An hdac6 inhibitor, its preparation method and use in anti-inflammatory and ulcerative colitis

Abstract

This invention provides an HDAC6 inhibitor, its preparation method, and its use in anti-inflammatory and ulcerative colitis treatment. By linking 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 exhibiting anti-ulcerative colitis activity have been developed. Experimental data show that the above compounds exhibit good inhibitory effects on HDAC6 at the enzyme level and can be used to prepare drugs for the prevention or treatment of ulcerative colitis.

Description

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to an HDAC6 inhibitor, its preparation method, and its use in anti-inflammatory and ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammation of the rectum and colon, characterized by a long and protracted course. It is more common in Europe and America, but its incidence in China has been increasing in recent years. Because the etiology and pathogenesis of UC are not fully understood, there are currently no specific drugs available.

[0003] Histone deacetylase 6 (HDAC6) is an important member of the class IIb HDAC family. By modifying specific substrates, including α-tubulin and heat shock protein 90 (HSP90), it participates in regulating important physiological processes such as cell growth, metastasis, and apoptosis. Studies have shown that HDAC6 overexpression dose-dependently increases α-tubulin levels, further upregulates NADPH oxidase expression, and induces reactive oxygen species (ROS) production, thereby reducing the levels of NF-κB-related proteins such as IKKα / β and IκB, ultimately leading to increased levels of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6. Currently, HDAC6 is receiving increasing attention in research on inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, and chronic asthma. Selective HDAC6 inhibitors, due to their minimal toxicity to normal cells, are gradually becoming a research hotspot. The design and development of novel selective HDAC6 inhibitors for inflammatory diseases has significant social value.

[0004] The pharmacophore model of selective HDAC6 inhibitors mainly consists of three parts: a zinc ion chelating group (ZBG), a linker occupying a hydrophobic channel, and a recognition region (Cap) on the surface of the active pocket. The affinity of HDAC6 inhibitors for different isoforms generally depends on the interaction of these three parts with the active site and their geometric conformation. Among them, the Cap group has the greatest impact on activity; this part is usually located on the protein surface and interacts with the edge region outside the pocket. The linker part occupies the protein channel and plays a supporting and connecting role in the stability of the overall molecular conformation. The ZBG group is generally located inside the protein pocket and interacts with the Zn in the protein. 2+ It forms chelation with other amino acid residues. Based on the different ZBG structures, HDAC6 inhibitors can be divided into hydroxamic acid, benzamide and fatty carboxylic acid. Among them, hydroxamic acid is the most widely used, such as vorinostat (SAHA), belistat (PXD101) and parbistat (LBH589).

[0005] Although significant progress has been made in the research of selective HDAC6 inhibitors, most are still in the clinical trial stage, and no drugs have yet been approved for marketing. Currently, the specific mechanisms by which HDAC6 inhibitors participate in inflammation are not fully understood, and most candidate drugs suffer from poor pharmacokinetics and metabolic instability, all of which limit their further development. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides an HDAC6 inhibitor, its preparation method, and its use in anti-inflammatory and ulcerative colitis treatments. This invention utilizes computer-aided drug design and skeletal transition principles to link an anti-inflammatory alkaloid structure with the hydroxamic acid group of an HDAC6 inhibitor, developing a novel HDAC6 inhibitor containing an alkaloid structure and exhibiting anti-ulcerative colitis activity.

[0007] The technical solution of this invention is as follows:

[0008] An HDAC6 inhibitor, with the following structure:

[0009]

[0010] R is selected from at least one of the following substituents: cycad, acridinone, indoline, 1,2,3,4-tetrahydro-9H-pyridin[3,4-B]indole, 1,2,3,4-tetrahydroisoquinoline, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, 5-aminoindole, 2-hydroxyquinoxaline, 2-hydroxyquinoline, etc.

[0011] X is a saturated or unsaturated alkane chain containing C0-C4.

