A colchicine binding site inhibitor prodrug, nanomedicine and its preparation and application

Abstract

This invention belongs to the field of pharmaceutical compound technology, and relates to the preparation of a ROS-responsive colchicine binding site inhibitor prodrug and nanomedicine, as well as its application in antitumor and anti-inflammatory drugs. The prodrug compound is obtained by conjugating a colchicine binding site inhibitor (CBSI) and biotin with a ROS-responsive thioacetate chain. This invention synthesizes a CBSI prodrug by linking CBSIs and biotin through a ROS-responsive thioacetate, and then prepares a CBSI prodrug nanomedicine by self-assembly using a nanoprecipitation method. This novel nanomedicine can significantly improve the water solubility of the CBSI prodrug. In application, it can target tumor or inflammatory sites and be cleaved by the high concentration of ROS in tumor or inflammatory tissues, thereby releasing the CBSIs to exert pharmacological activity while reducing the impact on normal tissues.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical compound technology, and relates to a ROS-responsive colchicine binding site inhibitor prodrug, nanomedicine preparation, and its application in antitumor and anti-inflammatory drugs. Background Technology

[0002] Malignant tumors pose a serious threat to human health, being the second leading cause of death after cardiovascular disease. Currently, commonly used chemotherapy drugs suffer from poor tolerability, numerous side effects, and high toxicity. Meanwhile, cytokine storms triggered by infections such as the novel coronavirus (2019-nCoV) pose a threat to human life, and existing drug treatments are ineffective. Therefore, the search for more effective and safer anti-tumor and anti-inflammatory drugs remains crucial.

[0003] Microtubules, formed by the polymerization of heterodimers of α- and β-tubulins, are essential components of the eukaryotic cytoskeleton, playing crucial roles in maintaining cell morphology, participating in cell division, assisting in nutrient transport, and signal transduction. Microtubules are also considered effective targets for antitumor and anti-inflammatory drug research. The colchicine site is located in the deep pocket of the β-tubulin subunit between the α- and β-tubulin heterodimers. Colchicine binding site inhibitors (CBSIs) are a class of microtubule destabilizers that interact with the colchicine binding site of tubulin. These compounds effectively prevent the formation of functional microtubules and further inhibit cell mitosis by inhibiting tubulin polymerization. CBSIs are characterized by their small molecular weight, simple structure, high activity, and numerous modifiable sites. However, CBSIs lack sufficient targeting specificity for tumor cells, leading to severe toxic side effects. Therefore, addressing the targeting issue of CBSIs is crucial for advancing CBSI research. Summary of the Invention

[0004] The purpose of this invention is to provide a ROS-responsive colchicine binding site inhibitor prodrug, nanomedicine preparation, and its application in antitumor and anti-inflammatory drugs.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

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

[0007] Preferably, the prodrug compound is a compound of Formula 1, or a salt or hydrate thereof.

[0008]

[0009] In Equation 1, R 1 R 2 R 3 R 4 R 5 It may be hydrogen, C1-C6 alkyl, C1-C6 alkyloxy, halogen atom, nitro, hydroxyl or amino, either the same or different;

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

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

[0012] R 7 R 8 It may be the same or different independently of hydrogen, C1-C6 alkyl, unsubstituted or substituted with at least one halogen or C1-C3 alkyl, and R 7 R 8 They are not both hydrogen;

[0013] Or, R 7 R 8 It forms five-, six-, seven-, or eight-membered saturated carbon rings with adjacent C atoms.

[0014] Further preferably, the prodrug compound is a compound of Formula 1, or its salt or hydrate.

[0015] In Equation 1, R 1 R 2 R 3 R 4 R 5 It may be hydrogen, C1-C3 alkyl, C1-C3 alkyloxy, halogen atom, nitro, hydroxyl or amino, either the same or different;

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

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

[0018] R 7 R 8 It may be the same or different independently of hydrogen, C1-C3 alkyl, unsubstituted or substituted with at least one halogen or C1-C3 alkyl, and R 7 R 8 They are not both hydrogen;

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

[0020] Further preferably, the prodrug compound is a compound of Formula 1, or its salt or hydrate.

