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

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

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

Background

Ulcerative colitis (Ulcerative colitis, UC) is a chronic, nonspecific inflammation of the rectum and colon, with a long course of disease, which is difficult to heal. The disease is seen in European and American regions, and the incidence rate in China has an increasing trend in recent years. Since the etiology and pathogenesis of UC are not yet fully elucidated, there is currently no specific drug.

Histone deacetylase 6 (Histone deacetylase) is an important member of the class IIb HDAC family, and is involved in important physiological processes such as regulation of cell growth, metastasis and apoptosis by modification of specific substrates including α -tubulin and heat shock protein 90 (HSP 90). Studies show that the over-expression of HDAC6 can increase the level of alpha-tubulin in a dose-dependent manner, further up-regulate the expression of NADPH oxidase and induce the generation of Reactive Oxygen Species (ROS), so as to reduce the level of NF- κB related proteins such as IKKα/β and IκB, and finally lead to the increase of the levels of pro-inflammatory factors such as TNF- α, IL-1β and IL-6. Currently, HDAC6 is gaining increasing attention in the research related to inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis and chronic asthma. The selective HDAC6 inhibitor gradually becomes a research hot spot because of being basically nontoxic to normal cells, and the design and development of a novel selective HDAC6 inhibitor for inflammatory diseases have important social value.

The pharmacophore model for selective HDAC6 inhibitors is largely divided into three parts: zinc ion chelating group (ZBG), linker occupying hydrophobic channel (linker), active pocket surface recognition region (Cap). The affinity of HDAC6 inhibitors for different subtypes generally depends on the interaction and geometric conformation of the above three moieties with the active site. Among these, the Cap group, which has the greatest effect on activity, is often located in the surface portion of the protein, and interacts with the edge region outside the pocket. Linker moiety is occupied in the channel of the protein, and plays a role in supporting and linking the stabilization of the whole molecular conformation. The ZBG group is generally located inside a protein pocket and forms a chelate with Zn ²⁺ and other amino acid residues in the protein, and HDAC6 inhibitors can be classified into hydroxamic acids, benzamides and fatty carboxylic acids according to the ZBG structure, wherein the hydroxamic acids are most widely used, such as vorinostat (SAHA), bei Lisi he (PXD 101) and panobinostat (LBH 589), etc.

Despite the significant progress in the current research of selective HDAC6 inhibitors, most are in the clinical trial stage and there are no approved drugs on the market. At present, the specific mechanism of the HDAC6 inhibitor in the aspect of participating in inflammation is not completely elucidated, and most candidate drugs have the problems of poor pharmacokinetics, unstable metabolism and the like, so that the further development of the candidate drugs is restricted.

Disclosure of Invention

In order to solve the problems, the invention provides an HDAC6 inhibitor, a preparation method thereof and application thereof in anti-inflammatory and ulcerative colitis. The invention connects the alkaloid structure with anti-inflammatory activity with the hydroxamic acid group of the HDAC6 inhibitor through the design of computer-aided drugs and the framework transition principle, and develops a novel HDAC6 inhibitor with anti-ulcerative colitis activity containing the alkaloid structure.

The technical scheme of the invention is as follows:

an HDAC6 inhibitor having 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.

Further preferred are the HDAC6 inhibitors of formula I or formula II, which are specifically represented by the following structures:

；

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.

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

a method of preparing the HDAC6 inhibitor 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.

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

In step (2), the alkaline conditions are 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) 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;

In the step (S1), the molar ratio of the alkaloid to the 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).

A pharmaceutical composition comprising the HDAC6 inhibitor.

The use of said HDAC6 inhibitors in the manufacture of a medicament for the treatment of inflammatory and ulcerative colitis.

