Preparation and application of biotin-conjugated dihydroartemisinin

Abstract

The application provides a preparation method and application of a biotin-dihydroartemisinin conjugate, and relates to the field of cancer treatment. The application esterifies -COOH of biotin and -OH of dihydroartemisinin through an esterification reaction, or reacts -COOH of biotin with -NH2 or -OH of a linker, and then esterifies -OH of dihydroartemisinin, so as to prepare several small-molecule prodrug conjugates. The application has mild reaction conditions, the prepared small-molecule conjugate enhances the targeting property of dihydroartemisinin, enhances the uptake rate of tumor cells to dihydroartemisinin, and improves the toxicity of dihydroartemisinin to tumor cells, so that the application has a wide application prospect in the field of anti-tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of anticancer pharmaceuticals and chemicals, specifically to the preparation and application of biotin-dihydroartemisinin conjugates. Background Technology

[0002] Dihydroartemisinin (structural formula A) is an active ingredient extracted from the traditional Chinese medicine Artemisia annua. In the late 1970s and early 1980s, Chinese scientist Tu Youyou and her research team successfully isolated the active ingredient from Artemisia annua through systematic screening and extraction of traditional Chinese medicine, and named it artemisinin. Artemisinin possesses broad-spectrum antimalarial activity against Plasmodium. However, the bioavailability of artemisinin itself is relatively low. Scientists have structurally modified artemisinin, ultimately successfully synthesizing dihydroartemisinin. Dihydroartemisinin is a derivative similar to artemisinin, obtained by reducing a double bond on the artemisinin lactone ring. This structural modification increases the stability and antimalarial activity of dihydroartemisinin, and improves its bioavailability. In addition to its antimalarial effects, dihydroartemisinin also exhibits certain antitumor activity. It can inhibit the growth and spread of tumor cells through multiple mechanisms, induce tumor cell apoptosis (programmed cell death), and inhibit angiogenesis. These properties make dihydroartemisinin a hot topic in antitumor research.

[0003] Drug targeting refers to the ability of a drug to act more effectively on tumor cells without producing significant toxic side effects on healthy cells. However, dihydroartemisinin has relatively poor targeting of tumor cells; there is no significant difference in its uptake by tumor cells and normal cells, which may lead to some toxicity to normal cells during treatment, limiting its application. Methods to improve the targeting of dihydroartemisinin should be explored to better facilitate its clinical application.

[0004] Biotin (structural formula B) is a water-soluble vitamin, also known as vitamin H or coenzyme R. It plays important biological functions in many organisms. Numerous studies have shown that biotin, as a targeting molecule, has significant advantages in enhancing the targeting of drug molecules. This is because biotin receptors are widely present on the surface of human cells, and these receptors have a high affinity for biotin. When drug molecules bind to biotin to form biotin conjugates, the drug molecules can specifically target to the surface of tumor cells through the mediation of biotin receptors. Small molecule drug conjugates formed by the conjugation of biotin and dihydroartemisinin can greatly enhance drug targeting and increase the uptake rate by tumor cells. Summary of the Invention

[0005] For cancer patients, their tumor cells have a low pH and a high expression of biotin receptors on the tumor cell membrane. Therefore, this invention can better achieve the effect of inhibiting tumors by directly coupling biotin to dihydroartemisinin or by coupling biotin to dihydroartemisinin through a series of linkers.

[0006] This invention provides a method for preparing a biotin-dihydroartemisinin conjugate, wherein the biotin-dihydroartemisinin conjugate has the structural formulas A1, A2, and A3. Intermediates in the reaction process include B1, B2, B3, and B4.

[0007] Terminology Explanation:

[0008] A1: Biotin is directly coupled with dihydroartemisinin.

[0009] A2: Biotin conjugated with dihydroartemisinin, linker is NH2-R-COOH (R=(-CH2-)). n (n=2, 3, 4, 5, 6, 7, 8, 9, 10).

[0010] A3: Biotin conjugated with dihydroartemisinin, linker is HO-R-COOH (R=(-CH2-)). n (n=2, 3, 4, 5, 6, 7, 8, 9, 10).

[0011] B1: Biotin-conjugated N-hydroxysuccinimide (NHS).

[0012] B2: Biotin-coupled aliphatic amino acid, with the linker being NH2-R-COOH (R=(-CH2-)). n (n=2, 3, 4, 5, 6, 7, 8, 9, 10).

[0013] B3: Biotin-coupled carboxylic acid methyl ester, linker is HO-R-COOCH3 (R=(-CH2-)). n (n=2, 3, 4, 5, 6, 7, 8, 9, 10).

