Colchicine derivatives, processes for their preparation and use thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a colchicine derivative, a preparation method and application thereof. The colchicine derivative has a structure as shown in formula I: wherein R1 is H or methyl; R2 is H or -C(=O)-R3, R3 is selected from non-steroidal anti-inflammatory drugs or phenylpropanoid small molecule groups, and R3 is selected from the following compounds: The colchicine derivative provided in the application can reduce the cytotoxicity of colchicine while maintaining the anti-inflammatory activity, and shows a dose-dependent anti-inflammatory effect on a zebra fish model.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to colchicine derivatives, their preparation methods, and applications. Background Technology

[0002] Colchicine is a classic anti-inflammatory drug that inhibits cytokine storms. Clinically, it is widely used to treat gouty arthritis and is also considered a second-line treatment for acute pericarditis. Clinical trials have demonstrated that colchicine can significantly reduce the incidence of cardiovascular and cerebrovascular diseases associated with chronic inflammatory diseases. However, along with its anti-inflammatory activity, colchicine also exhibits strong cytotoxicity, which limits its clinical application.

[0003] Although numerous scientific studies have shown that the main reason colchicine causes cytotoxicity is that it binds to tubulin, affecting microtubule function, inhibiting microtubule polymerization, and blocking mitosis. In his master's thesis on the design, synthesis, and antiarrhythmic activity of colchicine derivatives, Li Yuzhu modified the 10-position of colchicine, reducing cytotoxicity to varying degrees, but did not investigate its anti-inflammatory activity. Chinese invention patent CN 113024400 A modifies the 4 and 7 positions of colchicine, increasing its cytotoxicity. Existing structural optimization schemes for colchicine are insufficient to simultaneously reduce its cytotoxicity while maintaining its anti-inflammatory activity.

[0004] Furthermore, angiogenesis refers to the process by which new vascular systems emerge from primitive vascular plexuses or existing blood vessels. It is a necessary mechanism for maintaining circulatory system function and ensuring energy supply. Many important human diseases, such as cancer, cardiovascular and cerebrovascular diseases, and diabetic complications, are closely related to angiogenesis. Insufficient angiogenesis can lead to myocardial ischemia, cerebral ischemia, slow healing of trauma and fractures, occlusive vasculitis, and diabetic peripheral vascular disease. Finding therapeutic drugs that promote angiogenesis is an important strategy for treating these ischemic diseases and is currently a hot topic in clinical research. However, existing technologies rarely involve research on the pro-angiogenic activity of colchicine and its derivatives. Summary of the Invention

[0005] The purpose of this invention is to provide colchicine derivatives, their preparation methods, and applications. Based on the structure-activity relationship of colchicine, and guided by the principle of synergistic effect and toxicity reduction, molecular design is performed at the 2 and 10 positions of colchicine to increase the steric hindrance of the binding of colchicine derivatives to microtubules. This strategy not only significantly reduces the toxicity to cardiomyocytes and macrophages, but also maintains good anti-inflammatory activity in some compounds.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, embodiments of the present invention provide colchicine derivatives, characterized in that they have the structure shown in Formula I;

[0008]

[0009] Wherein, R1 is H or methyl; R2 is H or -C(=O)-R3, and R3 is selected from nonsteroidal anti-inflammatory drugs or phenylpropanoid small molecule groups.

[0010] In some embodiments, R3 is selected from the following compounds:

[0011] Nonsteroidal anti-inflammatory drugs

[0012]

[0013] Phenylpropanoids

[0014]

[0015] In some embodiments, the colchicine derivative is selected from the following compounds:

[0016]

[0017] Secondly, embodiments of the present invention provide a method for preparing the colchicine derivative described in the first aspect, comprising the following steps:

[0018] S1, colchicine undergoes a substitution reaction with N-Boc-ethylenediamine to synthesize 10C-NB;

[0019] S2,10C-NB reacts with trifluoroacetic acid to remove the protecting group, synthesizing 10C-N;

[0020] S3, 10C-N undergoes an amidation reaction with a group containing a carboxyl group on an aromatic ring to synthesize compound I;

[0021] The structures of 10C-NB and 10C-N are shown below:

[0022]

[0023] In some embodiments, in S1, the substitution reaction is carried out under the following conditions: the molar ratio of colchicine to Boc-ethylenediamine is 1:(1-3), and the reaction is refluxed at 80-85°C overnight.

[0024] In some embodiments, in S2, the reaction conditions for removing the protecting group are: room temperature and a volume ratio of dichloromethane to trifluoroacetic acid solvent of (1-3):1.

[0025] In some embodiments, in S3, the amidation reaction is carried out by mixing the aromatic ring containing a carboxyl group, a catalyst, and a condensing agent in a solvent under ice bath conditions to form a mixed solution; at room temperature, intermediate 10C-N is added to the mixed solution and the reaction is carried out overnight.

[0026] The molar ratio of intermediate 10C-N, aromatic ring containing carboxyl group, catalyst, and condensing agent is 1:(3-5):(2-10):(2-4);

[0027] The solvent is DMF or DCM;

[0028] The catalyst is DIPEA or DMAP;

[0029] The condensing agent is HATU or EDCI.

[0030] In some embodiments, when R1 in compound I is H, colchicine is dissolved in concentrated sulfuric acid and reacted in an oil bath in glacial acetic acid to remove the methoxy group at the 2-position of colchicine, thereby synthesizing DE-1.

[0031] The structure of DE-1 is shown below:

[0032]

[0033] Thirdly, embodiments of the present invention provide a pharmaceutical composition comprising the colchicine derivative described in the first aspect or a pharmaceutically acceptable salt, solvate, or prodrug molecule or metabolite thereof.

[0034] Fourthly, embodiments of the present invention provide the use of the colchicine derivatives described in the first aspect or the pharmaceutical compositions described in the third aspect in the preparation of anti-inflammatory drugs and / or pro-angiogenic activities.

[0035] In some embodiments, the anti-inflammatory drug is a drug for treating gouty arthritis, acute pericarditis, or chronic cardiovascular and cerebrovascular inflammation.

[0036] The beneficial effects of the above embodiments of the present invention are as follows:

[0037] This invention combines the structure-activity relationship of colchicine with the goal of enhancing efficacy and reducing toxicity. It designs molecules at the 2 and 10 positions of colchicine to increase the steric hindrance of colchicine derivatives binding to microtubules. This strategy not only significantly reduces the toxicity to cardiomyocytes and macrophages, but also allows some compounds to maintain good anti-inflammatory and angiogenesis activities.

