Colchicine binding site inhibitor prodrug, nano-drug as well as preparation and application of colchicine binding site inhibitor prodrug and nano-drug

Abstract

The invention belongs to the technical field of medical compounds, and relates to preparation of a colchicine binding site inhibitor prodrug and nano-drug with ROS response and application of the colchicine binding site inhibitor prodrug and nano-drug in anti-tumor and anti-inflammatory drugs. The prodrug compound is obtained by coupling a colchicine binding site inhibitor (CBSI) and biotin through a thioketal chain responded by ROS (reactive oxygen species). According to the invention, CBSIs and biotin are connected through ROS-responsive thioketal to synthesize a CBSIs prodrug, and then the CBSIs prodrug is self-assembled through a nano precipitation method to prepare the nano drug of the CBSIs prodrug. The novel nano-drug can obviously improve the water solubility of the CBSIs prodrug. When being applied, the compound can target tumor or inflammation parts and is split by high-concentration ROS in tumor or inflammation tissues, so that CBSIs are released to exert pharmacological activity, and meanwhile, the influence on normal tissues is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical compounds, and relates to a ROS-responsive prodrug of a colchicine binding site inhibitor, the preparation of a nano-drug, and its application in anti-tumor and anti-inflammatory drugs. Background Art

[0002] Malignant tumors seriously threaten human health and are the second leading cause of death after cardiovascular diseases. Currently, commonly used clinical chemotherapy drugs have problems such as poor tolerance, many side effects, and high toxicity. At the same time, cytokine storms caused by the infection of novel coronavirus (2019-nCoV) and others pose a threat to human life, and the existing drug treatment effects are not good. Therefore, it is still crucial to seek more efficient and safer anti-tumor and anti-inflammatory drugs.

[0003] Microtubules are polymerized from heterodimers of α- and β-tubulin and are important components of the eukaryotic cytoskeleton, with important functions such as maintaining cell morphology, participating in cell division, assisting in material transport, and signal transduction. Microtubules are one of the effective targets for anti-tumor and anti-inflammatory drug research. The colchicine site is located in a deep pocket of the β-tubulin subunit between α,β-tubulin heterodimers. Colchicine binding site inhibitors (CBSIs) are a class of microtubule destabilizers that act on the colchicine binding site of tubulin. These compounds can effectively prevent the formation of functional microtubules and further hinder the process of cell mitosis by inhibiting the polymerization of tubulin. CBSIs have the characteristics of small molecular weight, simple structure, outstanding activity, and many modifiable sites. However, CBSIs lack sufficient targeting to tumor cells and will cause serious toxic side effects. Therefore, solving the targeting problem of CBSIs is the key to promoting the research of CBSIs. Summary of the Invention

[0004] The purpose of the present invention is to provide a ROS-responsive prodrug of a colchicine binding site inhibitor, the preparation of a nano-drug, and its application in anti-tumor and anti-inflammatory drugs.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A prodrug compound of a colchicine binding site inhibitor, wherein the prodrug compound is obtained by coupling a colchicine binding site inhibitor (CBSI) and biotin with a ROS-responsive thioacetal chain.

[0007] Preferably, the prodrug compound is the compound shown in Formula 1, and its salts or hydrates.

[0008]

[0009] In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 can be the same or different and are independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, halogen atom, nitro, hydroxyl or amino;

[0010] R 6 is hydrogen, C1-C6 alkyl;

[0011] n and m are each independently 1 or 2;

[0012] R 7 , R 8 can be the same or different and are independently hydrogen, C1-C6 alkyl, phenyl which is unsubstituted or substituted by at least one halogen or C1-C3 alkyl, and, R 7 , R 8 are not both hydrogen at the same time;

[0013] or, R 7 , R 8 forms a five-membered, six-membered, seven-membered or eight-membered saturated carbon ring with the adjacent C.

[0014] More preferably, the prodrug compound is the compound represented by Formula 1, and its salt or hydrate,

[0015] In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 can be the same or different and are independently hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen atom, nitro, hydroxyl or amino;

[0016] R 6 is hydrogen, C1-C3 alkyl;

[0017] n and m are each independently 1 or 2;

[0018] R 7 , R 8 can be the same or different and are independently hydrogen, C1-C3 alkyl, phenyl which is unsubstituted or substituted by at least one halogen or C1-C3 alkyl, and, R 7 , R 8 are not both hydrogen at the same time;

[0019] or, R 7 , R 8 forms a five-membered, six-membered or seven-membered saturated carbon ring with the adjacent C.

[0020] More preferably, the prodrug compound is a compound represented by Formula 1, and its salt or hydrate.

[0021] In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 may be the same or different and are independently hydrogen, methoxy, ethoxy, amino, hydroxy, fluorine, chlorine or bromine;

[0022] R 6 is hydrogen, methyl, ethyl;

[0023] n and m are each independently 1 or 2;

[0024] R 7 , R 8 may be the same or different and are independently hydrogen, a C1-C3 alkyl group, a phenyl group which is unsubstituted or substituted by at least one halogen or a C1-C3 alkyl group, and R 7 , R 8 are not both hydrogen at the same time;

[0025] Or, R 7 , R 8 form a five-membered, six-membered or seven-membered saturated carbon ring with the adjacent C.

[0026] More preferably, the compound

[0027] Compound 1

[0028]

[0029] Compound 2

[0030]

[0031] Compound 3

[0032]

[0033] Compound 4

[0034]

[0035] Compound 5

[0036]

[0037] Compound 6

[0038]

[0039] Compound 7

[0040]

[0041] Compound 8

[0042]

[0043] The salt of the compound shown in Formula 1 is the salt formed by the compound shown in Formula 1 and an acid or a base; wherein, the acid is hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, malic acid; the base is sodium hydroxide, sodium carbonate, potassium hydroxide;

[0044] The hydrate of the compound shown in Formula 1, and the number of crystal water molecules of the hydrate is any real number from 0 to 16.