[0012] The structure of the HDAC6 inhibitor is further preferably of Formula I or Formula II, with the specific structure as follows:

[0013] ;

[0014] R1 and R2 are each independently selected from at least one of cycad, acridinone, 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.

[0015] The structure of the HDAC6 inhibitor is further preferably any of the following:

[0016]

[0017] The preparation method of the HDAC6 inhibitor includes the following steps:

[0018] (1) An intermediate is obtained by nucleophilic substitution reaction of alkaloids and methyl 4-bromomethylbenzoate or methyl 4-bromomethylcinnamate under alkaline conditions;

[0019] (2) The intermediate and an aqueous solution of hydroxylamine were subjected to hydroxylamine hydrolysis under alkaline conditions to obtain the HDAC6 inhibitor.

[0020] In step (1), the alkaline conditions are provided by potassium carbonate and / or sodium carbonate.

[0021] In step (2), the alkaline conditions are provided by sodium hydroxide and / or potassium hydroxide.

[0022] When the structure of the HDAC6 inhibitor is a compound of formula I, the specific steps are as follows:

[0023] (S1) An intermediate A is obtained by nucleophilic substitution reaction of an alkaloid and methyl 4-bromomethylbenzoate under alkaline conditions;

[0024] (S2) An aqueous solution of intermediate A and hydroxylamine is reacted with an aqueous solution of hydroxylamine under alkaline conditions to give compound I;

[0025]

[0026] In step (S1), the molar ratio of the alkaloid and methyl 4-bromomethylbenzoate is 1:(1-2);

[0027] In step (S2), the molar ratio of intermediate A to the aqueous solution of hydroxylamine is 1:(15-20).

[0028] A pharmaceutical composition comprising the HDAC6 inhibitor.

[0029] The use of the HDAC6 inhibitor in the preparation of anti-inflammatory and ulcerative colitis drugs.

[0030] The beneficial effects of this invention are as follows:

[0031] This invention provides an HDAC6 inhibitor by linking an anti-inflammatory alkaloid structure to the hydroxamic acid group of an HDAC6 inhibitor, thus developing a series of novel alkaloid-containing compounds with anti-ulcerative colitis activity. Experimental data show that these compounds exhibit good inhibitory effects on HDAC6 at the enzymatic level and can be used to prepare drugs for the prevention or treatment of ulcerative colitis. This is because the inventors discovered in their long-term research that alkaloids can reduce oxidative stress, decrease inflammatory mediators and MPO activity, maintain the integrity of the intestinal mucosa, and alter inflammatory responses by inhibiting the activation of inflammatory signaling pathways such as NF-κB / Nrf2. Alkaloid compounds have the effect of alleviating or treating ulcerative colitis, and their mechanism is related to regulating immune inflammatory responses, adjusting intestinal flora, and protecting the intestinal mucosal barrier. Therefore, this invention, through computer-aided drug design and skeletal transition principles, links an anti-inflammatory alkaloid structure to the hydroxamic acid group of an HDAC6 inhibitor to obtain an alkaloid-containing HDAC6 inhibitor with anti-ulcerative colitis activity. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The data represent the indices of a DSS-induced ulcerative colitis mouse model. A: mouse body weight change rate; B: disease activity index; C: mouse colon length change graph; all data are expressed as mean ± SD, with 6 mice per group. ***P < 0.001, ****P < 0.0001 represents a comparison with the Model group. ####P < 0.0001 represents a comparison with the Normal group;

[0034] Figure 2 AF shows an H&E staining image of isolated colon tissue from a mouse model of DSS-induced ulcerative colitis. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] The above technical solution will be described in detail below with reference to specific embodiments.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Example 1

[0039] 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), the structural formula of which is shown below:

[0040]

[0041] The specific steps for synthesizing the above-mentioned HDAC6 inhibitor are as follows:

[0042] (1) Weigh 2 mmol of cytisine and 3 mmol of methyl 4-bromomethylbenzoate and mix them into 20 mL of N,N-dimethylformamide. Then add 4 mmol of anhydrous potassium carbonate and stir at room temperature for 3 hours. After the reaction is complete by TLC monitoring, 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.