[0038] The method for synthesizing colchicine derivatives of the present invention is simple to operate, uses readily available raw materials, is highly practical, has a high yield, and is low in cost, which is of great significance for the industrial production of this type of substance. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 The 1H NMR spectrum of 10DN-T prepared in Example 3 of this invention;

[0041] Figure 2 The nuclear magnetic resonance carbon spectrum of 10DN-T prepared in Example 3 of this invention;

[0042] Figure 3 This is a high-resolution mass spectrum of 10DN-T prepared in Example 3 of the present invention;

[0043] Figure 4 The 1H NMR spectrum of 10CN-BLF prepared in Example 13 of this invention;

[0044] Figure 5 The carbon NMR spectrum of 10CN-BLF prepared in Example 13 of this invention;

[0045] Figure 6 This is a high-resolution mass spectrum of 10CN-BLF prepared in Example 13 of the present invention;

[0046] Figure 7 The 10CN-SLFS is a proton NMR spectrum prepared in Example 14 of this invention.

[0047] Figure 8 The carbon NMR spectrum of 10CN-SLFS prepared in Example 14 of this invention;

[0048] Figure 9 This is a high-resolution mass spectrum of 10CN-SLFS prepared in Example 14 of the present invention;

[0049] Figure 10 This is a standard curve of NO content in cell supernatant tested in Example 16 of the present invention;

[0050] Figure 11 The diagram shows how colchicine derivatives prepared in Examples 6 and 9-14 of this invention reduce the NO content in cell supernatant induced by LPS stimulation.

[0051] Figure 12 The effects of colchicine derivatives prepared in Examples 6 and 9-14 of this invention on zebrafish inflammatory cells;

[0052] Figure 13The graph shows the anti-inflammatory activity results of the colchicine derivatives prepared in Examples 6 and 9-14 of this invention when the addition amount is 50 μM;

[0053] Figure 14 The effects of different concentrations of the present invention on zebrafish inflammatory cells in Examples 10, 13 and 14 of this invention;

[0054] Figure 15 The graph shows the anti-inflammatory activity results at different concentrations in Examples 10, 13, and 14 of this invention.

[0055] Figure 16 Fluorescent images of zebrafish with different amounts of colchicine derivatives added, as shown in Examples 6 and 9-14 of this invention;

[0056] Figure 17 The bar chart shows the statistical analysis of ISVs (Intersegmental vessels) of zebrafish after treatment with the colchicine derivatives of Examples 6 and 9-13 of this invention. Detailed Implementation

[0057] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0058] The raw materials and reagents used in the following examples are all commercially available products and can be purchased. Some of the equipment used is shown below: Olympus IX73 inverted microscope, Olympus SZX16 microscope and DP2-BSW image acquisition system (Olympus Corporation, Japan); Forma 3111 water-jacketed CO2 incubator (Forma Corporation, USA); zebrafish farming and rearing equipment (Beijing Aisheng Technology Co., Ltd.).

[0059] Example 1

[0060] Preparation of compound 10C-N

[0061] The synthetic route for compound 10C-N is as follows:

[0062]

[0063] Dissolve 50 mg (0.125 mmol) of colchicine in 3 mL of acetonitrile and stir until dissolved. Add 60 mg (0.375 mmol) of Boc-ethylenediamine and reflux at 82 °C overnight. Monitor the reaction by TLC. After the reaction is complete, evaporate the acetonitrile to dryness, add 2 mL of DCM to dissolve, stir at room temperature, add 1 mL of TFA dropwise, and react for 2 h. Monitor the reaction by TLC until the reactant spots disappear, then stop the reaction. Evaporate the solvent under reduced pressure, add water, and extract three times with water:DCM = 1:1. Discard the organic phase, add saturated sodium bicarbonate until the solution is weakly alkaline, and extract three times with water:DCM = 1:1. Dry the solution with anhydrous Na₂SO₄ and separate by column chromatography to obtain 34 mg of yellow solid, yield 63.7%.

[0064] Spectral data of 10C-N: 1 H NMR (CD3OD, 600MHz): δ (ppm) 7.47 (d, J=11.1Hz, 1H, H-12), 7.31 (s, 1H, H-8), 6.91 (d, J=11.1Hz, 1H, H-11), 6.73 (s,1H,H-4),4.49(dd,J=12.2,6.4Hz,1H,H-7),3.89(s,3H,1-OCH3),3.88(s,3H,2-OCH3),3.83–3.72(m,2H,H-2 '),3.55(s,3H,3-OCH3),3.28(dd,J=6.1,1.9Hz,2H,H-1'),2.60(dd,J=13.3,6.3Hz,1H,H-5a),2.31(td,J=13.1 ,7.1Hz,1H,H-5b),2.20(tt,J=12.8,6.5Hz,1H,H-6a),1.99(s,3H,-COCH3),1.95(td,J=12.2,6.8Hz,1H,H-6b). 13 C NMR(150MHz,CD3OD)δ176.96(C-9),172.62(-NH-CO-),155.58,154.75,153.05,152.04,142.71,140.66,136.21,133.21,127 .76,124.75,110.44,108.83,61.68,61.64,56.68,53.89,40.96,39.01,37.95(C-6),30.68(C-5),22.43(-COCH3).HR-ESI-MS m / z:428.2168(calcd for C 23 H 30 O5N3 + [M+H] + ,428.2180).

[0065] Example 2

[0066] Preparation of compound 10D-N

[0067] The synthetic route for compound 10D-N is as follows:

[0068]

[0069] 400 mg (1 mmol) of colchicine was dissolved in 0.6 mL of glacial acetic acid, and 2 mL of concentrated sulfuric acid was added. The mixture was stirred at 60 °C for 5 h, and the reaction was monitored by TLC (DCM:MeOH 10:1). After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to 5-6 with NaOH, water was added, and the mixture was extracted three times with DCM. The solution was dried over anhydrous Na2SO4 and separated by column chromatography to obtain DE-1. 50 mg (0.13 mmol) of DE-1 was dissolved in 3 mL of acetonitrile and stirred until dissolved. 62.5 mg (0.39 mmol) of Boc-ethylenediamine was added, and the mixture was refluxed at 82 °C overnight. The reaction was monitored by TLC. After the reaction was completed, the acetonitrile was evaporated to dryness, 2 mL of DCM was added to dissolve the solution, and the mixture was stirred at room temperature. 1 mL of TFA was added dropwise, and the reaction was monitored by TLC for 2 h. The post-treatment was the same as in Example 1 (10C-N), yielding 27 mg of a deep yellow powder, with a yield of 50.3%.

[0070] Spectral data of 10D-N: 1 H NMR (CD3OD, 600MHz): δ (ppm) 7.48 (d, J=11.1Hz, 1H, H-12), 7.32 (s, 1H, H-8), 6.89 (d, J=11.3Hz, 1H, H-11), 6 .66(s,1H,H-4),4.51(dd,J=12.1,6.4Hz,1H,H-7),3.89(s,3H,1-OCH3),3.76(td,J=6.1,2.3Hz,2H,H-2'), 3.49(s,3H,3-OCH3),3.27(t,J=6.1Hz,2H,H-1'),2.54(dd,J=13.5,6.2Hz,1H,H-5a),2.27(td,J=13.0,6.6 Hz,1H,H-5b),2.19(tt,J=12.7,6.3Hz,1H,H-6a),1.99(s,3H,-COCH3),1.93(td,J=11.9,6.5Hz,1H,H-6b). 13C NMR(150MHz,CD3OD)δ176.87(C-9),172.60(-NH-CO-),155.55,153.21,149.76,146.35,140.52,139.66,133.53,131.03, 127.34,124.71,110.50,108.23,61.09,56.79,53.99,41.19,39.08,38.30(C-6),30.34(C-5),22.43(-COCH3).HR-ESI-MS m / z:412.1878(calcd for C 22 H 26 O5N3 - [MH] - ,412.1878).

