A β-galactose-modified chlorambucil-fluboron dipyrrole co-antitumor prodrug, its preparation method and application

Abstract

This invention discloses a β-galactose-modified chlorambucil-fluorobordipyrrole combined antitumor prodrug, its preparation, and its application. Due to the introduction of β-galactose, the prodrug can be rapidly and selectively taken up by tumor cells. Subsequently, the glycosidic bonds in the prodrug molecule are cleaved by β-galactosidase overexpressed in tumor cells. Through a series of rapid intramolecular conversion reactions, the original drug chlorambucil and the photosensitizer fluorobordipyrrole are released. Under light irradiation, selective chemotherapy-photodynamic therapy is achieved. Using human ovarian cancer cells A2780, mouse ovarian cancer cells ID8, and mouse embryonic fibroblasts 3T3 as test cell lines, this invention verified that the prodrug has the characteristic of actively targeting tumor cells and excellent combined anticancer activity. The synthesis method of this compound is simple, the raw materials are readily available, the cost is low, there are few side reactions, the yield is high, and it is easy to purify, which is conducive to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug design and synthesis, specifically involving a β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug and its preparation and application. Background Technology

[0002] Currently, cancer has become one of the major diseases seriously threatening human life and health worldwide. Photodynamic therapy (PDT) is a "green" treatment strategy, which has become a research hotspot due to its advantages such as minimal invasiveness, high selectivity, low toxicity, and no drug resistance. During PDT, photosensitizers exposed to specific wavelengths of light interact with molecular oxygen and other substances at the lesion site to produce highly oxidizing reactive oxygen species (ROS), which then kill tumor cells. Compared with traditional photosensitizers such as porphyrins and bacterial chlorophyll, fluoroboron dipyrrole photosensitizers (BODIPY) have advantages such as simple structure, stable chemical properties, and ease of modification. Specially modified BODIPY derivatives have strong light absorption in the phototherapy window (600-900nm), with the maximum emission wavelength in the near-infrared region and less susceptibility to background interference, thus becoming a promising second-generation anticancer photosensitizer. Currently, BODIPY and its derivatives have been applied in the fields of fluorescent probes and biolabeling, tumor diagnosis, etc.

[0003] β-galactosidase is a common enzyme in living organisms that catalyzes the hydrolysis of β-galactosidic bonds and plays an important role in carbohydrate metabolism. Abnormal intracellular β-galactosidase expression is directly associated with different cancer types, especially metastatic ovarian cancer. Because overexpressed β-galactosidase is a unique tumor marker that can actively target tumor cells through specific ligand-receptor interactions, it has attracted considerable attention in biological oncology research.

[0004] Chlorobutazone is a dichloroethylamine alkylating agent with broad-spectrum antitumor activity. It covalently binds to the nitrogen atom at the 7-position of guanine, leading to cross-linking of different bases within the same strand of DNA, further inducing apoptosis. It is commonly used to treat ovarian cancer, leukemia, glioma, lymphoma, and other diseases. Due to its poor water solubility and strong adverse reactions such as myelosuppression and neurotoxicity, covalently linking chlorambucil and β-galactose to form a prodrug can improve its bioavailability and reduce toxic side effects. Further covalently linking a fluoroboron dipyrrole derivative to the β-galactose chlorambucil prodrug also improves the targeting of photosensitizers, potentially enabling precise chemotherapy-photodynamic therapy for tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a combined antitumor prodrug, its preparation, and its application. Galactose is a hexacarbon sugar and one of the fastest-absorbed monosaccharides in the human intestine, providing energy for cell growth and proliferation. Tumor cells are highly proliferative, therefore their membrane surfaces highly express certain transporters to increase nutrient uptake. Galactolectins are one such transporter, capable of specifically recognizing and binding to galactose, mediating its intracellular transport. Furthermore, tumor cells contain overexpressed β-galactosidase. Therefore, the β-galactose-modified chlorambucil-fluboron dipyrrole prodrug reported in this invention can actively target tumor cells. After being taken up by tumor cells, the β-galactosidic bond in the prodrug is cleaved by β-galactosidase, releasing chlorambucil technical and fluboron dipyrrole photosensitizer. This novel anticancer drug, with high biocompatibility, good targeting, high efficiency, and low toxicity, can be used for targeted chemotherapy-photodynamic therapy. The compound synthesized by this invention has a simple synthesis method, few side reactions, high yield, readily available raw materials, and low cost, which is conducive to industrial production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A β-galactose-modified chlorambucil-fluboron dipyrrole co-antitumor prodrug has the following chemical structure: (A).

