A small molecule ligand-rucaparib conjugate and its preparation method

Abstract

The present invention belongs to the field of biomedicine technology and specifically relates to a small molecule ligand-rucaparib conjugate and a preparation method thereof. The small molecule ligand-rucaparib conjugate is a combination of one or both of a biotin-rucaparib conjugate and a lactobionic acid-rucaparib conjugate. Rucaparib is coupled to the small molecule ligand through an amidation reaction, thereby improving rucaparib's tumor targeting ability and thereby enhancing its inhibitory effect on tumor cells. In particular, the dual-targeting effect of biotin / lactobionic acid-rucaparib composite nanoparticles, which are self-assembled from the small molecule ligand complex, is more significantly inhibited against double-positive tumor cells such as biotin receptors and asialoglycoprotein receptors. Furthermore, the biotin / lactobionic acid-rucaparib composite nanoparticles have a significant application prospect due to their safety advantage over normal cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a small molecule ligand-rucaparib conjugate and a preparation method thereof. Background Art

[0002] The use of poly (ADP-ribose) polymerase (PARP) inhibitors is one of the most dynamic targeted therapies and is currently approved for the treatment of a range of tumor types. Rucaparib is an approved PARP inhibitor that blocks the repair of damaged DNA in cancer cells, leading to cancer cell death. However, patients taking rucaparib often experience adverse reactions such as nausea, fatigue, vomiting, anemia, abdominal pain, taste disturbances, constipation, decreased appetite, diarrhea, thrombocytopenia, and asthma. Laboratory abnormalities are common (≥35%), including increased creatinine, increased alanine aminotransferase, increased aspartate aminotransferase, decreased hemoglobin, increased lymphocytes and cholesterol, decreased platelets, and decreased neutrophils. These adverse reactions are mainly caused by the effects of rucaparib on normal tissues and cells. How to improve the targeting of rucaparib to tumor cells is a technical problem that urgently needs to be solved. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a small molecule ligand-rucaparib conjugate, which is a biotin-rucaparib conjugate and lactobionate-rucaparib conjugate By coupling rucaparib with a small molecule ligand, the tumor targeting of rucaparib is improved, thereby enhancing the inhibitory effect on tumor cells.

[0004] The present invention also provides a method for preparing the above-mentioned small molecule ligand-rucaparib conjugate:

[0005] The preparation method of the biotin-rucaparib conjugate comprises the following steps: adding biotin and rucaparib to a solvent at a molar ratio of 1 to 1.3:1, and simultaneously adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and p-dimethylaminopyridine, fully dissolving them, and reacting them by stirring at 40° C. to 50° C., adding the resulting reaction system to an organic solvent for precipitation, filtering, washing the resulting filter cake with an organic solvent, and fully dispersing it in ultrapure water, filtering the filter cake again, washing it with ultrapure water, and vacuum drying it to obtain the biotin-rucaparib conjugate;

[0006] The preparation method of the lactobionic acid-rucaparib conjugate comprises: adding lactobionic acid and rucaparib in a molar ratio of 1 to 1.3:1 to a solvent, and simultaneously adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and p-dimethylaminopyridine, fully dissolving and reacting with stirring at 40° C. to 50° C., adding the resulting reaction system to an organic solvent for precipitation, filtering, washing the resulting filter cake, and purifying by column chromatography to obtain the lactobionic acid-rucaparib conjugate;

[0007] The preparation method of the combination of a biotin-rucaparib conjugate and a lactobionic acid-rucaparib conjugate comprises dissolving the biotin-rucaparib conjugate and the lactobionic acid-rucaparib conjugate in an organic solvent to obtain a dispersed system of the conjugate, adding the dispersed system of the conjugate dropwise into ultrapure water under ultrasonication, continuing ultrasonication for a period of time after the dropwise addition is completed, and then dialyzing the obtained ultrasonic system in ultrapure water, and freeze-drying the dialyzed ultrasonic system to obtain biotin / lactobionic acid-rucaparib composite nanoparticles.

[0008] Preferably, in the method for preparing the biotin-rucaparib conjugate and the method for preparing the lactobionic acid-rucaparib conjugate, the solvent is one or a combination of N,N-dimethylformamide and dimethyl sulfoxide.