[0071] Example 3

[0072] Preparation of compound 10DN-T

[0073] The synthetic route for compound 10DN-T is as follows:

[0074]

[0075] The preparation method of 10D-N was the same as in Example 2, except that 24.6 mg (0.06 mmol) of 10D-N was weighed and dissolved in 2 mL of anhydrous dichloromethane. The mixture was stirred in an ice bath, and 25 μL of triethylamine (0.18 mmol) was added. 14 mg (0.06 mmol) of 3,4,5-trimethoxybenzoyl chloride was dissolved in 1 mL of DCM. The DCM solution of 3,4,5-trimethoxybenzoyl chloride was added dropwise to the reaction flask and allowed to react overnight. The reaction was monitored by TLC. When the product spot no longer increased, water was added to terminate the reaction. The mixture was extracted three times with dichloromethane:water at a ratio of 1:1. The organic phases were combined, dried over anhydrous Na2SO4, and separated by column chromatography to obtain 9.65 mg of a yellow solid powder, with a yield of 26.5%.

[0076] Spectral data of 10DN-T: 1H NMR (CD3OD, 600MHz): δ (ppm) 7.46 (d, J=11.3Hz, 1H, H-12), 7.28 (s, 1H, H-8), 7.14 (t, J=1.7Hz, 2H, H-5' ,H-9'),7.00(d,J=11.3Hz,1H,H-11),6.64(s,1H,H-4),4.50(dd,J=12.2,5.7Hz,1H,H-7),3.89(s,3H,1 -OCH3),3.84(s,6H,6'-OCH3,8'-OCH3),3.78(s,3H,3-OCH3),3.71(m,4H,H-1',H-2'),3.44(s,3H,7'- OCH3),2.52–2.47(m,1H,H-5a),2.24–2.14(m,2H,H-5b,H-6a),1.98(s,3H,-COCH3),1.92(m,1H,H-6b). 13 CNMR(150MHz,CD3OD)δ175.70(C-9),172.58(C-3'),170.32(-NHCO-),156.43 ,154.45,152.66,149.67,146.36,142.23,140.98,139.65,132.86,131.04,1 30.74,127.41,123.60,110.86,108.21,106.07,61.12,61.05,56.77,56.74, 53.93,42.99,39.97,38.47(C-6),30.37(C-5),22.43(-NHCOCH3).HR-ESI-MS m / z: 606.2457 (calcd for C 32 H 36 O9N3 - [MH] - ,606.2457).

[0077] Example 4

[0078] Preparation of compound 10CN-R

[0079] The synthetic route for compound 10CN-R is as follows:

[0080]

[0081] Weigh 56.8 mg (0.35 mmol) of 2-OH-cinnamic acid and dissolve it in 2 mL of DMF. Weigh 75 mg (0.58 mmol) of DIPEA and 87.8 mg (0.23 mmol) of HATU. Stir in an ice bath for 10-15 min. Add 49.3 mg (0.12 mmol) of 10C-N prepared in Example 1. Let the reaction proceed overnight. Monitor the reaction progress by TLC. When the reaction is complete, add water to stop the reaction. Extract three times with ethyl acetate. Combine the organic phases, back-extract with saturated brine, dry with anhydrous magnesium sulfate, and separate by column chromatography to obtain 26 mg of light yellow solid, with a yield of 37.8%.

[0082] Spectral data of 10CN-R: 1 H NMR (CD3OD, 600MHz): δ (ppm) 7.79 (d, J = 15.8Hz, 1H, H-4'), 7.40 (d, J = 11.4Hz, 1H, H-12), 7.37 (d, J = 8.4Hz, 1H, H-11'), 7.22 (s, 1H, H-8), 7 .12(t,J=7.8Hz,1H,H-9'),6.92(d,J=11.3Hz,1H,H-11),6.77(d,J=8.1Hz,2H,H-8',H-10'),6.66(d,J=17.9Hz,2H,H-5',H-4),4.44(dd, J=12.6,6.4Hz,1H,H-7),3.84(s,3H,1-OCH3),3.81(s,3H,2-OCH3),3.64–3.57(m,4H,H-1',H-2'),3.47(s,3H,3-OCH3),2.50(dd,J=13.6 ,6.3Hz,1H,H-5a),2.24(td,J=13.1,6.9Hz,1H,H-5b),2.13(tq,J=13.2,6.1Hz,1H,H-6a),1.94(s,3H,-COCH3),1.93–1.85(m,1H,H-6b). 13C NMR(150MHz,CD3OD)δ176.32(C-9),172.58(C-3'),170.21(-NH-CO-),158.03,156. 34,154.52,152.37,152.06,142.63,140.96,138.30,136.24,132.17,131.87,129. 94,127.99,123.67,123.14,121.32,120.67,116.97,110.49,108.72,61.68,61.56 ,56.66,53.78,43.09,39.38,38.11(C-6),30.74(C-5),22.45(-COCH3).HR-ESI-MS m / z:574.2543(calcd for C 32 H 36 O7N3 + [M+H] + ,574.2548).

[0083] Example 5

[0084] Preparation of compound 10CN-AWS

[0085] The preparation method of compound 10CN-AWS is the same as that of 10CN-R in Example 4. The difference is that the small molecule of phenylpropanoid is ferulic acid. The specific synthetic route is as follows, yielding 23.3 mg of yellow solid powder with a yield of 32.3%.