[0021] In Equation 1, R 1 R 2 R 3 R 4 R 5 They can be hydrogen, methoxy, ethoxy, amino, hydroxyl, fluorine, chlorine, or bromine, either the same or different.

[0022] R 6 It can be hydrogen, methyl, or ethyl.

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

[0024] R 7 R 8 It may be the same or different independently of hydrogen, C1-C3 alkyl, unsubstituted or substituted with at least one halogen or C1-C3 alkyl, and R 7 R 8 They are not both hydrogen;

[0025] Or, R 7 R 8 It forms a five-, six-, 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 a salt formed by the compound shown in Formula 1 and an acid or base; wherein the acid is hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, or malic acid; and the base is sodium hydroxide, sodium carbonate, or potassium hydroxide.

[0044] The hydrate of the compound shown in Formula 1 has any real number of water molecules between 0 and 16.

[0045] A method for preparing the colchicine binding site inhibitor prodrug compound described above.

[0046]

[0047] Specifically:

[0048] Step 1: Mix carbonyl compound (I) and mercaptoacetic acid in a molar ratio of 1:2 to 4. Add trifluoroacetic acid dropwise to the mixture while stirring. React at 15 to 40 °C for 0.5 to 12 h to obtain compound II.

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

[0050] Step 3: Under nitrogen protection, compound III and triethylamine were dissolved in anhydrous tetrahydrofuran. The tetrahydrofuran solution of p-nitrophenyl chloroformate was added to the mixed solution of compound III and triethylamine. The reaction was carried out at 0-30°C for 2-6 hours to obtain compound VI.

[0051] 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, compound VI reacts with compound V dissolved in tetrahydrofuran at 0–30 °C for 12–24 h to obtain compound IV;

[0053] 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 DMF in a molar ratio of 1:1 to 2:1 to 4:1 to 4, and react at 15 to 45 °C to obtain compound 1.

[0055] A nanomedicine, said nanomedicine being prepared from the prodrug compound via a nanoprecipitation method.

[0056] The nanomedicine has a particle size of approximately 130 nm.

[0057] The method for preparing nanomedicine involves dissolving the prodrug compound, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and egg yolk lecithin in an organic solvent to obtain a mixed solution. Then, under stirring conditions, the mixed solution is added to deionized water. After the reaction is completed by stirring, the organic solvent is removed, and the prodrug compound nanomedicine is self-assembled.

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

[0059] The organic solvent is one or more mixed solvents selected from ethanol, acetone, DMSO and acetonitrile.

[0060] The application of the aforementioned prodrug compound or nanomedicine, and the application of the aforementioned prodrug compound or nanomedicine in the preparation of antitumor drugs or anti-inflammatory drugs.

[0061] The beneficial effects of this invention are:

[0062] This invention synthesizes CBSI prodrugs by linking CBSIs and biotin via ROS-responsive thioacetates, and then prepares CBSI prodrug nanomedicines through a nanoprecipitation method. This novel nanomedicine significantly improves the water solubility of the CBSI prodrugs. In application, it can target tumor or inflammatory sites and be cleaved by the high concentration of ROS in tumor or inflammatory tissues, thereby releasing the CBSIs to exert pharmacological activity while reducing the impact on normal tissues. Attached Figure Description

[0063] Figure 1 It is compound 1 of Example 1 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

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

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

[0066] Figure 4 It is compound 4 of Example 4 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

[0067] Figure 5 It is compound 5 of Example 5 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

[0068] Figure 6 It is compound 6 of Example 6 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

[0069] Figure 7 It is compound 7 of Example 7 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

[0070] Figure 8 It is compound 8 of Example 8 of the present invention. 1 H-NMR and 13 C-NMR spectrum;

[0071] Figure 9 This refers to 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 nanomedicine prepared by this invention was measured by dynamic light scattering.