[0043] (2) Dissolve 2 mmol of intermediate A in 15 mL of methanol, add 2 mmol of sodium hydroxide and stir at 0 °C for 15 minutes, then add 2 mL of aqueous hydroxylamine solution dropwise. Monitor the reaction by TLC until complete, adjust the pH to 7-8 with hydrochloric acid, concentrate under reduced pressure, and purify by preparative thin-layer chromatography (DCM / MeOH = 7 / 1). After vacuum drying, the target compound is obtained.

[0044] Light brown solid, yield: 68%. mp: 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).

[0045] HRMS(ESI):C 19 H 21 N3O3[M+H] + The theoretical value is 340.1662, and the measured value is 340.1663.

[0046] Example 2

[0047] This embodiment provides another HDAC6 inhibitor, 4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-hydroxybenzamide (AK-03), the structural formula of which is shown below:

[0048]

[0049] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 1,2,3,4-tetrahydroisoquinoline.

[0050] Pale pink solid, yield: 66%. mp: 176.9-177.8; 1 H 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.

[0051] HRMS(ESI):C 17 H 18 N₂O₂[M+H] + The theoretical value is 283.1447, and the measured value is 283.1453.

[0052] Example 3

[0053] This embodiment provides another HDAC6 inhibitor, 4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-N-hydroxybenzamide (AK-05), with the following structure:

[0054]

[0055] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.

[0056] Pale pink solid, yield: 65%. mp: 108.5-110.2. 1 H NMR (400MHz, DMSO) δ7.72(d,J=7.8Hz,2H),7.41(d,J=7.8Hz,2H),6.65(s,1H),6.56(s,1 H),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-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.

[0057] HRMS(ESI):C 19 H 22 N₂O₄[M+H] + The theoretical value is 343.1659, and the measured value is 343.1665.

[0058] Example 4

[0059] This embodiment 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:

[0060]

[0061] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 1,2,3,4-tetrahydro-9H-pyridine[3,4-B]indole.

[0062] Light brown solid, yield: 70%. mp: 147.2-148.3. 1 H 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).

[0063] HRMS(ESI):C 19 H 19 N3O2[M+H] + The theoretical value is 322.1556, and the measured value is 322.1553.

[0064] Example 5

[0065] This embodiment 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:

[0066]

[0067] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 2-hydroxyquinoline.

[0068] Light brown solid, yield: 68%. mp: 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).

[0069] HRMS(ESI):C 17 H 16 N₂O₃[M+Na] + The theoretical value is 319.1059, and the measured value is 319.1063.

[0070] Example 6

[0071] This embodiment provides another HDAC6 inhibitor, N-hydroxy-4-(2-oxoquinoxalin-1(2H)-yl)methyl)benzamide (AK-11), whose structural formula is shown below:

[0072]

[0073] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 2-hydroxyquinoxaline.

[0074] Pale 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-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.

[0075] HRMS(ESI):C 16 H 13 N3O3[M+Na] +The theoretical value is 318.0855, and the measured value is 318.0854.

[0076] Example 7

[0077] This embodiment provides another HDAC6 inhibitor, 4-((1H-indol-5-yl)amino)methyl)-N-hydroxybenzamide (AK-13), whose structural formula is shown below:

[0078]

[0079] The synthesis steps are the same as in Example 1, except that cytisine is replaced with 5-aminoindole.

[0080] 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-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.

[0081] HRMS(ESI):C 16 H 15 N3O2[M+H] + The theoretical value is 282.1243, and the measured value is 282.1251.