[0008] The specific steps for preparing the above-mentioned β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug are as follows:

[0009] compound Dissolved in 150 mL of anhydrous toluene, as compound The molar quantity meter, then 2 equivalents Add the toluene to the reaction mixture and stir. Then add 1.2 mL piperidine, 1.0 mL glacial acetic acid, and anhydrous magnesium perchlorate, and reflux for 4 h. After the reaction is complete, remove most of the toluene using a rotary evaporator. The crude product is purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 50:1 as eluent. The solvent is removed by rotary evaporation under reduced pressure to obtain a dark green solid compound. (B);

[0010] (2) Under ice bath conditions, Dissolve in 20 mL of anhydrous dichloromethane solution, with Using a molar ratio, 7 equivalents of hydrobromoacetic acid solution were slowly added dropwise to the reaction system, and the mixture was rapidly stirred until homogeneous. The reaction was carried out in the dark for 3 hours. After the reaction, the organic phase was washed three times with 30 mL of ice-cold saturated sodium bicarbonate solution, followed by three times with 30 mL of ice water. The organic phase was dried over anhydrous sodium sulfate and then rotary evaporated under reduced pressure to obtain a transparent gel-like product. (E);

[0011] (3) Mix cesium carbonate with a mixed solvent of N,N-dimethylformamide / tetrahydrofuran at a volume ratio of 1:9 until homogeneous. Add dropwise a solution containing 0.25 equivalents of cesium carbonate. A mixed solvent of N,N-dimethylformamide / tetrahydrofuran with a volume ratio of 1:9 was added dropwise, followed by the addition of 0.25 equivalents of... A mixture of N,N-dimethylformamide and tetrahydrofuran (volume ratio 1:9) was used as a solvent, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, 50 mL of ethyl acetate was added and stirred for 10 min. The reaction solution was then washed three times with 50 mL of saturated brine each time. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (volume ratio 4:3) as eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound. (F);

[0012] (4) Dissolve completely in 5 mL of anhydrous tetrahydrofuran, Using a molar ratio, 4 equivalents of a tetrahydrofuran solution of lithium tri-tert-butyloxyaluminum hydride (LiAlH(OtBu)3) was added dropwise to the mixed solution under ice bath conditions. After reacting for 3 hours, 5 mL of saturated ammonium chloride aqueous solution and 10 mL of ethyl acetate were added to the mixture, and stirring was continued for 1 hour. After the reaction was completed, 10 mL of a prepared saturated potassium hydrogen tartrate aqueous solution was added, and the mixture was extracted three times with 20 mL of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powder as the compound. (G);

[0013] (5) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-pyrrolidinylpyridine (4-PPY) were dissolved in anhydrous dichloromethane in a molar ratio of 5:10:16 under ice bath conditions and stirred for 30 min. The molar quantity is 1.25 equivalent. The solvent was dissolved in 2 mL of anhydrous dichloromethane and added to the reaction system. The reaction was continued for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 100:1 as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound. (C);

[0014] (6) The dark green solid compound (B) prepared in step (1), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 4-pyrrolidinylpyridine (4-PPY) were dissolved in anhydrous dichloromethane in a molar ratio of 1:5:5 under ice bath and stirred for 30 min. Then, based on the molar amount of compound B, 1 equivalent of the white powder compound (C) prepared in step (5) was dissolved in anhydrous dichloromethane and added to the reaction system, and the reaction was continued for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 50:1 as the eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain the green powder compound. (D);

[0015] (7) The above compound (D) was dissolved in a mixed solvent of dichloromethane / methanol at a volume ratio of 1:4. 110 equivalents of sodium bicarbonate were added based on the molar amount of compound D, and the mixture was stirred for 4 hours. After the reaction was complete, 10 mL of ethyl acetate was added. The reaction solution was then washed three times with 20 mL of saturated brine each time. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 30:1, and the solvent was removed by rotary evaporation under reduced pressure to obtain a green powdery β-galactose-modified chlorambucil-fluoroboron dipyrrole co-antitumor prodrug. (A)

[0016] compound Synthesis reference: Zhang, H.-X., Lin, H.-H., Su,D., Yang, D.-C., Liu, J.-Y. Enzyme-Activated Multifunctional ProdrugCombining Site-Specific Chemotherapy with Light-Triggered PhotodynamicTherapy. Molecular Pharmaceutics, 2022, 19, 630–641.

[0017] compound Synthesis reference of (E): Mansour AK, Eid MM, KhalilN. Synthesis of some N-galactosides of 3-aryl-5-benzyl (or substitutedbenzyl)-1,2,4-triazin-6(1H)- / ones or thiones of expected biological activity. Nucleosides Nucleotides & Nucleic Acids, 2003, 22(9), 1825-1833.

[0018] The β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug is used to prepare an actively targeted anticancer drug for chemotherapy-photodynamic therapy combined with antitumor treatment.

