Colchicine derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to colchicine derivatives as well as a preparation method and application thereof. The colchicine derivative has a structure as shown in a formula I: # imgabs0 #, wherein R1 is H or methyl; r2 is H or-C (= O)-R3, R3 is selected from a non-steroidal anti-inflammatory drug or a phenylpropanoid small molecule group, and R3 is selected from the following compounds: # imgabs No.1. The colchicine derivative provided by the invention keeps anti-inflammatory activity while reducing the cytotoxicity of colchicine, and shows a dose-dependent anti-inflammatory effect on a zebra fish model.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to colchicine derivatives and preparation methods and applications thereof. Background Art

[0002] Colchicine is a classic anti-inflammatory drug that has the effect of inhibiting inflammatory storms. It is widely used in the treatment of gouty arthritis in clinical practice, and is also used as a second-line drug for the treatment of acute pericarditis. Clinical trials have shown that colchicine can significantly reduce the incidence of chronic inflammatory diseases and cardiovascular and cerebrovascular diseases. However, colchicine's anti-inflammatory activity coexists with its strong cytotoxicity. The toxicity of colchicine limits its clinical application.

[0003] Although a large number of scientific studies have shown that the main reason for colchicine to cause cytotoxicity is that colchicine binds to tubulin to affect microtubule function, inhibit microtubule polymerization, and block the mitotic process. In Li Yuzhu's master's thesis on the design, synthesis, and antiarrhythmic activity of colchicine derivatives, although the 10th position of colchicine was modified to reduce cytotoxicity to varying degrees, no research on anti-inflammatory activity was conducted. The Chinese invention patent with publication number CN 113024400 A modifies the 4th and 7th positions of colchicine, and the cytotoxicity of colchicine is increased. The existing optimization scheme of the structure of colchicine is difficult to achieve while reducing the cytotoxicity of colchicine and maintaining anti-inflammatory activity.

[0004] In addition, angiogenesis refers to the process of sprouting new vascular systems based on primitive vascular plexuses or existing blood vessels. It is an essential 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. When angiogenesis is insufficient, it can cause myocardial ischemia, cerebral ischemia, slow healing of trauma and fractures, occlusive vasculitis, diabetic peripheral vascular disease and other diseases. Finding therapeutic drugs that promote angiogenesis is an important strategy for treating these ischemic diseases, which is also a hot topic in clinical research today. There are few studies in the prior art involving the angiogenic activity of colchicine and its derivatives. Summary of the invention

[0005] The purpose of the present invention is to provide colchicine derivatives and their preparation methods and applications. Combining the structure-activity relationship of colchicine, with the orientation of enhancing efficacy and reducing toxicity, molecular design is carried out on the 2nd and 10th positions of colchicine, and a strategy of increasing the steric hindrance of the binding of colchicine derivatives to microtubules is adopted. This strategy not only significantly reduces the toxicity to cardiomyocytes and macrophages, but also some compounds maintain good anti-inflammatory activity.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] In the first aspect, embodiments of the present invention provide colchicine derivatives, characterized in that they have a 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 non-steroidal 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] Phenylpropanoid compounds

[0014]

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

[0016]

[0017] In a second aspect, an embodiment of the present invention provides a method for preparing the colchicine derivative described in the first aspect, comprising the following steps:

[0018] S1, colchicine and N-Boc-ethylenediamine undergo substitution reaction to synthesize 10C-NB;

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

[0020] S3, 10C-N and the group containing carboxyl on the aromatic ring undergo amidation reaction to synthesize the compound of formula I;

[0021] The structures of 10C-NB and 10C-N are as follows:

[0022]

[0023] In some embodiments, in S1, the conditions for the substitution reaction are that 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 the volume ratio of dichloromethane to trifluoroacetic acid solvent is (1-3):1.

[0025] In some embodiments, in S3, the amidation reaction is: in an ice bath, the group containing a carboxyl group on the aromatic ring, the catalyst, and the condensation agent are mixed in a solvent to form a mixed solution; at room temperature, the intermediate 10C-N is added to the mixed solution and reacted overnight;

[0026] The molar ratio of the intermediate 10C-N, the group containing a carboxyl group on the aromatic ring, the catalyst, and the condensation 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 the compound of formula I is H, colchicine and concentrated sulfuric acid are dissolved in glacial acetic acid in an oil bath for reaction, and the methoxy group at the 2-position of colchicine is removed to synthesize DE-1;

[0031] Among them, the structure of DE-1 is as follows:

[0032]

[0033] In a third aspect, an embodiment of the present invention provides a pharmaceutical composition comprising the colchicine derivative described in the first aspect or a pharmaceutically acceptable salt, solvate or a prodrug molecule or metabolite thereof.