[0009] Preferably, in the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added at a molar ratio of 2 to 8:1 to rucaparib.

[0010] Preferably, in the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, dimethylaminopyridine is added at a molar ratio of 0.1 to 0.5:1 to rucaparib.

[0011] Preferably, in the method for preparing the biotin-rucaparib conjugate and the method for preparing the lactobionic acid-rucaparib conjugate, the stirring reaction time is 12 to 48 hours.

[0012] Preferably, in the method for preparing the biotin-rucaparib conjugate and the method for preparing the lactobionic acid-rucaparib conjugate, the organic solvent is a mixture of ether and alcohol in a volume ratio of 1 to 9:1.

[0013] Furthermore, the alcohol is one or a combination of isopropyl alcohol and ethanol.

[0014] Preferably, in the preparation method of the combination of biotin-rucaparib conjugate and lactobionic acid-rucaparib conjugate, the organic solvent is dimethyl sulfoxide, and the dispersed system of the conjugate is added dropwise into ultrapure water with a volume of 5 to 20 times that of dimethyl sulfoxide.

[0015] The present invention couples rucaparib with a small molecule ligand through an amidation reaction, which can enhance rucaparib's tumor targeting and thus improve its inhibitory effect on tumor cells. It has potential application value in the treatment of biotin receptor-positive and asialoglycoprotein receptor-positive tumors. DETAILED DESCRIPTION

[0016] The preparation method of the biotin-rucaparib conjugate comprises: adding biotin and rucaparib at a molar ratio of 1 to 1.3:1 to a solvent, wherein the solvent is one or a combination of N,N-dimethylformamide and dimethyl sulfoxide; simultaneously adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a molar ratio of 2 to 8:1 to rucaparib; and adding p-dimethylaminopyridine at a molar ratio of 0.1 to 0.5:1 to rucaparib; fully stirring to dissolve; and stirring at 40° C. to 50° C. for 12 to 48 hours. The resulting reaction system is added to a mixed solvent of ether / isopropanol (1 to 9:1, v / v) for precipitation, filtering, washing the resulting filter cake with the mixed solvent, and then fully dispersing it in ultrapure water. The filter cake is filtered again, washed with ultrapure water, and vacuum dried to obtain the biotin-rucaparib conjugate.

[0017] The preparation method of the lactobionic acid-rucaparib conjugate comprises: adding lactobionic acid and rucaparib in a molar ratio of 1 to 1.3:1 to a solvent, wherein the solvent is one or a combination of N,N-dimethylformamide and dimethyl sulfoxide; simultaneously, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a molar ratio of 2 to 8:1 to rucaparib; and adding p-dimethylaminopyridine in a molar ratio of 0.1 to 0.5:1 to rucaparib; stirring and dissolving the mixture, and reacting the mixture at 40° C. to 50° C. for 12 to 48 hours; then, adding the obtained reaction system to a mixed solvent of ether / ethanol (1 to 9:1, v / v) for precipitation, filtering, washing the obtained filter cake with ether, and purifying the mixture by column chromatography to obtain the lactobionic acid-rucaparib conjugate;

[0018] The preparation method of the combination of biotin-rucaparib conjugate and lactobionic acid-rucaparib conjugate is as follows: dissolving the biotin-rucaparib conjugate and the lactobionic acid-rucaparib conjugate in dimethyl sulfoxide at a molar ratio of 1:1 to obtain a dispersed system of the conjugate; adding the dispersed system of the conjugate dropwise into ultrapure water with a volume of 5 to 20 times that of dimethyl sulfoxide under ultrasonication; continuing ultrasonication for 3 minutes after the dropwise addition is completed; then dialyzing the obtained ultrasonic system in ultrapure water for 48 hours; and freeze-drying the dialyzed ultrasonic system to obtain biotin / lactobionic acid-rucaparib composite nanoparticles.

[0019] Example 1

[0020] Preparation of biotin-rucaparib conjugate

[0021] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred for dissolution. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature (25°C, the same below) and added to 50 mL of a 1:1, v / v diethyl ether / isopropanol mixed solvent for precipitation. The mixture was filtered and the filter cake was washed with a 1:1, v / v diethyl ether / isopropanol mixed solvent (3 × 20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield (i.e., conversion of rucaparib, the same below) 87%, purity 96%).