[0073] Figure 11 The zeta potential of the nanomedicine prepared by this invention was measured. Detailed Implementation

[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 reagents used in this invention are commercially available. The nuclear magnetic resonance spectra were measured using a Bruker AVANCE 600 nuclear magnetic resonance spectrometer, and the high-resolution mass spectrometry was measured using 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-triazacyclopentanyl-4-yl}hexahydropyridin-4-yl)amino]methane-10,10-dimethyl-5-oxylidene-1-[(3aS,4S,6aR)-2-oxylidene-2,3,3a,4,6,6a-hexahydro-1H-thiopheno[4,3-d]imidazol-4-yl]-9,11-dithia-6-oxatridecane-13-yl ester (compound 1)

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

[0078] Acetone (5.00 g, 86.14 mmol) and mercaptoacetic acid (15.85 g, 172.28 mmol) were added to a 100 mL round-bottom flask. Trifluoroacetic acid (0.89 g, 8.61 mmol) was added dropwise while stirring. The reaction was carried out at room temperature for 6 h to obtain a white solid. The crude product was washed with n-hexane and cold water, dried, and used directly in the next step without purification. 1 H NMR (400MHz, DMSO-d6) δ3.37 (s, 4H), 1.56 (s, 6H). 13 C NMR (100MHz, DMSO-d) 6) δ171.8(2C),56.7,33.3(2C),30.6(2C).MS(ESI):m / z calcd.for C7H 12 O4S2:224.0 Found:222.8 [MH] - .

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

[0080] ({2-[(carboxymethyl)thio]propyl-2-yl}thio)acetic acid (5.00 g, 22.29 mmol) and sodium borohydride (5.06 g, 133.76 mmol) were added to a 250 mL round-bottom flask, dissolved in 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 to the round-bottom flask. After the addition was complete, stirring was continued until the solution became colorless. Then, the round-bottom flask was placed in an oil bath and refluxed for 24 h. After the reaction was completed as detected by TLC, anhydrous methanol was added to the reaction flask until no more bubbles were generated, and the solvent was evaporated off under reduced pressure. The crude product was added to a 10% NaOH solution, extracted three times with ethyl acetate, and the organic phases were combined and the ethyl acetate was evaporated off under reduced pressure. The product was obtained by column chromatography purification, a colorless oily liquid, 2.93 g, yield 67%.1 H NMR (400MHz, CDCl3) δ4.78 (s, 2H), 3.53 (t, J = 4.76Hz, 4H), 2.66 (t, J = 4.76Hz, 4H), 1.53 (s, 6H). 13 C NMR(100MHz, CDCl3)δ60.3(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 [(4-nitrophenyl)oxy]methane-7-hydroxy-4,4-dimethyl-3,5-dithiahept-1-yl ester

[0082] Under nitrogen protection, 2-({2-[(2-hydroxyethyl)thio]propyl-2-yl}thio)ethanol-1-ol (0.50 g, 2.55 mmol) and triethylamine (0.35 mL, 2.55 mmol) were dissolved in 100 mL of anhydrous tetrahydrofuran. The mixture was stirred at 0 °C. A tetrahydrofuran solution of p-nitrophenyl chloroformate (0.51 mg, 2.55 mmol) was added dropwise over 5 min to a mixture of compound III and triethylamine. The reaction was allowed to proceed at room temperature for 8 h. After the reaction was complete as detected by TLC, the pH was adjusted to neutral by adding saturated NaHCO3 solution. The mixture was extracted with dichloromethane, and the organic layers were combined, washed with saturated brine (150 mL), dried over anhydrous magnesium chloride, and the dichloromethane was removed under reduced pressure to obtain the crude product. The crude product, compound VI, was obtained by column chromatography as a colorless oily liquid, 0.39 g, yield 42%. 1 H NMR (400MHz, CDCl3) δ8.19(d,J=9.20Hz,2H),7.32(d,J=9.20Hz,2H),4.37(t,J=7.20Hz, 2H), 3.71 (t, J = 6.40Hz, 2H), 2.93 (t, J = 7.20Hz, 2H), 2.77 (t, J = 6.00Hz, 2H), 1.56 (s, 6H). 13 C NMR (100MHz, CDCl3) δ155.4,152.3,145.4,125.3(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 NO6S2:361.1Found:384.0[M+Na] + .