[0045] A preparation method of the prodrug compound of the colchicine binding site inhibitor described above,

[0046]

[0047] Specifically:

[0048] Step 1: Mix a carbonyl compound (I) and mercaptoacetic acid in a molar ratio of 1:2 to 4, and drop trifluoroacetic acid into the mixed solution under stirring, and react at a temperature of 15 to 40 °C for 0.5 to 12 h to obtain Compound II;

[0049] Step 2: Dissolve Compound II, sodium borohydride and iodine in a molar ratio of 1:1 to 6:2 to 6 in tetrahydrofuran at a temperature of -20 to 0 °C, and reflux for 12 to 24 h to obtain Compound III;

[0050] Step 3: Under nitrogen protection, dissolve Compound III and triethylamine in anhydrous tetrahydrofuran, and add a tetrahydrofuran solution of p-nitrophenyl chloroformate to the mixed solution of Compound III and triethylamine, and react at a temperature of 0 to 30 °C for 2 to 6 h to obtain Compound VI;

[0051] Among them, the molar ratio of Compound III, triethylamine and p-nitrophenyl chloroformate is 1:1 to 1.2:1 to 1.2;

[0052] Step 4, under nitrogen protection, react Compound VI with Compound V dissolved in tetrahydrofuran at 0 to 30 °C for 12 to 24 h to obtain Compound IV;

[0053] Among them, the molar ratio of Compound VI to Compound V is 1:1 to 2;

[0054] Step 5: Dissolve Compound VI, biotin, DMAP, and DCC in a molar ratio of 1:1 to 2:1 to 4:1 to 4 in DMF, and react at 15 to 45 °C to obtain the compound of Formula 1.

[0055] A nano-drug, which is prepared from the prodrug compound by the nanoprecipitation method.

[0056] The particle size of the nano-drug is about 130 nm.

[0057] A method for preparing a nano-drug, wherein the prodrug compound, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and egg yolk lecithin are dissolved in an organic solvent to obtain a mixed solution, and then the mixture solution is added to deionized water under stirring conditions. After continuing the stirring reaction, the organic solvent is removed, and the prodrug compound nano-drug is self-assembled.

[0058] The mass ratio of the prodrug compound to DSPE-PEG2000 is 1:0.1 - 1, and the mass ratio of the prodrug compound to egg yolk lecithin is 1:0.1 - 1.

[0059] The organic solvent is one or a mixture of ethanol, acetone, DMSO and acetonitrile.

[0060] An application of the prodrug compound and the nano-drug, the application of the prodrug compound and the nano-drug in the preparation of anti-tumor drugs or anti-inflammatory drugs.

[0061] Advantages of the present invention:

[0062] In the present invention, CBSIs and biotin are connected by a ROS-responsive thioacetal to synthesize a CBSIs prodrug, and then the CBSIs prodrug is self-assembled into a nano-drug of the CBSIs prodrug by the nanoprecipitation method. This novel nano-drug can significantly improve the water solubility of the CBSIs prodrug. When applied, it can target tumor or inflammation sites and be cleaved by high concentrations of ROS in tumor or inflammatory tissues, thereby releasing CBSIs to exert pharmacological activity while reducing the impact on normal tissues. Description of the drawings

[0063] Figure 1 is the 1 H-NMR and 13 C-NMR spectrum of Compound 1 in Example 1 of the present invention;

[0064] Figure 2 is the 1 H-NMR and 13 C-NMR spectrum of Compound 2 in Example 2 of the present invention;

[0065] Figure 3 is the 1 H-NMR and 13 C-NMR spectrum of Compound 3 in Example 3 of the present invention;

[0066] Figure 4 The 1 H-NMR and 13 C-NMR spectra of Compound 4 in Example 4 of the present invention;

[0067] Figure 5 The 1 H-NMR and 13 C-NMR spectra of Compound 5 in Example 5 of the present invention;

[0068] Figure 6 The 1 H-NMR and 13 C-NMR spectra of Compound 6 in Example 6 of the present invention;

[0069] Figure 7 The 1 H-NMR and 13 C-NMR spectra of Compound 7 in Example 7 of the present invention;

[0070] Figure 8 The 1 H-NMR and 13 C-NMR spectra of Compound 8 in Example 8 of the present invention;

[0071] Figure 9 The cumulative release rate of CBSIs in the ROS responsiveness experiment of the CBSIs prodrug in Example 10 of the present invention;

[0072] Figure 10 The average particle size of the nano-drug prepared in the present invention measured by dynamic light scattering;

[0073] Figure 11 The zeta potential measured for the nano-drug prepared in the present invention. Detailed implementation manners

[0074] The following examples will help to understand the present invention, but the content of the present invention is not limited to the examples given.

[0075] All the reagents used in the present invention are commercially available. The nuclear magnetic resonance spectra were measured by a Bruker AVANCE 600 nuclear magnetic resonance spectrometer, and the high-resolution mass spectra were measured by an Agilent Accurate-Mass Q-TOF 6530 (Agilent, Santa Clara, CA, USA) mass spectrometer.