The beneficial effects of the invention are as follows:

The application provides an HDAC6 inhibitor, which is characterized in that an alkaloid structure with anti-inflammatory activity is connected with a hydroxamic acid group of the HDAC6 inhibitor to develop a series of novel compounds with the alkaloid structure and anti-ulcerative colitis activity, 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. This is because the present inventors have found in long-term studies that alkaloids can reduce oxidative stress, reduce inflammatory mediators and MPO activity, maintain intestinal mucosa integrity, and alter inflammatory responses by inhibiting activation of inflammatory signaling pathways such as (NF- κb/Nrf 2). The alkaloid compounds have the effect of relieving or treating ulcerative colitis, and the mechanism of the alkaloid compounds is related to regulating immune inflammatory response, regulating intestinal flora and protecting intestinal mucosa barrier. Therefore, the application connects the alkaloid structure with anti-inflammatory activity with the hydroxamic acid group of the HDAC6 inhibitor through the design of computer-aided drugs and the framework transition principle, and obtains the HDAC6 inhibitor with anti-ulcerative colitis activity containing the alkaloid structure.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Figure 1 shows the index of mice model of DSS-induced ulcerative colitis. Wherein A: rate of change of body weight of mice; b: disease activity index; c: a plot of colon length change in mice; all data are expressed as mean±sd with 6 mice per group. * P < 0.001 and P < 0.0001 represent a comparison to the Model group. # # P < 0.0001, representing a comparison to Normal group;

Figures 2A-F show H & E staining patterns of colon tissue ex vivo for a mouse model of DSS-induced ulcerative colitis.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

The following describes the above technical scheme in detail with reference to specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

The present example 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), having the following structural formula:

the synthesis procedure of the above HDAC6 inhibitor is specifically as follows:

(1) 2mmol of cytisine and 3mmol of methyl 4-bromomethylbenzoate were weighed and mixed and added to 20mL of N, N-dimethylformamide, followed by addition of 4mmol of anhydrous potassium carbonate and stirring at room temperature for 3 hours. After TLC monitoring the reaction was completed, the reaction was extracted with water, concentrated under reduced pressure, and separated by thin layer preparative chromatography (developer PE/ea=1/1), and dried to give intermediate a.

(2) 2Mmol of intermediate A was dissolved in 15mL of methanol, 2mmol of sodium hydroxide was added and stirred at 0deg.C for 15 minutes, and 2mL of aqueous hydroxylamine solution was added dropwise. TLC was monitored until the reaction was complete, pH was adjusted to 7-8 with addition of hydrochloric acid, concentrated under reduced pressure, and purified by preparative thin layer chromatography (DCM/meoh=7/1) to give the title compound after drying in vacuo.

Light brown solid, yield ：68％.m.p:82.6-83.7;¹H NMR(400MHz,DMSO)δ11.14(s,1H),8.99(s,1H),7.58(d,J＝7.8Hz,2H),7.35(dd,J＝8.9,6.7Hz,1H),7.01(d,J＝7.9Hz,2H),6.28(d,J＝8.8Hz,1H),6.03(d,J＝6.6Hz,1H),3.87(d,J＝15.2Hz,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) C ₁₉H₂₁N₃O₃[M+H]⁺ found 340.1662 and 340.1663.

Example 2

This example provides another HDAC6 inhibitor, 4- ((3, 4-dihydroisoquinolin-2 (1H) -yl) methyl) -N-hydroxybenzamide (AK-03), having the structural formula:

The synthesis procedure is as in example 1, except that cytisine is replaced by 1,2,3, 4-tetrahydroisoquinoline.

Pale pink solid, yield ：66％.m.p:176.9-177.8;¹H NMR(400MHz,DMSO)δ7.73(d,J＝7.4Hz,2H),7.42(d,J＝7.3Hz,2H),7.13-7.04(m,3H),6.98(d,J＝6.7Hz,1H),3.67(s,2H),3.53(s,2H),2.80(t,J＝5.7Hz,2H),2.66(t,J＝5.7Hz,2H).¹³C NMR(101MHz,DMSO-d₆)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) C ₁₇H₁₈N₂O₂[M+H]⁺ found 283.1447 and 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), having the structure:

the synthesis procedure was as in example 1, except that cytisine was replaced with 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline.

Pale pink solid, yield ：65％.m.p:108.5-110.2;¹H NMR(400MHz,DMSO)δ7.72(d,J＝7.8Hz,2H),7.41(d,J＝7.8Hz,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).¹³C NMR(101MHz,DMSO-d₆)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) C ₁₉H₂₂N₂O₄[M+H]⁺ found 343.1659 and 343.1665.