[0082] Example 8

[0083] This embodiment 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), the structural formula of which is shown below:

[0084]

[0085] The specific steps for synthesizing the above-mentioned HDAC6 inhibitor are as follows:

[0086] (1) Weigh 2 mmol of cytisine and 3 mmol of methyl 4-bromomethylcinnamate and mix them into 20 mL of N,N-dimethylformamide. Then add 4 mmol of anhydrous potassium carbonate and stir at room temperature for 3 hours. After the reaction is complete by TLC monitoring, 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.

[0087] (2) Dissolve 2 mmol of intermediate B in 15 mL of methanol, add 2 mmol of sodium hydroxide and stir at 0 °C for 15 minutes, then add 2 mL of aqueous hydroxylamine solution dropwise. Monitor the reaction by TLC until complete, adjust the pH to 7-8 with hydrochloric acid, concentrate under reduced pressure, and purify by preparative thin-layer chromatography (DCM / MeOH = 7 / 1). After vacuum drying, the target compound is obtained.

[0088] Pale 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), 6.2 7(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-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.

[0089] HRMS(ESI):C 21 H 23 N3O3[M+H] +The theoretical value is 366.1818, and the measured value is 366.1815.

[0090] Example 9

[0091] This embodiment provides another HDAC6 inhibitor, (E)-3-(4-((3,4-dihydroisoquinolin-2(1H)-yl)methyl)phenyl)-N-hydroxyacrylamide (AK-04), whose structure is as follows:

[0092]

[0093] The synthesis steps are the same as in Example 8, except that cytisine is replaced with 1,2,3,4-tetrahydroisoquinoline.

[0094] Pale 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-d6)δ163.24,140.51,138.55,135.19,134.53,134.10,129.6 9,128.92,127.93,126.82,126.45,125.94,119.15,61.94,55.89,50.71,29.14.

[0095] HRMS(ESI):C 19 H 20 N₂O₂[M+H] + The theoretical value is 390.1604, and the measured value is 309.1608.

[0096] Example 10

[0097] This embodiment 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:

[0098]

[0099] The synthesis steps are the same as in Example 8, except that cytisine is replaced with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.

[0100] Light brown solid, yield: 58%. mp: 119.4–120.6. 1 H 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). 13 C 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.

[0101] HRMS(ESI):C 21 H 24 N₂O₄[M+H] + The theoretical value is 369.1815, and the measured value is 369.1822.

[0102] Example 11

[0103] This embodiment 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:

[0104]

[0105] The synthesis steps are the same as in Example 8, except that cytisine is replaced with 1,2,3,4-tetrahydro-9H-pyridine[3,4-B]indole.

[0106] Light brown solid, yield: 80%. mp: 145.6-146.3. 1 H 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). 13 C NMR(101MHz,DMSO-d6)δ140.80,136.30,134.14,133.19,129.74,127.94,127.1 6,120.78,119.13,118.74,117.81,111.34,106.79,61.42,51.11,50.37,21.61.

[0107] HRMS(ESI):C 21 H 21 N3O2[M+H] + The theoretical value is 348.1713, and the measured value is 348.1713.

[0108] Example 12

[0109] This embodiment 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:

[0110]

[0111] The synthesis steps are the same as in Example 8, except that cytisine is replaced with 2-hydroxyquinoxaline.

[0112] Pale pink solid, yield: 66%. mp: 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).

[0113] HRMS(ESI):C 18 H 15 N3O3[M+Na] + The theoretical value is 344.1011, and the measured value is 344.1009.

[0114] Example 13

[0115] This embodiment provides another HDAC6 inhibitor, (E)-N-hydroxy-3-(4-(indolin-1-ylmethyl)phenyl)acrylamide (AK-12), the structure of which is shown below:

[0116]

[0117] The synthesis steps are the same as in Example 8, except that cytisine is replaced with indoline.

[0118] Dark brown solid, yield: 40%. mp: 10⁴.8–10⁵.1. 1 H 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).

[0119] HRMS(ESI):C 18 H 18 N₂O₂[M+H] + The theoretical value is 295.1447, and the measured value is 295.1446.