[0076] Example 1: Preparation of [(1-{2-phenyl-5-[(3,4,5-trimethoxyphenyl)carbonyl]-1,2,3-triazepan-4-yl}piperidin-4-yl)amino]methanoic acid-10,10-dimethyl-5-oxo-1-[(3aS,4S,6aR)-2-oxo-2,3,3a,4,6,6a-hexahydro-1H-thieno[4,3-d]imidazol-4-yl]-9,11-dithia-6-oxatridecan-13-yl ester (Compound 1)

[0077] Step 1: Preparation of ({2-[(carboxymethyl)thio]propan-2-yl}thio)acetic acid

[0078] Acetone (5.00 g, 86.14 mmol) and mercaptoacetic acid (15.85 g, 172.28 mmol) were added into a 100 mL eggplant-shaped flask. Trifluoroacetic acid (0.89 g, 8.61 mmol) was added dropwise under stirring, and the reaction was carried out at room temperature for 6 h to obtain a white solid. The obtained crude product was washed with n-hexane and cold water, dried, and used directly in the next step without purification. 1 1H NMR (400 MHz, DMSO-d6) δ 3.37 (s, 4H), 1.56 (s, 6H). 13 13C NMR (100 MHz, DMSO-d 6) δ 171.8 (2C), 56.7, 33.3 (2C), 30.6 (2C). MS (ESI): m / z calcd. for C7H 12 O4S2: 224.0 Found: 222.8 [M-H] - .

[0079] Step 2: Preparation of 2-({2-[(2-hydroxyethyl)thio]propan-2-yl}thio)ethan-1-ol

[0080] ({2-[(Carboxymethyl)thio]propan-2-yl}thio)acetic acid (5.00 g, 22.29 mmol) and sodium borohydride (5.06 g, 133.76 mmol) were added into a 250 mL eggplant-shaped flask, dissolved with tetrahydrofuran, and placed in a cold well at -10 °C. Iodine (14.14 g, 55.73 mmol) was weighed, dissolved in an appropriate amount of anhydrous tetrahydrofuran, and added dropwise into the eggplant-shaped flask. After the addition was completed, stirring was continued until the solution was colorless. Then, the eggplant-shaped flask was placed in an oil bath and refluxed for 24 h. After the reaction was completed by TLC detection, anhydrous methanol was added to the reaction flask until no bubbles were generated, and the solvent was evaporated under reduced pressure. 10% NaOH solution was added to the obtained crude product, and the mixture was extracted with ethyl acetate three times. The organic phases were combined and the ethyl acetate was evaporated under reduced pressure. The product could be obtained by column chromatography separation and purification, which was a colorless oily liquid, 2.93 g, with a yield of 67%.1 1H NMR (400 MHz, CDCl3) δ 4.78 (s, 2H), 3.53 (t, J = 4.76 Hz, 4H), 2.66 (t, J = 4.76 Hz, 4H), 1.53 (s, 6H). 13 13C NMR (100 MHz, CDCl3) δ 60.3 (2C), 54.9, 32.6 (2C), 30.2 (2C). MS (ESI): m / z calcd. for C7H 16 O2S2: 196.1 Found: 219.0 [M+Na] + .

[0081] Step 3: Preparation of 7-Hydroxy-4,4-dimethyl-3,5-dithiaheptan-1-yl [(4-nitrophenyl)oxy]methanecarboxylate

[0082] Under nitrogen protection, 2-({2-[(2-Hydroxyethyl)thio]propan-2-yl}thio)ethan-1-ol (0.50 g, 2.55 mmol) and triethylamine (0.35 mL, 2.55 mmol) were dissolved in 100 mL of anhydrous tetrahydrofuran, stirred at 0 °C, and a solution of p-nitrophenyl chloroformate (0.51 mg, 2.55 mmol) in tetrahydrofuran was added dropwise to the mixed solution of Compound III and triethylamine within 5 min. The reaction was carried out at room temperature for 8 h. After the reaction was completed as detected by TLC, saturated NaHCO3 solution was added to adjust the pH to neutral, extracted with dichloromethane, the organic layers were combined, washed with saturated brine (150 mL), dried over anhydrous magnesium chloride, and dichloromethane was removed by distillation under reduced pressure to obtain a crude product. The product Compound VI was obtained by column chromatography separation, a colorless oily liquid, 0.39 g, yield 42%. 1 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 9.20 Hz, 2H), 7.32 (d, J = 9.20 Hz, 2H), 4.37 (t, J = 7.20 Hz, 2H), 3.71 (t, J = 6.40 Hz, 2H), 2.93 (t, J = 7.20 Hz, 2H), 2.77 (t, J = 6.00 Hz, 2H), 1.56 (s, 6H). 13 13C NMR (100 MHz, CDCl3) δ 155.4, 152.3, 145.4, 125.3 (2C), 121.8 (2C), 68.0, 61.5, 56.4, 33.5, 31.0 (2C), 28.8. MS (ESI): m / z calcd. for C 14 H 19 NO6S2: 361.1 Found: 384.0 [M+Na] + .

[0083] Step 4: Preparation of [(1-{2-phenyl-5-[(3,4,5-trimethoxyphenyl)carbonyl]-1,2,3-triazepan-4-yl}piperidin-4-yl)amino]methanesulfonic acid 7-hydroxy-4,4-dimethyl-3,5-dithiahept-1-yl ester