[0019] Photodynamic therapy (PDT) is a "green" anti-tumor strategy. Its three essential elements include a photosensitizer, light of a specific wavelength, and oxygen. The photosensitizer is the core of PDT, and an ideal photosensitizer should ideally meet the following characteristics: single component, well-defined structure, stable properties, strong targeting, strong phototoxicity, no dark toxicity, high singlet oxygen quantum yield, and a maximum absorption peak located within the PDT window (600-900 nm). Fluoroboropyridine (BODIPY) derivatives are considered ideal second-generation photosensitizers due to their excellent photophysical and photochemical properties. This invention synthesizes a β-galactose-modified chlorambucil-fluoroboroboropyridine combined anti-tumor prodrug that actively targets tumor cells.

[0020] The significant advantages of this invention are:

[0021] (1) Galactose agglutinin and β-galactosidase are known tumor biomarkers. Therefore, tumor cells can selectively take up compounds containing β-galactose. This prodrug can precisely target tumor cells. Combined with the fluorescence properties of BODIPY, it can achieve PDT-chemotherapy synergistic treatment under the guidance of fluorescence imaging.

[0022] (2) Chlorobutachlor is a chemotherapeutic drug with severe toxic side effects. Preparing it as a prodrug can significantly reduce its toxicity. β-galactosidase overexpressed in tumor cells can cleave the β-galactosidic bond of the prodrug, thus activating and releasing the original drug in tumor cells, improving the selectivity of tumor treatment. At the same time, the introduction of β-galactose can improve the bioavailability of the original drug chlorbutachlor.

[0023] (3) Fluoroboropyrrole is a photosensitizer with a rigid large π system. It has poor water solubility and serious aggregation in water. Combining it with β-galactose can improve the solubility of the photosensitizer and its targeting to tumors, thereby improving the bioavailability of the photosensitizer.

[0024] (4) The target compound has a simple structure and no isomers; the synthesis method is simple, the product is easy to purify, there are few side reactions, the raw materials are readily available, the cost is low, and it is conducive to industrial production. Attached Figure Description

[0025] Figure 1 Fluorescence intensity curves and photofluorescence intensity comparisons of compound A (1 μM) in cancer cells A2708, ID8, and normal cells 3T3; where (a) and (b) are the fluorescence intensity curves and photofluorescence intensity comparisons of A2708 cells, (c) and (d) are the fluorescence intensity curves and photofluorescence intensity comparisons of ID8 cells, and (e) and (f) are the fluorescence intensity curves and photofluorescence intensity comparisons of 3T3 cells.

[0026] Figure 2 Fluorescence imaging of compound A (1 μM) co-cultured with cancer cells ID8 and normal cells 3T3, with the upper left portion of each image showing ID8 cells and the lower right portion showing normal cells 3T3.

[0027] Figure 3 The fluorescence intensity curves and photofluorescence intensity comparison diagrams of the inhibitory targeted uptake of compound A (1 μM) in cancer cells A2708 and ID8 are shown. (a) and (b) are the fluorescence intensity curves and photofluorescence intensity comparison diagrams of A2708 cells, respectively, and (c) and (d) are the fluorescence intensity curves and photofluorescence intensity comparison diagrams of ID8 cells, respectively.

[0028] Figure 4 Killing curves of compound A and chlorambucil (MN) on A2708 and ID8 cells under both illumination and darkness; (a) represents A2708 cells and (b) represents ID8 cells. Light source: 660nm LED light panel, illumination time 2 min, light energy density 2.4 J•cm². -2 .

[0029] Figure 5 The effects of compound A (1 μM) on reactive oxygen species (ROS) production in cancer cells A2708 and ID8 are shown in (a) and (b), respectively. (c) and (d) are the fluorescence intensity curves and photofluorescence intensity comparisons of A2708 cells, respectively. Light source: 660nm LED panel, illumination for 2 min, light energy density: 2.4 J•cm². -2 .

[0030] Figure 6 Fluorescence imaging of live and dead cells stained with compound A (0.5 μM) in cancer cells A2708 and ID8, where (a) represents A2708 cells and (b) represents ID8 cells. Light source: 660nm LED light panel, illumination for 2 min, light energy density 2.4 J•cm². -2 ;

[0031] Figure 7 For compound A 1 H NMR spectrum;

[0032] Figure 8 For compound A 13 C NMR spectrum;

[0033] Figure 9 This is the high-resolution mass spectrum of compound A;

[0034] Figure 10 For compound D 1 H NMR spectrum;

[0035] Figure 11 For compound D 13 C NMR spectrum;

[0036] Figure 12 For compound C 1 H NMR spectrum;

[0037] Figure 13 For compound C 13 C NMR spectrum;

[0038] Figure 14 This is the high-resolution mass spectrum of compound C;

[0039] Figure 15 For compound G 1 H NMR spectrum;

[0040] Figure 16 For compound G 13 C NMR spectrum;

[0041] Figure 17 This is the high-resolution mass spectrum of compound G;

[0042] Figure 18 For compound F 1 H NMR spectrum;

[0043] Figure 19 For compound F 13 C NMR spectrum;

[0044] Figure 20 This is the high-resolution mass spectrum of compound F. Detailed Implementation

[0045] To further disclose, and not limit, the present invention, the invention will be further described in detail below with reference to examples.