[0022] 1 H NMR (300MHz, DMSO) δ11.72(d,J=9.8Hz,1H),8.26(s,1H),7.63(dd,J=15.9,7.9H z,2H),7.48–7.24(m,4H),6.59–6.22(m,2H),4.61(d,J=25.5Hz,2H),4.30(d,J=4 .7Hz,1H),4.14(d,J=6.8Hz,1H),3.05(s,4H),2.97(s,2H),2.85(s,2H),2.58(d d,J=12.3,6.7Hz,1H),2.39(dd,J=15.3,7.9Hz,2H),1.70–1.27(m,6H).HRMS:m / z calculatedfor C 29 H 32 FN5O3S 550.2288([M+H] + ),found550.228.

[0023] Example 2

[0024] Preparation of biotin-rucaparib conjugate

[0025] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (383 mg, 2 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 67%, purity 92%).

[0026] Example 3

[0027] Preparation of biotin-rucaparib conjugate

[0028] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.53 g, 8 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 90%, purity 94%).

[0029] Example 4

[0030] Preparation of biotin-rucaparib conjugate

[0031] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (12 mg, 0.1 mmol) were added thereto and stirred for dissolution. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the resulting filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 82%, purity 94%).

[0032] Example 5

[0033] Preparation of biotin-rucaparib conjugate

[0034] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (49 mg, 0.4 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 90%, purity 95%).

[0035] Example 6

[0036] Preparation of biotin-rucaparib conjugate

[0037] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of dimethyl sulfoxide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 84%, purity 93%).

[0038] Example 7

[0039] Preparation of biotin-rucaparib conjugate

[0040] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (2:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (2:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 91%, purity 87%).

[0041] Example 8

[0042] Preparation of biotin-rucaparib conjugate

[0043] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / ethanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / ethanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 84%, purity 92%).

[0044] Example 9

[0045] Preparation of biotin-rucaparib conjugate

[0046] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 12 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 75%, purity 94%).

[0047] Example 10

[0048] Preparation of biotin-rucaparib conjugate

[0049] Biotin (1 mmol, 244 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (767 mg, 4 mmol) and p-dimethylaminopyridine (24 mg, 0.2 mmol) were added thereto and stirred to dissolve. The mixture was heated to 40°C and stirred for 48 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / isopropanol (1:1, v / v) for precipitation. The mixture was filtered and the filter cake was washed with a mixed solvent of ether / isopropanol (1:1, v / v) (3×20 mL). The filter cake was then dispersed in 20 mL of ultrapure water and stirred for 30 min. The filter cake was filtered again and washed with ultrapure water and vacuum dried to obtain a light yellow powdery solid (yield 88%, purity 92%).

[0050] Example 11

[0051] Preparation of lactobionic acid-rucaparib conjugate

[0052] Lactobionic acid (1 mmol, 358 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (6 mmol, 1.15 g) and p-dimethylaminopyridine (0.5 mmol, 61 mg) were added thereto and stirred to dissolve. The mixture was heated to 50 ° C. and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / ethanol (9:1, v / v) for precipitation. The mixture was filtered and the resulting filter cake was washed with ether (3×20 mL). The filter cake was then purified by column chromatography to obtain a light yellow powdery solid (purity 91%, yield 73%).

[0053] 1 H NMR(300MHz,DMSO)δ11.97(s,1H),8.30(s,1H),7.52-8.00(m,3H),7.43(d,J=11.5Hz,2H ),6.14(d,J=36.2Hz,1H),4.12-5.53(m,11H),2.82-3.97(m.17H),2.73(m,2H).HRMS:m / z of[M+H] + calculated for C31H38FN3O12 664.2518,found664.2512.

[0054] Example 12

[0055] Preparation of lactobionic acid-rucaparib conjugate

[0056] Lactobionic acid (1 mmol, 358 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3 mmol, 575 mg) and p-dimethylaminopyridine (0.5 mmol, 61 mg) were added thereto and stirred to dissolve. The mixture was heated to 50 ° C. and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / ethanol (9:1, v / v) for precipitation. The mixture was filtered and the resulting filter cake was washed with ether (3×20 mL). The filter cake was then purified by column chromatography to obtain a light yellow powdery solid (purity 88%, yield 59%).