[0084] Under nitrogen protection, compound VI (0.20 g, 0.46 mmol) and [(4-nitrophenyl)oxy]methanesulfonic acid 7-hydroxy-4,4-dimethyl-3,5-dithiahept-1-yl ester (Compound V) (0.17 g, 0.46 mmol) were dissolved in 10 ml of tetrahydrofuran and reacted at room temperature for 12 h. After the reaction was completed by TLC detection, saturated NaHCO3 solution was added to adjust the pH to neutral, and the mixture was extracted with dichloromethane. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium chloride, and the dichloromethane was removed under reduced pressure to obtain a crude product. The product was separated by column chromatography to obtain a yellow solid, 0.26 g, with a yield of 87%. 1 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.20 Hz, 2H), 7.53 (s, 2H), 7.47 (t, J = 7.60 Hz, 2H), 7.35 (t, J = 7.44 Hz, 1H), 5.06 (d, J = 7.56 Hz, 1H), 4.24 (t, J = 6.24 Hz, 2H), 3.96 (s, 3H), 3.93 (s, 6H), 3.88 (m, 2H), 3.81 (t, J = 6.00 Hz, 2H), 3.72 (m, 1H), 3.04 (t, J = 11.28 Hz, 2H), 2.92 (t, J = 6.56 Hz, 2H), 2.88 (t, J = 5.96 Hz, 2H), 2.08 (dd, J1 = 12.68 Hz, J2 = 2.76 Hz, 2H), 1.68 (m, 2H), 1.63 (s, 6H). 13 13C NMR (100 MHz, CDCl3) δ 185.68, 158.13, 152.74 (2C), 139.29, 133.98, 132.77, 129.39 (2C), 127.83, 126.20 (2C), 118.54 (2C), 108.07 (2C), 63.51, 61.29, 61.03, 56.29 (2C), 63.51, 61.29, 61.04 (2C), 56.29 (3C), 48.41 (2C), 33.67 (2C), 31.88, 31.08 (2C), 29.69. HRMS calcd for C 31 H 41 N5NaO7S2 [M + Na] + 682.2345, found 682.2357.

[0085] Step 5: Preparation of [(1-{2-phenyl-5-[(3,4,5-trimethoxyphenyl)carbonyl]-1,2,3-triazacyclopent-4-yl}piperidin-4-yl)amino]methanoic acid 10,10-dimethyl-5-oxo-1-[(3aS,4S,6aR)-2-oxo-2,3,3a,4,6,6a-hexahydro-1H-thieno[4,3-d]imidazol-4-yl]-9,11-dithia-6-oxatridecan-13-yl ester

[0086] Dissolve biotin (0.22 g, 0.91 mmol), [(1-{2-phenyl-5-[(3,4,5-trimethoxyphenyl)carbonyl]-1,2,3-triazacyclopent-4-yl}piperidin-4-yl)amino]methanoic acid 7-hydroxy-4,4-dimethyl-3,5-dithiahept-1-yl ester (0.50 g, 0.76 mmol), DMAP (0.05 g, 0.38 mmol), and DCC (0.47 g, 2.27 mmol) in 10 mL of N,N-dimethylformamide, react at room temperature for 24 h. After the reaction is completed by TLC detection, filter, add dichloromethane to the filtrate, wash three times with water, evaporate dichloromethane under reduced pressure to obtain the crude product, and separate the target product compound 1 by column chromatography, yellow solid, 0.41 g, yield 61% (see Figure 1 ). 1 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.16 Hz, 2H), 7.49 (s, 2H), 7.44 (t, J = 7.58 Hz, 2H), 7.31 (t, J = 7.30 Hz, 1H), 6.01 (s, 1H), 5.64 (s, 1H), 4.44 (t, J = 7.01 Hz, 1H), 4.26 (t, J = 6.43 Hz, 1H), 4.23 (t, J = 6.72 Hz, 2H), 4.20 (m, 2H), 3.92 (s, 3H), 3.90 (s, 6H), 3.86 (s, 2H), 3.10 (m, 1H), 3.01 (t, J = 11.69 Hz, 2H), 2.84 (m, 4H), 2.69 (d, J = 13.15 Hz, 1H), 2.31 (t, J = 7.60 Hz, 2H), 2.04 (d, J = 12.56 Hz, 2H), 1.67 (m, 2H), 1.64 (m, 4H), 1.58 (s, 6H), 1.42 (m, 2H). 1313C NMR (100 MHz, CDCl3) δ 185.48, 173.49, 163.82, 158.10, 155.51, 152.71 (2C), 142.44, 139.29, 133.95, 132.79, 129.26 (2C), 127.73, 118.49 (2C), 108.02 (2C), 63.50, 63.41, 60.96, 60.91 (2C), 60.13, 56.42, 56.27 (3C), 55.51, 53.49, 48.43, 48.10, 40.54, 33.81, 31.86, 30.95 (2C), 29.62, 29.08, 28.37, 28.23, 24.75. HRMS calculated for C 41 H 55 N7NaO9S3 [M+Na] + 908.3121, found 908.3118.

[0087] Examples 2 - 8

[0088] Examples 2 - 8 are the synthetic methods of Compounds 2 - 8 respectively, all referring to Example 1.

[0089] Compounds 2 - 8 were prepared in the same manner as in Example 1, except that the corresponding carbonyl compounds were used in Step 1.

[0090] Compound 2 (see Figure 2 ): 1 1H NMR (400 MHz, CDCl3) δ = 7.95 (2H d, J = 7.84 Hz), 7.45 (2H s), 7.39 (2H t, J = 7.62 Hz), 7.27 (1H t, J = 7.24 Hz), 6.03 (1H s), 5.65 (1H s), 4.39 (1H t, J = 6.65 Hz), 4.20 (1H t, J = 4.81 Hz), 4.18 (4H t, J = 6.85 Hz), 3.88 (3H s), 3.85 (6H s), 3.81 (2H s), 3.04 - 3.07 (1H m), 2.84 (2H t, J = 11.25 Hz), 2.80 - 2.81 (4H m), 2.63 - 2.66 (1H m), 2.25 - 2.29 (2H m), 1.99 (2H d, J = 9.62 Hz), 1.98 - 2.01 (4H m), 1.75 - 1.87 (4H m), 1.40 - 1.73 (6H m), 1.36 - 1.38 (2H m), 1313C NMR (150 MHz, CDCl3) δ = 185.42, 173.50, 164.04, 158.02, 154.05, 152.65 (2C), 142.45, 139.23, 133.90, 132.72, 129.29 (2C), 127.67, 118.42 (2C), 108.07 (2C), 66.32, 63.37, 61.97, 61.80, 60.16, 57.79 (3C), 56.20, 55.53, 50.08, 48.37, 41.61, 40.39, 34.64, 33.78, 32.38, 31.72, 30.32, 30.10, 29.61, 28.37, 28.17, 26.82, 26.03.