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

[0084] Compound VI (0.20 g, 0.46 mmol) and [(4-nitrophenyl)oxy]methane-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 under nitrogen protection and reacted at room temperature for 12 h. After the reaction was completed as detected by TLC, 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 the crude product. The product was separated by column chromatography to obtain 0.26 g of yellow solid, with a yield of 87%. 1 H NMR (400MHz, CDCl3) δ8.02(d,J=8.20Hz,2H),7.53(s,2H),7.47(t,J=7.60Hz,2H),7.35(t,J =7.44Hz,1H),5.06(d,J=7.56Hz,1H),4.24(t,J=6.24Hz,2H),3.96(s,3H),3.93(s,6H),3.8 8(m,2H),3.81(t,J=6.00Hz,2H),3.72(m,1H),3.04(t,J=11.28Hz,2H),2.92(t,J=6.56Hz,2 H), 2.88 (t, J = 5.96Hz, 2H), 2.08 (dd, J1 = 12.68Hz, J2 = 2.76Hz, 2H), 1.68 (m, 2H), 1.63 (s, 6H). 13 C NMR(100MHz, CDCl3)δ185.68,158.13,152.74(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-triazacyclopentanyl-4-yl}hexahydropyridin-4-yl)amino]methane-10,10-dimethyl-5-oxylidene-1-[(3aS,4S,6aR)-2-oxylidene-2,3,3a,4,6,6a-hexahydro-1H-thieno[4,3-d]imidazol-4-yl]-9,11-dithia-6-oxatridecane-13-yl ester]

[0086] Biotin (0.22 g, 0.91 mmol), [(1-{2-phenyl-5-[(3,4,5-trimethoxyphenyl)carbonyl]-1,2,3-triazacyclopentanyl-4-yl}hexahydropyridin-4-yl)amino]methane-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) were dissolved in 10 mL of N,N-dimethylformamide and reacted at room temperature for 24 h. After the reaction was completed as detected by TLC, the mixture was filtered, and dichloromethane was added to the filtrate. The mixture was washed three times with water, and the dichloromethane was removed by vacuum distillation to obtain the crude product. The target product compound 1 was separated by column chromatography, yielding 0.41 g of a yellow solid, with a yield of 61% (see [link to product description]). Figure 1 ). 1 H NMR (400MHz, CDCl3) δ7.98(d,J=8.16Hz,2H),7.49(s,2H),7.44(t,J=7.58Hz,2H),7.31(t,J=7.30Hz,1H),6. 01(s,1H),5.64(s,1H),4.44(t,J=7.01Hz,1H),4.26(t,J=6.43Hz,1H),4.23(t,J=6.72Hz,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=11.69Hz,2H),2.84(m,4H),2.69(d,J=13.15H z,1H),2.31(t,J=7.60Hz,2H),2.04(d,J=12.56Hz,2H),1.67(m,2H),1.64(m,4H),1.58(s,6H),1.42(m,2H). 13C NMR (100MHz, CDCl3) δ185.48,173.49,163.82,158.10,155.51,152.71(2C),142. 44,139.29,133.95,132.79,129.26(2C),127.73,118.49(2C),108.02(2C),63.5 0,63.41,60.96,60.91(2C),60.13,56.42,56.27(3C),55.51,53.49,48.43,48.1 0,40.54,33.81,31.86,30.95(2C),29.62,29.08,28.37,28.23,24.75.HRMScalcd for C 41 H 55 N7NaO9S3[M+Na] + 908.3121, found 908.3118.

[0087] Examples 2-8

[0088] Examples 2-8 describe the synthesis methods of compounds 2-8, all of which are based on Example 1.

[0089] Compounds 2-8 were prepared using the same method as in Example 1, except that the corresponding carbonyl compound was used in step 1.