Example 4

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

The procedure is as in example 1, except that cytisine is replaced by 1,2,3, 4-tetrahydro-9H-pyrido [3,4-B ] indole.

Light brown solid, yield ：70％.m.p:147.2-148.3;¹H NMR(400MHz,DMSO)δ7.73(d,J＝7.7Hz,2H),7.44(d,J＝7.7Hz,2H),7.35(d,J＝7.7Hz,1H),7.24(d,J＝7.9Hz,1H),6.99(t,J＝7.4Hz,1H),6.93(t,J＝7.3Hz,1H),3.76(s,2H),3.56(s,2H),2.80(t,J＝5.6Hz,2H),2.69(t,J＝5.6Hz,2H).

HRMS (ESI) C ₁₉H₁₉N₃O₂[M+H]⁺ found 322.1556 and 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), having the following structural formula:

The synthesis procedure is as in example 1, except that cytisine is replaced by 2-hydroxyquinoline.

Light brown solid, yield ：68％.m.p:115.0-116.8;¹H NMR(400MHz,DMSO)δ7.67(d,J＝7.7Hz,2H),7.28(d,J＝7.7Hz,2H),7.22(d,J＝7.1Hz,1H),7.10(t,J＝7.6Hz,1H),6.95(t,J＝7.2Hz,1H),6.86(d,J＝7.9Hz,1H),5.17(s,2H),2.94(t,J＝7.0Hz,2H),2.70(dd,J＝8.4,5.8Hz,2H).

HRMS (ESI) C ₁₇H₁₆N₂O₃[M+Na]⁺ found 319.1059 and 319.1063.

Example 6

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

The synthesis procedure is as in example 1, except that cytisine is replaced by 2-hydroxyquinoxaline.

Pale pink solid, yield ：60％.m.p:181.5-183.3;¹H 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).¹³C NMR(101MHz,DMSO-d₆)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) C ₁₆H₁₃N₃O₃[M+Na]⁺ found 318.0855 and 318.0854.

Example 7

This example provides another HDAC6 inhibitor, 4- ((1H-indol-yl) amino) methyl) -N-hydroxybenzamide (AK-13), having the structural formula:

the synthesis procedure is as in example 1, except that cytisine is replaced by 5-aminoindole.

Brown solid, yield ：50％.m.p:185.2-186.8;¹H 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).¹³C NMR(101MHz,DMSO-d₆)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) C ₁₆H₁₅N₃O₂[M+H]⁺ found 282.1243 and 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), having the following structural formula:

the synthesis procedure of the above HDAC6 inhibitor is specifically as follows:

(1) 2mmol of cytisine and 3mmol of methyl 4-bromomethylcinnamate were weighed and mixed and added to 20mL of N, N-dimethylformamide, followed by addition of 4mmol of anhydrous potassium carbonate and stirring at room temperature for 3 hours. After TLC monitoring the reaction was completed, the reaction was extracted with water, concentrated under reduced pressure, and separated by thin layer preparative chromatography (developer PE/ea=1/1), and dried to give intermediate B.

(2) 2Mmol of intermediate B was dissolved in 15mL of methanol, 2mmol of sodium hydroxide was added and stirred at 0deg.C for 15 minutes, and 2mL of aqueous hydroxylamine solution was added dropwise. TLC was monitored until the reaction was complete, pH was adjusted to 7-8 with addition of hydrochloric acid, concentrated under reduced pressure, and purified by preparative thin layer chromatography (DCM/meoh=7/1) to give the title compound after drying in vacuo.

Pale pink solid, yield ：71％.m.p:157.5-159.1;¹H 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).¹³C NMR(101MHz,DMSO-d₆)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) C ₂₁H₂₃N₃O₃[M+H]⁺ found 366.1818 and 366.1815.

Example 9

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

The synthesis procedure is as in example 8, except that cytisine is replaced by 1,2,3, 4-tetrahydroisoquinoline.

Pale pink solid, yield ：60％.m.p:185.0-186.9;¹H 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).¹³C NMR(101MHz,DMSO-d₆)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) C ₁₉H₂₀N₂O₂[M+H]⁺ found 390.1604 and 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), having the structure shown below:

the procedure is as in example 8, except that cytisine is replaced by 6, 7-dimethoxy-1, 2,3, 4-tetrahydroisoquinoline.