[0046] Example 1

[0047] (1) Accurate weighing (130 mg, 0.210 mmol) was dissolved in 150 mL of anhydrous toluene in a 250 mL round-bottom flask, and then added... (134 mg, 0.500 mmol) 1.2 mL piperidine, 1.0 mL glacial acetic acid, and a catalytic amount of anhydrous magnesium perchlorate were added under stirring, and the mixture was refluxed for 4 h. After the reaction was complete, most of the toluene was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (v / v) as eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain a dark green solid compound. (B) (235 mg, 50.1%).

[0048] Under ice bath conditions, (1.62 g, 4.15 mmol) was dissolved in 20 mL of anhydrous dichloromethane solution. A solution of hydrobromic acid (33% w / w, 5 mL, 27.81 mmol) was slowly added dropwise to the reaction system, stirred thoroughly, and reacted in the dark for three hours. After the reaction was complete, the mixture was washed three times with 30 mL of ice-cold saturated sodium bicarbonate solution, followed by three times with 30 mL of ice water. The organic phase was dried over anhydrous sodium sulfate and then rotary evaporated under reduced pressure to obtain a transparent gel-like product. (E) (1.64 g, 96.0%).

[0049] (3) Mix cesium carbonate (2.70 g, 8.29 mmol) with DMF / THF (1 / 9, 10 mL) thoroughly, and add dropwise the solution containing... (0.25 g, 2.04 mmol) of a DMF / THF (1 / 9, 7 mL) mixed solvent, then added dropwise to a solution containing... (999 mg, 2.44 mmol) of a DMF / THF (1 / 9, 7 mL) mixed solvent was added. The reaction was stirred at room temperature for 24 h. After the reaction was complete, 50 mL of ethyl acetate was added and stirred for 10 min. The reaction solution was then washed with saturated brine (50 mL x 3), and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate at a volume ratio of 4:3 as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound. (F) (339 mg, 33.7%).

[0050] 1 H NMR (500 MHz, CDCl3): δ (ppm) = 10.55 (s, 2H), 7.92 (s, 2H), 5.60 (dd, J = 10.5, 8.0 Hz, 1H), 5.40 (dd, J = 3.5, 1.0 Hz, 1H), 5.08 (dd, J =10.5, 3.5 Hz, 1H), 4.87 (d, J = 8.0 Hz, 1H), 4.04 (d, J = 6.5 Hz, 2H), 3.85-3.79 (m, 1H), 2.44 (s, 3H), 2.22 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H), 1.95(s, 3H); 13 C NMR (125 MHz, CDCl3): δ (ppm) = 188.71, 170.18, 170.12, 169.93,169.39, 157.44, 136.65, 134.44, 130.53, 103.56, 77.40, 71.35, 70.69, 69.06,66.75, 60.72, 20.78, 20.67, 20.50, 20.44; HRMS-ESI (m / z): calculated forC 23 H 26 O 12 [M+Na] + : 517.1316; found 517.1307.

[0051] Compound F 1 H NMR spectrum as shown Figure 18 As shown, compound F 13 The C NMR spectrum is as follows Figure 19 As shown, the high-resolution mass spectrum of compound F is as follows: Figure 20 As shown.

[0052] (4) (0.23 g, 0.51 mmol) was completely dissolved in 5 mL of anhydrous tetrahydrofuran. A 1.0 M LiAlH(OtBu)3 THF solution (2.0 mL, 2.0 mmol) was added to the above solution under ice bath conditions. After reacting for 3 hours, a saturated ammonium chloride aqueous solution (5 mL) and ethyl acetate (10 mL) were added to the mixture, and stirring was continued for 1 hour. After the reaction was complete, 10 mL of a prepared saturated potassium hydrogen tartrate aqueous solution was added, and the mixture was extracted three times with 20 mL of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powdery compound. (G) (239 mg, 94%).

