8.97(s,1H,NH),8.24(s,1H,Ar-H),7.67(d ,J=7.7Hz,2H,Ar-H),7.50(dd,J=12.7,7.9Hz,5H,Ar-H),7.37(d,J=7.6Hz,2H,Ar-H) ,7.26(d,J=7.6Hz,2H,Ar-H),7.09(d,J=8.0Hz,2H,Ar-H),6.43(s,1H,NH),4.60(d,J =4.5Hz,2H,CH2),3.81(s,3H,CH3),2.93–2.84(m,1H,CH),1.21(d,J=6.6Hz,6H,CH3). 13 C NMR(101MHz,DMSO-d6)δ164.29,158.79,154.36,146.59,144.74,143.91,135.33,135.18,130.79,130.11, 129.62,126.77,126.73,125.59,124.74,121.32,114.51,55.17,44.03,33.02,23.85.HR-MS(ESI),calcd.C 29 H 29 N3O3,[M+H] + m / z:468.2287,found:468.2290.

[0045] Example 3

[0046] The application of the pyridine compound containing N-hydroxy-benzamide in this embodiment is in the preparation of HDAC6 inhibitors or anti-tumor drugs.

[0047] In different embodiments, when the HDAC6 inhibitor is specifically prepared, the present invention has no special requirements for the content of the active component in the HDAC6 inhibitor, and those skilled in the art can select it based on experience. Similarly, there are no special requirements for the preparation method of the HDAC6 inhibitor, and any drug preparation method known in the art can be used. In other embodiments, there are no special requirements for the dosage form of the HDAC6 inhibitor, and those skilled in the art can select the dosage form of the HDAC6 inhibitor according to actual needs.

[0048] In other embodiments, when preparing an antitumor drug, the antitumor drug may include the pyridine compound containing N-hydroxy-benzamide of Example 1 and a pharmaceutically acceptable excipient. The present invention has no special requirements on the specific type of the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0049] Test example

[0050] Experimental Example 1 CCK-8 method to detect the inhibitory effect of compounds on the proliferation of ovarian cancer cells SKOV3 and A2780

[0051] The experimental process is as follows: (1) Plating: SKOV3 and A2780 cells were digested and resuspended into single cell suspensions, and plated in 96-well plates, with 5 × 10 cells per well. 3 cells, and cultured them in a 37°C incubator overnight; (2) Preparation of drugs: The compound of Example 1 was prepared into a 10mM stock solution with DMSO, and diluted in two-fold gradients starting from 100μM, 100μL per well, and 3 replicates for each gradient; (3) Dosing: Aspirate the cell supernatant of the 96-well plate, add 100μL of the compound of Example 1 of different concentrations to each well, and culture them in a 37°C incubator for 24 to 72h; (4) Cell viability detection: Dilute the CCK-8 stock solution with serum-free cell culture medium at a ratio of 1:10, aspirate the cell supernatant, add CCK-8 detection solution, 100μL per well, incubate at 37°C for 1h, and detect the absorbance at 450nm with an enzyme marker. The results are as follows Figure 3 shown.

[0052] Depend on Figure 3 It can be seen that with the extension of time and the increase of drug concentration, the cell viability is significantly reduced, indicating that the drug has a significant inhibitory effect on the proliferation of ovarian cancer cells, and the inhibitory effect is concentration- and time-dependent. Among them, when treated for 72 hours, the IC50 of the compound of Example 1 for A2780 is 5.40±0.53μM, and the IC50 for SKOV3 is 0.85±0.02μM.

[0053] Experimental Example 2 Inhibitory Effect of Compounds on Colony Formation of Ovarian Cancer Cell A2780

[0054] The experimental process is as follows: (1) Plating: A2780 cells were digested and resuspended into a single cell suspension, and then plated on a 6-well plate, with about 1,000 cells per well, and cultured in a 37°C incubator overnight; (2) Drug preparation: The compound of Example 1 was prepared with DMSO at 2 μM, 4 μM, and 6 μM, 2 mL per well, and the group without drug addition was used as a blank control. Each gradient had 3 replicate wells, and was cultured in a 37°C incubator for 10 days; (3) The supernatant was discarded, fixed with 4% formaldehyde for 10 minutes, stained with crystal violet for 10 minutes, and the number of clones formed was counted after washing with running water. The results are shown in Figure 2. Figure 4 shown.