[0014] B4: Biotin-coupled carboxylic acid derivative, linker is HO-R-COOH (R=(-CH2-)). n (n=2, 3, 4, 5, 6, 7, 8, 9, 10).

[0015] In this invention, the compound numbers and structural formula numbers correspond completely and have the same referential relationship, based on the compound structural formula.

[0016] This invention provides the following technical solutions:

[0017] A method for preparing a biotin-dihydroartemisinin conjugate, wherein the method for preparing the biotin-dihydroartemisinin conjugate shown in A1 includes the following steps: dissolving biotin and dihydroartemisinin in a reaction solvent, and directly esterifying them in the presence of a catalyst and a dehydrating agent to obtain A1, with the following structural formula:

[0018] The preparation method of the biotin-dihydroartemisinin conjugate shown in A2 includes the following steps: (1) Biotin and N-hydroxysuccinimide (NHS) were dissolved in a reaction solvent and esterified in the presence of a dehydrating agent to obtain B1; (2) NH2-R-COOH (R=(-CH2-) n (n=2~10) and B1 are dissolved in the reaction solvent and reacted in the presence of an organic base to obtain B2; (3) B2 and dihydroartemisinin were dissolved in a reaction solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain A2, with the following structural formula:

[0019] The preparation method of the biotin-dihydroartemisinin conjugate shown in A3 includes the following steps: (1) Combine biotin with HO-R-COOCH3 (R=(-CH2-) n (n = 2 to 8) are dissolved in the reaction solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain B3; (2) Remove the methyl ester from B3 in the presence of an inorganic base to obtain B4; (3) B4 was dissolved in a reaction solvent with dihydroartemisinin and esterified in the presence of a catalyst and a dehydrating agent to obtain A3, with the following structural formula:

[0020] Preferably, the catalyst for the reaction of the above-mentioned coupling A1 is selected from one of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), or N-methylpyrrolidone; the dehydrating agent is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 2-(1H-benzotriazolyl-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).

[0021] Preferably, the catalyst for the reaction of the above-mentioned coupling A2 is selected from one of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), or N-methylpyrrolidone; the dehydrating agent is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 2-(1H-benzotriazolyl-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU); and the organic base is selected from triethylamine (TEA), pyridine, or N,N-diisopropylethylamine (DIPEA).

[0022] Preferably, the catalyst for the reaction of the above-mentioned coupling A3 is selected from one of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), or N-methylpyrrolidone; the dehydrating agent is selected from one of N,N'-dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 2-(1H-benzotriazolyl-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), or 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).

[0023] Preferably, the reaction of the above-mentioned coupling A1 is carried out in an inert solvent; the inert solvent is selected from, but is not limited to, dichloromethane, trichloromethane, dioxane, or N,N-dimethylformamide.

[0024] The reactions in steps (1) and (3) of the above-mentioned coupling A2 are carried out in an inert solvent; the inert solvent is selected from, but is not limited to, dichloromethane, trichloromethane, dioxane, or N,N-dimethylformamide. The reaction in step (2) is carried out in an inorganic weakly basic solvent, the inorganic weakly basic solvent is selected from, but is not limited to, sodium bicarbonate (NaHCO3) solution, sodium carbonate (Na2CO3) solution, sodium bisulfite solution (NaHSO3) or disodium hydrogen phosphate (Na2HPO4) solution.

[0025] The reactions in steps (1) and (3) of the above-mentioned coupling A3 are carried out in an inert solvent; the inert solvent is selected from, but is not limited to, dichloromethane, trichloromethane, dioxane, or N,N-dimethylformamide. The reaction in step (2) is carried out in an inorganic base solution; the inorganic base is selected from sodium hydroxide (NaOH), potassium hydroxide (KOH), sodium bicarbonate (NaHCO3), and sodium carbonate (Na2CO3).

[0026] The reaction temperature for the steps described above is 0–60°C.

[0027] The beneficial effects of this invention are as follows: This invention provides a method for preparing biotin-dihydroartemisinin conjugates and their applications. The method involves directly esterifying biotin with dihydroartemisinin via esterification, or reacting the -COOH group of biotin with an aliphatic amino acid containing -NH2 and then esterifying it with dihydroartemisinin, or esterifying the -COOH group of biotin with a carboxylic acid linker containing -OH and then esterifying it with dihydroartemisinin, thereby preparing a series of small-molecule prodrug conjugates. The prepared small-molecule prodrug conjugates exhibit significant beneficial effects. They can specifically target biotin receptors on the surface of tumor cells through the targeting action of biotin, thereby achieving tumor-targeting effects. Furthermore, the preparation method of this invention uses mild conditions, which enhances the targeting of dihydroartemisinin, increases the uptake of dihydroartemisinin by tumor cells, and improves the inhibitory rate of dihydroartemisinin on tumor cells. Therefore, this invention has significant application prospects and beneficial effects. Attached Figure Description

[0028] Figure 1 Fluorescence images showing the inhibitory effect of different concentrations of biotin (0.001 mM, 0.005 mM, 0.01 mM) on targeted uptake inhibition in HeLa cells.