[0053] 1 H NMR (400 MHz, CDCl3): δ (ppm) =7.19 (s, 2H), 5.52 (dd, J = 10.4,8.0 Hz, 1H), 5.40 (d, J = 3.6 Hz, 1H,), 5.09 (dd, J = 10.4, 3.6 Hz, 1H), 5.05(d, J = 8.4 Hz, 1H, CH), 4.77 (d, J = 12.0 Hz, 2H), 4.53 (d, J = 12.0 Hz, 2H), 4.20 - 4.06 (m, 2H), 3.86 (t, J =6.8 Hz, 1H), 2.34 (s, 3H), 2.22 (s,3H), 2.19 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H). 1H NMR (400 MHz, CDCl3) δ =7.19 (s, 2H), 5.52 (dd, J = 10.4, 8.0 Hz, 1H), 5.40 (d, J = 3.6 Hz, 1H,),5.09 (dd, J = 10.4, 3.6 Hz, 1H), 5.05 (d, J = 8.4 Hz, 1H, CH), 4.77 (d, J =12.0 Hz, 2H), 4.53 (d, J = 12.0 Hz, 2H), 4.20 - 4.06 (m, 2H), 3.86 (t, J =6.8Hz, 1H), 2.34 (s, 3H), 2.22 (s, 3H), 2.19 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H); 13C NMR (125 MHz, CDCl3): δ (ppm) = 170.52, 170.43, 170.26, 169.76,149.40, 135.61, 134.27, 130.46, 102.52, 71.23, 70.82, 69.23, 67.18, 61.43,60.02, 20.84, 20.78, 20.69, 20.55, 20.49; HRMS-ESI (m / z): calculated forC 23 H 30 O 12 [M+Na] + : 521.1629; found 521.1622.

[0054] Compound G 1 H NMR spectrum as shown Figure 15 As shown, compound G's 13 The C NMR spectrum is as follows Figure 16 As shown, the high-resolution mass spectrum of compound G is as follows: Figure 17 As shown.

[0055] (5) Chlorobasilar integriate (48 mg, 0.16 mmol) was dissolved in 3 mL of anhydrous dichloromethane with EDCI (53 mg, 0.30 mmol) and 4-PPY (72 mg, 0.49 mmol) in an ice bath and stirred for 30 min. (100 mg, 0.20 mmol) was dissolved in 2 mL of anhydrous dichloromethane and added to the reaction system. The reaction was continued for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (100 / 1, v / v) as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound. (C) (42 mg, 33%).

[0056] 1H NMR (500 MHz, CDCl3): δ (ppm) = 7.25 (d, J = 2.0 Hz, 1H), 7.15 (d,J = 2.0 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.5 Hz, 2H), 5.51(dd, J = 10.5, 8.0 Hz, 1H), 5.36 (dd, J = 3.5, 1.5 Hz, 1H), 5.17 (d, J = 12.5Hz, 1H), 5.07 (d, J = 12.5 Hz, 1H), 5.02 (dd, J = 10.5, 3.5 Hz, 1H), 4.92 (d,J = 8.0 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H), 4.17 –4.06 (m, 2H), 3.77 (ddd, J = 7.0, 6.0, 1.5 Hz, 1H), 3.72 – 3.68 (m, 4H), 3.64– 3.59 (m, 4H), 2.57 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 2.34 (s,3H), 2.21 (s, 3H), 2.17 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.94 (t, J = 7.5Hz, 2H); 13 C NMR (151 MHz, CDCl3): δ = 173.27, 170.39, 170.34, 170.11, 169.65,149.90, 144.45, 135.86, 135.62, 131.42, 130.85, 130.58, 129.80, 128.65,112.27, 102.85, 71.38, 70.94, 69.03, 67.05, 61.42, 61.38, 60.38, 53.67,40.57, 34.06, 33.80, 27.04, 20.92, 20.88, 20.79, 20.63, 20.62; HRMS-ESI (m / z)calculated for C 37 H 47 C l2 NO 13 [M+H] +: 784.2489; found 784.2497.

[0057] Compound C 1 H NMR spectrum as shown Figure 12 As shown, compound C 13 The C NMR spectrum is as follows Figure 13 As shown, the high-resolution mass spectrum of compound C is as follows: Figure 14 As shown.

[0058] (6) The dark green solid compound (B) (30 mg, 0.026 mmol) prepared in step (1) was dissolved in 3 mL of anhydrous dichloromethane with EDCI (25 mg, 0.13 mmol) and 4-PPY (20 mg, 0.13 mmol) in an ice bath and stirred for 30 min. The white powder compound (C) (20 mg, 0.026 mmol) prepared in step (5) was dissolved in 2 mL of anhydrous dichloromethane and added to the reaction system. The reaction was continued for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (50 / 1, v / v) as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain the green powder compound. (D) (24 mg, 50%).