[0120] Example 14

[0121] This embodiment provides another HDAC6 inhibitor, N-hydroxy-4-((9-oxoacridin-10(9H)-yl)methyl)benzamide (AK-14), the structure of which is shown below:

[0122]

[0123] The specific steps for synthesizing the above-mentioned HDAC6 inhibitor are as follows:

[0124] (1) Weigh 2 mmol of acridinone and dissolve it in 10 mL of N,N-dimethylformamide. Then add 3 mmol of sodium hydride and stir at 0 °C for 10 minutes. Next, add 3 mmol of methyl 4-bromomethylbenzoate and gradually raise the temperature to room temperature. Stir for 2 hours. Monitor the reaction until it is complete by TLC. After quenching the reaction, extract, concentrate under reduced pressure, and separate by preparative thin-layer chromatography (developing solvent PE / EA = 1 / 1). After drying, obtain intermediate C.

[0125] (2) Dissolve 2 mmol of intermediate C in 15 mL of methanol, add 2 mmol of sodium hydroxide and stir at 0 °C for 15 minutes, then add 2 mL of aqueous hydroxylamine solution dropwise. Monitor the reaction by TLC until complete, adjust the pH to 7-8 with hydrochloric acid, concentrate under reduced pressure, and purify by preparative thin-layer chromatography (DCM / MeOH = 7 / 1). After vacuum drying, the target compound is obtained.

[0126] Pale yellow solid, yield: 80%. mp: 226.9-227.8; 1 H 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). 13 C 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.

[0127] HRMS(ESI):C 21 H 16 N₂O₃[M+Na] + The theoretical value is 367.1059, and the measured value is 367.1061.

[0128] Experimental Example 1

[0129] The inhibition rates of 14 compounds against HDAC1 and HDAC6 enzymes at a concentration of 50 nM were screened in vitro using a fluorescence method, with SAHA as the positive compound. The results are as follows:

[0130] (1) Prepare 1x experimental buffer (modified Tris buffer);

[0131] (2) Tandem dilution: The compound was transferred to the assay plate in 100% DMSO using an Echo. The final fraction of dimethyl sulfoxide was 1%.

[0132] (3) Prepare enzyme solution: Prepare enzyme solution in 1x experimental buffer;

[0133] (4) Prepare substrate solution: Add trypsin and ac peptide substrate to 1x experimental buffer to prepare substrate solution;

[0134] (5) Transfer 15 μL of enzyme solution to the assay plate, or transfer 15 μL of 1x assay buffer to the low control;

[0135] (6) Incubate at room temperature for 15 minutes;

[0136] (7) Add 10 μL of substrate solution to each well to start the reaction;

[0137] (8) Dynamically read the flat plate on Envision, with an excitation wavelength of 355nm and an emission wavelength of 460nm;

[0138] (9) Curve fitting: Fit the data in Excel and use equation (1) to obtain the suppression value.

[0139] Formula (1): Inh%=(Max-Signal) / (Max-Min)*100

[0140] The data was fitted in xml-Fit, and the IC50 value was obtained using equation (2).

[0141] Equation (2): Y = Bottom + (Top - Bottom) / (1 + (IC50 / X) * HillSlope) where Y is the inhibition rate %, and X is the composite concentration.

[0142] Table 1—In vitro HDAC1 and HDAC6 enzyme inhibitory activities of the compounds

[0143]

[0144]

[0145] As shown in Table 1, except for AK-12, all the compounds synthesized from Formulas I-II (i.e., those containing N-hydroxycinnamonamide groups) exhibited strong inhibitory activity against HDAC1 and HDAC6 at a concentration of 50 nM. Among the compounds synthesized from Formula I (i.e., those containing N-hydroxy-4-methylbenzamide groups), AK-11, AK-13, and AK-14 showed strong HDAC6 inhibitory activity. It can be inferred that AK-14 exhibited the best selectivity for the HDAC1 / 6 isoforms.