[0034] In a fourth aspect, embodiments of the present invention provide use of the colchicine derivative described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of anti-inflammatory drugs and / or pro-angiogenic drugs.

[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] The present invention combines the structure-activity relationship of colchicine, takes increasing efficacy and reducing toxicity as the guide, conducts molecular design at positions 2 and 10 of colchicine, and adopts a strategy to increase the steric hindrance of the binding of colchicine derivatives to tubulin. This strategy not only significantly reduces the toxicity to cardiomyocytes and macrophages, but also some compounds still maintain good anti-inflammatory activity and pro-angiogenic activity.

[0038] The method for synthesizing colchicine derivatives of the invention has simple operation, readily available raw materials, strong practicability, high yield and low cost, and is of great significance for the industrial production of such substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0040] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of 10DN-T prepared in Example 3 of the present invention;

[0041] Figure 2 This is the carbon nuclear magnetic resonance spectrum of 10DN-T prepared in Example 3 of the present 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 This is the hydrogen nuclear magnetic resonance spectrum of 10CN-BLF prepared in Example 13 of the present invention;

[0044] Figure 5 This is the carbon nuclear magnetic resonance spectrum of 10CN-BLF prepared in Example 13 of the present 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 This is the hydrogen nuclear magnetic resonance spectrum of 10CN-SLFS prepared in Example 14 of the present invention;

[0047] Figure 8 This is the carbon nuclear magnetic resonance spectrum of 10CN-SLFS prepared in Example 14 of the present invention;

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

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

[0050] Fig.11 This is a graph showing that the colchicine derivatives prepared in Examples 6 and 9-14 of the present invention reduce the NO content in the cell supernatant caused by LPS stimulation;

[0051] Fig.12 Effects of colchicine derivatives prepared in Example 6 and Examples 9-14 of the present invention on zebrafish inflammatory cells;

[0052] Fig.13This is a graph showing the anti-inflammatory activity results of colchicine derivatives prepared in Examples 6 and 9-14 of the present invention when the addition amount is 50 μM;

[0053] Fig.14 The effects of different concentrations of Example 10, Example 13 and Example 14 of the present invention on zebrafish inflammatory cells;

[0054] Fig.15 The anti-inflammatory activity results at different concentrations of Example 10, Example 13 and Example 14 of the present invention are shown;

[0055] Fig.16 Fluorescence photos of zebrafish at different addition amounts of colchicine derivatives prepared in Examples 6 and 9-14 of the present invention;

[0056] Fig.17 This is a bar graph showing the statistics of ISVs (Intersegmental vessel length) of zebrafish after the zebrafish were treated with the colchicine derivatives of Examples 6 and Examples 9-13 of the present invention. DETAILED DESCRIPTION

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] The raw materials and reagents used in the following examples are all conventional commercially available products and can be purchased. Among them, some of the equipment used are as follows: IX73 Olympus inverted microscope, SZX16 Olympus microscope and DP2-BSW image acquisition system (Olympus, Japan); Forma 3111 water-jacketed CO2 incubator (Forma, USA); zebrafish breeding equipment (Beijing Aisheng Technology Co., Ltd.).

[0059] Example 1

[0060] Preparation of compound 10C-N

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

[0062]

[0063] Take 50mg (0.125mmol) of colchicine and dissolve it in 3mL acetonitrile, stir to dissolve it, add 60mg (0.375mmol) of Boc-ethylenediamine, place it at 82℃ for reflux reaction, react overnight, monitor the reaction by TLC, evaporate acetonitrile after the reaction, add 2mL DCM to dissolve it, stir at room temperature, add 1mL TFA dropwise, react for 2h, monitor the reaction by TLC, and terminate the reaction when the reaction point disappears. Evaporate the solvent under reduced pressure, add water, extract three times with water: DCM = 1: 1, discard the organic phase, add saturated sodium bicarbonate until the solution is weakly alkaline, extract three times with water: DCM = 1: 1, dry with anhydrous Na2SO4, separate by column chromatography, and obtain 34mg of yellow solid with a yield of 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 of compound 10D-N is as follows:

[0068]

[0069] Take 400 mg (1 mmol) of colchicine and dissolve it in 0.6 mL of glacial acetic acid, add 2 mL of concentrated sulfuric acid, stir at 60 ° C for 5 h, and monitor the reaction by TLC (DCM: MeOH 10: 1). After completion, cool to room temperature, adjust pH 5-6 with NaOH, add water, extract with DCM 3 times, dry with anhydrous Na2SO4, and separate by column chromatography to obtain DE-1. Take 50 mg (0.13 mmol) of DE-1 and dissolve it in 3 mL of acetonitrile, stir to dissolve it, add 62.5 mg (0.39 mmol) of Boc-ethylenediamine, place the device at 82 ° C for reflux reaction, react overnight, and monitor the reaction by TLC. After the reaction is completed, evaporate the acetonitrile, add 2 mL of DCM to dissolve it, stir at room temperature, add 1 mL of TFA dropwise, react for 2 h, and monitor the reaction by TLC. The post-treatment is the same as 10C-N in Example 1, and 27 mg of dark yellow powder is obtained, 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 of compound 10DN-T is as follows:

[0074]

[0075] The preparation method of 10D-N is the same as that in Example 2, except that 24.6 mg (0.06 mmol) of 10D-N was weighed and dissolved in 2 mL of anhydrous dichloromethane, stirred under an ice bath, 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, and the DCM solution of 3,4,5-trimethoxybenzoyl chloride was added dropwise to the reaction flask. The reaction was allowed to react overnight and monitored by TLC. When the product point no longer increased, water was added to terminate the reaction, and the mixture was extracted three times with dichloromethane: water = 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 of compound 10CN-R is as follows:

[0080]

[0081] 56.8 mg (0.35 mmol) of 2-OH-cinnamic acid was weighed and dissolved in 2 mL of DMF. 75 mg (0.58 mmol) of DIPEA and 87.8 mg (0.23 mmol) of HATU were weighed and stirred for 10-15 min in an ice bath. 49.3 mg (0.12 mmol) of 10C-N prepared in Example 1 was added and reacted overnight. The progress of the reaction was monitored by TLC. After the reaction was completed, water was added to terminate the reaction. The reaction was extracted three times with ethyl acetate, and the organic phases were combined, back-extracted with saturated brine, dried over anhydrous magnesium sulfate, and separated by column chromatography to obtain 26 mg of a 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 the preparation method of 10CN-R in Example 4, except that the phenylpropanoid small molecule is ferulic acid. The specific synthesis route is as follows, and 23.3 mg of yellow solid powder is obtained with a yield of 32.3%.

[0086]

[0087] 10CN-AWS spectrum data: 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 the preparation method of 10CN-R in Example 4, except that the phenylpropanoid small molecule is dihydrocaffeic acid. The specific synthesis route is as follows, and 22.5 mg of light yellow solid powder is obtained 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, except that the raw material 10D-N and the phenylpropanoid small molecule are 3-(2-hydroxyphenyl)-propionic acid. The specific synthesis route is as follows, and 23.2 mg of yellow solid powder is obtained with a yield of 67.7%.

[0096]

[0097] Spectral data of 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, except that the raw material 10D-N and the phenylpropanoid small molecule are 2-OH-cinnamic acid. The specific synthesis route is as follows, and 12 mg of yellow solid powder is obtained 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, except that the raw material 10D-N is different from the carboxylic acid small molecule naproxen. The specific synthesis route is as follows, and 6.1 mg of yellow solid powder is obtained with a yield of 18%.