[0059] 1H NMR (500 MHz, CDCl3): δ = 8.25 (d, J = 8.5 Hz, 2H), 8.15 (d, J =16.5 Hz, 2H), 7.63 – 7.56 (m, 6H), 7.44 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 2.0Hz, 1H), 7.21 (d, J = 2.0 Hz, 1H), 7.08 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5Hz, 4H), 6.63 (d, J = 9.0 Hz, 2H), 5.60 – 5.51 (m, 2H), 5.47 (d, J = 13.0 Hz,1H), 5.38 (dd, J = 3.5, 1.0 Hz, 1H), 5.26 (d, J = 12.5 Hz, 1H), 5.15 (d, J =13.0 Hz, 1H), 5.03 (dd, J = 10.5, 3.5 Hz, 1H), 4.91 (d, J = 8.0 Hz, 1H), 4.21– 4.18 (m, 4H), 4.12 (dd, J = 7.0, 1.0 Hz, 2H), 3.91 – 3.88 (m, 4H), 3.80 –3.74 (m, 5H), 3.72 – 3.66 (m, 12H), 3.62 (ddd, J = 8.0, 6.5, 1.0 Hz, 4H),3.59 – 3.55 (m, 4H), 3.39 (s, 6H), 2.59 (t, J = 7.5 Hz, 2H), 2.42 (t, J = 7.5Hz, 2H), 2.38 (s, 3H), 2.23 (s, 3H), 2.19 (s, 3H), 2.02 (s, 3H), 1.99 (s,3H), 1.98 – 1.90 (m, 2H), 1.43 (s, 6H); 13C NMR (151 MHz, CDCl3): δ = 173.23,170.30, 170.26, 170.03, 169.51, 165.61, 160.07, 150.87, 149.84, 145.26,144.36, 140.33, 139.53, 136.59, 135.73, 132.33, 131.17, 130.74, 130.66,130.47, 130.34, 130.08, 129.86, 129.69, 129.60, 129.32, 128.99, 116.61,115.00, 112.18, 103.22, 83.03, 71.93, 70.96, 70.91, 70.87, 70.66, 70.58, 69.66, 69.04, 67.55, 66.73, 62.44, 61.25, 60.71, 59.05, 53.57, 40.50, 33.96, 33.66, 26.83, 21.04, 20.80, 20.74, 20.62, 20.57, 17.71.

[0060] Compound D 1 H NMR spectrum as shown Figure 10 As shown, compound D 13 The C NMR spectrum is as follows Figure 11 As shown.

[0061] (7) The green powder compound (D) (40 mg, 0.021 mmol) prepared in step (6) was dissolved in 2 mL of a mixed solvent of dichloromethane / methanol (1 / 4, v / v), and sodium bicarbonate (200 mg, 2.38 mmol) was added and stirred for 4 hours. After the reaction was completed, 10 mL of ethyl acetate was added, and the reaction solution was washed three times with 20 mL of saturated brine each time. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 30:1 as the eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain the green powder compound. (A) (12 mg, 32%).

[0062] 1H NMR (500 MHz, CDCl3): δ = 8.23 (d, J = 8.5 Hz, 2H), 8.15 (d, J =16.5 Hz, 2H), 7.64 – 7.53 (m, 6H), 7.42 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 2.0Hz, 1H), 7.18 (d, J = 2.0 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 9.0Hz, 3H), 6.61 (d, J = 8.7 Hz, 2H), 5.87 (d, J = 13.0 Hz, 1H), 5.60 (d, J =13.0 Hz, 1H), 5.49 (d, J = 13.0 Hz, 1H), 5.22 (d, J = 13.0 Hz, 1H), 4.72 (d,J = 8.0 Hz, 1H), 4.21 – 4.17 (m, 4H), 4.08 – 4.00 (m, 2H), 3.96 (dd, J =12.5, 5.0 Hz, 1H), 3.92 – 3.88 (m, 4H), 3.85 (d, J = 12.5 Hz, 1H), 3.80 –3.73 (m, 4H), 3.73 – 3.65 (m, 13H), 3.63 – 3.59 (m, 4H), 3.59 – 3.54 (m, 5H),3.45 – 3.40 (m, 1H), 3.39 (s, 6H), 2.56 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 7.5Hz, 2H), 2.37 (s, 3H), 1.93 (p, J = 7.5 Hz, 2H), 1.41 (s, 6H); 13C NMR (151MHz, CDCl3): δ = 173.95, 166.05, 160.07, 150.84, 150.30, 145.22, 144.38,140.47, 139.53, 136.47, 135.31, 132.30, 130.98, 130.71, 130.35, 130.25,130.17, 129.97, 129.75, 129.67, 129.59, 129.33, 128.99, 116.59, 115.00,112.14, 106.11, 83.07, 74.73, 73.80, 71.96, 71.92, 70.86, 70.65, 70.56,69.66, 69.59, 67.54, 62.81, 62.57, 61.69, 59.05, 53.53, 40.54, 33.92, 33.68,26.72, 21.08, 17.75; HRMS(ESI): m / z calculated for C 77 H 90 BC l2 F2I2N3O 18 [M+H] + :1718.3812; found 1718.3820.

[0063] Compound A 1 H NMR spectrum as shown Figure 7 As shown, compound A 13 The C NMR spectrum is as follows Figure 8 As shown, the high-resolution mass spectrum of compound A is as follows: Figure 9 As shown.