Light brown solid, yield ：58％.m.p:119.4-120.6;¹H NMR(400MHz,DMSO)δ7.51(d,J＝7.5Hz,2H),7.43(d,J＝14.9Hz,1H),7.37(d,J＝7.4Hz,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＝5.6Hz,2H),2.63(t,J＝5.7Hz,2H).¹³C NMR(101MHz,DMSO-d₆)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) C ₂₁H₂₄N₂O₄[M+H]⁺ found 369.1815 and 369.1822.

Example 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), having the structure shown below:

the procedure is as in example 8, except that cytisine is replaced by 1,2,3, 4-tetrahydro-9H-pyrido [3,4-B ] indole.

Light brown solid, yield ：80％.m.p:145.6-146.3;¹H NMR(400MHz,DMSO-d₆)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).¹³C NMR(101MHz,DMSO-d₆)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) C ₂₁H₂₁N₃O₂[M+H]⁺ found 348.1713 and 348.1713.

Example 12

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

The synthesis procedure is as in example 8, except that cytisine is replaced by 2-hydroxyquinoxaline.

Pale pink solid, yield ：66％.m.p:146.2-147.7;¹H NMR(400MHz,DMSO)δ8.36(s,1H),7.86(d,J＝7.8Hz,1H),7.56(t,J＝7.6Hz,1H),7.50(d,J＝7.8Hz,2H),7.42(d,J＝9.5Hz,1H),7.39-7.35(m,1H),7.30(d,J＝7.7Hz,2H),6.41(d,J＝15.8Hz,1H),5.50(s,2H),4.34(t,J＝5.2Hz,1H).

HRMS (ESI) C ₁₈H₁₅N₃O₃[M+Na]⁺ found 344.1011 and 344.1009.

Example 13

This example provides another HDAC6 inhibitor, (E) -N-hydroxy-3- (4- (indolin-1-ylmethyl) phenyl) acrylic amide (AK-12) having the structure shown below:

The procedure is as in example 8, except that cytisine is replaced by indoline.

Dark brown solid, yield ：40％.m.p:104.8-105.1;¹H NMR(400MHz,DMSO-d₆)δ7.52(s,2H),7.39(t,J＝12.0Hz,3H),7.08-6.93(m,2H),6.57(q,J＝7.2Hz,3H),4.26(s,2H),3.24(d,J＝8.1Hz,2H),2.88(t,J＝8.1Hz,2H).

HRMS (ESI) C ₁₈H₁₈N₂O₂[M+H]⁺ found 295.1447 and 295.1446.

Example 14

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

the synthesis procedure of the above HDAC6 inhibitor is specifically as follows:

(1) 2mmol of acridone is weighed, added into 10mL of N, N-dimethylformamide for dissolution, then 3mmol of sodium hydride is added, stirring is carried out for 10 minutes at 0 ℃, 3mmol of methyl 4-bromomethylbenzoate is added, the temperature is gradually raised to room temperature, and stirring is carried out for 2 hours. After TLC monitoring to completion of the reaction, the reaction was quenched, extracted, concentrated under reduced pressure, and separated by thin layer preparative chromatography (developing solvent PE/ea=1/1) to give intermediate C after drying.

(2) 2Mmol of intermediate C was dissolved in 15mL of methanol, 2mmol of sodium hydroxide was added and stirred at 0deg.C for 15 minutes, and 2mL of aqueous hydroxylamine solution was added dropwise. TLC was monitored until the reaction was complete, pH was adjusted to 7-8 with addition of hydrochloric acid, concentrated under reduced pressure, and purified by preparative thin layer chromatography (DCM/meoh=7/1) to give the title compound after drying in vacuo.

Pale yellow solid, yield ：80％.m.p:226.9-227.8;¹H NMR(400MHz,DMSO)δ11.15(s,1H),9.09(s,1H),8.40(d,J＝7.8Hz,2H),7.93-7.67(m,4H),7.63(d,J＝8.6Hz,2H),7.36(t,J＝7.4Hz,2H),7.23(d,J＝7.8Hz,2H),5.84(s,2H).¹³C NMR(101MHz,DMSO-d₆)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) C ₂₁H₁₆N₂O₃[M+Na]⁺ found 367.1059 and 367.1061.