[0086]

[0087] Spectral data from 10CN-AWS: 1H NMR (CD3OD, 600MHz): δ (ppm) 7.38 (m, 1H, H-4'), 7.40 (d, J = 11.4Hz, 1H, H-12), 7.22 (s, 1H, H-8), 7.05 (d, J = 1.9Hz, 1H, H-7'), 6.96 (dd, J = 8.2, 1.8Hz, 1H ,H-11'),6.93(d,J=11.4Hz,1H,H-11),6.74(d,J=8.2Hz,1H,H-10'),6.64( s,1H,H-4),6.35(d,J=15.6Hz,1H,H-5'),4.44(dd,J=12.1,6.3Hz,1H,H-7) ,3.85(s,3H,1-OCH3),3.82(s,6H,H-12',2-OCH3),3.63(dd,J=8.4,5.4Hz, 2H,H-2'),3.59(t,J=4.1Hz,2H,H-1'),3.47(s,3H,3-OCH3),2.47(dd,J=13 .2,6.2Hz,1H,H-5a),2.20(td,J=13.0,6.8Hz,1H,H-5b),2.12(tt,J=12.8, 6.1Hz,1H,H-6a),1.95(s,3H,-COCH3),1.88(td,J=12.1,6.7Hz,1H,H-6b). 13 C NMR(150MHz,CD3OD)δ176.44(C-9),172.72(C-3'),170.01(-NH-CO-),156.53,154.6 6,152.50,152.17,150.10,149.44,142.74,142.64,141.05,136.37,132.29,128.33, 128.07,123.79,123.43,118.57,116.65,111.80,110.62,108.83,61.82,61.71,56. 79,56.56,53.92,43.19,39.65,38.23(C-6),30.87(C-5),22.59(-COCH3).HR-ESI-MS m / z:602.2507(calcd for C 33 H 36 O8N3 - [MH] - ,602.2508).

[0088] Example 6

[0089] Preparation of compound 10CN-HKFS

[0090] The preparation method of compound 10CN-HKFS is the same as that of 10CN-R in Example 4, except that the small molecule of phenylpropanoid is dihydrocaffeic acid. The specific synthetic route is as follows, yielding 22.5 mg of pale yellow solid powder with a yield of 29.2%.

[0091]

[0092] Spectral data of 10CN-HKFS: 1 H NMR (CD3OD, 600MHz): δ (ppm) 7.46 (dd, J=11.2, 2.2Hz, 1H, H-12), 7.28 (s, 1H, H-8), 6.90 (dd, J=11.4, 2.4Hz, 1H, H-11), 6.72 (s, 1H, H-4), 6.63 (d,J=2.2Hz,1H,H-7'),6.61(d,J=8.1Hz,1H,H-10'),6.50(dd,J=8.1,2.2Hz,1H,H-11'),4.51(dd,J=12.1,6.4Hz,1H,H-7),3.90(s,3H,1-OCH 3),3.87(s,3H,2-OCH3),3.54(s,3H,3-OCH3),3.53–3.46(m,4H,H-1',H-2'),2.75(t,J=7.6Hz,2H,H-4'),2.57(dd,J=13.2,6.5Hz,1H,H-5a) ,2.42(t,J=7.7Hz,2H,H-5'),2.32(m,1H,H-5b),2.20(tt,J=12.7,6.5Hz,1H,H-6a),2.01(s,3H,-COCH3),1.96(td,J=12.2,7.1Hz,1H,H-6b). 13 C NMR(150MHz,CD3OD)δ174.07(C-9),173.95(C-3'),170.41(-NH-CO-),154.06,152.32 ,150.17,149.84,143.96,142.40,140.42,138.72,134.04,131.44,129.88,125.78,12 1.41,118.36,114.32,114.10,108.18,106.50,59.47,59.39,54.42,51.60,40.91,37 .13,36.78(C-6),35.84(C-5'),30.09(C-5),28.51(C-4'),20.21(-COCH3).HR-ESI-MS m / z:590.2508(calcdfor C 32 H36 O8N3 - [MH] - ,590.2508).

[0093] Example 7

[0094] Preparation of compound 10DN-B

[0095] The preparation method of compound 10DN-B is the same as that of 10CN-R in Example 4. The difference is that the active pharmaceutical ingredient 10D-N and the small molecule of phenylpropanol are 3-(2-hydroxyphenyl)-propionic acid. The specific synthetic route is as follows, yielding 23.2 mg of yellow solid powder with a yield of 67.7%.

[0096]

[0097] Spectral data for 10DN-B: 10DN-B: 1 H NMR (CD3OD, 600MHz): δ (ppm) 7.41 (d, J = 10.9Hz, 1H, H-12), 7.23 (s, 1H, H-8), 6.96 (d, J = 7.5Hz, 1H, H-10'), 6.90 (t, J = 7.8Hz, 1H, H-8'), 6.81 ( d,J=10.9Hz,1H,H-11),6.66(d,J=8.0Hz,1H,H-7'),6.61(s,1H,H-4),6.58(t,J=7.5Hz,1H,H-9'),4.48(dd,J=12.2,6.4Hz,1H,H-7),3.85(s ,3H,1-OCH3),3.53–3.43(m,4H,H-1',H-2'),3.42(s,3H,3-OCH3),2.83(t,J=7.4Hz,2H,H-3'),2.46(ddd,J=15.2,11.8,6.9Hz,3H,H-4',H-5 a),2.23(td,J=13.2,6.9Hz,1H,H-5b),2.13(tt,J=12.4,6.2Hz,1H,H-6a),1.95(d,J=1.9Hz,3H,-COCH3),1.89(tt,J=11.9,6.2Hz,1H,H-6b). 13CNMR(150MHz,CD3OD)δ176.53(C-9),176.29(-NH-CO-),172.57(-COCH3),156.32,15 6.23,152.59,149.60,146.40,140.75,139.62,132.49,131.18,131.05,128.47,128. 24,127.55,123.69,120.54,116.10,110.44,108.23,61.07,56.78,53.91,43.22,39. 00,38.44(C-4'),37.28(C-6),30.41(C-5),27.65(C-3'),22.45(-COCH3).HR-ESI-MS m / z:562.2543(calcdfor C 31 H 36 O7N3 + [M+H] + ,562.2548).

[0098] Example 8

[0099] Preparation of compound 10DN-R

[0100] The preparation method of compound 10DN-R is the same as that of 10CN-R in Example 4. The difference is that the active pharmaceutical ingredient 10D-N and the small molecule of phenylpropanol are 2-OH-cinnamic acid. The specific synthetic route is as follows, yielding 12 mg of yellow solid powder with a yield of 33.4%.

[0101]

[0102] Spectral data of 10DN-R: 1H NMR (CD3OD, 600MHz): δ (ppm) 7.83 (d, J = 15.9Hz, 1H, H-4'), 7.49 (d, J = 11.2Hz, 1H, H-12), 7.42 (d, J = 7.8Hz, 1H, H-11'), 7.27 (s, 1H, H-8), 7 .16(t,J=7.8Hz,1H,H-9'),6.96(d,J=11.2Hz,1H,H-11),6.82(d,J=7.8Hz,2H,H-8',H-10'),6.71(d,J=15.8Hz,1H,H-5'),6.63(s,1H,H-4 ),4.51(dd,J=12.3,6.3Hz,1H,H-7),3.89(s,3H,1-OCH3),3.66–3.62(m,4H,H-1',H-2'),3.47(s,3H,3-OCH3),2.50(dd,J=13.4,6.4Hz,1 H,H-5a),2.26(td,J=13.2,6.9Hz,1H,H-5b),2.17(tt,J=12.9,6.5Hz,1H,H-6a),1.99(s,3H,-COCH3),1.92(td,J=12.5,7.0Hz,1H,H-6b). 13 C NMR(150MHz,CD3OD)δ176.45(C-9),172.73(C-3'),170.33(-NH-CO-),158.19,15 6.39,152.77,149.74,146.54,140.94,139.74,138.44,132.74,132.01,131.21,1 30.10,127.65,123.88,123.27,121.46,120.81,117.11,110.71,108.36,61.21, 56.91,54.05,43.28,39.41,38.59(C-6),30.54(C-5),22.58(-COCH3).HR-ESI-MS m / z:560.2388(calcd for C 31 H 34 O7N3 + [M+H] + ,560.2391).