[0090] Compound 2 (see Figure 2 ): 1 H NMR (400MHz, CDCl3) δ = 7.95 (2H d, J = 7.84Hz), 7.45 (2Hs), 7.39 (2H t, J = 7.62Hz), 7.27 (1H t, J = 7.24Hz), 6.03 (1H s), 5.65 (1H s), 4.39 (1H t,J=6.65Hz),4.20(1H t,J=4.81Hz),4.18(4H t,J=6.85Hz),3.88(3H s),3.85(6H s),3.81(2H s),3.04-3.07(1H m),2.84(2H t,J=11.25Hz),2.80-2.81(4H m),2.63-2.66(1H m),2.25-2.29(2H m),1.99(2H d,J=9.62Hz),1.98-2.01(4H m),1.75-1.87(4H m),1.40-1.73(6H m),1.36-1.38(2H m), 13C NMR (150MHz, 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),66.3 2,63.37,61.97,61.80,60.16,57.79(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 H NMR (400MHz, CDCl3) δ = 8.00 (2H d, J = 7.84Hz), 7.50 (2Hs), 7.45 (2H t, J = 7.84Hz), 7.32 (1H t, J = 7.24Hz), 5.84 (1H s), 5.49 (1H s), 4.45 (1H t,J=5.43Hz),4.27(1H t,J=4.83Hz),4.21(4H t,J=7.24Hz),3.93(3H s),3.90(6H s),3.86(2H s),3.09-3.14(1H m),3.02(2H t,J=11.46Hz),2.84(4H t J=4.84Hz),2.68-2.72(1H m),2.32(2H t,J=7.24Hz),2.04(2H d,J=10.86Hz),1.82(4H t,J=5.12Hz),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), 13C NMR (150MHz, CDCl3) δ = 183.83, 171.83, 162.03, 156.43, 153.87, 151.04 (2C), 140.7 7,137.61,132.28,131.11,127.68(2C),126.05,116.82(2C),106.34(2C),61.92,60 .74,60.28,59.30,58.54,54.59,(3C),53.79,51.79,49.89,48.79,46.76,46.40,3 8.85,36.36,32.14,30.19,27.99,26.68,26.54,26.35,25.82,23,80,23.07,20.70.

[0092] Compound 4 (see Figure 4 ): 1 H NMR (400MHz, CDCl3) δ = 8.01 (2H d, J = 7.84Hz), 7.70 (2Hd, J = 7.84Hz), 7.51 (2H s, J = 8.1), 7.46 (2H t, J = 8.15Hz), 7.34 (2H t, J = 8.15Hz), 7.24 (1H s),5.73(1H s),5.06(1H t,J=6.64Hz),4.45(1H t,J=6.64Hz),4.28(1H d,J=3.58Hz),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=12.54Hz),2.31(2Ht,J=7.16Hz),2.03-2.06(2H m),2.01(3H s),1.71(2H s),1.67(3H d,J=6.14Hz),1.63(2H s),1.43(2H d,J=7.68Hz). 13C NMR (150MHz, CDCl3) δ = 185.52, 173.39, 158.12, 155.42, 152.74 (2C), 143.08, 142.4 7,139.32,133.98,132.81,129.74,129.38(2C),128.44(2C),127.75(2C),126.93(2 C),118.52(2C),108.03(2C),63.28,61.95,61.57,61.00(2C),60.10,56.29(3C),5 5.41,53.46,50.80,48.44,48.09,40.55,33.79,31.90,29.92,29.70,28.33,24.73.