Experimental example 1

Inhibition of HDAC1 and HDAC6 enzymes by 14 compounds at 50nM concentration was screened in vitro using fluorescence, with SAHA as positive compound, as follows:

(1) Preparing 1x assay buffer (modified Tris buffer);

(2) Compound serial dilutions compounds were transferred to assay plates with Echo in 100% dmso. The final fraction of dimethyl sulfoxide was 1%;

(3) Preparing enzyme solution, namely preparing the enzyme solution in a 1x experiment buffer solution;

(4) Preparing a substrate solution by adding trypsin and ac peptide substrate into a 1x experiment buffer solution to prepare a substrate solution;

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

(6) Incubating for 15 minutes at room temperature;

(7) 10 mu L of substrate solution is added to each well to start the reaction;

(8) Dynamically reading the plate on Envision, wherein the excitation wavelength is 355nm, and the emission wavelength is 460nm;

(9) Curve fitting the data was fitted in Excel and inhibition values were obtained using equation (1).

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

The data were Fit in xml-Fit and IC50 values were obtained using equation (2).

Equation (2) y=bottom+ (Top-Bottom)/(1+ (IC 50/X) HillSlope) Y is the inhibition and X is the recombination concentration.

TABLE 1 in vitro HDAC1 and HDAC6 enzyme inhibitory Activity

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As can be seen from Table 1, all of the 14 compounds synthesized from formulas I-II (i.e., containing N-hydroxycinnamamide groups) showed potent inhibitory activity against HDAC1 and HDAC6 at a concentration of 50nM, except AK-12. Of the compounds synthesized from formula I (i.e., containing N-hydroxy-4-methylbenzamide groups), AK-11, AK-13 and AK-14 exhibited strong HDAC6 inhibitory activity, among which it is presumed that the HDAC1/6 subtype of AK-14 has the best selectivity.

Experimental example 2

The inhibition rate of NO production of the 14 compounds on the mouse macrophage RAW264.7 was determined by MTT method, and the result is shown in Table 2 by taking dexamethasone as a positive control.

RAW264.7 cells were incubated in DMEM medium, 10% fetal bovine serum (Biological Industries), 100U/mL penicillin and 100mg/mL streptomycin (Beyotime) were added, transferred to 37℃and cultured in a humidified environment with 5% CO ₂.

Log-grown RAW264.7 cells were collected in 15mL centrifuge tubes, centrifuged at 800rpm for 5min, the supernatant discarded, and diluted to the appropriate density with fresh medium for use. A special cover slip was placed in the center of the cell counter plate, 10. Mu.L of suspension was aspirated from the centrifuge tube, and added between the counter plate and the special cover slip, and the cover slip was filled with liquid by siphoning. The counting plate was placed under a microscope for counting.

The counting formula: (number of cells per well×100×96 well plates)/(number of 4 large cells×10 ⁴) =suspension volume (mL)

The cell suspension volume was calculated and by calculating the cell suspension density, 8000-12000 cells per well were plated, i.e.: a 96-well plate was plated to aspirate cell suspension volume (mL) = (number of cells per well×96×1)/cell suspension density. The desired cell suspension and culture medium were mixed well, and the suspension was applied to a 96-well plate using a discharge gun at 100. Mu.L per well, and the cell suspension was mixed once every three times. After the cell suspension was added, the plate was covered and the cell density and uniformity were observed under an inverted microscope.

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

Control, DMSO solution treated with LPS only;

compound, LPS and Compound treated solution;

blanc-DMSO solution without LPS.

Table 2-inhibitory Activity of Compounds on NO production by mouse macrophage RAW264.7

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As can be seen from Table 2, the 14 compounds AK-04, AK-06, AK-08 and AK-14 showed good inhibitory activity (inhibition ratio is more than 50%) on the NO production of mouse macrophage RAW264.7 at the concentrations of 5. Mu.M and 10. Mu.M, and were similar to dexamethasone as a positive drug.