[0029] Figure 2 Fluorescence intensity extraction images of the inhibitory effect of different concentrations of biotin (0.001mM, 0.005mM, 0.01mM) on the targeted uptake of HeLa cells.

[0030] Figure 3 Fluorescence diagram of HeLa cells uptake of 7-hydroxycoumarin and biotin-7-hydroxycoumarin.

[0031] Figure 4 The image shows the fluorescence intensity extraction of 7-hydroxycoumarin and biotin-7-hydroxycoumarin in HeLa cells.

[0032] Figure 5 The effects of dihydroartemisinin, A1 of Example 1, A2a of Example 2, and A3a of Example 5, on the survival rate of HeLa, H460, and A549 cells at the same concentration were investigated.

[0033] Figure 6 The effects of dihydroartemisinin, Example 1 (A1), Example 2 (A2a), and Example 5 (A3a) on the survival rate of HeLa cells at the same biotin-inhibiting concentration. Detailed Implementation

[0034] The present invention will be further described below through specific embodiments, but the scope of protection of the present invention is not limited thereto. Example 1

[0035] A method for preparing a biotin-dihydroartemisinin conjugate and its application, wherein the direct conjugation of biotin to dihydroartemisinin includes the following steps: (1) Weigh 146.5 mg (0.6 mmol) of biotin, 170.4 mg (0.6 mmol) of dihydroartemisinin, 88 mg (1.2 eq) of 4-dimethylaminopyridine, and 172.8 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and add them to a 50 ml round-bottom flask. Add 5 ml of N,N-dimethylformamide and stir at 30 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution and three times with deionized water, separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate, and remove the solvent by rotary evaporation to obtain biotin-dihydroartemisinin conjugate A1. A1 NMR data: 1 H NMR(400MHz,DMSO-d6)δ6.46 -6.41(m,1H),5.68(d,J=9.7Hz,1H),5.57(s,1H),4.31(dd,J=7.7,4.9Hz,1H),4.14(dd,J=7.8,4.3Hz,1H),3.11(ddd,J=8 .6,6.1,4.3Hz,1H),2.83(dd,J=12.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.47-2.24(m,2H),2.19(ddd,J=16.8,10.8,3.7 Hz,1H),2.11-1.95(m,1H),1.82(ddt,J=13.4,6.7,3.6Hz,1H),1.62(ddq,J=18.5,10.7,4.6,3.7Hz,4H),1.59-1.30(m,4H ),1.29(s,3H),1.18(td,J=11.3,6.6Hz,1H),0.97(td,J=13.2,12.6,4.5Hz,1H),0.89(d,J=6.3Hz,3H),0.87-0.75(m,3H). Example 2