[0103] Example 9

[0104] Preparation of compound 10DN-NPS

[0105] The preparation method of compound 10DN-NPS is the same as that of 10CN-R in Example 4. The difference lies in the difference between the active pharmaceutical ingredient 10D-N and the small molecule carboxylic acid naproxen. The specific synthetic route is as follows, yielding 6.1 mg of yellow solid powder with a yield of 18%.

[0106]

[0107] Spectral data of 10DN-NPS: 1 H NMR (CD3OD, 400MHz): δ (ppm) 7.64 (s, 1H, H-8), 7.58 (t, J=7.9Hz, 2H, H-11', H-12'), 7.35 (dd, J=8.5, 1.8Hz, 1H, H-7'), 7.26 (s, 1H, H-6'), 7 .20(d,J=11.2Hz,1H,H-12),7.06(d,J=2.4Hz,1H,H-10'),7.02(dd,J=8.9,2.5Hz,1H,H-8'),6.73(d,J=11.3Hz,1H,H-11),6.66(s,1H,H-4 ),4.50(dd,J=11.9,5.8Hz,1H,H-7),3.91(s,3H,1-OCH3),3.85(s,3H,H-14'),3.71(q,J=7.1Hz,1H,H-4'),3.64–3.45(m,2H),3.42(s,3H, 3-OCH3),2.52(m,1H,H-5a),2.31–2.10(m,2H,H-5b,H-6a),1.99(s,3H,-COCH3),1.94(m,J=6.1Hz,1H,H-6b),1.49(d,J=7.0Hz,3H,H-13'). 13 C NMR(150MHz, CDCl3)δ175.40(C-9),175.09(C-3'),169.81(-NH-CO-),157.94,154.60,1 51.08,147.23,144.78,139.00,137.87,136.30,133.93,130.11,129.32,129.11,127.79 ,126.32,125.94,123.41,119.32,108.83,106.98,105.86,61.12,56.46,55.47,52.74,4 7.11,42.36,38.76,37.90(C-6),29.87(C-5),23.17(-COCH3),18.65(C-13').HR-ESI-MS m / z:624.2714(calcd forC 36H 38 O7N3 - [MH] - ,624.2715).

[0108] Example 10

[0109] Preparation of compound 10DN-Y

[0110] The preparation method of compound 10DN-Y is the same as that of 10CN-R in Example 4. The difference lies in the difference between the active pharmaceutical ingredient 10D-N and the small molecule carboxylic acid indomethacin. The specific synthetic route is as follows, yielding 21 mg of yellow solid powder with a yield of 51.7%.

[0111]

[0112] Spectral data of 10DN-Y: 1 H NMR (CD3OD, 400MHz): δ (ppm) 7.66 (d, J = 8.4Hz, 2H, H-15'), 7.55 (d, J = 8.2Hz, 2H, H-16'), 7.30 (d, J = 11.3Hz, 1H, H-12), 7.23 (s, 1H, H-8), 6.98 (d ,J=2.6Hz,1H,H-7'),6.86(d,J=9.0Hz,1H,H-10'),6.80(d,J=11.3Hz,1H,H-11),6.64(s,1H,H-4),6.59(dd,J=9.0,2.5Hz,1H,H-9'),4.50(dd, J=12.0,6.0Hz,1H,H-7),3.89(s,3H,1-OCH3),3.72(s,3H,8'-OCH3),3.62(s,2H,H-4'),3.61–3.47(m,4H,H-1',H-2'),3.47(s,3H,3-OCH3),2. 50(dd,J=12.7,5.7Hz,1H,H-5a),2.26(s,3H,12'-CH3),2.24–2.07(m,2H,H-5b,H-6a),1.98(s,3H,-COCH3),1.90(td,J=11.8,6.2Hz,1H,H-6b). 13C NMR(150MHz, CDCl3)δ171.26(C-9),169.78(-NH-CO-),168.46(C-13'),156.43,154.55,147.34,1 44.74,139.65,137.86,136.65,133.78,133.19,131.37(C-15'),131.10,130.53,130.16,129.35( C-16'),125.78,123.30,115.25,112.78,112.26,106.97,104.59,101.18,61.16,56.46,55.94,5 2.75,42.65,38.55,37.88(C-6),32.31,29.85(C-5),23.16(-COCH3),13.47(12'-CH3).HR-ESI-MS m / z:751.2540(calcd for C 41 H 40 O8N4Cl - [MH] - ,751.2540).

[0113] Example 11

[0114] Preparation of compound 10CN-NPS

[0115] The synthetic route for preparing compound 10CN-NPS is as follows:

[0116]

[0117] 37 mg (0.087 mmol) of 10C-N prepared in Example 1 was dissolved in 3 mL of dichloromethane. 21.2 mg (0.17 mmol) of DMAP and 66.5 mg (0.35 mmol) of EDCI were added under ice bath conditions. After complete dissolution, 100 mg (0.43 mmol) of naproxen was added. The reaction was allowed to proceed overnight, and the reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to dryness, and the solution was dissolved in water. The solution was extracted three times with dichloromethane:water at a ratio of 1:1. The organic phases were combined, dried over anhydrous Na2SO4, filtered, evaporated to dryness, and separated by column chromatography to obtain 25.9 mg of a yellow solid, with a yield of 46.5%.

[0118] Spectral data of 10CN-NPS: 1H NMR (CDCl3, 600MHz): δ (ppm) 7.65 (t, J = 9.1Hz, 2H, H-11', H-12'), 7.62 (s, 1H, H-8), 7.49 (s, 1H, H-6'), 7.38 (d, J = 11.1Hz, 1H, H-12), 7.33 (d, J = 8.5H z,1H,H-7'),7.10(d,J=8.6Hz,1H,H-8'),7.07(s,1H,H-10'),6.65(d,J=1 1.2Hz,1H,H-11),6.51(s,1H,H-4),6.08(s,1H,NH),4.67(dt,J=12.7,6.9 Hz,1H,H-7),3.93(s,3H,1-OCH3),3.89(s,3H,H-14'),3.88(s,3H,2-OCH3 ),3.70(q,J=7.3Hz,1H,H-4'),3.58(s,3H,3-OCH3),3.49(s,4H,H-1',H-2 '),2.43(m,1H,H-5a),2.33(m,1H,H-5b),2.23(dt,J=12.6,6.4Hz,1H,H-6 a),1.99(s,3H,-COCH3),1.90(m,1H,H-6b),1.58(d,J=7.1Hz,3H,H-13'). 13 C NMR(150MHz, CDCl3)δ175.64(C-9),170.02(-NH-CO-),158.14,154.81,153.40,151.45,14 2.04,139.84,136.48,134.72,134.14,129.54,129.32,127.99,127.07,126.71,126.55,12 6.54,123.55,119.53,109.20,107.69,106.07,106.02,61.72,61.60,56.51,55.67,52.84 ,47.31,42.58,38.93,37.90(C-6),30.40(C-5),23.39(-COCH3),18.82(C-13').HR-ESI-MS m / z:638.2871(calcd for C 37 H 40 O7N3 - [MH] - ,638.2872).