[0055] Depend on Figure 4 It can be seen that as the concentration of the compound of Example 1 increases, the number of clones formed by ovarian cancer cell A2780 significantly decreases, indicating that the compound of the present invention can effectively inhibit the clone formation of ovarian cancer cells.

[0056] Experimental Example 3 Flow cytometry detection of the apoptosis-inducing effect of compounds on ovarian cancer cells SKOV3 and A2780

[0057] The experimental process is as follows: (1) Plating: SKOV3 and A2780 cells were digested and resuspended into single cell suspensions, and plated in 6-well plates with 2-4×10 5 cells, and cultured them in a 37°C incubator overnight; (2) Adding drugs: Aspirate and discard the supernatant of the 6-well plate cells, add 2 mL of the compound of Example 1 at different concentrations to each well, and culture them in a 37°C incubator for 72 h; (3) Detection of apoptotic cells: Digest the cells with trypsin without EDTA, stain the cells according to the instructions of the Biyuntian apoptosis kit, and detect them on the machine; the results are as follows: Figure 5 shown.

[0058] Depend on Figure 5 It can be seen that as the drug concentration of the compound of Example 1 increases, the percentages of early apoptosis and late apoptosis of ovarian cancer cell A2780 and SKOV3 cells are significantly increased, indicating that the compound of the present invention can effectively promote the apoptosis of ovarian cancer cells.

[0059] Experimental Example 4 WB detection of the regulation of the action of compounds on intracellular HDAC6

[0060] The experimental process is as follows: (1) Plating: SKOV3 and A2780 cells were digested and resuspended into single cell suspensions, and plated in 6-well plates with 2-4×10 5 cells, and cultured them in a 37°C incubator overnight; (2) Adding drugs: Aspirate the supernatant of the 6-well plate cells, add 2 mL of the compound of Example 1 at different concentrations to each well, and culture them in a 37°C incubator for 24 hours; (3) Collecting proteins: Wash the cells twice with pre-cooled PBS, add RIPA containing PMFS and phosphatase inhibitors, lyse on ice for 30 minutes, scrape the cells to collect proteins, centrifuge at 12,000 rpm for 10 minutes, aspirate the supernatant, add loading buffer, and boil for 10 minutes; (4) Electrophoresis: Separate with 10% SDS page gel electrophoresis; (5) Transfer to membrane: After transferring to PVDF membrane, block with 5% BSA for 2 hours, add 1:1000 dilution of anti-HDAC6, α-tubulin, acetylated tubulin, H3, acetylated H3 and GAPDH, and incubate at 4°C overnight; (6) Wash with PBST three times, add 1:1000 dilution of secondary antibody; wash with PBST three times, add ECL luminescent solution for development and exposure. The results are as follows Figure 6 shown.

[0061] Depend on Figure 6 It can be seen that the addition of the compound of Example 1 of the present invention has no effect on the acetylation level of H3 histone; however, the compound of Example 1 can significantly reduce the expression level of HDAC6 and significantly increase the acetylation level of tubulin, indicating that the compound of Example 1 of the present invention can effectively reduce the expression level and activity of HDAC6 in ovarian cancer cells.

[0062] Based on the above results, it can be seen that the pyridine compound containing N-hydroxy-benzamide of the present invention can effectively inhibit HDAC6 in ovarian cancer cells, and can inhibit the proliferation and cloning of ovarian cancer cells, and promote apoptosis of ovarian cancer cells, so it can be used to prepare HDAC6 inhibitors, and can also be used as a new compound for the preparation of anticancer drugs. Therefore, the present invention can provide a new research and development direction for the development of HDAC6 inhibitors, and can provide a new structural selection for the development of new anti-tumor drugs.

Claims

1. The use of pyridine compounds containing N-hydroxy-benzamide, characterized in that: The application is the application of pyridine compounds containing N-hydroxy-benzamide in the preparation of anti-tumor drugs; the anti-tumor drugs are drugs that inhibit tumor cell proliferation and cloning and promote tumor cell apoptosis; the tumor cells are ovarian cancer cells; the ovarian cancer cells are one or both of A2780 ovarian cancer cells and SKOV3 ovarian cancer cells; The pyridine compound containing N-hydroxy-benzamide is a compound shown in Formula I:

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