[0106]

[0107] Spectrum 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, except that the raw material 10D-N is different from the carboxylic acid small molecule indomethacin. The specific synthesis route is as follows, and 21 mg of yellow solid powder is obtained 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] Embodiment 11

[0114] Preparation of compound 10CN-NPS

[0115] The preparation method of compound 10CN-NPS, the synthetic route is as follows:

[0116]

[0117] 37 mg (0.087 mmol) of 10C-N prepared in Example 1 was dissolved in 3 mL of dichloromethane, and 21.2 mg (0.17 mmol) of DMAP and 66.5 mg (0.35 mmol) of EDCI were added under an ice bath. After sufficient dissolution, 100 mg (0.43 mmol) of naproxen was added, and the mixture was reacted overnight. The reaction was monitored by TLC. After the reaction was completed, the solvent was evaporated, the mixture was dissolved in water, and extracted with dichloromethane: water = 1:1. The extraction was performed three times, and 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, except that the raw material non-steroidal anti-inflammatory drug indomethacin is different. The specific synthesis route is as follows, and 33.2 mg of yellow crystalline powder is obtained 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] Embodiment 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, except that the raw material non-steroidal anti-inflammatory drug ibuprofen is used. The specific synthesis route is as follows, and 21 mg of yellow solid powder is obtained 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] Embodiment 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, except that the raw material non-steroidal anti-inflammatory drugs are different. The specific synthesis route is as follows, and 31 mg of yellow crystalline solid powder is obtained 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] Embodiment 15:

[0135] Evaluation of cytotoxicity of colchicine derivatives

[0136] In order to verify whether colchicine derivatives reduce cytotoxicity, this example uses MTT colorimetry to determine the inhibition rate of mouse macrophage leukemia cells RAW264.7 and rat cardiomyocytes H9C2, and sets up DMSO and colchicine raw material drug groups for control experiments.

[0137] Cell culture: Rat cardiomyocytes H9C2 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), and mouse macrophage leukemia cells RAW264.7 were cultured in a DMEM medium specifically for RAW264.7 at 37° C. in an incubator with 5% CO 2 .

[0138] MTT colorimetric method: H9C2 cells were plated at 8×10 4 / mL density was inoculated in a 96-well plate, 100μL was added to each well, and incubated overnight in a 5% CO2, 37°C incubator until the cells adhered to the wall and covered the bottom of the well, and the drug was added to the well to a concentration of 10μM. The drug was gently shaken evenly to mix the drug and the culture medium. A blank control and 3 replicate wells were set up for each concentration. After incubation in the incubator for 72h, 10μL of 0.5mg / mL MTT was added to each well. After continuing to incubate for 4h, all the culture medium in the well plate was removed. 100μL of dimethyl sulfoxide was added to each well to dissolve the water-insoluble blue-purple crystalline formazan, and detected at a wavelength of 570nm on an enzyme reader. The experimental method for the inhibition rate of colchicine derivatives on RAW264.7 is the same as that for H9C2, except that the cell density is 1×10 5 pcs / mL. See Table 1 for specific data.

[0139] Table 1 Inhibitory effect 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 different degrees of reduced toxicity. It can be seen from the table that the cell inhibition rate of colchicine on RAW264.7 was 93.9% at 10 μM, and the cell inhibition rate on H9C2 was 34.5%, while the derivatives significantly reduced the cell inhibition rate.

[0142] Embodiment 16:

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

[0144] In order to explore whether colchicine derivatives still have anti-inflammatory activity while reducing cytotoxicity, this example uses LPS stimulation to establish a macrophage inflammation model, and uses CuSO4 to treat healthy inflammatory cells fluorescently labeled transgenic zebrafish to establish a zebrafish inflammation model to study the anti-inflammatory activity of colchicine derivatives. The nitric oxide (NO) content in the cell supernatant is often used as a biomarker of the degree of inflammation. NO is a small molecule free radical, which is catalyzed by nitric oxide synthase (iNOS) in the body. It plays an important role in various biological processes, especially in the function and inflammatory response of the immune system. In the inflammatory process, immune cells such as macrophages and neutrophils will be activated, and the production of NO will be increased by inducible nitric oxide synthase (iNOS). The production of NO is often positively correlated with the activity of inflammation. Therefore, measuring the content of NO in the cell supernatant can provide a direct indicator of the inflammatory state.

