CN118027033A - HDAC6 inhibitor, preparation method thereof and application thereof in anti-inflammatory and ulcerative colitis - Google Patents

Abstract

The invention provides an HDAC6 inhibitor, a preparation method thereof and application thereof in anti-inflammatory and ulcerative colitis, wherein a series of novel compounds containing alkaloid structures and having anti-ulcerative colitis activity are developed by connecting an alkaloid structure with a hydroxamic acid group of the HDAC6 inhibitor, experimental data show that the compounds have better inhibition effect on the HDAC6 at enzyme level, and can be used for preparing medicines for preventing or treating ulcerative colitis.

Description

A HDAC6 inhibitor and its preparation method and use in anti-inflammatory and ulcerative colitis

The invention belongs to the technical field of drug synthesis, and particularly relates to an HDAC6 inhibitor and a preparation method thereof and use thereof in anti-inflammatory and ulcerative colitis.

Background Art

Ulcerative colitis (UC) is a chronic, nonspecific inflammation of the rectum and colon that lasts a long time and is difficult to cure. The disease is more common in Europe and the United States, and the incidence in China has been on the rise in recent years. As the etiology and pathogenesis of UC have not been fully elucidated, there is currently a lack of specific drugs.

Histone deacetylase 6 is an important member of the class IIb HDAC family. It participates in regulating important physiological processes such as cell growth, metastasis and apoptosis by modifying specific substrates including α-tubulin and heat shock protein 90 (HSP90). Studies have shown that overexpression of HDAC6 increases the level of α-tubulin in a dose-dependent manner, further upregulates the expression of NADPH oxidase, and induces the production of reactive oxygen species (ROS), thereby reducing the levels of NF-κB-related proteins such as IKKα/β and IκB, and finally leading to increased levels of pro-inflammatory factors such as TNF-α, IL-1β and IL-6. At present, HDAC6 has received increasing attention in the research of inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis and chronic asthma. Selective HDAC6 inhibitors have gradually become a research hotspot because they are basically non-toxic to normal cells. The design and development of new selective HDAC6 inhibitors for inflammatory diseases has important social value.

The pharmacophore model of selective HDAC6 inhibitors is mainly divided into three parts: zinc ion chelating group (ZBG), linker group occupying hydrophobic channel, and active pocket surface recognition region (Cap). The affinity of HDAC6 inhibitors to different subtypes usually depends on the interaction and geometric conformation of the above three parts with the active site. Among them, the Cap group has the greatest impact on activity. This part is often located on the surface of the protein and interacts with the edge area outside the pocket. The Linker part occupies the channel of the protein and plays a supporting and connecting role in the stability of the entire molecular conformation. The ZBG group is generally located inside the protein pocket and forms a chelation with Zn ²⁺ and other amino acid residues in the protein. According to the different ZBG structures, HDAC6 inhibitors can be divided into hydroxamic acids, benzamides and fatty carboxylic acids. Among them, hydroxamic acids are the most widely used, such as vorinostat (SAHA), belinostat (PXD101) and panobinostat (LBH589).

Although the research on selective HDAC6 inhibitors has made important progress, most of them are still in the clinical trial stage, and no drug has been approved for marketing. At present, the specific mechanism of HDAC6 inhibitors in inflammation has not been fully elucidated, and most candidate drugs have problems such as poor pharmacokinetics and metabolic instability, which restrict their further development.

Summary of the invention

In order to solve the above problems, the present invention provides an HDAC6 inhibitor and a preparation method thereof and use thereof in anti-inflammatory and ulcerative colitis. The present invention connects an alkaloid structure with anti-inflammatory activity with the hydroxamic acid group of the HDAC6 inhibitor through computer-aided drug design and skeleton transition principles, and develops a new type of HDAC6 inhibitor containing an alkaloid structure and having anti-ulcerative colitis activity.

The technical solution of the present invention is:

An HDAC6 inhibitor having the following structure:

R is at least one selected from the group consisting of cytisine, acridone, indoline, 1,2,3,4-tetrahydro-9H-pyrido[3,4-B]indole, 1,2,3,4-tetrahydroisoquinoline, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, 5-aminoindole, 2-hydroxyquinoxaline, and 2-hydroxyquinoline;

X is a saturated or unsaturated alkane chain containing C0-C4.

It is further preferred that the structure of the HDAC6 inhibitor is Formula I or Formula II, and the specific structure is as follows:

;

Wherein, R1 and R2 are independently selected from at least one of cytisine, acridone, indoline, 1,2,3,4-tetrahydro-9H-pyrido[3,4-B]indole, 1,2,3,4-tetrahydroisoquinoline, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, 5-aminoindole, 2-hydroxyquinoxaline, and 2-hydroxyquinoline.