[0093] Compound 5 (see Figure 5 ): 1 H NMR (400MHz, CDCl3) δ = 7.95 (2H d, J = 7.84Hz), 7.83 (1Hdd, J1 = 1.81Hz, J2 = 6.94Hz), 7.45 (2H s), 7.39 (2H t, J = 7.84Hz), 7.34 (1H dd,J1=1.81Hz,J2=6.94Hz),7.27(1H t,J=7.24Hz),7.15-7.18(2H m),5.87(1H s),5.46(1Hs),4.39(1H t,J=6.34Hz),4.21(1H t,J=5.73Hz),4.07(4H t,J=6.34Hz),3.88(3H s),3.85(6H s),3.81(2H s),3.05-3.06(1H m),2.97(2H t,J=11.46Hz),2.78-2.79(1H m),2.71-2.72(4H m),2.63-2.64(1H m),2.24(2H t,J=7.54Hz),2.18(3H s),1.97(2H d,J=3.32Hz),1.62(2H s),1.60(3H d,J=6.14Hz),1.58(2H s),1.35(2H d,J=2.72Hz). 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),1 14.26,114.11,107.01(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). 13C NMR(150MHz, 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 H NMR (400MHz, CDCl3) δ = 7.99 (2H d, J = 8.15Hz), 7.63 (2Hd, J = 8.75Hz), 7.50 (2H s), 7.44 (2H t, J = 7.54Hz), 7.34 (1H t, J = 7.24Hz), 7.29 (2H d,J=8.45Hz),5.98(1H s),5.58(1H s),4.43(1H t,J=6.64Hz),4.25(1H t,J=4.53Hz),4.14(4H t,J=6.64Hz),3.93(3H s),3.90(6H s),3.86(2H s),3.06-3.12(1H m),3.02(2Ht,J=11.46Hz),2.82-2.86(1H m),2.73-2.79(4H m),2.67-2.70(1H m),2.29(2H t,J=7.54Hz),2.02(2H t,J=3.92Hz),1.97(3H s), 1.68 (2H s), 1.66 (3H d, J = 2.72Hz), 1.62 (2H d, J = 4.52Hz), 1.40 (2H t, J = 7.54Hz). 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). 13C NMR(150MHz, CDCl3)δ=184.49,172.41,162.64,157.09,154.43,151.71(2C),141.44,1 39.02,138.29,136.45,132.95,131.79,128.35(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),54.45,52.4 5,47.41,47.06,39.52,32.77,30.86,29.60,29.37,28.89,28.66,27.33,23.71,19.95.

[0097] Example 9: In vitro antitumor activity test of the compounds involved in this invention

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

[0099] Screening method: MTT (microtetrozolium) reduction method

[0100] Cell lines: MCF-7 (human breast cancer), SGC-7901 (human gastric adenocarcinoma), and A949 (human non-small cell lung cancer).

[0101] Duration of action: 72 hours

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

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

[0104] Table 1

[0105]

[0106] Example 10: ROS responsiveness of CBSI prodrugs

[0107] Accurately weigh 1.0 mg of compound 4 and dissolve it completely in an appropriate amount of acetonitrile. Slowly add the prepared acetonitrile solution of prodrug compound 4 to PBS buffer (pH 7.2-7.4) to achieve a CBSIs prodrug concentration of 5 μM, ensuring no solid precipitation. Add 30% H2O2 aqueous solution to the PBS buffer (pH 7.2-7.4) solution of the CBSIs prodrug to achieve H2O2 concentrations of 10 mM and 100 mM, respectively, and add one drop of FeSO4 aqueous solution. Incubate the prepared mixed solutions in a constant temperature water bath at 37°C for 3 hours, and take samples every 30 minutes to determine the changes in solution composition using HPLC.

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

[0109] Example 11: Preparation of Nanomedicines

[0110] A mixed solution was prepared by dissolving 6 mg of the general formula compound prepared in the above examples, 1.2 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 0.6 mg of egg yolk lecithin in 1 ml of ethanol. The general formula compound prepared in the above examples can be the product prepared in Examples 1-8.

[0111] This embodiment uses the compound prepared in Example 4 (compound 4). The mixture was then stirred vigorously at 1200 rpm and then added to 4 ml of deionized water at 1000 rpm. After the reaction was completed, the organic solvent was evaporated, and the nanomedicine (4NPs) was self-assembled and stored at 4°C in the dark for later use.