[0064] Application Example 1

[0065] The tumor-targeting properties of the prepared β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug (A) were investigated. Based on the significantly higher content of glucose transporter receptors on the surface of tumor cells compared to normal cells, tumor cells showed a higher uptake of this compound, resulting in a significantly higher fluorescence intensity in tumor cells. Therefore, this β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug can be used to track tumor cells, enabling tumor imaging and early diagnosis.

[0066] Tumor targeting assay: Human ovarian cancer cells A2780, mouse ovarian cancer cells ID8, and normal cells 3T3 in good growth condition were digested with trypsin (containing 0.25% EDTA) and then cultured in DMEM medium (containing 10% fetal bovine serum) to prepare 1×10⁻⁶ cells / day. 5 Cell suspension of cells / mL was evenly spread in 12-well plates and incubated overnight in an incubator. The plates were then removed and washed three times with PBS buffer. Cell culture medium (containing 0.9% DMSO and 0.1% Tween 80) containing a certain concentration (1 μM) of β-galactose-modified chlorambucil-fluoroboropyrrole combined with an antitumor prodrug was added to each well, and the plates were incubated for 1 min, 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, and 2 h. The plates were then removed and washed three times with PBS buffer. 1 mL of trypsin was added to each well for digestion, and fluorescence detection was performed using flow cytometry: the APC channel (excitation wavelength 633 nm, emission wavelength 690 nm) was used, with an injection volume of 50 μL per well at a medium injection rate. 10,000 events were performed per sample. The cell control group and the drug experimental group were detected separately. The results are presented as a histogram, where the x-axis represents the fluorescence intensity of drug uptake by cells, and the y-axis represents the cell number.

[0067] This invention used flow cytometry to determine the fluorescence intensity of the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug prepared in Example 1 in human ovarian cancer cells A2780, mouse ovarian cancer cells ID8, and normal cells 3T3. The excitation wavelength was 633 nm. Figure 1 ). Flow cytometry histograms and fluorescence intensity comparisons of compound A prepared in Example 1 in tumor cells A2780, ID8, and normal cells 3T3.

[0068] Experimental data showed that in tumor cells A2780 and ID8, the fluorescence intensity of drug A increased with increasing incubation time, reaching saturation at 2 hours; however, no significant uptake was observed in normal cells 3T3. These results indicate that the combination of β-galactose-modified chlorambucil-fluoroboropyrrole and the antitumor prodrug A can rapidly target tumor cells. This imaging technique can be used for tumor diagnosis and to guide photodynamic therapy and surgery.

[0069] Application Example 2

[0070] The targeted uptake of the prepared β-galactose-modified chlorambucil-fluboron dipyrrole antitumor prodrug (A) was studied through co-culture. To further illustrate the targeting effects of the drug on cancer cells, a competitive uptake experiment was conducted.

[0071] Co-culture targeted uptake experiment: Well-grown mouse ovarian cancer cells ID8 and normal cells 3T3 were digested, pipetted, and counted, and then diluted to 8×10⁻⁶. 4 cells / mL, 1.2×10 5 Cell suspension at 0.5 mL / mL was added to the same well of a 12-well plate and mixed thoroughly. The plates were incubated overnight at 37°C with 5% CO2. Once the cells adhered and spread out, the culture medium was discarded, and a cell culture medium containing a specific concentration (1 μM) of β-galactose-modified chlorambucil-fluoroboropyrrole combined with an antitumor prodrug (containing 0.9% DMSO and 0.1% Tween 80) was added. The cells were incubated for 2 h. After washing four times with PBS, serum-free and phenol red-free medium was added, and images were taken using a fluorescence microscope. The emission wavelength receiving range was 650-750 nm (633 nm excitation).

[0072] This invention used fluorescence microscopy to determine the difference in fluorescence intensity between mouse ovarian cancer cells ID8 and normal cells 3T3 when the β-galactose-modified chlorambucil-fluoroboropyrrole combined antitumor prodrug prepared in Example 1 was co-cultured. The excitation wavelength was 633 nm. Figure 2 ).

[0073] Experimental data show that the fluorescence intensity of drug A in tumor cells (ID8) is significantly greater than that in normal cells (3T3). These results further demonstrate that the combination of β-galactose-modified chlorambucil-fluoroboropyrrole and the antitumor prodrug A can selectively target tumor cells. This imaging technique can be used to diagnose tumors and guide photodynamic therapy and surgery.

[0074] Application Example 3

[0075] The targeted uptake inhibition of the prepared β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug (A) was investigated. By adding β-galactose, the drug competitively inhibited the cellular uptake of the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug. Intracellular drug concentration was detected by flow cytometry, and the fluorescence intensity of BODIPY cells was compared between the groups with and without added β-galactose to further reflect the drug's targeting.