[0091] Compound 3 (see Figure 3 ): 1 1H NMR (400 MHz, CDCl3) δ = 8.00 (2H d, J = 7.84 Hz), 7.50 (2Hs), 7.45 (2H t, J = 7.84 Hz), 7.32 (1H t, J = 7.24 Hz), 5.84 (1H s), 5.49 (1H s), 4.45 (1H t, J = 5.43 Hz), 4.27 (1H t, J = 4.83 Hz), 4.21 (4H t, J = 7.24 Hz), 3.93 (3H s), 3.90 (6H s), 3.86 (2H s), 3.09 - 3.14 (1H m), 3.02 (2H t, J = 11.46 Hz), 2.84 (4H t J = 4.84 Hz), 2.68 - 2.72 (1H m), 2.32 (2H t, J = 7.24 Hz), 2.04 (2H d, J = 10.86 Hz), 1.82 (4H t, J = 5.12 Hz), 1.67 - 3.68 (2H m), 1.65 - 1.67 (4H m), 1.62 - 1.63 (4H m), 1.40 - 1.73 (6H m), 1.40 - 1.44 (4Hm), 1313C NMR (150 MHz, CDCl3) δ = 183.83, 171.83, 162.03, 156.43, 153.87, 151.04 (2C), 140.77, 137.61, 132.28, 131.11, 127.68 (2C), 126.05, 116.82 (2C), 106.34 (2C), 61.92, 60.74, 60.28, 59.30, 58.54, 54.59, (3C), 53.79, 51.79, 49.89, 48.79, 46.76, 46.40, 38.85, 36.36, 32.14, 30.19, 27.99, 26.68, 26.54, 26.35, 25.82, 23, 80, 23.07, 20.70.

[0092] Compound 4 (see Figure 4 ): 1 1H NMR (400 MHz, CDCl3) δ = 8.01 (2H d, J = 7.84 Hz), 7.70 (2H d, J = 7.84 Hz), 7.51 (2H s, J = 8.1), 7.46 (2H t, J = 8.15 Hz), 7.34 (2H t, J = 8.15 Hz), 7.24 (1H s), 5.73 (1H s), 5.06 (1H t, J = 6.64 Hz), 4.45 (1H t, J = 6.64 Hz), 4.28 (1H d, J = 3.58 Hz), 4.12 - 4.16 (4H m), 3.95 (3H s), 3.92 (6H s), 3.87 (2H s), 3.10 - 3.14 (1H m), 2.30 - 3.05 (2H m), 2.85 - 2.89 (1H m), 2.74 - 2.82 (4H m), 2.70 (1H d, J = 12.54 Hz), 2.31 (2H t, J = 7.16 Hz), 2.03 - 2.06 (2H m), 2.01 (3H s), 1.71 (2H s), 1.67 (3H d, J = 6.14 Hz), 1.63 (2H s), 1.43 (2H d, J = 7.68 Hz). 1313C NMR (150 MHz, CDCl3) δ = 185.52, 173.39, 158.12, 155.42, 152.74 (2C), 143.08, 142.47, 139.32, 133.98, 132.81, 129.74, 129.38 (2C), 128.44 (2C), 127.75 (2C), 126.93 (2C), 118.52 (2C), 108.03 (2C), 63.28, 61.95, 61.57, 61.00 (2C), 60.10, 56.29 (3C), 55.41, 53.46, 50.80, 48.44, 48.09, 40.55, 33.79, 31.90, 29.92, 29.70, 28.33, 24.73.

[0093] Compound 5 (see Figure 5 ): 1 1H NMR (400 MHz, CDCl3) δ = 7.95 (2H d, J = 7.84 Hz), 7.83 (1H dd, J1 = 1.81 Hz, J2 = 6.94 Hz), 7.45 (2H s), 7.39 (2H t, J = 7.84 Hz), 7.34 (1H dd, J1 = 1.81 Hz, J2 = 6.94 Hz), 7.27 (1H t, J = 7.24 Hz), 7.15 - 7.18 (2H m), 5.87 (1H s), 5.46 (1H s), 4.39 (1H t, J = 6.34 Hz), 4.21 (1H t, J = 5.73 Hz), 4.07 (4H t, J = 6.34 Hz), 3.88 (3H s), 3.85 (6H s), 3.81 (2H s), 3.05 - 3.06 (1H m), 2.97 (2H t, J = 11.46 Hz), 2.78 - 2.79 (1H m), 2.71 - 2.72 (4H m), 2.63 - 2.64 (1H m), 2.24 (2H t, J = 7.54 Hz), 2.18 (3H s), 1.97 (2H d, J = 3.32 Hz), 1.62 (2H s), 1.60 (3H d, J = 6.14 Hz), 1.58 (2H s), 1.35 (2H d, J = 2.72 Hz). 13CNMR(150MHz,CDCl3) δ = 184.53, 172.33, 161.75, 160.11, 157.10, 154.39, 151.73(2C), 141.46, 138.30, 138.01, 132.97, 131.78, 128.36(2C), 127.86, 126.74, 117.50(3C), 114.26, 114.11, 107.01(2C), 62.18, 60.96, 59.99(2C), 59.13, 55.27(3C), 54.30, 49.82, 47.42, 47.09, 39.51, 32.73, 30.88, 29.71, 29.41, 28.94, 28.64, 27.30, 23.67, 21.67.