[0036] A method for preparing a biotin-dihydroartemisinin conjugate and its application, wherein the biotin-dihydroartemisinin conjugate includes the following steps: (1) Weigh 146.5 mg (0.6 mmol) of biotin, 71.3 mg (0.62 mmol) of N-hydroxysuccinimide, and 185.7 mg (1.5 eq) of N,N'-dicyclohexylcarbodiimide and add them to a 50 ml round-bottom flask. Add 2 ml of N,N-dimethylformamide and stir at 50 °C for 3 h. After centrifugation to remove the solid, take the supernatant and slowly drop it into a beaker containing 50 ml of diethyl ether. After a large amount of precipitate is precipitated, filter to obtain the solid and dry it to obtain biotin-N-hydroxysuccinimide ester B1. B1 NMR data: 1 H NMR (400MHz, DMSO-d6) δ6.43(t,J=1.9Hz,1H),6.37(s,1H),4.36-4.28(m,1H),4.15(ddd,J=7.8,4.4,1.8Hz,1H),3.11(ddd,J=8.1,6.3 ,4.3Hz,1H),2.92-2.81(m,1H),2.82(s,4H),2.76-2.63(m,2H),2.59(d,J=12.4Hz,1H),1.65(h,J=6.4,5.6Hz,3H),1.57-1.36(m,3H). (2) Weigh 68.2 mg (0.2 mmol) of the biotin-N-hydroxysuccinimide ester obtained above into a 50 ml round-bottom flask, add 26.5 mg (0.2 mmol) of 6-aminohexanoic acid, add 10 ml of 0.5 mmol / L sodium bicarbonate aqueous solution, stir the reaction at 25 °C for 12 h to obtain a white suspension reaction solution, filter the reaction solution, add 1 mol / L hydrochloric acid to the filtrate to adjust the pH to below 3, and a large amount of precipitate is obtained. Filter the precipitate, collect the white solid, and dry it to obtain 6-biotin-aminohexanoic acid B2a. B2a NMR data: 1H NMR (400MHz, DMSO-d6) δ11.99(s,1H),7.73(t,J=5.7Hz,1H),6.44(s,1H),6.37(s,1H),4.31( dd,J=7.8,5.0Hz,1H),4.13(dd,J=8.3,4.4Hz,1H),3.10(dt,J=8.8,5.5Hz,1H),3.01(q,J=6. 5Hz,2H),2.82(dd,J=12.4,5.0Hz,1H),2.58(d,J=12.4Hz,1H),2.19(t,J=7.4Hz,2H),2.04(t ,J=7.4Hz,2H),1.61(ddt,J=12.5,9.6,6.1Hz,1H),1.49(h,J=7.9Hz,5H),1.46-1.18(m,6H). (3) Weigh 71.5 mg (0.2 mmol) of the obtained 6-biotin-aminohexanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate. When the remaining 0.5 ml of solvent is evaporated by rotary evaporation, petroleum ether is precipitated. Filter and collect the white solid. Wash the solid three times with petroleum ether and remove the solvent by rotary evaporation to obtain biotin-6-aminohexanoic acid-dihydroartemisinin conjugate A2a. A2a NMR data: 1H NMR(400MHz,Chloroform-d)δ6.63(s,1H),6.43(t,J=5.7Hz,1H),5.78(d,J=1 0.0Hz,1H),5.73(s,1H),5.45(d,J=7.2Hz,1H),4.55-4.43(m,1H),4.32(dd,J= 8.0,4.5Hz,1H),3.30-3.10(m,3H),2.91(dd,J=12.9,4.9Hz,1H),2.76(d,J=12 .8Hz,1H),2.66-2.47(m,1H),2.47-2.39(m,1H),2.37(ddd,J=17.5,9.4,3.3Hz ,2H),2.22(t,J=7.5Hz,2H),2.17-1.98(m,3H),1.89(dtt,J=16.7,6.8,3.4Hz, 2H),1.83-1.73(m,1H),1.71(ddt,J=10.7,7.2,3.3Hz,2H),1.64(ddd,J=18.2, 8.6,4.6Hz,3H),1.58-1.22(m,9H),1.35(s,2H),1.22-1.03(m,1H),1.06-0.96 (m,1H),0.96(dd,J=5.9,3.3Hz,4H),0.96-0.84(m,1H),0.84(d,J=7.1Hz,3H).

[0037] Example 3 (1) Repeat step (1) of Example 2. (2) Weigh 68.2 mg (0.2 mmol) of biotin-N-hydroxysuccinimide ester into a 50 ml round-bottom flask, add 29.04 mg (0.2 mmol) of 7-aminoheptanoic acid, add 10 ml of 0.5 mmol / L sodium bicarbonate aqueous solution, stir the reaction at 25 °C for 12 h to obtain a white suspension reaction solution, filter the reaction solution, add 1 mol / L hydrochloric acid to the filtrate to adjust the pH to below 3, and a large amount of precipitate is obtained. Filter the precipitate, collect the white solid, and dry it to obtain 7-biotin-aminoheptanoic acid B2b. B2b's NMR data: 1H NMR (400MHz, DMSO-d6) δ11.97(s,1H),7.72(t,J=5.6Hz,1H),6.42(s,1H),6.36(s,1H),4.30(dd,J=7.8,4.9 Hz,1H),4.13(dd,J=7.9,4.4Hz,1H),3.09(ddd,J=8.6,6.1,4.3Hz,1H),3.00(q,J=6.6Hz,2H),2.82(dd,J=1 2.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.18(t,J=7.4Hz,2H),2.04(t,J=7.4Hz,2H),1.61(ddt,J=12.4,9. 5,6.1Hz,1H),1.48(qd,J=11.4,4.5Hz,5H),1.36(h,J=6.7Hz,2H),1.28(s,1H),1.25(dp,J=7.3,3.2Hz,4H). (3) Weigh 74.24 mg (0.2 mmol) of the obtained 7-biotinaminoheptanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate. When the remaining 0.5 ml of solvent is evaporated by rotary evaporation, petroleum ether is precipitated. Filter and collect the white solid. Wash the solid three times with petroleum ether and remove the solvent by rotary evaporation to obtain biotin-7-aminoheptanoic acid-dihydroartemisinin conjugate A2b. A2b NMR data: 1H NMR (400MHz, DMSO-d6) δ7.72(t,J=5.6Hz,1H),6.41(d,J=2.8Hz,1H),6.35(s,1H),5.67(d,J=9.7Hz,1H),5.56(s,1H),4.30(dd,J =7.7,5.1Hz,1H),4.12(ddd,J=7.5,4.5,1.8Hz,1H),4.03(q,J=7.1Hz,1H),3.09(ddd,J=8.5,6.1,4.3Hz,1H),3.01(q,J=6.6Hz,2H ),2.82(dd,J=12.4,5.1Hz,1H),2.57(d,J=12.4Hz,1H),2.47-2.23(m,3H),2.23-2.12(m,1H),2.11-1.94(m,4H),1.86-1.76(m,1H ),1.68-1.42(m,7H),1.46-1.35(m,1H),1.37-1.30(m,1H),1.28(s,4H),1.33-1.23(m,4H),1.23-1.10(m,2H),1.02-0.74(m,8H).