[0119] Example 12

[0120] Preparation of compound 10CN-Y

[0121] The preparation method of compound 10CN-Y is the same as that of 10CN-NPS in Example 11, the difference being the difference in the active pharmaceutical ingredient, the nonsteroidal anti-inflammatory drug indomethacin. The specific synthetic route is as follows, yielding 33.2 mg of yellow crystalline powder, with a yield of 54.2%.

[0122]

[0123] Spectral data of 10CN-Y: 1 H NMR (CD3OD, 400MHz): δ (ppm) 7.65 (d, J = 8.3Hz, 2H, H-15'), 7.54 (d, J = 8.3Hz, 2H,H-16'),7.28(d,J=11.2Hz,1H,H-12),7.22(s,1H,H-8),6.97(d,J=2.5Hz, 1H,H-7'),6.88(d,J=9.0Hz,1H,H-10'),6.81(d,J=11.3Hz,1H,H-11),6.71(s ,1H,H-4),6.58(dd,J=9.0,2.5Hz,1H,H-9'),4.48(dd,J=12.1,6.2Hz,1H,H-7 ),3.89(s,3H,1-OCH3),3.87(s,3H,2-OCH3),3.72(s,3H,8'-OCH3),3.61(s,2 H,H-4'),3.61–3.53(m,4H,H-1',H-2'),3.53(s,3H,3-OCH3),2.55(dd,J=13. 1,5.9Hz,1H,H-5a),2.31(m,1H,H-5b),2.25(s,3H,12'-CH3),2.16(dt,J=12. 5,6.1Hz,1H,H-6a),1.98(s,3H,-COCH3),1.91(td,J=12.0,6.6Hz,1H,H-6b). 13C NMR(150MHz, CDCl3)δ173.83(C-9),171.17(C-3'),169.80(-NH-CO-),168.46(C-13'),156.43,156.21,154 .59,153.25,151.22,141.83,139.80,139.62,136.64,134.48(C-15'),133.82,131.32(C-16'),130.54,129 .30,126.73,123.36,115.25,115.08,112.80,112.26,111.64,107.50,101.71,101.16,61.50,61.39,56.29 ,55.90,52.57,42.49,38.50,37.72(C-6),32.27,30.16(C-5),23.08(-COCH3),13.42(12'-CH3).HR-ESI-MS m / z:765.2697(calcd for C 42 H 42 O8N4Cl - [MH] - ,765.2697).

[0124] Example 13

[0125] Preparation of compound 10CN-BLF

[0126] The preparation method of compound 10CN-BLF is the same as that of 10CN-NPS in Example 11, the difference being the difference in the active pharmaceutical ingredient, the nonsteroidal anti-inflammatory drug ibuprofen. The specific synthetic route is as follows, yielding 21 mg of yellow solid powder with a yield of 50.5%.

[0127]

[0128] Spectral data of 10CN-BLF: 1H NMR (CDCl3, 600MHz): δ (ppm) 7.46 (s, 1H, NH), 7.44 (s, 1H, H-8), 7.41 (d, J=11.3Hz, 1H, H-12), 7.21–7.13 (m, 2H, H-7'), 7.11–7.04 (m, 2H, H-8'), 6.67(d,J=11.3Hz,1H,H-11),6.52(s,1H,H-4),4.67(dq,J=11.9,7.0Hz,1H,H-7),3.93(s,3H,1-OCH3),3.89(s,3H,2-OCH3),3.60(s,3H,3-OCH 3),3.52–3.38(m,4H,H-1',H-2'),2.47(dd,J=13.6,6.4Hz,1H,H-5a),2 .42(d,J=7.2Hz,1H,H-4'),2.39(d,J=7.2Hz,2H,H-10'),2.34(m,1H,H- 11'),2.25(m,1H,H-5b),1.97(s,3H,-COCH3),1.91(m,1H,H-6a),1.81( m,1H,H-6b),1.50(d,J=7.2Hz,3H,H-5'),0.87(t,J=6.0Hz,6H,H-12'). 13 C NMR (151MHz, CDCl3) δ175.48,175.03,169.82,154.58,153.17,151.27,151.14,141.84,140.98,139.57,138.54,138.37,134.51,129.82,1 27.48,126.92,123.33,108.90,107.47,61.40,56.29,52.66,46.81,4 5.12,42.36,38.72,38.58,37.64,30.26,23.15,22.51,18.70,18.62. HR-ESI-MS m / z:616.3362(calcd for C 36 H 44 O6N3 + [M+H] + ,616.3386).

[0129] Example 14

[0130] Preparation of compound 10CN-SLFS

[0131] The preparation method of compound 10CN-SLFS is the same as that of 10CN-NPS in Example 11, the difference being the nonsteroidal anti-inflammatory drug (NSAID) as the active pharmaceutical ingredient. The specific synthetic route is as follows, yielding 31 mg of yellow crystalline solid powder with a yield of 51.8%.

[0132]

[0133] Spectral data of 10CN-SLFS: 1 H NMR (CD3OD, 400MHz): δ (ppm) 8.69 (d, J = 7.0Hz, 1H, H-9'), 7.37 (d, J = 8.1Hz, 2 H,H-13'),7.28–7.21(m,2H,H-12,H-11),7.13(dd,J=7.5,1.6Hz,1H,H-7'),7 .05(t,J=8.1Hz,1H,H-14'),6.89(s,1H,H-8),6.75(td,J=7.4,1.2Hz,1H,H-8 '),6.71(s,1H,H-4),6.31(dd,J=7.9,1.2Hz,1H,H-6'),4.50(ddd,J=11.7,6. 5,4.2Hz,1H,H-7),3.89(s,3H,1-OCH3),3.86(s,3H,2-OCH3),3.65(d,J=10. 0Hz,2H,H-4'),3.62–3.57(m,4H,H-1',H-2'),3.53(s,3H,3-OCH3),2.56(dd, J=13.0,6.0Hz,1H,H-5a),2.29(td,J=12.9,6.9Hz,1H,H-6a),2.18(tt,J=12. 8,6.3Hz,1H,H-5b),1.99(s,3H,-COCH3),1.93(td,J=11.9,6.7Hz,1H,H-6b). 13C NMR(150MHz, CDCl3)δ175.03(C-9),172.51(C-3'),169.89(-NH-CO-),154.70,153.21, 151.23,151.14,143.16,139.80,137.58,134.46,130.95,130.31,129.02(C-13'),128 .16,126.78,124.53,124.16,123.31,122.18,121.54,117.49,107.46,61.50,61.39,5 6.29,52.62,42.50,41.07,38.58,37.77(C-6),30.18(C-5),23.07(-COCH3).HR-ESI-MS m / z:703.2096(calcd for C 37 H 37 O6N4Cl2 - [MH] - ,703.2096).