[0112] For the characterization of the obtained nanomedicines, see [link to documentation]. Figure 10 and Figure 11 ,

[0113] The particle size and zeta potential of nanoparticles were measured using a Bruke particle size and zeta potential analyzer based on the principle of dynamic light scattering. The prepared CBSI prodrug nanoparticles were diluted with ultrapure water to a suitable concentration (0.5 mg / mL) and placed in the nanoparticle size analyzer to measure the light intensity distribution. The results showed that the CBSI prodrug nanoparticles had a particle size of approximately 130 nm, a PDI of 0.082, and a zeta potential of -26.2 mV.

[0114] Example 12: Study on the antitumor activity of the compounds involved in this invention in animals

[0115] The nanomedicine of compound 4 prepared in the above embodiments was used to test its antitumor activity in animals. The model used was the mouse S-180 sarcoma model, and the positive control drug was fluorouracil (5-Fu), a commonly used antitumor drug in clinical practice.

[0116] Experimental methods: Female Kunming mice weighing 18-22 grams and well-developed S-180 tumor cells aged 7-11 days were selected. The tumor tissue was prepared into a cell suspension and inoculated subcutaneously into the right axilla of the mice, at a dose of approximately 1.0-2.0 × 10⁶ cells / day. 6 Cells / animal, randomly divided into cages 24 hours after inoculation, and administered the drug via tail vein injection for 7 consecutive days. Animals were sacrificed 24 hours after drug withdrawal, and their body weight and tumor weight were measured. The average tumor weight of each group was calculated, and the tumor inhibition rate was calculated using the following formula and a t-test was performed.

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

[0118] The experimental results are shown in Table 2.

[0119] Table 2

[0120]

[0121] As shown in Table 2, the tumor inhibition rate of the nanomedicine of compound 4 was 66.5%, which was comparable to that of the positive control fluorouracil. However, the mice in the positive control fluorouracil group gained weight slowly, while the mice in the compound 4 nanomedicine group had a weight comparable to that of the blank control group at the end of the treatment, demonstrating better safety.

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

[0123] The nanomedicine containing compound 4 obtained above was selected for in vivo anti-inflammatory activity testing in animals. The model used was an acute inflammation model of mice induced by phorbol ester (TPA), and the positive control drug was indomethacin, a commonly used anti-inflammatory drug in clinical practice.

[0124] Experimental Methods: Forty female Kunming mice weighing 18-22 grams were randomly divided into groups of 10 each. Each group received either a tail vein injection of compound 4 nanomedicine (3 mg / kg), a control (physiological saline), or an intraperitoneal injection of indomethacin (3 mg / kg, positive control). Thirty minutes later, TPA (2.5 μg / ear, 20 μL acetone) was applied topically to both sides of the right ear of each mouse to induce edema. Six hours after inflammation, the mice were euthanized by cervical dislocation. Ear flaps were then punched symmetrically from the left and right ears using a 7 mm diameter punch. The flaps were weighed, recorded, and the degree of swelling and swelling inhibition rate were calculated.

[0125] Swelling degree = Average mass of right earpiece - Average mass of left earpiece

[0126] Swelling inhibition rate = [(swelling degree in blank group - swelling degree in treatment group) / (swelling degree in 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 and biotin with a ROS-responsive thioacetate chain. The prodrug compound is the compound shown in Formula 1 and its salt. In Formula 1, R 1 , R 2 , R 3 , R 4 , R 5 may be the same or different and are independently hydrogen, C1-C6alkyl, a halogen atom, a hydroxyl group, or an amino group; R 6 is hydrogen, C1-C6alkyl; n and m are each independently 2; R 7 , R 8 may be the same or different and are independently hydrogen, C1-C6alkyl, phenyl unsubstituted or substituted by at least one halogen or C1-C3alkyl, and, R 7 , R 8 are not simultaneously hydrogen; or, R 7 or, R 8 with the adjacent C forms a five-, six-, seven- or eight-membered saturated carbocyclic ring.