[0038] Example 4 (1) Repeat step (1) of Example 2. (2) Weigh 68.2 mg (0.2 mmol) of biotin-N-hydroxysuccinimide ester into a 50 ml round-bottom flask, add 31.846 mg (0.2 mmol) of 8-aminooctanoic acid, add 10 ml of 0.5 mmol / L sodium bicarbonate aqueous solution, stir the reaction at 25 °C for 12 h to obtain a white suspension reaction solution, filter the reaction solution, add 1 mol / L hydrochloric acid to the filtrate to adjust the pH to below 3, and a large amount of precipitate is obtained. Filter the precipitate, collect the white solid, and dry it to obtain 8-biotin-aminooctanoic acid B2c. B2C NMR data: 1H NMR (400MHz, DMSO-d6) δ11.98(s,1H),7.72(t,J=5.6Hz,1H),6.42(s,1H),6.36(s,1H),4.30(dd,J=7.7, 4.9Hz, 1H), 4.13 (dd, J=7.8, 4.4Hz, 1H), 3.09 (ddd, J=8.5, 6.1, 4.4Hz, 1H), 3.00 (q, J=6.6Hz, 2H), 2.82 (d d,J=12.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.18(t,J=7.4Hz,2H),2.04(t,J=7.4Hz,2H),1.68-1.52(m ,1H),1.47(tq,J=11.0,5.3,4.5Hz,5H),1.36(h,J=6.7Hz,2H),1.33-1.24(m,1H),1.24(d,J=3.7Hz,6H). (2) Weigh 77.046 mg (0.2 mmol) of the obtained 8-biotin-aminooctanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate. When the remaining 0.5 ml of solvent is evaporated by rotary evaporation, petroleum ether is precipitated. Filter and collect the white solid. Wash the solid three times with petroleum ether and remove the solvent by rotary evaporation to obtain biotin-8-aminooctanoic acid-dihydroartemisinin conjugate A2c. A2c NMR data: 1H NMR (400MHz, DMSO-d6) δ7.71(t,J=5.6Hz,1H),6.41(s,1H),6.35(s,1H),5.67(d,J=9.8Hz,1H),5.56(s,1H),4.30(dd,J=7.8,5.0Hz,1H ),4.12(ddd,J=7.3,4.5,1.8Hz,1H),4.03(q,J=7.1Hz,1H),3.09(ddd,J=8.5,6.1,4.3Hz,1H),3.00(q,J=6.5Hz,2H),2.82(dd,J=12.4,5 .1Hz,1H),2.58(d,J=12.4Hz,1H),2.47-2.24(m,2H),2.18(td,J=14.0,3.9Hz,1H),2.01(d,J=19.2Hz,5H),1.81(ddd,J=13.5,5.8,3.3H z,1H),1.68-1.30(m,7H),1.27(d,J=9.6Hz,9H),1.18(t,J=7.2Hz,2H),1.02-0.91(m,1H),0.89(d,J=6.4Hz,3H),0.77(d,J=7.1Hz,3H).