It is further preferred that the structure of the HDAC6 inhibitor is any one of the following:

The preparation method of the HDAC6 inhibitor comprises the following steps:

(1) subjecting an alkaloid and methyl 4-bromomethylbenzoate or methyl 4-bromomethylcinnamate to a nucleophilic substitution reaction under alkaline conditions to obtain an intermediate;

(2) The intermediate and an aqueous solution of hydroxylamine are subjected to a hydroxylamine hydrolysis reaction under alkaline conditions to obtain the HDAC6 inhibitor.

In step (1), the alkaline condition is provided by potassium carbonate and/or sodium carbonate.

In step (2), the alkaline condition is provided by sodium hydroxide and/or potassium hydroxide.

When the structure of the HDAC6 inhibitor is a compound of formula I, the specific steps are:

(S1) reacting an alkaloid with methyl 4-bromomethylbenzoate under alkaline conditions through a nucleophilic substitution reaction to obtain an intermediate A;

(S2) intermediate A and an aqueous solution of hydroxylamine are subjected to a hydroxylamine hydrolysis reaction under alkaline conditions to obtain a compound of formula I;

In step (S1), the molar ratio of the alkaloid to methyl 4-bromomethylbenzoate is 1:(1-2);

In step (S2), the molar ratio of the intermediate A to the aqueous solution of hydroxylamine is 1:(15-20).

A pharmaceutical composition comprising the HDAC6 inhibitor.

Use of the HDAC6 inhibitor in preparing anti-inflammatory and ulcerative colitis drugs.

The beneficial effects of the present invention are:

The present invention provides an HDAC6 inhibitor, by connecting an alkaloid structure with anti-inflammatory activity to the hydroxamic acid group of the HDAC6 inhibitor, a series of novel compounds containing alkaloid structures and having anti-ulcerative colitis activity are developed, and experimental data show that the above-mentioned compounds show good inhibitory effects on HDAC6 at the enzyme level, and can be used to prepare drugs for preventing or treating ulcerative colitis. This is because the inventors of the present application have found in long-term studies that alkaloids can reduce oxidative stress reactions, reduce inflammatory mediators, MPO activity, maintain the integrity of the intestinal mucosa, and change inflammatory reactions by inhibiting the activation of inflammatory signaling pathways such as (NF-κB/Nrf2). Alkaloid compounds have the effect of reducing or treating ulcerative colitis, and their mechanism is related to regulating immune inflammatory reactions, adjusting intestinal flora, and protecting intestinal mucosal barriers. Therefore, the present invention connects an alkaloid structure with anti-inflammatory activity to the hydroxamic acid group of the HDAC6 inhibitor through the principle of computer-aided drug design and skeleton transition, and obtains an HDAC6 inhibitor containing an alkaloid structure and having anti-ulcerative colitis activity.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the indicators of the DSS-induced ulcerative colitis mouse model. A: Mouse weight change rate; B: Disease activity index; C: Mouse colon length change diagram; All data are expressed as mean±SD, 6 mice per group. ***P＜0.001, ****P＜0.0001, compared with the Model group. ####P＜0.0001, compared with the Normal group;

FIG. 2A-F shows H&E staining images of isolated colon tissues from a DSS-induced ulcerative colitis mouse model. FIG.

DETAILED DESCRIPTION

To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

The above technical solution is described in detail below in conjunction with specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

Example 1

This embodiment provides an HDAC6 inhibitor, N-hydroxy-4-(((1R,5S)-8-oxo-1,5,6,8-tetrahydro-2H-1,5-methanopyrido[1,2-a][1,5]diazocin-3(4H)-yl)methyl)benzamide (AK-01), whose structural formula is shown below:

The synthesis steps of the above HDAC6 inhibitor are as follows:

(1) Weigh 2 mmol of cytisine and 3 mmol of methyl 4-bromomethylbenzoate, mix and add to 20 mL of N,N-dimethylformamide, then add 4 mmol of anhydrous potassium carbonate, and stir at room temperature for 3 hours. After TLC monitoring until the reaction is complete, add water to extract the reaction solution, concentrate under reduced pressure, separate by thin layer preparative chromatography (developing solvent PE/EA=1/1), and dry to obtain intermediate A.

(2) Dissolve 2 mmol of intermediate A in 15 mL of methanol, add 2 mmol of sodium hydroxide, stir at 0°C for 15 minutes, and dropwise add 2 mL of hydroxylamine aqueous solution. Monitor by TLC until the reaction is complete, add hydrochloric acid to adjust the pH to 7-8, concentrate under reduced pressure, separate and purify by preparative thin layer chromatography (DCM/MeOH=7/1), and dry in vacuo to obtain the target compound.