Experimental example 3

In vivo animal experimental study on the therapeutic effect of compound AK-14 on mice model of DSS induced ulcerative colitis shows the results in FIG. 1 and FIG. 2. The results show that: the compound AK-14 has good treatment effect on the ulcerative colitis induced by DSS, 15mg/kg or 30mg/kg of administration can obviously reduce the influence of the disease on the weight of mice (figure 1A), the disease activity index can also obviously reduce (figure 1B), compared with a DSS modeling group, the colon length of AK-14 treatment group mice is longer (figure 1C), no obvious ulcer and inflammatory cell infiltration are seen in the colon (figure 2), and the effect is superior to that of a positive drug Tofacitinib. Thus, compound AK-14 is considered to have a good therapeutic effect on DSS-induced ulcerative colitis in mice.

(1) Animal modeling and index evaluation method

C57BL/6J mice were randomly divided into a normal group, a DSS model group, and an experimental group (4 experimental groups are AK-1415mg/kg, AK-1430mg/kg, respectively) and a positive drug group (Tofacitinib 40 mg/kg) according to body weight. On day 1, each group was free to drink 2.5% aqueous DSS solution except the normal group, and the normal control group was given sterile water without DSS, and after 6 days of continuous drinking, molding was successful. All mice were changed to sterilized water without DSS on day seven and dosing was started, with 4 experimental groups each being given by intraperitoneal injection and the positive group (Tofacitinib) by gavage, for 6 consecutive days, 1 time per day.

From the modeling, the eating, activity and hair conditions of the mice were observed daily, the body weight was weighed, and the presence or absence of hematochezia and the like of the mice were observed, so as to evaluate the degree of colitis disease. The severity of the disease was quantified by a disease activity index (DISEASE ACTIVITY index, DAI), which was comprehensively evaluated by weight loss, stool shape and rectal bleeding, and specific indices are shown in table 3. The corresponding compound medicines are orally taken by the treatment group. On day 8 of dosing, all experimental mice were sacrificed, the free colon and distal ileum of each mouse were taken, the general changes in the colon of each group of mice were observed, the entire colon and rectal length were measured, the intestinal contents were removed, and the intestinal contents were removed, rinsed with normal saline, and fixed as specimens with 10% formalin solution. Tissue specimens were taken for visual and histopathological examination.

TABLE 3 evaluation index of Disease Activity Index (DAI)

(2) Mouse colon tissue section and analysis method

Drawing materials: fresh tissue is fixed for more than 24 hours by using a fixing liquid. And taking out the tissue from the fixing solution, trimming the tissue of the target part in a fume hood by using a surgical knife, and placing the trimmed tissue and a corresponding label in a dehydration box.

Dehydrating: and placing the dehydration box into a basket, and sequentially carrying out gradient alcohol dehydration in a dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1 h-absolute alcohol I30 min-absolute alcohol II 30 min-alcohol benzene 5-10 min-xylene I5-10 min-xylene II 5-10 min-wax I1 h-wax II 1 h-wax III 1h.

Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, putting melted wax into an embedding frame, taking out tissues from a dehydration box before the wax is solidified, putting the tissues into the embedding frame according to the requirement of an embedding surface, and attaching corresponding labels. Cooling at-20 deg.c, solidifying, taking out the wax block from embedding frame and trimming.

Slicing: the trimmed wax block was sliced in a paraffin slicer to a thickness of 3 μm. The slices float on warm water at 40 ℃ of a slice spreading machine to flatten the tissues, the glass slide drags the tissues out, and the slices are baked in a baking oven at 60 ℃. And (5) baking the water, drying the wax, baking, taking out and preserving at normal temperature for standby.

Paraffin sections dewaxed to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water.

Hematoxylin staining: hematoxylin dyeing for 3-5min, hydrochloric acid aqueous solution differentiation, ammonia aqueous solution blue returning and water washing;

eosin staining: the slices are dehydrated in gradient alcohol of 85% and 95% in sequence and then are dyed in eosin dye solution for 5min.

And (3) removing the water sealing piece: sequentially slicing, adding absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II 5min, and sealing with neutral resin.

Photographing: an optical microscope was used to collect images (microscope: NIKON ECLIPSE CI, imaging system: NIKON DIGITAL SIGHT DS-FI2, MADE IN JAPAN, magnification of film: 100X 200X).

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to 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.