[0094] Compound 6 (see Figure 6 ): 1 H NMR(600MHz,CDCl3) δ = 8.01(2H d,J = 7.84Hz), 7.68(2Hdd,J1 = 5.43Hz,J2 = 9.05Hz), 7.51(2H s), 7.46(2H t,J = 7.24Hz), 7.34(1H t,J = 7.24Hz), 7.02(2H t,J = 9.05Hz), 5.60(1H s), 5.22(1H s), 4.46(1H t,J = 6.64Hz), 4.28(1H t,J = 4.22Hz), 4.16(4H t,J = 6.03Hz), 3.95(3H s), 3.92(6H s), 3.88(2H s), 3.12 - 3.13(1H m), 3.04(2H t,J = 12.07Hz), 2.86 - 2.87(1H m), 2.77 - 2.79(4H m), 2.69 - 2.72(1Hm), 2.04(2H t,J = 3.02Hz), 2.00(3H s), 1.69(2H d,J = 3.32Hz), 1.66(3H d,J = 6.54Hz), 1.64(2H s), 1.43(2H d,J = 9.65Hz). 1313C NMR (150 MHz, CDCl3) δ = 188.53, 172.44, 169.94, 162.62, 157.11, 154.42, 152.21, 151.72 (2C), 141.37, 138.29, 137.42, 132.96, 131.94, 129.22, 128.36 (2C), 126.75, 125.50, 117.50 (2C), 107.03 (2C), 106.27, 62.85, 62.47, 60.93, 59.99, 59.11, 55.27 (3C), 54.42, 52.45, 49.67, 47.43, 39.85, 39.52, 32.76, 30.86, 29.63, 28.67, 27.33, 26.71, 25.07, 23.73.

[0095] Compound 7 (see Figure 7 ): 1 1H NMR (400 MHz, CDCl3) δ = 7.99 (2H d, J = 8.15 Hz), 7.63 (2H d, J = 8.75 Hz), 7.50 (2H s), 7.44 (2H t, J = 7.54 Hz), 7.34 (1H t, J = 7.24 Hz), 7.29 (2H d, J = 8.45 Hz), 5.98 (1H s), 5.58 (1H s), 4.43 (1H t, J = 6.64 Hz), 4.25 (1H t, J = 4.53 Hz), 4.14 (4H t, J = 6.64 Hz), 3.93 (3H s), 3.90 (6H s), 3.86 (2H s), 3.06 - 3.12 (1H m), 3.02 (2H t, J = 11.46 Hz), 2.82 - 2.86 (1H m), 2.73 - 2.79 (4H m), 2.67 - 2.70 (1H m), 2.29 (2H t, J = 7.54 Hz), 2.02 (2H t, J = 3.92 Hz), 1.97 (3H s), 1.68 (2H s), 1.66 (3H d, J = 2.72 Hz), 1.62 (2H d, J = 4.52 Hz), 1.40 (2H t, J = 7.54 Hz). 1313C NMR (150 MHz, CDCl3) δ = 185.46, 173.36, 163.72, 158.08, 155.38, 152.74 (2C), 142.54, 141.87, 139.32, 133.99, 133.51, 132.80, 129.36 (2C), 128.43 (3C), 127.73 (2C), 118.51 (2C), 108.10 (2C), 63.13, 62.94, 61.97 (2C), 60.97, 60.13, 56.29 (3C), 55.47, 53.47, 48.41, 48.12, 40.52, 33.78, 31.87, 30.55, 30.44, 29.96, 28.35, 28.24, 24.74.

[0096] Compound 8 (see Figure 8 ): 1 1H NMR (400 MHz, CDCl3) δ = 8.00 (2H d, J = 7.84 Hz), 7.57 (2H d, J = 8.45 Hz), 7.51 (2H s), 7.45 (2H t, J = 7.84 Hz), 7.33 (1H t, J = 7.24 Hz), 7.13 (2H d, J = 8.19 Hz), 5.86 (1H s), 5.45 (1H s), 4.44 (1H t, J = 6.65 Hz), 4.26 (1H t, J = 4.61 Hz), 4.14 (4H t, J = 6.65 Hz), 3.94 (3H s), 3.91 (6H s), 3.86 (2H s), 3.08 - 3.13 (1H m), 3.03 (2H t, J = 11.26 Hz), 2.83 - 2.88 (1H m), 2.74 - 2.80 (4H m), 2.74 - 2.80 (4H m), 2.31 (3H s), 2.29 (2H d, J = 8.19 Hz), 2.05 (2H d, J = 10.75 Hz), 1.99 (3H s), 1.70 (2H s), 1.66 (3H d, J = 8.19 Hz), 1.63 (2H s), 1.41 - 1.45 (2H m). 1313C NMR (150 MHz, CDCl3) δ = 184.49, 172.41, 162.64, 157.09, 154.43, 151.71 (2C), 141.44, 139.02, 138.29, 136.45, 132.95, 131.79, 128.35 (2C), 128.07 (2C), 126.72, 125.81 (2C), 117.49 (2C), 107.01 (2C), 62.29, 62.14, 60.93, 59.97, 59.10, 55.26 (3C), 54.45, 52.45, 47.41, 47.06, 39.52, 32.77, 30.86, 29.60, 29.37, 28.89, 28.66, 27.33, 23.71, 19.95.

[0097] Example 9: In vitro anti-tumor activity test of the compounds involved in the present invention

[0098] The in vitro activity test methods and results are as follows: Among them, colchicine is the positive experimental group.