[0146] Experiment Example 2

[0147] The inhibition rate of 14 compounds on NO production in mouse macrophage RAW264.7 was determined by the MTT assay, with dexamethasone as a positive control. The results are shown in Table 2.

[0148] RAW264.7 cells were incubated in DMEM medium with the addition of 10% fetal bovine serum (Biological Industries), 100 U / mL penicillin and 100 mg / mL streptomycin (Beyotime), and then transferred to a humidified environment at 37°C containing 5% CO2.

[0149] Log-grown RAW264.7 cells were collected in 15 mL centrifuge tubes, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were then diluted with fresh culture medium to a suitable density and set aside. A special coverslip was placed in the center of a cell counting chamber. 10 μL of the suspension was aspirated from the centrifuge tube and added between the counting chamber and the coverslip, allowing the coverslip to be filled with liquid through siphoning. The counting chamber was then placed under a microscope for counting.

[0150] Counting formula: (Number of cells per well × 100 × Number of wells in a 96-well plate) ÷ (Number of cells in 4 large squares × 10) 4 = Suspension volume (mL)

[0151] Calculate the cell suspension volume by calculating the cell suspension density, and seed the plate with 8000-12000 cells per well. That is: the volume of cell suspension (mL) aspirated from one 96-well plate = (number of cells per well × 96 × 1) / cell suspension density. Mix the required cell suspension and culture medium thoroughly. Add 100 μL of suspension to each well using a pipette, mixing the suspension after every three additions. After adding all the cell suspension, cap the plate and observe the cell density and uniformity under an inverted microscope.

[0152] In 96-well plates, experimental groups were treated with different compounds for 48 hours. Three wells served as the control group, with an equal volume of DMSO added. Three wells served as the LPS group, with 100 μL of culture medium added to each well, and no drug added to the side wells. After 48 hours, the cell suspensions of each group were transferred to new cell counting plates. Solutions A and B from the NO kit were then added to each well. After the solution changed from colorless to pink, the culture medium was collected, and the nitrite level (an indicator of NO production) was measured using the Griess method. The absorbance (OD540) of the samples was measured at 540 nm using a microplate reader.

[0153]

[0154] Control: DMSO solution treated with LPS only;

[0155] Compound: LPS and the solution after compound treatment;

[0156] Blank: DMSO solution without LPS.

[0157] Table 2—Inhibitory activity of compounds on NO production in mouse macrophage RAW264.7 cells.

[0158]

[0159]

[0160]

[0161] 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 against NO production in mouse macrophages RAW264.7 at concentrations of 5 μM and 10 μM (inhibition rates were all greater than 50%), similar to the positive control drug dexamethasone.

[0162] Experimental Example 3

[0163] In vivo animal experiments were conducted to investigate the therapeutic effect of compound AK-14 on a mouse model of DSS-induced ulcerative colitis. The results are as follows: Figure 1 and Figure 2 As shown. The results indicate that the compound AK-14 has a good therapeutic effect on DSS-induced ulcerative colitis, and administration of 15 mg / kg or 30 mg / kg can significantly reduce the effect of the disease on the body weight of mice. Figure 1 A), the disease activity index can also be significantly reduced ( Figure 1 B), and compared to the DSS model group, the mice in the AK-14 treatment group had a longer colon ( Figure 1 C) No obvious ulcers or inflammatory cell infiltration were observed in the colon. Figure 2 Furthermore, its effects were superior to those of the positive control drug Tofacitinib. Therefore, compound AK-14 is considered to have good therapeutic effects on DSS-induced ulcerative colitis in mice.