[0145] The Griess method was used to detect the NO content in the cell supernatant. The NaNO2 concentration (0, 6.58, 13.17, 19.75, 29.63, 44.44, 66.67, 100 μM) was used as the X-axis, and the corresponding OD value at a wavelength of 540 nm on the microplate reader was used as the Y-axis to make a standard curve. Fig.10 As shown, the standard curve is y = 0.0042x + 0.0587, R 2 =0.9943, with a linear range of 0-100 μM. The low-toxic compounds in Example 15 were further screened for anti-inflammatory activity. After the cells were pre-protected with drugs for 1 hour, LPS was added for 24 hours, and the cell supernatant was aspirated. The measured OD value was substituted into the standard curve, and the NO content in the cell supernatant was calculated. The measured concentration was normalized with the LPS stimulation group. The results are shown in Fig.11 It can be seen that compared with the LPS group, the NO content in the cell supernatant of the drug-added group was reduced to varying degrees. Taking the toxicity and anti-inflammatory activity of colchicine into consideration, the reduction of NO content at 0.37 μM colchicine 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 is 10CN-HKFS (abbreviated as CH), Example 13 is 10CN-BLF (abbreviated as CBL), Example 12 is 10CN-Y (abbreviated as CY), Example 11 is 10CN-NPS (abbreviated as CN), Example 9 is 10DN-NPS (abbreviated as DN), and Example 10 is 10DN-T (abbreviated as DT); it can significantly reduce the NO content in the cell supernatant caused by LPS stimulation.

[0146] In order to evaluate the anti-inflammatory activity of colchicine derivatives, transgenic zebrafish (Tg:zlyz-EGFP) with fluorescently labeled healthy inflammatory cells at 72 hpf (hour postfertilization) were used as experimental animals. Seven compound (CH, CY, CN, CBL, CSL, DN, DT) samples (50 μM) and positive control colchicine (20, 50 μM) were administered in 24-well plates and placed in a light incubator (28°C) to allow the embryos to continue to develop. After 2 hours, all the above groups of zebrafish except the blank control group were 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 line was calculated and statistically analyzed. The experimental results are shown in Fig.12 and Fig.13Compared with the blank control group, the number of zebrafish inflammatory cell migration in the model group increased significantly, indicating that the inflammation model was successfully established; compared with the model group, the number of zebrafish inflammatory cell migration in the positive control group and the 7 sample groups decreased significantly, which was statistically significant, indicating that they all had significant anti-inflammatory activity. Among them, the anti-inflammatory activity of the CSL, CBL, and DT groups was stronger. 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 inflammatory cell fluorescent transgenic zebrafish (Tg:zlyz-EGFP) at 72 hpf (hour post fertilization) were used as experimental animals. Three compound samples CSL, CBL, DT (12.5, 25, 50 μM) and the positive control colchicine (Colchicine) (12.5, 25, 50 μM) were respectively administered in a 24-well plate. After adding culture water to 2.0 mL, the plates were placed in a light incubator (28°C) to allow the embryos to continue to develop. After 2 hours, the zebrafish in each of the above-mentioned treatment groups 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 line was calculated and statistically analyzed. The results are shown in Fig.14 and Fig.15 Compared with the blank control group, the number of inflammatory cell migration in the model group increased significantly, indicating that the inflammation model was successfully established; compared with the model group, the positive control group had statistical differences. As the concentration of the sample treatment group increased, the number of inflammatory cell migration decreased, indicating that the compound samples CBL, CSL, and DT could exert their anti-inflammatory effects in a dose-dependent manner.

[0148] The discovery of angiogenic drugs requires the help of angiogenesis models. Common angiogenesis models include in vitro models (endothelial cell model and rat artery ring model), in vivo models (corneal microcapsules, chicken embryo chorioallantoic membrane, disc angiogenesis model and sponge-matrigel model, etc.), and whole animal models (zebrafish model and clawed frog tadpole model). Zebrafish has many characteristics such as small size, high egg laying, fast development, transparent embryos, and easy breeding. It has high throughput of cells and high content of whole animals. It has become an important link in the drug screening and evaluation system in the world to connect cell and mammalian models. Transgenic zebrafish marked with green vascular fluorescence make it more intuitive and quick to observe the process of sample action on angiogenesis in living embryos. Therefore, the zebrafish model is an advantageous model for evaluating angiogenic activity.