[0099] Screening method: Microculture tetrazolium (MTT) reduction method

[0100] Cell lines: MCF-7 (human breast cancer), SGC-7901 (human gastric adenocarcinoma), and A549 (human non-small cell lung cancer)

[0101] Action time: 72 hours

[0102] The inhibition rates of each compound on the growth of three tumor cells (10 μg / mL) are shown in Table-1.

[0103] The inhibition rates of each compound on tumor growth (μg / mL) are shown in Table-1.

[0104] Table-1

[0105]

[0106] Example 10: ROS responsiveness of CBSIs prodrugs

[0107] It should be noted that in the original text, the cell line "A949" is corrected to "A549" in the translation for accuracy.Accurately weigh 1.0 mg of Compound 4, and dissolve it completely with an appropriate amount of acetonitrile. Slowly drop the prepared acetonitrile solution of prodrug Compound 4 into PBS buffer (pH 7.2 - 7.4) respectively, so that the concentration of CBSIs prodrug is 5 μΜ, and ensure that no solid precipitates. Drop 30% H2O2 aqueous solution into the PBS buffer (pH 7.2 - 7.4) solution of CBSIs prodrug to make the concentration of H2O2 be 10 mM and 100 mM respectively, and drop a drop of FeSO4 aqueous solution; place the prepared mixed solution in a constant temperature water bath and incubate at 37 °C for 3 hours, then take samples every 30 min and determine the change of solution composition by HPLC.

[0108] From Figure 9 It can be seen that prodrug Compound 4 can release anti-tumor active substances under the action of H2O2, and the release rate is concentration-dependent. Under the condition that the concentration of H2O2 is 10 mM, about 40% of the active substances can be released in 3 hours. Under the condition that the concentration of H2O2 is 100 mM, the release rate in 3 hours can reach 80%.

[0109] Example 11: Preparation of nano-drugs

[0110] Dissolve 6 mg of the general formula compound prepared in the above example, 1.2 mg of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and 0.6 mg of egg yolk lecithin in 1 ml of ethanol to obtain a mixed solution. Among them, the general formula compound prepared in the above example can be the product prepared in Examples 1 - 8.

[0111] In this example, the compound prepared in Example 4 (Compound 4) is selected. Then, under the condition of vigorously stirring at a speed of 1200 rpm, add the mixed solution to 4 ml of deionized water at 1000 rpm. After continuing to stir the reaction until completion, spin-dry the organic solvent, and self-assemble to obtain nano-drugs (4NPs), which are stored in the dark at 4 °C for standby.

[0112] For the characterization of the obtained nano-drugs, see Figure 10 and Figure 11 ,

[0113] Use a nano-particle size analyzer (Bruke particle size and Zeta potential analyzer) to measure the particle size and Zeta potential of the nanoparticles by the principle of dynamic light scattering. Dilute the prepared CBSIs prodrug nano-drugs with ultrapure water to an appropriate concentration (0.5 mg / mL), put them into the nano-particle size analyzer to measure the light intensity distribution map. The measurement results show that the particle size of the CBSIs prodrug nano-drugs is about 130 nm, the PDI is 0.082, and the Zeta potential is -26.2 mV.

[0114] Example 12: Study on the anti-tumor activity of the compounds involved in the present invention in animals

[0115] The nano-drug of compound 4 prepared by the above-mentioned examples was tested for anti-tumor activity in animals. The model used was the mouse S-180 sarcoma model, and the positive control drug was the commonly used anti-tumor drug fluorouracil (5-Fu) in clinic.

[0116] Experimental method: Female Kunming mice weighing 18 - 22 g and well-grown S-180 tumor seeds for 7 - 11 days were selected. The tumor tissue was made into cell suspension and inoculated subcutaneously into the right axilla of the mice, about 1.0 - 2.0×10 6 cells / mouse. After 24 hours of inoculation, the mice were randomly caged and administered by tail vein injection for 7 consecutive days. The animals were sacrificed 24 hours after stopping the drug, and the body weight and tumor weight were measured. The average tumor weight of each group was calculated, and the tumor inhibition rate was calculated according to the following formula and a t-test was performed.

[0117] Tumor inhibition rate = [(average tumor weight of blank control group - average tumor weight of treatment group) / (average tumor weight of blank control group)]×100%

[0118] The experimental results are shown in Table 2

[0119] Table 2

[0120]

[0121] As can be seen from Table 2, the tumor inhibition rate of the nano-drug of compound 4 was 66.5%, which was comparable to that of the positive control fluorouracil. However, the body weight of the mice in the positive control fluorouracil group increased slowly, while the body weight of the mice in the nano-drug group of compound 4 was equivalent to that of the mice in the blank control group at the end, indicating better safety.

[0122] Example 13: Study on anti-inflammatory activity of the compounds involved in the present invention in animals

[0123] The nano-drug of compound 4 obtained above was tested for anti-inflammatory activity in animals. The model used was the mouse acute inflammation model induced by phorbol ester (TPA), and the positive control drug was the commonly used anti-inflammatory drug indomethacin in clinic.

[0124] Experimental method: 40 female Kunming mice weighing 18 - 22 g were selected and randomly divided into groups of 10 each. The mice in each group were administered the nano-drug of compound 4 (3 mg / Kg), the control (saline) or indomethacin (3 mg / Kg, positive control) by tail vein injection or intraperitoneal injection. 30 minutes later, TPA (2.5 μg / ear, 20 μL acetone) was topically applied to both sides of the right ear of each mouse to induce edema. 6 hours after inflammation induction, the mice in each group were sacrificed by cervical dislocation. Then, ear pieces were punched out at symmetric positions on the left and right ears with a 7-mm diameter puncher, weighed with a balance, the mass was recorded, and the swelling degree and swelling inhibition rate were calculated.