[0134] Example 15:

[0135] Cytotoxicity evaluation of colchicine derivatives

[0136] To verify whether colchicine derivatives reduced cytotoxicity, this embodiment used the MTT assay to determine their inhibition rate on mouse macrophage leukemia cells RAW264.7 and rat cardiomyocytes H9C2. A control experiment was conducted using DMSO and colchicine raw material groups.

[0137] Cell culture: Rat cardiomyocytes H9C2 were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and mouse macrophage leukemia cells RAW264.7 were cultured in DMEM medium specifically for RAW264.7. Culture conditions were 37°C in an incubator with 5% CO2.

[0138] MTT assay: H9C2 cells were prepared at 8 × 10⁻⁶ 4Cells were seeded at a density of 100 μL / mL in 96-well plates. Incubation was carried out overnight at 37°C with 5% CO2 until cells adhered and covered the bottom of the wells. Drug concentration was then increased to 10 μM, and the plates were gently shaken to mix the drug with the culture medium. A blank control and three replicates for each concentration were included. After 72 hours of incubation, 10 μL of 0.5 mg / mL MTT was added to each well, and incubation continued for 4 hours. The culture medium was then completely removed from the plates. 100 μL of dimethyl sulfoxide was added to each well to dissolve water-insoluble blue-purple formazan crystals, and the cells were detected at 570 nm using a microplate reader. The inhibition rate of colchicine derivatives against RAW264.7 was determined using the same method as that against H9C2, except that the cell density was 1 × 10⁻⁶ cells / mL. 5 Cells / mL. See Table 1 for specific data.

[0139] Table 1. Inhibition rate of 10 μM colchicine and its derivatives on RAW264.7 and H9C2 cells

[0140]

[0141] The results are shown in Table 1. Compared with colchicine, the colchicine derivatives all showed varying degrees of reduced toxicity. As shown in the table, colchicine inhibited RAW264.7 cells by 93.9% and H9C2 cells by 34.5% at 10 μM, while the derivatives significantly reduced the inhibition rate of cells.

[0142] Example 16:

[0143] Evaluation of the anti-inflammatory activity of colchicine derivatives

[0144] To investigate whether colchicine derivatives retain anti-inflammatory activity while reducing cytotoxicity, this study established a macrophage inflammation model using LPS stimulation and a zebrafish inflammation model using CuSO4-treated fluorescently labeled transgenic zebrafish to investigate the anti-inflammatory activity of colchicine derivatives. The nitric oxide (NO) content in cell supernatants is often used as a biomarker of inflammation severity. NO is a small free radical catalyzed by nitric oxide synthase (iNOS) in vivo and plays an important role in various biological processes, especially in immune system function and inflammatory responses. During inflammation, immune cells such as macrophages and neutrophils are activated and increase NO production through inducible nitric oxide synthase (iNOS). NO production is often positively correlated with inflammatory activity. Therefore, measuring the NO content in cell supernatants can provide a direct indicator of the inflammatory state.

[0145] The NO content in cell supernatant was detected using the Griess method. A standard curve was plotted with NaNO2 concentrations (0, 6.58, 13.17, 19.75, 29.63, 44.44, 66.67, 100 μM) on the X-axis and the corresponding OD values ​​at 540 nm wavelength using a microplate reader on the Y-axis. Figure 10 As shown, the standard curve is y = 0.0042x + 0.0587, R0 2 =0.9943, linear range 0-100 μM. Further anti-inflammatory activity screening was performed on the low-toxicity compounds from Example 15. Cells were pre-protected with the drug for 1 h, then stimulated with LPS for 24 h. Cell supernatant was collected, and the measured OD values ​​were substituted into the standard curve to calculate the NO content in the cell supernatant. The measured concentration was normalized to the LPS-stimulated group. The results are shown below. Figure 11 As can be seen, compared with the LPS group, the NO content in the cell supernatant of the drug-treated groups was reduced to varying degrees. Considering the toxicity and anti-inflammatory activity of colchicine, the reduction of NO content by colchicine at 0.37 μM was used as a positive control. The experimental results showed that the colchicine derivatives prepared in Examples 6 and 9-13 reduced the NO content in the cell supernatant caused by LPS stimulation. Among them, Example 6 was 10CN-HKFS (abbreviated as CH), Example 13 was 10CN-BLF (abbreviated as CBL), Example 12 was 10CN-Y (abbreviated as CY), Example 11 was 10CN-NPS (abbreviated as CN), Example 9 was 10DN-NPS (abbreviated as DN), and Example 10 was 10DN-T (abbreviated as DT); all of which could significantly reduce the NO content in the cell supernatant caused by LPS stimulation.

[0146] To evaluate the anti-inflammatory activity of colchicine derivatives, transgenic zebrafish (Tg:zlyz-EGFP) with fluorescently labeled healthy inflammatory cells developed for 72 hours postfertilization were used as experimental animals. Seven compounds (CH, CY, CN, CBL, CSL, DN, DT) samples (50 μM) and the positive control colchicine (20, 50 μM) were administered to 24-well plates, and the embryos were placed in a light incubator (28℃) to allow further embryonic development. After 2 hours, each group of zebrafish except the blank control group was treated with 20 μM CuSO4 for 1 hour. The zebrafish were then washed. The inflammatory response was observed under a fluorescence microscope, and the number of inflammatory cells migrating to the zebrafish lateral lines was counted and statistically analyzed. The experimental results are shown below. Figure 12 and Figure 13Compared with the blank control group, the number of migrating inflammatory cells in the model group of zebrafish was significantly increased, indicating successful inflammation modeling. Compared with the model group, the number of migrating inflammatory cells in the positive control group and the seven sample groups of zebrafish was significantly reduced, which was statistically significant, indicating that they all had significant anti-inflammatory activity. Among them, the CSL, CBL, and DT groups had stronger anti-inflammatory activity. The CH and CY groups had certain lethality rates (16.7% and 10%, respectively) and teratogenicity rates (3.3% and 6.7%, respectively).