2. The prodrug compound according to claim 1, characterized in that: The prodrug compound is the compound shown in Formula 1 and its salt. In Equation 1, R 1 R 2 R 3 R 4 R 5 They may be hydrogen, C1-C3 alkyl, halogen atom, hydroxyl or amino, either the same or different; R 6 It is hydrogen or C1-C3 alkyl; n and m are each independently 2; R 7 R 8 It may be the same or different independently of hydrogen, C1-C3 alkyl, unsubstituted or substituted with at least one halogen or C1-C3 alkyl, and R 7 R 8 They are not both hydrogen; Or, R 7 R 8 It forms a five-, six-, or seven-membered saturated carbon ring with the adjacent C.

3. The prodrug compound according to claim 2, characterized in that: The prodrug compound is the compound shown in Formula 1 and its salt. In Equation 1, R 1 R 2 R 3 R 4 R 5 They can be hydrogen, amino, hydroxyl, fluorine, chlorine, or bromine, either the same or different; R 6 It can be hydrogen, methyl, or ethyl. n and m are each independently 2; R 7 R 8 It may be the same or different independently of hydrogen, C1-C3 alkyl, unsubstituted or substituted with at least one halogen or C1-C3 alkyl, and R 7 R 8 They are not both hydrogen; Or, R 7 R 8 It forms a five-, six-, or seven-membered saturated carbon ring with the adjacent C.

4. The prodrug compound according to any one of claims 1-3, characterized in that: The salt of the compound shown in Formula 1 is a salt formed by the compound shown in Formula 1 with an acid or base; wherein the acid is hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, or malic acid; and the base is sodium hydroxide, sodium carbonate, or potassium hydroxide.

5. A method for preparing a colchicine binding site inhibitor prodrug compound according to claim 1, characterized in that: 。 6. The method for preparing the colchicine binding site inhibitor prodrug compound according to claim 5, characterized in that: Step 1: Mix carbonyl compound I and mercaptoacetic acid in a molar ratio of 1:2~4. Add trifluoroacetic acid dropwise to the mixture while stirring. React at 15~40℃ for 0.5~12 h to obtain compound II. Step 2: Compound II, sodium borohydride and iodine in a molar ratio of 1:1~6:2~6 are dissolved in tetrahydrofuran at a temperature of -20~0℃ and refluxed for 12~24 h to obtain compound III; Step 3: Under nitrogen protection, compound III and triethylamine were dissolved in anhydrous tetrahydrofuran. The tetrahydrofuran solution of p-nitrophenyl chloroformate was added to the mixed solution of compound III and triethylamine. The reaction was carried out at 0~30℃ for 2~6 h to obtain compound VI. The molar ratio of compound III, triethylamine, and p-nitrophenyl chloroformate is 1:1~1.2:1~1.2; Step 4: Under nitrogen protection, compound VI reacts with compound V dissolved in tetrahydrofuran at 0~30℃ for 12~24 h to obtain compound IV; The molar ratio of compound VI to compound V is 1:1~2; Step 5: Dissolve compound VI, biotin, DMAP, and DCC in DMF in a molar ratio of 1:1~2:1~4:1~4 and react at 15~45℃ to obtain compound 1.

7. A nanomedicine, characterized in that, The nanomedicine is prepared from the prodrug compound described in claims 1 to 5 by a nanoprecipitation method.

8. The method for preparing nanomedicine according to claim 7, characterized in that: The prodrug compound of any one of claims 1-6, distearate phosphatidylethanolamine-polyethylene glycol 2000 and egg yolk lecithin are dissolved in an organic solvent to obtain a mixed solution. Then, the mixed solution is added to deionized water under stirring. After the reaction is completed, the organic solvent is removed, and the prodrug compound nanomedicine is self-assembled.

9. The application of a prodrug compound or nanomedicine as described in claim 1 or 7, characterized in that, The application of the prodrug compounds and nanomedicines in the preparation of antitumor drugs or anti-inflammatory drugs; The tumors mentioned are breast cancer, gastric adenocarcinoma, non-small cell lung cancer, and sarcoma.

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