Light brown solid, yield: 68%. mp: 82.6-83.7; ¹ H NMR (400MHz, DMSO) δ11.14 (s, 1H), 8.99 (s, 1H), 7.58 (d, J = 7.8 Hz, 2H), 7.35 (dd, J = 8.9, 6.7 Hz, 1H), 7.01 (d, J = 7.9 Hz, 2H), 6.28 (d, J = 8.8 Hz, 1H), 6.03 (d, J = 6.6 Hz, 1H), 3.87 (d, J = 15.2 Hz, 1H), 3.70 (dd ,J＝15.3,6.3Hz,1H),3.46(dd,J＝35.6,14.3Hz,2H),3.01(s,1H),2.88(d,J＝10.5Hz,1H),2.73(d,J＝10.4Hz,1H),2.39(s,1H),2.31(dd,J＝23.9,10.5Hz, 2H), 1.84 (d, J = 12.6Hz, 1H), 1.76-1.66 (m, 1H).

HRMS (ESI): The theoretical value for C ₁₉ H ₂₁ N ₃ O ₃ [M+H] ⁺ is 340.1662, the measured value is 340.1663.

Example 2

This embodiment provides another HDAC6 inhibitor, 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-hydroxybenzamide (AK-03), whose structural formula is shown below:

The synthesis steps are the same as those in Example 1, except that cytisine is replaced by 1,2,3,4-tetrahydroisoquinoline.

Light pink solid, yield: 66%. mp: 176.9-177.8; ¹ H NMR (400MHz, DMSO) δ7.73 (d, J = 7.4 Hz, 2H), 7.42 (d, J = 7.3 Hz, 2H), 7.13-7.04 (m, 3H), 6.98 (d, J = 6.7 Hz, 1H), 3.67 (s, 2H), 3.53 (s, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.66 (t, J = 5.7 Hz, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ142.22,135.16,134.51,129.03,128.93,127.34,126.81,126.47,125.95,61.86,55.89,50.73,29.14.

HRMS (ESI): Calculated value for C ₁₇ H ₁₈ N ₂ O ₂ [M+H] ⁺ is 283.1447. Found value is 283.1453.

Example 3

This example provides another HDAC6 inhibitor, 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-hydroxybenzamide (AK-05), the structure of which is as follows:

The synthesis steps are the same as those of Example 1, except that cytisine is replaced by 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.

Light pink solid, yield: 65%. mp:108.5-110.2; ¹ H NMR (400MHz, DMSO) δ7.72 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 6.65 (s, 1H), 6.56 (s ,1H),3.68(s,3H),3.65(s,5H),3.42(s,2H),2.72(t,J＝5.8Hz,2H),2.64(t,J＝5.8Hz,2H). ¹³ C NMR(101MHz,DMSO-d ₆ )δ147.62,147.38,142.25,132.09,129.06,127.31,126.88,126.18,112.25,110.36,61.94,55.92,55.91,55.48,51.01,28.73,26.81.

HRMS (ESI): Calculated value for C ₁₉ H ₂₂ N ₂ O ₄ [M+H] ⁺ is 343.1659. Found value is 343.1665.

Example 4

This example provides another HDAC6 inhibitor, N-hydroxy-4-((1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methyl)benzamide (AK-07), the structure of which is shown below:

The synthesis steps were the same as those in Example 1, except that cytisine was replaced by 1,2,3,4-tetrahydro-9H-pyridine[3,4-B]indole.

Light brown solid, yield: 70%. mp: 147.2-148.3; ¹ H NMR (400 MHz, DMSO) δ 7.73 (d, J = 7.7 Hz, 2H), 7.44 (d, J = 7.7 Hz, 2H), 7.35 (d, J = 7.7 Hz, 1H), 7.24 (d, J = 7.9 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.93 (t, J = 7.3 Hz, 1H), 3.76 (s, 2H), 3.56 (s, 2H), 2.80 (t, J = 5.6 Hz, 2H), 2.69 (t, J = 5.6 Hz, 2H).

HRMS (ESI): Calculated value for C ₁₉ H ₁₉ N ₃ O ₂ [M+H] ⁺ is 322.1556. Found value is 322.1553.

Example 5

This example provides another HDAC6 inhibitor, N-hydroxy-4-(2-oxo-3,4-dihydroquinolin-1(2H)-yl)methyl)benzamide (AK-09), whose structural formula is shown below:

The synthesis steps are the same as those in Example 1, except that cytisine is replaced by 2-hydroxyquinoline.

Light brown solid, yield: 68%. mp: 115.0-116.8; ¹ H NMR (400 MHz, DMSO) δ 7.67 (d, J = 7.7 Hz, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.22 (d, J = 7.1 Hz, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 5.17 (s, 2H), 2.94 (t, J = 7.0 Hz, 2H), 2.70 (dd, J = 8.4, 5.8 Hz, 2H).