[0164] (1) Animal modeling and indicator evaluation methods

[0165] C57BL / 6J mice were randomly divided into four groups according to body weight: a normal control group, a DSS model group, experimental groups (AK-14 15 mg / kg, AK-14 30 mg / kg, and a positive control group (Tofacitinib 40 mg / kg). On day 1, except for the normal control group, all other groups had free access to 2.5% DSS aqueous solution. The normal control group was given sterile water without DSS. After 6 consecutive days of drinking, the model was successfully established. On day 7, all mice were given sterile water without DSS and drug administration began. The four experimental groups were administered intraperitoneally, while the positive control group (Tofacitinib) was administered by gavage. The administration was carried out once daily for 6 consecutive days.

[0166] From the start of modeling, mice were observed daily for their food intake, activity, fur condition, and weight. The formation of feces and the presence of blood in the stool were also monitored to assess the severity of colitis. The Disease Activity Index (DAI) was used to quantify disease severity; it is a comprehensive evaluation based on weight loss, stool shape, and rectal bleeding. Specific indicators are shown in Table 3. The treatment group received the corresponding compound medication orally. On day 8 of administration, all experimental mice were sacrificed. The free colon and distal ileum of each mouse were harvested. Gross changes in the colon were observed in each group, and the length of the entire colon and rectum was measured. Intestinal contents were removed, the intestines were rinsed with physiological saline, and fixed in 10% formalin solution as specimens. Tissue specimens were taken for macroscopic and histopathological examination.

[0167] Table 3—Disease Activity Index (DAI) Evaluation Indicators

[0168]

[0169] (2) Mouse colon tissue sections and analysis methods

[0170] Sampling: Fresh tissue is fixed in fixative for at least 24 hours. The tissue is removed from the fixative and trimmed smooth in a fume hood using a scalpel. The trimmed tissue and corresponding labels are then placed in a dehydration box.

[0171] Dehydration: Place the dehydration box into the basket and then into the dehydrator for sequential dehydration with alcohol in a gradient. 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - benzene 5-10min - xylene I 5-10min - xylene II 5-10min - wax I 1h - wax II 1h - wax III 1h.

[0172] Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and labeled accordingly. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0173] Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 3μm. Float the sections on 40℃ warm water in a slide spreader to flatten the tissue. Remove the tissue from the slide and bake it in a 60℃ oven. After the wax has melted in the water, remove the slide and store it at room temperature for later use.

[0174] Dewaxing 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, and 75% ethanol for 5 min, then wash with tap water.

[0175] Hematoxylin staining: stain with hematoxylin for 3-5 minutes, differentiate with hydrochloric acid solution, turn blue with ammonia solution, and wash with water;

[0176] Eosin staining: The sections were dehydrated by sequentially immersing them in a gradient of 85% and 95% alcohol, and then stained in eosin staining solution for 5 minutes.

[0177] Dehydration and mounting: The sections were sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After clearing, the sections were mounted with neutral resin.

[0178] Photography: Optical microscope, image acquisition (microscope: NIKON Eclipse ci, imaging system: NIKON digital sight DS-FI2, MADE IN JAPAN, magnification: 100×200×).

[0179] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A compound, characterized in that, The structure can be any of the following: 。 2. A method for preparing a compound, characterized in that, The specific steps are as follows: (S1) An intermediate A is obtained by nucleophilic substitution reaction of an alkaloid and methyl 4-bromomethylbenzoate under alkaline conditions; (S2) An aqueous solution of intermediate A and hydroxylamine is reacted with hydroxylamine under alkaline conditions to give compound I; ； The structure of the compound of formula I is any of the following: 。 3. The method for preparing the compound according to claim 2, characterized in that, In step (S1), the molar ratio of the alkaloid and methyl 4-bromomethylbenzoate is 1:(1-2).

4. The method for preparing the compound according to claim 2, characterized in that, In step (S2), the molar ratio of intermediate A to the aqueous solution of hydroxylamine is 1:(15-20).

5. A pharmaceutical composition comprising the compound of claim 1.

6. The use of the compound of claim 1 in the preparation of HDAC6 inhibitors.

7. The use of the compound in the preparation of anti-inflammatory and ulcerative colitis drugs, characterized in that, The structure of the compound is as follows: 。

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