[0149] In order to evaluate the angiogenic activity of colchicine derivatives, vascular fluorescent transgenic zebrafish (CZ62 strain) were used as experimental animals. Embryos that developed normally to 24 hpf (hour post fertilization) were demembraned with 1 mg / mL pronase E, and zebrafish were randomly grouped in 24-well plates (10 fish per well plate). In addition to the blank control group, the model group and the drug group were added with a final concentration of 0.25 μg / mL of modeling drug PTK787, and the drug group was given 12.5, 25 and 50 μM concentration samples, respectively, and placed in a 28±5℃ constant temperature and light incubator to allow embryos to continue to develop. At 48 hpf, the zebrafish were placed under a fluorescent inverted microscope to observe changes. The length of the intersegmental vessels (ISV) of the zebrafish was counted and statistically analyzed. Among them, zebrafish was provided by the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences. Vatalanib (PTK787) was used to construct a zebrafish angiogenesis injury model. On this basis, the angiogenesis-promoting activity of compounds CH, CY, CN, CBL, CSL, DN, and DT was evaluated.

[0150] Fig.16 These are zebrafish fluorescence photographs at different addition amounts of colchicine derivatives prepared in Examples 6 and 9-14 of the present 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. Fig.16 The middle red line is an intersegmental blood vessel, Control is the blank control group, PTK787 is the model group, and the rest are different dosing groups. The concentration unit is μM. The zebrafish in each concentration group of the compound CSL prepared in Example 14 died, and the zebrafish in the 50 μM CY dosing group were deformed.

[0151] Fig.17 This is a bar graph showing the statistics of ISVs (Intersegmental vessel) of zebrafish after the colchicine derivatives prepared in Examples 6 and 9-13 of the present invention were treated. Among them, Example 6 is CH, Example 13 is CBL, Example 12 is CY, Example 11 is CN, Example 9 is DN, and Example 10 is DT.

[0152] from Fig.16 , 17 It can be seen that DN and DT have the best angiogenic activity, and the activity is obvious at all administration concentrations; compound CH has angiogenic activity at 25 and 50 μM, and compound CN has angiogenic activity at 25 μM. Among them, compound CSL at all concentrations and 50 μM CY are more toxic to zebrafish.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A colchicine derivative, characterized in that Having the structure shown in formula I; Wherein, R1 is H or methyl; R2 is H or -C(=O)-R3, and R3 is selected from non-steroidal anti-inflammatory drugs or phenylpropanoid small molecule groups.

2. The colchicine derivative according to claim 1, characterized in that R3 is selected from the following compounds: Nonsteroidal anti-inflammatory drugs Phenylpropanoid compounds 3. The colchicine derivative according to claim 1, characterized in that The colchicine derivative is selected from the following compounds:

4. A method for preparing the colchicine derivative according to claim 1, characterized in that: The following steps are involved: S1, colchicine and N-Boc-ethylenediamine undergo substitution reaction to synthesize 10C-NB; S2, 10C-NB reacts with trifluoroacetic acid to remove the protecting group and synthesize 10C-N; S3, 10C-N and the group containing carboxyl on the aromatic ring undergo amidation reaction to synthesize the compound of formula I; The structures of 10C-NB and 10C-N are as follows:

5. The preparation method according to claim 4, characterized in that: In S1, the conditions for the substitution reaction are: the molar ratio of colchicine to Boc-ethylenediamine is 1:(1-3), and the reaction is refluxed at 80-85°C overnight; In S2, the reaction conditions for removing the protecting group are room temperature and a volume ratio of dichloromethane to trifluoroacetic acid of (1-3):

1.

6. The preparation method according to claim 4, characterized in that: In S3, the amidation reaction is to mix the carboxyl group on the aromatic ring, the catalyst, and the condensation agent in a solvent in an ice bath to form a mixed solution; and add the intermediate 10C-N to the mixed solution at room temperature to react overnight; The molar ratio of the intermediate 10C-N, the group containing a carboxyl group on the aromatic ring, the catalyst, and the condensation 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.

7. The preparation method according to claim 4, characterized in that: When R1 in the compound of formula I is H, colchicine and concentrated sulfuric acid are dissolved in glacial acetic acid in an oil bath for reaction, and the methoxy group at the 2-position of colchicine is removed to synthesize DE-1; Among them, the structure of DE-1 is as follows:

8. A pharmaceutical composition, characterized in that Contains the colchicine derivative according to claim 1 or its pharmaceutically acceptable salt, solvate or prodrug molecule or metabolite.

9. Use of the colchicine derivative according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of anti-inflammatory drugs and / or pro-angiogenic drugs.

10. The use according to claim 9, characterized in that The anti-inflammatory drug is a drug for treating gouty arthritis, acute pericarditis or chronic cardiovascular and cerebrovascular inflammation.

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