HRMS (ESI): The theoretical value of C ₁₇ H ₁₆ N ₂ O ₃ [M+Na] ⁺ is 319.1059, the measured value is 319.1063.

Example 6

This example provides another HDAC6 inhibitor, N-hydroxy-4-(2-oxoquinoxalin-1(2H)-yl)methyl)benzamide (AK-11), whose structural formula is shown below:

The synthesis steps are the same as those in Example 1, except that cytisine is replaced by 2-hydroxyquinoxaline.

Light pink solid, yield: 60%. mp: 181.5-183.3; ¹ H NMR (400MHz, DMSO) δ8.36 (s, 1H), 7.86 (d, J = 7.5Hz, 1H), 7.68 (d, J = 7.4Hz, 2H), 7.55 (t, J = 7.5Hz, 1H), 7.42 (d, J = 7.9Hz, 1H), 7.35 (dd, J = 14.5, 7.3Hz, 3H), 5.74 (s, 1H), 5.52 (s, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ164.27,154.97,150.84,139.29,133.46,132.66,132.46,131.56,130.35,127.78,127.25,124.18,115.59,44.79.

HRMS (ESI): the theoretical value for C ₁₆ H ₁₃ N ₃ O ₃ [M+Na] ⁺ is 318.0855. The found value is 318.0854.

Example 7

This embodiment provides another HDAC6 inhibitor, 4-((1H-indol-5-yl)amino)methyl)-N-hydroxybenzamide (AK-13), whose structural formula is shown below:

The synthesis steps are the same as those in Example 1, except that cytisine is replaced by 5-aminoindole.

Brown solid, yield: 50%. mp:185.2-186.8; ¹ H NMR (400MHz, DMSO) δ10.62 (s, 1H), 7.68 (d, J = 7.8Hz, 2H), 7.44 (d, J = 7.8Hz, 2H), 7.10 (d ,J＝8.4Hz,2H),6.57(dd,J＝8.0,2.8Hz,1H),6.52(d,J＝6.9Hz,1H),6.10(t,J＝3.8Hz,1H),5.69(s ,1H),4.29(s,2H). ¹³ CNMR(101MHz,DMSO-d ₆ )δ145.04,142.33,131.50,130.06,128.80,127.52,127.26,125.14,112.17,111.76,101.04,100.43,47.88.

HRMS (ESI): Calculated value for C ₁₆ H ₁₅ N ₃ O ₂ [M+H] ⁺ is 282.1243. Found value is 282.1251.

Example 8

This example provides another HDAC6 inhibitor, (E)-N-hydroxy-3-(4-((1R,5S)-8-oxo-1,5,6,8-tetrahydro-2H-1,5-methanopyrido-[1,2-a][1,5]diazocin-3(4H)-yl)methyl)phenyl)acrylamide (AK-02), whose structural formula is shown below:

The synthesis steps of the above HDAC6 inhibitor are as follows:

(1) Weigh 2 mmol of cytisine and 3 mmol of methyl 4-bromomethylcinnamate, mix and add to 20 mL of N,N-dimethylformamide, then add 4 mmol of anhydrous potassium carbonate, and stir at room temperature for 3 hours. After TLC monitoring until the reaction is complete, add water to extract the reaction solution, concentrate under reduced pressure, separate by thin layer preparative chromatography (developing solvent PE/EA=1/1), and dry to obtain intermediate B.

(2) Dissolve 2 mmol of intermediate B in 15 mL of methanol, add 2 mmol of sodium hydroxide, stir at 0°C for 15 minutes, and dropwise add 2 mL of hydroxylamine aqueous solution. Monitor by TLC until the reaction is complete, add hydrochloric acid to adjust the pH to 7-8, concentrate under reduced pressure, separate and purify by preparative thin layer chromatography (DCM/MeOH=7/1), and dry in vacuo to obtain the target compound.

Light pink solid, yield: 71%. mp: 157.5-159.1; ¹ H NMR (400MHz, DMSO) δ7.59-7.23 (m, 4H), 6.99 (d, J = 7.5Hz, 2H), 6.41 (d, J = 15.7Hz, 1H), 6.27 (d, J = 8.8Hz, 1H), 6.03 (d, J = 6.6Hz, 1H), 3.86 (d, J = 15.3Hz, 1H), 3.70 (dd, J = 15.3, 6.3Hz, 1H),3.47(d,J＝14.2Hz,2H),3.01(s,1H),2.88(d,J＝10.6Hz,1H),2.74(d,J＝10.4Hz,1H),2.39(s,1H),2.29(dd,J＝16.8,10.6Hz,2H),1.84(d,J＝12.6Hz ,1H),1.71(d,J=12.7Hz,1H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ163.20,162.69,152.51,140.30,139.24,138.19,133.96,128.86,127.66,119.21,115.72,104.34,61.20,60.14,59.97 ,50.09,35.02,27.89,25.62.