[0057] Example 13

[0058] Preparation of lactobionic acid-rucaparib conjugate

[0059] Lactobionic acid (1 mmol, 358 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3 mmol, 575 mg) and p-dimethylaminopyridine (0.2 mmol, 24 mg) were added thereto and stirred to dissolve. The mixture was heated to 50 ° C. and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / ethanol (9:1, v / v) for precipitation. The mixture was filtered and the resulting filter cake was washed with ether (3×20 mL). The filter cake was then purified by column chromatography to obtain a light yellow powdery solid (purity 90%, yield 51%).

[0060] Example 14

[0061] Preparation of lactobionic acid-rucaparib conjugate

[0062] Lactobionic acid (1 mmol, 358 mg) and rucaparib (1 mmol, 323 mg) were dissolved in 5 mL of N,N-dimethylformamide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3 mmol, 575 mg) and p-dimethylaminopyridine (0.5 mmol, 61 mg) were added thereto and stirred to dissolve. The mixture was heated to 50 ° C. and stirred for 24 h. The reaction system was then cooled to room temperature and added to 50 mL of a mixed solvent of ether / ethanol (5:1, v / v) for precipitation. The mixture was filtered and the resulting filter cake was washed with ether (3×20 mL). The filter cake was then purified by column chromatography to obtain a light yellow powdery solid (purity 87%, yield 38%).

[0063] Example 15

[0064] Preparation of biotin-rucaparib nanoparticles

[0065] The biotin-rucaparib conjugate prepared in Example 1 (219.6 mg, 0.4 mmol) was dissolved in 6 mL of dimethyl sulfoxide to obtain a dispersed conjugate. The dispersed conjugate was then added dropwise to 60 mL of ultrapure water under ultrasound. After the addition was complete, ultrasound was continued for 3 minutes. The resulting ultrasound system was then dialyzed in excess ultrapure water for 48 hours. The dialyzed ultrasound system was freeze-dried to obtain biotin-rucaparib nanoparticles.

[0066] Example 16

[0067] Preparation of lactobionic acid-rucaparib nanoparticles

[0068] The lactobionic acid-rucaparib conjugate (265.2 mg, 0.4 mmol) prepared in Example 11 was dissolved in 6 mL of dimethyl sulfoxide to obtain a dispersed conjugate. The dispersed conjugate was then added dropwise to 60 mL of ultrapure water under ultrasound. After the addition was complete, ultrasound was continued for 3 minutes. The resulting ultrasonic system was then dialyzed in excess ultrapure water for 48 hours. The dialyzed ultrasonic system was freeze-dried to obtain lactobionic acid-rucaparib nanoparticles.

[0069] Example 17

[0070] Preparation of biotin / lactobionic acid-rucaparib composite nanoparticles

[0071] The biotin-rucaparib conjugate (109.8 mg, 0.2 mmol) prepared in Example 1 and the lactobionic acid-rucaparib conjugate (132.6 mg, 0.2 mmol) prepared in Example 11 were dissolved in 6 mL of dimethyl sulfoxide to obtain a dispersed conjugate system. The dispersed conjugate system was then added dropwise to 60 mL of ultrapure water under ultrasonication. After the addition was complete, ultrasonication was continued for 3 minutes. The obtained ultrasonic system was then dialyzed in excess ultrapure water for 48 hours. The dialyzed ultrasonic system was freeze-dried to obtain biotin / lactobionic acid-rucaparib composite nanoparticles.

[0072] NIH3T3 cell inhibition assay

[0073] Preparation of test solutions: The biotin-rucaparib nanoparticles prepared in Example 15, the lactobionic acid-rucaparib nanoparticles prepared in Example 16, and the biotin / lactobionic acid-rucaparib composite nanoparticles prepared in Example 17 were dispersed in ultrapure water, respectively. Free rucaparib was dissolved in dimethyl sulfoxide to prepare respective drug stock solutions containing 4 mM rucaparib. These were then diluted with culture medium to obtain test solutions of various drug concentrations.