[0039] Example 5 (1) Weigh 146.5 mg (0.6 mmol) of biotin, 86 μL (0.6 mmol) of methyl 6-hydroxyhexanoate, 88 mg (1.2 eq) of 4-dimethylaminopyridine, and 172.8 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and add them to a 50 ml round-bottom flask. Add 2 ml of N,N-dimethylformamide and stir at 35 °C for 12 h to obtain a clear reaction solution. Purify the solution by silica gel column chromatography (dichloromethane:methanol = 8:1) and remove the solvent by rotary evaporation to obtain methyl 6-biotin-hydroxyhexanoate conjugate B3a. (2) Weigh 74.4 mg (0.2 mmol) of the obtained methyl 6-biotinylated hydroxyhexanoate into a 50 ml round-bottom flask, add 5 ml of 0.1 M sodium bicarbonate aqueous solution, stir at 45 °C for 12 h, and after the solid disappears, add 1 M hydrochloric acid to adjust the pH to below 3. After a large amount of precipitate is formed, filter, wash with water, collect the solid and dry it in an oven to obtain 6-biotinylated hydroxyhexanoate B4a. (3) Weigh 72 mg (0.2 mmol) of the obtained 6-biotin-hydroxyhexanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate. Remove the solvent by rotary evaporation to obtain biotin-6-hydroxyhexanoic acid-dihydroartemisinin A3a. A3a NMR data: 1 H NMR (400MHz, DMSO-d6) δ6.43(s,1H),6.35(s,1H),5.67(d,J=9.7Hz,1H),5.56(s,1H),4.31(dd,J=7.7,4.9Hz,1H),4.13(dd,J=7.8, 4.4Hz,1H),4.00(t,J=6.4Hz,2H),3.10(ddd,J=8.5,6.1,4.3Hz,1H),2.83(dd,J=12.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.47-2.1 3(m,6H),2.13-1.99(m,1H),2.03-1.93(m,1H),1.81(ddt,J=14.0,6.7,3.6Hz,1H),1.68-1.58(m,3H),1.61-1.54(m,4H),1.57-1.45 (m,3H),1.49-1.37(m,1H),1.34(dp,J=13.4,4.9,4.2Hz,5H),1.29(s,3H),1.29-1.18(m,1H),1.21-1.09(m,1H),1.05-0.74(m,9H).

[0040] Example 6 (1) Weigh 146.5 mg (0.6 mmol) of biotin, 96 mg (0.6 mmol) of methyl 7-hydroxyheptanoate, 88 mg (1.2 eq) of 4-dimethylaminopyridine, and 172.8 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and add them to a 50 ml round-bottom flask. Add 2 ml of N,N-dimethylformamide and stir at 35 °C for 12 h to obtain a clear reaction solution. Purify the solution by silica gel column chromatography (dichloromethane:methanol = 8:1) and remove the solvent by rotary evaporation to obtain 7-biotin-methyl hydroxyheptanoate conjugate B3b. (2) Weigh 77.2 mg (0.2 mmol) of the obtained methyl 7-biotinylated hydroxyheptanate into a 50 ml round-bottom flask, add 5 ml of 0.1 M sodium bicarbonate aqueous solution, stir at 45 °C for 12 h, and after the solid disappears, add 1 M hydrochloric acid to adjust the pH to below 3. After a large amount of precipitate is formed, filter, wash with water, collect the solid and dry it in an oven to obtain 7-biotinylated hydroxyheptanate B4b. (3) Weigh 74.4 mg (0.2 mmol) of the obtained 7-biotin-hydroxyheptanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate, and remove the solvent by rotary evaporation to obtain biotin-7-hydroxyheptanoic acid-dihydroartemisinin A3b. A3b NMR data: 1 H NMR (400MHz, DMSO-d6) δ6.43(s,1H),6.35(s,1H),5.67(d,J=9.7Hz,1H),5.56(s,1H),4.31(dd,J=7.7,4.9Hz,1H),4.13(dd,J=7.8, 4.4Hz,1H),4.00(t,J=6.4Hz,2H),3.10(ddd,J=8.5,6.1,4.3Hz,1H),2.83(dd,J=12.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.47-2.1 3(m,6H),2.13-1.99(m,1H),2.03-1.93(m,1H),1.81(ddt,J=14.0,6.7,3.6Hz,1H),1.68-1.58(m,3H),1.61-1.54(m,6H),1.57-1.45 (m,3H),1.49-1.37(m,1H),1.34(dp,J=13.4,4.9,4.2Hz,5H),1.29(s,3H),1.29-1.18(m,1H),1.21-1.09(m,1H),1.05-0.74(m,9H).