[0147] Healthy transgenic zebrafish (Tg:zlyz-EGFP) with inflammatory cells developed for 72 hours post-fertilization were used as experimental animals. Three compound samples (CSL, CBL, DT) (12.5, 25, 50 μM) and the positive control colchicine (12.5, 25, 50 μM) were administered to 24-well plates, respectively. After adding culture water to a final volume of 2.0 mL, the plates were placed in a light incubator (28℃) to allow the embryos to continue developing. Two hours later, the zebrafish in each treatment group were treated with 40 μM CuSO4 for 1 hour. The zebrafish were then washed. The inflammatory response was observed under a fluorescence microscope, and the number of inflammatory cells migrating to the zebrafish lateral lines was counted and statistically analyzed. The results are shown in [Figure number missing]. Figure 14 and Figure 15 Compared with the blank control group, the number of migrating inflammatory cells in the model group of zebrafish was significantly increased, indicating that the inflammation model was successfully established; the positive control group showed a statistically significant difference compared with the model group. As the concentration of the sample increased, the number of migrating inflammatory cells decreased, indicating that the compounds CBL, CSL, and DT could exert their anti-inflammatory effects in a dose-dependent manner.

[0148] The discovery of angiogenesis drugs relies on angiogenesis models. Common angiogenesis models include in vitro models (endothelial cell models and rat arterial ring models), in vivo models (corneal microcapsules, chicken embryo chorioallantoic membrane, disc angiogenesis models, and sponge-stromal gel models), and whole animal models (zebrafish models and Xenopus tadpole models). Zebrafish, with their small size, high egg production, rapid development, transparent embryos, and ease of rearing, possess both high cell throughput and high comprehensiveness as whole animals, making them a crucial link between cell and mammalian models in international drug screening and evaluation systems. Furthermore, transgenic zebrafish labeled with green vascular fluorescence allow for a more direct and rapid observation of the angiogenesis process in live embryos. Therefore, the zebrafish model is a superior model for evaluating angiogenesis activity.

[0149] To evaluate the pro-angiogenic activity of colchicine derivatives, transgenic zebrafish (CZ62 strain) with vascular fluorescence were used as experimental animals. Embryos that had developed normally to 24 hpf (hour post-fertilization) were demembranes treated with 1 mg / mL streptomycin E. The zebrafish were randomly divided into groups (10 fish per well) in 24-well plates. Except for the blank control group, the model group and the treatment group were treated with the modeling agent PTK787 at a final concentration of 0.25 μg / mL. The treatment groups were also given samples at concentrations of 12.5, 25, and 50 μM, respectively. The embryos were incubated at 28±5℃ under constant temperature and light to allow further development. At 48 hpf, changes in the zebrafish were observed under a fluorescence inverted microscope. The length of intersegmental vessels (ISVs) in the zebrafish was counted and statistically analyzed. Among them, zebrafish were provided by the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences. A zebrafish angiogenesis injury model was constructed using Vatalanib (PTK787). Based on this, the pro-angiogenic activities of compounds CH, CY, CN, CBL, CSL, DN, and DT were evaluated.

[0150] Figure 16 The images show the fluorescence of zebrafish with different amounts of colchicine derivatives prepared in Examples 6 and 9-14 of this invention; wherein, Example 6 is CH, Example 13 is CBL, Example 12 is CY, Example 11 is CN, Example 9 is DN, Example 10 is DT, and Example 14 is CSL. Figure 16 The red line represents an intersegmental vessel. Control is the blank control group, PTK787 is the model group, and the rest are different drug administration groups. The concentration unit is μM. In Example 14, zebrafish in all concentration groups of compound CSL died, and zebrafish in the 50 μM CY drug administration group were deformed.

[0151] Figure 17 The bar chart shows the statistical results of ISVs (Intersegmental vessels) of zebrafish after treatment with the colchicine derivatives prepared in Examples 6 and 9-13 of this invention; wherein, CH is used in Example 6, CBL in Example 13, CY in Example 12, CN in Example 11, DN in Example 9, and DT in Example 10.

[0152] from Figure 16 , 17 It can be seen that DN and DT exhibited the best angiogenic activity, with significant activity at all administered concentrations. Compound CH showed angiogenic activity at 25 and 50 μM, while compound CN showed angiogenic activity at 25 μM. Among these, compounds CSL at all concentrations and CY at 50 μM were highly toxic to zebrafish.

[0153] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A colchicine derivative, characterized in that, It has the structure shown in Formula I; Wherein, R1 is H or methyl; R2 is -C(=O)-R3; The colchicine derivatives are selected from the following compounds: 。 2. A method for preparing the colchicine derivative according to claim 1, characterized in that, Includes the following steps: S1, when R1 in compound I is methyl, colchicine undergoes a substitution reaction with N-Boc-ethylenediamine to synthesize 10C-NB; S2,10C-NB reacts with trifluoroacetic acid to remove the protecting group, synthesizing 10C-N; S3,10C-N reacts with the corresponding compound containing a carboxylic acid group to undergo an amidation reaction, synthesizing compound I; The structures of 10C-NB and 10C-N are shown below: 。 3. The preparation method according to claim 2, characterized in that, In S1, the substitution reaction conditions are: the molar ratio of colchicine to Boc-ethylenediamine is 1:(1~3), and the reaction is carried out under reflux at 80~85℃ overnight; In S2, the reaction condition for removing the protecting group is room temperature.

4. The preparation method according to claim 2, characterized in that, In S3, the amidation reaction is carried out by mixing the corresponding compound containing a carboxylic acid group, the catalyst, and the condensing agent in a solvent under ice bath conditions to form a mixed solution; at room temperature, intermediate 10C-N is added to the mixed solution and the reaction is carried out overnight. The molar ratio of intermediate 10C-N, the corresponding compound containing a carboxylic acid group, the catalyst, and the condensing agent is 1:(3~5):(2~10):(2~4). The solvent is DMF or DCM; The catalyst is DIPEA or DMAP; The condensing agent is HATU or EDCI.

5. A method for preparing the colchicine derivative according to claim 1, characterized in that, Includes the following steps: S1, when R1 in compound I is H, colchicine is dissolved in concentrated sulfuric acid and reacted in an oil bath in glacial acetic acid to remove the methoxy group at the 2-position of colchicine, thus synthesizing DE-1; DE-1 undergoes a substitution reaction with N-Boc-ethylenediamine to synthesize 10D-NB. S2, 10D-NB reacts with trifluoroacetic acid to remove the protecting group, synthesizing 10D-N; S3,10D-N reacts with the corresponding compound containing a carboxylic acid group via an amidation reaction to synthesize compound I; The structure of DE-1 is shown below: ； The structure of the 10D-NB is shown below: ； The structure of 10D-N is shown below: 。 6. A pharmaceutical composition, characterized in that, Contains the colchicine derivative of claim 1 or a pharmaceutically acceptable salt thereof.

7. The use of a colchicine derivative of claim 1 or a pharmaceutical composition of claim 6 in the preparation of anti-inflammatory drugs and / or angiogenic active drugs.

8. The application as described in claim 7, characterized in that, The anti-inflammatory drug is used to treat gouty arthritis, acute pericarditis, or chronic inflammation of the cardiovascular and cerebrovascular systems.

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