[0074] CCK8 assay: NIH3T3 cells in the logarithmic phase were digested with trypsin and collected by centrifugation after termination to prepare a cell suspension. The cell count was adjusted to a concentration of 10 × 10 4 / ml. After the cell suspension is prepared, mix gently, add 100uL to each well, and fill the edge wells with sterile PBS. Place the inoculated cell culture plate in an incubator and culture until the cell monolayer covers the bottom of the well (96-well flat-bottom plate). Add different concentrations of each drug test solution, culture for different times, add 10uL CCK8 to each well, and continue to culture for 4 hours. Measure the absorbance of each well at OD450nm using an enzyme-linked immunosorbent assay, set the cell inhibition rate of blank culture medium to 0%, and calculate the cell inhibition rate of each drug at different concentrations.

[0075] The cell inhibition rates of biotin-rucaparib nanoparticles, lactobionic acid-rucaparib nanoparticles, biotin / lactobionic acid-rucaparib composite nanoparticles, and free rucaparib were determined on biotin receptor-negative and asialoglycoprotein receptor-negative NIH3T3 fibroblasts, as shown in Tables 1 and 2.

[0076] Table 1. Cell inhibition rate after 24 hours of culture

[0077]

[0078] Table 2. Inhibition rate of cells cultured for 48 hours

[0079]

[0080]

[0081] As shown in the table above, at 24 and 48 hours of culture, the cell inhibition rates of biotin-rucaparib nanoparticles, lactobionic acid-rucaparib nanoparticles, and biotin / lactobionic acid-rucaparib composite nanoparticles were all lower than those of free rucaparib. These results indicate that the inhibitory effects of biotin-rucaparib conjugates, lactobionic acid-rucaparib conjugates, and biotin / lactobionic acid-rucaparib composite nanoparticles on normal cells were lower than those of free rucaparib.

[0082] Furthermore, compared with the nanoparticles self-assembled from either the biotin-rucaprib conjugate or the lactobionic acid-rucaprib conjugate alone, the biotin / lactobionic acid-rucaprib composite nanoparticles have a lower degree of inhibition on normal cells. This is probably because during the mixed self-assembly process of the biotin-rucaprib conjugate and the lactobionic acid-rucaprib conjugate, better mutual binding occurs between the biotin segments and the lactobionic acid segments, which further reduces the overall activity of the self-assembled composite nanoparticles and thereby reduces the uptake by normal cells.

[0083] Hela cell inhibition assay

[0084] Preparation of test solutions: The biotin-rucaparib nanoparticles prepared in Example 15 and the biotin / lactobionic acid-rucaparib composite nanoparticles prepared in Example 17 were separately dispersed in ultrapure water, and free rucaparib was dissolved in dimethyl sulfoxide to prepare respective drug stock solutions containing 4 mM rucaparib. These were then diluted with culture medium to obtain test solutions of various drug concentrations.

[0085] CCK8 assay: Hela cells in the logarithmic phase were digested with trypsin and collected by centrifugation after termination to prepare a cell suspension. The cell count was adjusted to a concentration of 10 × 10 4 / ml. After the cell suspension is prepared, mix gently, add 100uL to each well, and fill the edge wells with sterile PBS. Place the inoculated cell culture plate in an incubator and culture until the cell monolayer covers the bottom of the well (96-well flat-bottom plate). Add different concentrations of each drug test solution, culture for different times, add 10uL CCK8 to each well, and continue to culture for 4 hours. Measure the absorbance of each well at OD450nm using an enzyme-linked immunosorbent assay, set the cell inhibition rate of blank culture medium to 0%, and calculate the cell inhibition rate of each drug at different concentrations.

[0086] The cell inhibition rates of biotin-rucaparib nanoparticles, biotin / lactose-rucaparib composite nanoparticles, and free rucaparib were determined on biotin receptor-positive ovarian cancer Hela cells, as shown in Tables 3 and 4.