HRMS (ESI): Calcd. for C ₂₁ H ₂₃ N ₃ O ₃ [M+H] ⁺ : 366.1818, found: 366.1815.

Embodiment 9

This example provides another HDAC6 inhibitor, (E)-3-(4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)-N-hydroxyacrylamide (AK-04), whose structure is as follows:

The synthesis steps are the same as those of Example 8, except that cytisine is replaced by 1,2,3,4-tetrahydroisoquinoline.

Light pink solid, yield: 60%. mp: 185.0-186.9; ¹ H NMR (400MHz, DMSO) δ10.63 (s, 1H), 7.53 (d, J = 7.5Hz, 1H), 7.41 (d, J = 7.2Hz, 2H), 7.34 (d, J = 7.7Hz, 1H), 7.24 (d, J = 8.0Hz, 1H), 6.96 (dt, J = 25.9, 7.3Hz, 2H), 6.46 (d, J = 15.8Hz, 1H), 3.74 (s, 2H), 3.55 (s, 2H), 2.80 (t, J = 5.6Hz, 2H), 2.69 (t, J = 5.6Hz, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ163.24,140.51,138.55,135.19,134.53,134.10,129.69,128.92,127.93,126.82,126.45,125.94,119.15,61.94,55.89,50.71,29.14.

HRMS (ESI): Calculated value for C ₁₉ H ₂₀ N ₂ O ₂ [M+H] ⁺ is 390.1604. Found value is 309.1608.

Example 10

This example provides another HDAC6 inhibitor, (E)-3-(4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)-N-hydroxyacrylamide (AK-06), the structure of which is shown below:

The synthesis steps are the same as those of Example 8, except that cytisine is replaced by 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.

Light brown solid, yield: 58%. mp: 119.4-120.6; ¹ H NMR (400MHz, DMSO) δ7.51 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 14.9 Hz, 1H), 7.37 (d, J = 7.4 Hz, 2H), 6.64 (s, 1H), 6.56 (s, 1H), 6.52-6.23 (m, 1H), 3.68 (s, 3H), 3.64 (s, 3H), 3.61 (s, 2H), 3.41 (s, 2H), 2.71 (t, J = 5.6 Hz, 2H), 2.63 (t, J = 5.7 Hz, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ147.62,147.38,134.15,129.72,127.89,126.92,126.20,112.24,110.36,62.04,55.92,55.89,55.49,55.36,51.00,28.75.

HRMS (ESI): theoretical value for C ₂₁ H ₂₄ N ₂ O ₄ [M+H] ⁺ is 369.1815. found value is 369.1822.

Embodiment 11

This example provides another HDAC6 inhibitor, (E)-N-hydroxy-3-(4-((1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methyl)-phenyl)acrylamide (AK-08), the structure of which is shown below:

The synthesis steps were the same as those of Example 8, except that cytisine was replaced by 1,2,3,4-tetrahydro-9H-pyridine[3,4-B]indole.

Light brown solid, yield: 80%. mp:145.6-146.3; ¹ H NMR (400MHz, DMSO-d ₆ ) δ10.63 (s, 1H), 7.53 (d, J = 7.5Hz, 2H), 7.42 (t, J = 10.1Hz, 3H), 7.34 (d, J = 7.7Hz, 1H), 7.24 (d, J = 8.0Hz, 1H), 6.99 (t,J＝7.4Hz,1H),6.92(t,J＝7.3Hz,1H),6.46(d,J＝15.7Hz,1H),3.74(s,2H),3.55(s,2H),2.80(t,J＝5.6Hz,2H),2.70(d,J＝5.4Hz,2H). ¹³ C NMR (101MHz, DM SO-d ₆ )δ140.80,136.30,134.14,133.19,129.74,127.94,127.16,120.78,119.13,118.74,117.81,111.34,106.79,61.42,51.11,50.37,21.61.

HRMS (ESI): Calcd. for C ₂₁ H ₂₁ N ₃ O ₂ [M+H] ⁺ : 348.1713. Found: 348.1713.

Example 12

This example provides another HDAC6 inhibitor, (E)-N-hydroxy-3-(4-(2-oxoquinoxalin-1(2H)-yl)methyl)phenyl)acrylamide (AK-10), the structure of which is shown below:

The synthesis steps are the same as those of Example 8, except that cytisine is replaced by 2-hydroxyquinoxaline.

Pale pink solid, yield: 66%. mp: 146.2-147.7; ¹ H NMR (400 MHz, DMSO) δ 8.36 (s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.42 (d, J = 9.5 Hz, 1H), 7.39-7.35 (m, 1H), 7.30 (d, J = 7.7 Hz, 2H), 6.41 (d, J = 15.8 Hz, 1H), 5.50 (s, 2H), 4.34 (t, J = 5.2 Hz, 1H).