[0041] Example 7 (1) Weigh 146.5 mg (0.6 mmol) of biotin, 104.5 mg (0.6 mmol) of methyl 8-hydroxyoctanoate, 88 mg (1.2 eq) of 4-dimethylaminopyridine, and 172.8 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and add them to a 50 ml round-bottom flask. Add 2 ml of N,N-dimethylformamide and stir at 35 °C for 12 h to obtain a clear reaction solution. Purify the solution by silica gel column chromatography (dichloromethane:methanol = 8:1) and remove the solvent by rotary evaporation to obtain 8-biotin-methyl 8-hydroxyoctanoate conjugate B3c. (2) Weigh 80 mg (0.2 mmol) of the obtained methyl 8-biotinylated hydroxyoctanoate into a 50 ml round-bottom flask, add 5 ml of 0.1 M sodium bicarbonate aqueous solution, stir at 45 °C for 12 h, and after the solid disappears, add 1 M hydrochloric acid to adjust the pH to below 3. After a large amount of precipitate is formed, filter, wash with water, collect the solid and dry it in an oven to obtain 8-biotinylated hydroxyoctanoate B4c. (3) Weigh 77.2 mg (0.2 mmol) of the obtained 8-biotin-hydroxyoctanoic acid into a 50 ml round-bottom flask, add 56.87 mg (0.2 mmol) of dihydroartemisinin, 29.28 mg (1.2 eq) of 4-dimethylaminopyridine, 57.6 mg (1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3 ml of N,N-dimethylformamide. Stir the reaction at 35 °C for 12 h to obtain a clear reaction solution. Extract the solution three times with ethyl acetate and dilute hydrochloric acid solution, wash three times with saturated saline solution, and wash three times with deionized water. Separate the ethyl acetate layer, dry it with anhydrous sodium sulfate, filter and collect the filtrate, and remove the solvent by rotary evaporation to obtain biotin-8-hydroxyoctanoic acid-dihydroartemisinin A3b. A3c NMR data: 1H NMR (400MHz, DMSO-d6) δ6.43(s,1H),6.35(s,1H),5.67(d,J=9.7Hz,1H),5.56(s,1H),4.31(dd,J=7.7,4.9Hz,1H),4.13(dd,J=7.8, 4.4Hz,1H),4.00(t,J=6.4Hz,2H),3.10(ddd,J=8.5,6.1,4.3Hz,1H),2.83(dd,J=12.4,5.1Hz,1H),2.58(d,J=12.4Hz,1H),2.47-2.1 3(m,6H),2.13-1.99(m,1H),2.03-1.93(m,1H),1.81(ddt,J=14.0,6.7,3.6Hz,1H),1.68-1.58(m,3H),1.61-1.54(m,8H),1.57-1.45 (m,3H),1.49-1.37(m,1H),1.34(dp,J=13.4,4.9,4.2Hz,5H),1.29(s,3H),1.29-1.18(m,1H),1.21-1.09(m,1H),1.05-0.74(m,9H).

[0042] Example 8

[0043] Cell uptake experiment Studies have found that many tumor cells take up large amounts of biotin because they express high levels of biotin receptors. Since dihydroartemisinin is non-fluorescent, we used 7-hydroxycoumarin as a fluorescent reagent to evaluate drug uptake by tumor cells. Through competitive inhibition assays, we observed cell fluorescence to determine whether cell uptake was inhibited by biotin, thus investigating the role of biotin receptors in tumor cell uptake. HeLa cells were harvested by trypsin digestion and resuspended in DMEM medium at a density of 3 × 10⁶ cells / well. 5 Cells were seeded into 6-well cell culture plates (each well pre-placed with a round glass slide). After cell attachment, the original culture medium was replaced with DMEM containing different concentrations of biotin (50 μM, 100 μM, 200 μM), with a blank control included. Cells were incubated at 37°C for 1 h, followed by the addition of 5 μg / ml of 7-hydroxycoumarin-labeled biotin, and incubation continued for another 1 h. The glass slides were then removed, cells were washed with cold PBS, fixed with 4% paraformaldehyde for 10 minutes, and the paraformaldehyde was removed followed by washing with PBS. Fluorescence micrographs were acquired using a fluorescence microscope. Cells were treated with the same concentrations of 7-hydroxycoumarin and 7-hydroxycoumarin-labeled biotin using the same procedure, and fluorescence was observed.

[0044] according to Figure 1 and Figure 2 As shown, fluorescence patterns at three different magnifications for HeLa cells with added 50 μM, 100 μM, and 200 μM biotin revealed that, compared to the uninhibited group, HeLa cells exhibited lower blue fluorescence, and the fluorescence intensity gradually decreased with increasing biotin concentration. This indicates that biotin can competitively inhibit the uptake of biotinylated 7-hydroxycoumarin by HeLa cells, and the higher the biotin concentration, the more significant the inhibitory effect. This demonstrates that HeLa cells possess a large number of biotin receptors, and the introduction of biotin helps improve drug targeting.

[0045] like Figure 3 and Figure 4 As shown, at three magnifications, the fluorescence intensity of the biotin group labeled with 7-hydroxycoumarin was significantly higher than that of the 7-hydroxycoumarin group. This further demonstrates that the introduction of biotin can enhance the drug uptake rate by tumor cells.