[0087] Table 3. Cell inhibition rate after 24 hours of culture

[0088]

[0089] Table 4. Inhibition rate of cells cultured for 48 hours

[0090]

[0091] As shown in the table above, after 24 hours of culture, at low concentrations, the cell inhibition rates of biotin-rucaparib nanoparticles and biotin / lactobionic acid-rucaparib composite nanoparticles were comparable to those of free rucaparib. At concentrations above 8 μmol / L, the cell inhibition rates of biotin-rucaparib nanoparticles and biotin / lactobionic acid-rucaparib composite nanoparticles were significantly higher than those of free rucaparib. As shown in Table 4, after 48 hours of culture, within the measured concentration range, the cell inhibition rates of biotin-rucaparib nanoparticles and biotin / lactobionic acid-rucaparib composite nanoparticles were significantly higher than those of free rucaparib. These results indicate that biotin-rucaparib conjugates and biotin / lactobionic acid-rucaparib composite nanoparticles can significantly enhance the inhibitory effect on biotin receptor-positive tumor cells.

[0092] Furthermore, the table above reveals that while biotin, as a targeting small molecule ligand, enhances rucaparib's targeting and thus its inhibitory effect on HeLa ovarian cancer cells after being conjugated to rucaparib, a comparison of biotin-rucaparib nanoparticles and biotin / lactobionic acid-rucaparib composite nanoparticles reveals that, at the same rucaparib molar concentration, the molar amount of biotin in the biotin / lactobionic acid-rucaparib composite nanoparticles is roughly half that of the biotin-rucaparib nanoparticles. However, the cancer cell inhibitory effect of the biotin / lactobionic acid-rucaparib composite nanoparticles is only slightly lower than that of the biotin-rucaparib nanoparticles, with no significant difference. This is because the molecular structures of the two conjugates in this scheme have a hydrophobic end at rucaparib and a hydrophilic end at biotin or lactobionic acid. Therefore, both conjugates have an ideal self-assembly effect. After mixing and self-assembling, the biotin / lactobionic acid is located on the surface of the nanoparticles. The presence of biotin makes the entire colloid targeted to ovarian cancer HeLa cells, thus also endowing the lactobionic acid and rucaparib conjugate in the colloid with this targeting effect. Therefore, the targeting properties reflected in Tables 3 and 4 are the targeting properties of the self-assembled colloids, not the individual conjugate molecules.

[0093] HepG2 cell inhibition assay

[0094] Preparation of test solutions: The biotin-rucaparib nanoparticles prepared in Example 15, the lactobionic acid-rucaparib nanoparticles prepared in Example 16, and the biotin / lactobionic acid-rucaparib composite nanoparticles prepared in Example 17 were dispersed in ultrapure water, respectively. Free rucaparib was dissolved in dimethyl sulfoxide to prepare respective drug stock solutions containing 4 mM rucaparib. These were then diluted with culture medium to obtain test solutions of various drug concentrations.

[0095] CCK8 assay: HepG2 cells in the logarithmic phase were digested with trypsin and collected by centrifugation after termination. Cell suspension was prepared and the cell count was adjusted to a concentration of 10 × 10 4 / ml. After the cell suspension is prepared, mix gently, add 100uL to each well, and fill the edge wells with sterile PBS. Place the inoculated cell culture plate in an incubator and culture until the cell monolayer covers the bottom of the well (96-well flat-bottom plate). Add different concentrations of each drug test solution, culture for different times, add 10uL CCK8 to each well, and continue to culture for 4 hours. Measure the absorbance of each well at OD450nm using an enzyme-linked immunosorbent assay, set the cell inhibition rate of blank culture medium to 0%, and calculate the cell inhibition rate of each drug at different concentrations.

[0096] The cell inhibition rates of biotin-rucaparib nanoparticles, lactobionic acid-rucaparib nanoparticles, biotin / lactobionic acid-rucaparib composite nanoparticles, and free rucaparib were determined on HepG2 cells, which are double positive for biotin receptor and asialoglycoprotein receptor, as shown in Tables 5 and 6.