HRMS (ESI): the theoretical value for C ₁₈ H ₁₅ N ₃ O ₃ [M+Na] ⁺ is 344.1011. The found value is 344.1009.

Embodiment 13

This example provides another HDAC6 inhibitor, (E)-N-hydroxy-3-(4-(indolin-1-ylmethyl)phenyl)acrylamide (AK-12), the structure of which is shown below:

The synthesis steps are the same as those of Example 8, except that cytisine is replaced by indoline.

Dark brown solid, yield: 40%. mp: 104.8-105.1; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ7.52 (s, 2H), 7.39 (t, J=12.0 Hz, 3H), 7.08-6.93 (m, 2H), 6.57 (q, J=7.2 Hz, 3H), 4.26 (s, 2H), 3.24 (d, J=8.1 Hz, 2H), 2.88 (t, J=8.1 Hz, 2H).

HRMS (ESI): Calculated value for C ₁₈ H ₁₈ N ₂ O ₂ [M+H] ⁺ is 295.1447. Found value is 295.1446.

Embodiment 14

This example provides another HDAC6 inhibitor, N-hydroxy-4-((9-oxoacridin-10(9H)-yl)methyl)benzamide (AK-14), the structure of which is shown below:

The synthesis steps of the above HDAC6 inhibitor are as follows:

(1) Weigh 2 mmol of acridone, add it to 10 mL of N,N-dimethylformamide to dissolve it, then add 3 mmol of sodium hydride, stir at 0°C for 10 minutes, then add 3 mmol of methyl 4-bromomethylbenzoate, gradually warm to room temperature, and stir for 2 hours. Monitor by TLC until the reaction is complete, quench the reaction, extract, concentrate under reduced pressure, separate by thin layer preparative chromatography (developing solvent PE/EA=1/1), and dry to obtain intermediate C.

(2) Dissolve 2 mmol of intermediate C in 15 mL of methanol, add 2 mmol of sodium hydroxide, stir at 0°C for 15 minutes, and dropwise add 2 mL of hydroxylamine aqueous solution. Monitor by TLC until the reaction is complete, add hydrochloric acid to adjust the pH to 7-8, concentrate under reduced pressure, separate and purify by preparative thin layer chromatography (DCM/MeOH=7/1), and dry in vacuo to obtain the target compound.

Pale yellow solid, yield: 80%. mp: 226.9-227.8; ¹ H NMR (400MHz, DMSO) δ 11.15 (s, 1H), 9.09 (s, 1H), 8.40 (d, J = 7.8 Hz, 2H), 7.93-7.67 (m, 4H), 7.63 (d, J = 8.6 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 5.84 (s, 2H). ¹³ C NMR (101MHz, DMSO-d ₆ )δ177.18,142.53,140.07,134.84,132.46,127.98,127.23,126.34,122.20,122.12,116.58,49.35.

HRMS (ESI): The theoretical value of C ₂₁ H ₁₆ N ₂ O ₃ [M+Na] ⁺ is 367.1059, the measured value is 367.1061.

Experimental Example 1

The inhibition rate of 14 compounds on HDAC1 and HDAC6 enzymes at a concentration of 50nM was screened in vitro by fluorescence method, with SAHA as the positive compound. The results are as follows:

(1) Prepare 1x experimental buffer (modified Tris buffer);

(2) Compound serial dilution: Transfer the compounds to the assay plate in 100% DMSO using Echo. The final fraction of DMSO is 1%;

(3) Prepare enzyme solution: Prepare enzyme solution in 1x assay buffer;

(4) Prepare substrate solution: Add trypsin and ac peptide substrate to 1x experimental buffer to make substrate solution;

(5) Transfer 15 μL of enzyme solution to the assay plate, or transfer 15 μL of 1x assay buffer for the low control;

(6) Incubate at room temperature for 15 minutes;

(7) Add 10 μL of substrate solution to each well to start the reaction;

(8) The plate was dynamically read on Envision with an excitation wavelength of 355 nm and an emission wavelength of 460 nm;

(9) Curve Fitting The data were fitted in Excel and the inhibition value was obtained using formula (1).

Formula (1): Inh% = (Max-Signal)/(Max-Min)*100

The data were fitted in xml-Fit and the IC50 value was obtained using formula (2).

Equation (2): Y = Bottom + (Top-Bottom) / (1 + (IC50/X) * HillSlope) Y is the inhibition rate, X is the compound concentration.