[0125] Degree of swelling = Average mass of the right ear piece - Average mass of the left ear piece

[0126] Swelling inhibition rate = [(Degree of swelling in the blank group - Degree of swelling in the drug-administered group) / (Degree of swelling in the blank group)] × 100%

[0127] The experimental results are shown in Table - 3

[0128] Table - 3

[0129]

Claims

1. A colchicine-binding site inhibitor prodrug compound, characterized in that, The prodrug compound is obtained by conjugating a colchicine binding site inhibitor (CBSI) and biotin with a ROS-responsive thioacetal chain.

2. The prodrug compound according to claim 1, wherein The prodrug compound is the compound shown in Formula 1, and its salt or hydrate, In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 can be the same or different and independently are hydrogen, C1-C6 alkyl, C1-C6 alkoxy, halogen atom, nitro, hydroxyl or amino; R 6 is hydrogen, C1-C6 alkyl; n and m are each independently 1 or 2; R 7 and R 8 may independently be the same or different and are each hydrogen, C1-C6 alkyl, or phenyl which is unsubstituted or substituted by at least one halogen or C1-C3 alkyl, and R 7 and R 8 are not both hydrogen at the same time; Or, R 7 , R 8 forms a five-, six-, seven- or eight-membered saturated carbocyclic ring with the adjacent C.

3. The prodrug compound according to claim 2, wherein: The prodrug compound is the compound shown in Formula 1, and its salt or hydrate, In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 can be the same or different and independently be hydrogen, C1-C3 alkyl, C1-C3 alkoxy, halogen atom, nitro, hydroxy or amino; R 6 is hydrogen, C1-C3 alkyl; n and m are each independently 1 or 2; R 7 and R 8 may independently be the same or different and each is hydrogen, C1-C3 alkyl, or phenyl which is unsubstituted or substituted by at least one halogen or C1-C3 alkyl, and R 7 and R 8 are not simultaneously hydrogen; Or, R 7 , R 8 forms a five-, six- or seven-membered saturated carbon ring with the adjacent C.

4. The prodrug compound according to claim 3, wherein: The prodrug compound is the compound shown in Formula 1, and its salt or hydrate, In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 can be the same or different and independently be hydrogen, methoxy, ethoxy, amino, hydroxy, fluorine, chlorine or bromine; R 6 is hydrogen, methyl, or ethyl; n and m are each independently 1 or 2; R 7 、R 8 may be the same or different and independently are hydrogen, C1-C3 alkyl, phenyl which is unsubstituted or substituted by at least one halogen or C1-C3 alkyl, and R 7 、R 8 are not simultaneously hydrogen; Or, R 7 , R 8 forms a five-, six- or seven-membered saturated carbon ring with the adjacent C.

5. The prodrug compound according to any one of claims 1-4, characterized in that: The salt of the compound shown in Formula 1 is a salt formed by the compound shown in Formula 1 and an acid or a base; wherein, the acid is hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, malic acid; the base is sodium hydroxide, sodium carbonate, potassium hydroxide; The hydrate of the compound shown in Formula 1, the number of crystal water molecules of the hydrate is any real number from 0 to 16.

6. A method for preparing the colchicine binding site inhibitor prodrug compound according to claim 1, characterized in that: 。 7. According to the method for preparing the colchicine binding site inhibitor prodrug compound according to claim 6, characterized in that: Step 1: Mix a carbonyl compound (I) and mercaptoacetic acid in a molar ratio of 1:2 to 4, and drop trifluoroacetic acid into the mixed solution under stirring, and react at a temperature of 15 to 40 °C for 0.5 to 12 h to obtain Compound II; Step 2: Dissolve Compound II, sodium borohydride and iodine in a molar ratio of 1:1 to 6:2 to 6 in tetrahydrofuran at a temperature of -20 to 0 °C, and reflux for 12 to 24 h to obtain Compound III; Step 3: Under nitrogen protection, dissolve Compound III and triethylamine in anhydrous tetrahydrofuran, and add a tetrahydrofuran solution of p-nitrophenyl chloroformate to the mixed solution of Compound III and triethylamine, and react at a temperature of 0 to 30 °C for 2 to 6 h to obtain Compound VI; Among them, the molar ratio of Compound III, triethylamine and p-nitrophenyl chloroformate is 1:1 to 1.2:1 to 1.2; Step 4, under nitrogen protection, react Compound VI with Compound V dissolved in tetrahydrofuran at 0 to 30 °C for 12 to 24 h to obtain Compound IV; Among them, the molar ratio of Compound VI to Compound V is 1:1 to 2; Step 5, dissolve Compound VI, biotin, DMAP, and DCC in a molar ratio of 1:1 to 2:1 to 4:1 to 4 in DMF, and react at 15 to 45 °C to obtain the compound of Formula 1.

8. A nano-drug, characterized in that, The nano-drug is prepared by the nano-precipitation method from the prodrug compound according to claims 1 to 5.

9. The preparation method of the nano-drug according to claim 8, characterized in that: Dissolve the prodrug compound according to any one of claims 1-6, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and egg yolk lecithin in an organic solvent to obtain a mixed solution, and then add the mixture solution to deionized water under stirring conditions, and continue to stir. After the reaction is completed, remove the organic solvent, and self-assemble to obtain the prodrug compound nano-drug.

10. Use of the prodrug compound or the nano-drug according to claim 1 or 8, characterized in that, The application of the prodrug compound and the nano-drug in the preparation of anti-tumor drugs or anti-inflammatory drugs.

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