[0097] Table 5. Cell inhibition rate after 24 hours of culture

[0098]

[0099] Table 6. Cell inhibition rate after 48 hours of culture

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[0102] As shown in the table above, after 24 hours of culture, the cell inhibition rates of biotin-rucapab nanoparticles, lactobionic acid-rucapab nanoparticles, and biotin / lactobionic acid-rucapab composite nanoparticles were slightly higher than those of free rucapab, with the biotin / lactobionic acid-rucapab composite nanoparticles having the highest cell inhibition rate. As shown in Table 6, after 48 hours of culture, the cell inhibition rates of biotin-rucapab nanoparticles, lactobionic acid-rucapab nanoparticles, and biotin / lactobionic acid-rucapab composite nanoparticles were significantly higher than those of free rucapab, with the biotin / lactobionic acid-rucapab composite nanoparticles having the highest cell inhibition rate. These results indicate that biotin / lactobionic acid-rucapab composite nanoparticles significantly enhance the inhibitory effect against biotin receptor and asialoglycoprotein receptor double-positive tumor cells, primarily due to the dual targeting effect of biotin / lactobionic acid-rucapab composite nanoparticles.

[0103] Combined with the safety advantages of biotin / lactobionic acid-rucaparib composite nanoparticles on normal cells as reflected in Tables 1 and 2 above, biotin / lactobionic acid-rucaparib composite nanoparticles have considerable application prospects.

Claims

1. A small molecule ligand-rucaparib conjugate, characterized in that: The small molecule ligand-rucaparib conjugate is a biotin-rucaparib conjugate and lactobionic acid-rucaparib conjugate One or a combination of two.

2. A method for preparing the small molecule ligand-rucaparib conjugate according to claim 1, characterized in that: The preparation method of the biotin-rucaparib conjugate comprises: adding biotin and rucaparib in a molar ratio of 1 to 1.3:1 to a solvent, and simultaneously adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and p-dimethylaminopyridine, fully dissolving them, and stirring and reacting at 40° C. to 50° C., adding the resulting reaction system to an organic solvent for precipitation, filtering, washing the resulting filter cake with an organic solvent, and fully dispersing it in ultrapure water, filtering the filter cake again, washing it with ultrapure water, and vacuum drying, thereby obtaining the biotin-rucaparib conjugate; The preparation method of the lactobionic acid-rucaparib conjugate comprises adding lactobionic acid and rucaparib in a molar ratio of 1 to 1.3:1 to a solvent, and simultaneously adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and p-dimethylaminopyridine, fully dissolving and stirring at 40° C. to 50° C. to react, adding the resulting reaction system to an organic solvent for precipitation, filtering, washing the resulting filter cake, and purifying by column chromatography to obtain the lactobionic acid-rucaparib conjugate; The preparation method of the combination of the biotin-rucaprib conjugate and the lactobionic acid-rucaprib conjugate is as follows: dissolving the biotin-rucaprib conjugate and the lactobionic acid-rucaprib conjugate in an organic solvent to obtain a dispersed system of the conjugate; adding the dispersed system of the conjugate dropwise into ultrapure water under ultrasonication; continuing ultrasonication for a period of time after the dropwise addition is completed; then dialyzing the obtained ultrasonic system in ultrapure water; and freeze-drying the dialyzed ultrasonic system to obtain biotin / lactobionic acid-rucaprib composite nanoparticles.

3. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, the solvent is one or a combination of N,N-dimethylformamide and dimethyl sulfoxide.

4. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added at a molar ratio of 2 to 8:1 to the rucaparib.

5. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, the p-dimethylaminopyridine is added at a molar ratio of 0.1 to 0.5:1 to the rucaparib.

6. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the method for preparing the biotin-rucaparib conjugate and the method for preparing the lactobionic acid-rucaparib conjugate, the stirring reaction time is 12 to 48 hours.

7. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the preparation method of the biotin-rucaparib conjugate and the preparation method of the lactobionic acid-rucaparib conjugate, the organic solvent is prepared by mixing ether and alcohol in a volume ratio of 1 to 9:

1.

8. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 7, wherein: The alcohol is one or a combination of isopropyl alcohol and ethanol.

9. The method for preparing the small molecule ligand-rucaparib conjugate according to claim 2, wherein: In the preparation method of the combination of the biotin-rucaparib conjugate and the lactobionic acid-rucaparib conjugate, the organic solvent is dimethyl sulfoxide, and the dispersed system of the conjugate is added dropwise into ultrapure water with a volume 5 to 20 times that of the dimethyl sulfoxide.

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