Table 1—In vitro HDAC1 and HDAC6 enzyme inhibitory activities of compounds

As can be seen from Table 1, except for AK-12, all the compounds synthesized by Formula I-II (i.e., containing N-hydroxycinnamic acid amide groups) of the above 14 compounds showed strong inhibitory activity against HDAC1 and HDAC6 at a concentration of 50 nM. Among the compounds synthesized by Formula I (i.e., containing N-hydroxy-4-methylbenzamide groups), AK-11, AK-13 and AK-14 showed strong HDAC6 inhibitory activity, among which it can be inferred that AK-14 has the best HDAC1/6 subtype selectivity.

Experimental Example 2

The inhibition rate of 14 compounds on NO production in mouse macrophage RAW264.7 was determined by MTT method, with dexamethasone as positive control. The results are shown in Table 2.

RAW264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (Biological Industries), 100 U/mL penicillin and 100 mg/ml streptomycin (Beyotime), and transferred to a humidified environment at 37°C containing 5% _CO2 for culture.

Collect the logarithmically growing RAW264.7 cells in a 15 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, dilute with fresh culture medium to a suitable density, and set aside. Place a special cover glass in the center of the cell counting plate, aspirate 10 μL of suspension from the centrifuge tube, add it between the counting plate and the special cover glass, and fill the cover glass with liquid through siphoning. Place the counting plate under a microscope for counting.

Counting formula: (number of cells per well × 100 × number of 96-well plates) ÷ (number of cells in 4 large grids × 10 ⁴ ) = volume of suspension (mL)

Calculate the volume of the cell suspension, and by calculating the cell suspension density, plate 8000-12000 cells per well, that is: plate a 96-well plate and aspirate the cell suspension volume (mL) = (number of cells per well × 96 × 1) / cell suspension density. Mix the required cell suspension and culture medium, add the suspension to the 96-well plate with a spray gun, 100 μL per well, and mix the cell suspension once every three additions. After the cell suspension is added, cover the plate and observe the cell density and uniformity under an inverted microscope.

In a 96-well plate, the experimental group was treated for 48 hours by adding different compounds, 3 wells were used as blank groups, and an equal volume of DMSO was added, and 3 wells were used as LPS groups, and 100 μL of culture medium was added to each well, and no drug was added to the side wells. After 48 hours, the cell suspensions of each group were transferred to a new cell counting plate, and then the A solution and B solution in the NO kit were added to each well, respectively. After the solution changed from colorless to pink, the culture medium was collected, and the nitrite level (an indicator of NO production) was determined by the Griess method, and the absorbance of the sample was determined at a wavelength of 540 nm (OD540) using an enzyme reader.

Control: DMSO solution treated with LPS only;

Compound: solution after LPS and compound treatment;

Blank: DMSO solution without LPS.

Table 2 - Inhibitory activity of compounds on NO production in mouse macrophage RAW264.7 cells

As can be seen from Table 2, the above 14 compounds, AK-04, AK-06, AK-08, and AK-14, all showed good inhibitory activity on the NO production of mouse macrophages RAW264.7 at concentrations of 5 μM and 10 μM (the inhibition rate was greater than 50%), which was similar to the positive drug dexamethasone.

Experimental Example 3

The results of in vivo animal experiments on the therapeutic effect of compound AK-14 on the DSS-induced ulcerative colitis mouse model are shown in Figures 1 and 2. The results show that the compound AK-14 has a good therapeutic effect on DSS-induced ulcerative colitis. Administration of 15 mg/kg or 30 mg/kg can significantly reduce the effect of the disease on the body weight of mice (Figure 1A), and the disease activity index can also be significantly reduced (Figure 1B). Compared with the DSS modeling group, the colon length of mice in the AK-14 treatment group is longer (Figure 1C), and no obvious ulcers and inflammatory cell infiltration are observed in the colon (Figure 2). In addition, the effects are better than the positive drug Tofacitinib. Therefore, compound AK-14 is considered to have a good therapeutic effect on DSS-induced ulcerative colitis in mice.

(1) Animal modeling and index evaluation methods

C57BL/6J mice were randomly divided into normal group, DSS model group, experimental group (4 experimental groups were AK-1415mg/kg, AK-1430mg/kg), and positive drug group (Tofacitinib 40mg/kg) according to body weight. On the first day, except for the normal group, all other groups were free to drink 2.5% DSS aqueous solution, and the normal control group was given sterile water without DSS. After drinking for 6 consecutive days, the model was successfully established. On the seventh day, all mice were replaced with sterile water without DSS and began to be administered. Among them, the 4 experimental groups were intraperitoneally injected, and the positive drug group (Tofacitinib) was administered by gavage for 6 consecutive days, once a day.

From the beginning of modeling, the mice were observed for eating, activity, hair, and body weight every day. The mice were observed for the formation of feces and the presence of blood in the stool to evaluate the degree of colitis. The disease activity index (DAI) was used to quantify the severity of the disease. It was a comprehensive evaluation of weight loss, stool shape, and rectal bleeding. The specific indicators are shown in Table 3. The treatment group was given the corresponding compound drug orally. On the 8th day of administration, all experimental mice were killed, and the free colon and distal ileum of each mouse were taken to observe the gross changes in the colon of each group of mice, measure the length of the entire colon and rectum, remove the intestinal contents, rinse with saline, and fix with 10% formalin solution as specimens. Tissue specimens were taken for macroscopic and histopathological examinations.