[0046] Cytotoxicity assay HeLa cells were seeded in 96-well plates at a density of 5 × 10⁶ cells per well. 3 Cells were incubated for 24 hours. Then, dihydroartemisinin, conjugates A1, A2a, and A3a were prepared in DMEM medium at a concentration of 160 μM. 100 μL of the drug was added to each well of a 96-well plate, with three replicates, and a control group was included. The 96-well plates were then incubated at 37°C in a CO2 incubator for another 24 hours. After incubation, 20 μL of LTT (5 mg / ml) was added to each well, and incubation continued at 37°C for 4 hours. The medium was then removed, and 150 μL of DMSO was added. Cell viability was calculated by measuring absorbance at 570 nm using a microplate reader. The same procedure was used to treat two other cell types, H460 and A549 cells, except that 1640 medium was used instead of DMEM medium. To further validate the tumor-targeting activity of biotin, different concentrations of biotin (0.01, 0.05, and 0.1 mM) were added to the culture medium, and the cells were then cultured at 37°C with 5% CO2 for 12 hours to allow the biotin receptors on the surface of cancer cells to bind to different concentrations of biotin. Subsequently, dihydroartemisinin, conjugates A1, A2a, and A3a were prepared using DMEM medium at a concentration of 160 μM. 100 μL of the drug was added to each well of a 96-well plate, with three replicates, and a control group was included. The cells were then cultured for another 24 hours. Finally, the absorbance was measured using a microplate reader at a wavelength of 570 nm to calculate cell viability.

[0047] according to Figure 5The results showed that, at the same concentration, the survival rates of the three types of tumor cells treated with conjugates A1, A2a, and A3a were significantly lower than those treated with dihydroartemisinin. In particular, cells treated with A2a exhibited the lowest survival rate, further confirming that the synthesized biotin-dihydroartemisinin conjugates have a stronger toxic effect on tumor cells than dihydroartemisinin alone.

[0048] Figure 6 The effects of four conjugates on HeLa cell survival under different concentrations of biotin inhibition were clearly demonstrated. The results showed that, regardless of the presence of biotin, treatment with dihydroartemisinin had little impact on HeLa cell survival, which remained at approximately 52%. With increasing biotin concentration, the survival rate of HeLa cells treated with conjugates A1, A2a, and A3a showed a certain upward trend. For biotin-conjugated dihydroartemisinin, the inhibitory effect on cells gradually weakened with increasing biotin concentration, leading to a gradual increase in HeLa cell survival. However, for dihydroartemisinin without targeting ability, biotin had almost no effect. In summary, biotin-conjugated dihydroartemisinin A1, A2a, and A3a all exhibited significant tumor targeting, improving the inhibition rate of tumor cells.

Claims

1. A biotin-dihydroartemisinin conjugate, characterized in that, The conjugate is a compound with the structure shown in A2 and A3 below: A2: , where R = (-CH2-) n n=2~5; A3: , where R = (-CH2-)n, n = 2~5.

2. The method for preparing A2 according to claim 1, characterized in that, Includes the following steps: (1) Biotin and N-hydroxysuccinimide were dissolved in a reaction solvent and esterified in the presence of a dehydrating agent to obtain biotin-N-hydroxysuccinimide ester B1. ; (2) Dissolve NH2-R-COOH and B1 in a reaction solvent and react in the presence of an organic base to obtain B2. , where R = (-CH2-) n n=2~5; (3) Dissolve B2 and dihydroartemisinin in a reaction solvent and esterify them in the presence of a catalyst and a dehydrating agent to obtain A2.

3. The method for preparing A3 according to claim 1, characterized in that, Includes the following steps: (1) Biotin and HO-R-COOCH3 were dissolved in a reaction solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain B3. , where R = (-CH2-) n n=2~5; (2) Remove the methyl ester from B3 in the presence of an inorganic base to obtain B4. ; (3) B4 and dihydroartemisinin were dissolved in a reaction solvent and esterified in the presence of a catalyst and a dehydrating agent to obtain A3.

4. The preparation method according to any one of claims 2 and 3, characterized in that: The catalyst is selected from 4-dimethylaminopyridine or 1-hydroxybenzotriazole; the dehydrating agent is selected from N,N'-dicyclohexylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

5. The preparation method according to claim 2, characterized in that: The organic base mentioned in step (2) is selected from triethylamine, pyridine, and N,N-diisopropylethylamine.

6. The preparation method according to claim 3, characterized in that: The inorganic base in step (2) is selected from one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, and sodium carbonate.

7. The preparation method according to any one of claims 2 and 3, characterized in that: The reaction in step (1) or (3) is carried out in an inert solvent; the inert solvent is selected from one of dichloromethane, trichloromethane, dioxane or N,N-dimethylformamide.

8. The preparation method according to any one of claims 2 and 3, characterized in that: The reaction temperature for the above steps is 0~60℃.

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