Table 3—Disease Activity Index (DAI) evaluation indicators

(2) Mouse colon tissue sections and analysis methods

Sampling: Fresh tissues are fixed with fixative for more than 24 hours. Take the tissue out of the fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue and the corresponding label in a dehydration box.

Dehydration: Place the dehydration box in the hanging basket and dehydrate in the dehydrator with gradient alcohols. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h-absolute ethanol I 30min-absolute ethanol II 30min-alcohol benzene 5-10min-xylene I 5-10min-xylene II 5-10min-wax I 1h-wax II 1h-wax III 1h.

Embedding: Embed the wax-soaked tissue in the embedding machine. First, put the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and affix the corresponding label. Cool it in a -20° freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

Sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 3 μm. Float the slices on the 40°C warm water of the slice spreader to flatten the tissue, pick up the tissue with a slide, and bake the slices in a 60°C oven. After the wax is baked dry, take it out and store it at room temperature for later use.

Dewax the paraffin sections to water: place the sections in xylene I for 20 min - xylene II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water.

Hematoxylin staining: Hematoxylin staining for 3-5 minutes, differentiate with hydrochloric acid aqueous solution, turn blue with ammonia aqueous solution, and wash with water;

Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol in turn, and then stained in eosin solution for 5 minutes.

Dehydration and sealing: the slices were placed in anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - anhydrous ethanol III for 5 min - xylene I for 5 min - xylene II for 5 min, and then transparent and sealed with neutral gum.

Photography: optical microscope, image acquisition (microscope: NIKON Eclipse ci, imaging system: NIKONdigital sight DS-FI2, MADE IN JAPAN, film magnification: 100×200×).

The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (10)

1. An HDAC6 inhibitor characterized by the structure:

R is at least one substituent selected from cytisine, acridone, indoline, 1,2,3, 4-tetrahydro-9H-pyridine [3,4-B ] benzidine, 1,2,3, 4-tetrahydroisoquinoline, 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline, 5-aminoindole, 2-hydroxyquinoxaline, 2-hydroxyquinoline and the like;

x is a saturated or unsaturated alkane containing from 0 to 4 carbon atoms.

2. The HDAC6 inhibitor according to claim 1, having the structure of formula I or formula II, in particular:

wherein R1 and R2 are respectively and independently selected from at least one of cytisine, acridone, indoline, 1,2,3, 4-tetrahydro-9H-pyridine [3,4-B ] indole, 1,2,3, 4-tetrahydroisoquinoline, 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline, 5-aminoindole, 2-hydroxyquinoxaline and 2-hydroxyquinoline.

3. The HDAC6 inhibitor according to claim 1, characterized by any one of the following structures:

4. The method of preparing an HDAC6 inhibitor according to claim 1, comprising the steps of:

(1) Carrying out nucleophilic substitution reaction on alkaloid and 4-bromomethyl benzoate or 4-bromomethyl cinnamate under alkaline conditions to obtain an intermediate;

(2) The intermediate and aqueous solution of hydroxylamine undergo hydroxylamine hydrolysis under alkaline conditions to obtain the HDAC6 inhibitor.

5. The method of preparing an HDAC6 inhibitor according to claim 4, wherein in step (1), the alkaline condition is provided by potassium carbonate and/or sodium carbonate.

6. The method of preparing an HDAC6 inhibitor according to claim 4, wherein in step (2), the alkaline condition is provided by sodium hydroxide and/or potassium hydroxide.

7. The method of preparing an HDAC6 inhibitor according to claim 4, wherein when the HDAC6 inhibitor has the structure of a compound of formula I, the specific steps are:

(S1) carrying out nucleophilic substitution reaction on alkaloid and 4-bromomethyl benzoate under alkaline conditions to obtain an intermediate A;

(S2) obtaining a compound of the formula I through hydroxylamine hydrolysis reaction of an intermediate A and an aqueous solution of hydroxylamine under alkaline conditions;

8. The method of preparing an HDAC6 inhibitor according to claim 7, wherein in step (S1), the molar ratio of the alkaloid to methyl 4-bromomethylbenzoate is 1: (1-2);

In the step (S2), the molar ratio of the intermediate a to the aqueous solution of hydroxylamine is 1: (15-20).

9. A pharmaceutical composition comprising the HDAC6 inhibitor of any one of claims 1-6.

10. Use of an HDAC6 inhibitor according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment of inflammatory and ulcerative colitis.