[0076] Inhibition of targeted uptake assay: Human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8 in good growth condition were digested with trypsin (containing 0.25% EDTA by mass) and then prepared into 1×10⁻⁶ cells / day using DMEM medium (containing 10% fetal bovine serum by volume). 5Cell suspension of cells / mL was evenly spread in 12-well plates and incubated overnight in an incubator. The plates were then removed, washed three times with PBS buffer, pretreated with β-galactose (10 mM) for 2 h, and then each well was added to cell culture medium (containing 0.9% DMSO, 0.1% Tween 80) containing a certain concentration (1 μM) of β-galactose-modified chlorambucil-fluoroboropyrrole combined with an antitumor prodrug. The plates were co-cultured for 2 h in an incubator. The plates were then removed, washed three times with PBS buffer, and 1 mL of trypsin was added to each well for digestion. Fluorescence detection was performed using flow cytometry: the APC channel (excitation wavelength 633 nm, emission wavelength 690 nm) was used for detection, and the results are presented as a histogram.

[0077] This invention used flow cytometry to determine the fluorescence intensity of the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug prepared in Example 1 in human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8, with an excitation wavelength of 633 nm. Figure 3 ). Flow cytometry histograms and fluorescence intensity comparison diagrams of compound A prepared in Example 1 in A2780 and ID8 cells.

[0078] The experimental data show that the uptake of drug A by both cell types was significantly reduced after treatment with β-galactose. This phenomenon fully demonstrates that drug A can enter cells through the glucose transport receptor on the cell surface, showing that the targeting of tumor cells is achieved by targeting glucose transport receptors.

[0079] Application Example 4

[0080] The cytotoxicity of the prepared β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug (A) was investigated. Drug cytotoxicity typically includes phototoxicity and dark toxicity, generally determined using the MTT assay (tetraazole salt reduction method). The principle is that succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetraazole bromide) to a water-insoluble blue-purple crystalline substance called formazan, which is then deposited in the cells. Dead cells lack succinate dehydrogenase and therefore do not produce formazan. The formazan produced in living cells is dissolved in DMSO (dimethyl sulfoxide), and its absorbance at 570 nm is measured using a microplate reader, indirectly reflecting the number of living cells. Within a certain cell number range, the amount of formazan formed is directly proportional to the number of living cells.

[0081] MTT experiment:

[0082] a. Cell Plating: Select healthy human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8, respectively. Remove the old culture medium from the culture flasks, wash three times with PBS, add 1 mL of trypsin, and incubate for 2 min. Remove the flasks, add 2 mL of fresh Dulbecco's modified Eagle's medium (DMEM) to stop the digestion, and carefully pipette the cells onto the flask walls until the flask walls are clear. Continue pipetting to homogenize the cell suspension, then transfer aliquots into three 1.5 mL centrifuge tubes and centrifuge. Remove the old culture medium, add 1 mL of fresh DMEM to resuspend the cells, and count them. Dilute the cells to a density of 5 × 10⁻⁶ cells / mL. 4 Cells were added evenly to a 96-well plate using a pipette, with 6 replicates for each concentration. 100 μL of cell suspension was added to each well.

[0083] b. Drug Addition: Prepare DMSO stock solutions (containing 5% Tween 80) with drug concentrations of 1 mM, 0.316 mM, 0.1 mM, 0.0316 mM, 0.01 mM, 0.00316 mM, and 0.001 mM. Dilute 10 μL of the stock solution to 1 mL of fresh DMEM medium to obtain logarithmic concentrations {log[conc.(M)]} of -5.0, -5.5, -6.0, -6.5, -7.0, -7.5, and -8.0, respectively. Remove the old medium from the 96-well plate using a pipette and wash three times with PBS. Add 100 μL of the corresponding drug concentration to each well. Incubate the 96-well plate overnight in a cell culture incubator to allow cells to take up the drug.

[0084] c. Phototoxicity and Dark Toxicity Assays: After removing the old culture medium and washing three times with PBS, add 100 μL of fresh culture medium to each well. In the phototoxicity assay, illuminate the well with a 660nm LED light for 2 minutes, then incubate overnight. For the dark toxicity assay, immediately place the well in the incubator after replacing the culture medium with fresh one, avoiding light exposure throughout the process.

[0085] d. OD value detection: After culture, MTT solution (5 mg / mL, 10 μL) was added to each well using a pipette. The 96-well plate was then incubated for 4 hours to allow MTT to react with the cells. The culture medium was removed, and 100 μL of DMSO was added to each well to lyse the cells and dissolve the intracellular formazan. The 96-well plate was then shaken on a shaker for 15 minutes to fully dissolve the formazan. The OD value of the solution at 570 nm was then measured using a microplate reader.

[0086] This invention used the MTT assay to determine the β-galactose-modified chlorambucil-fluoroboropyrrole combined antitumor prodrug prepared in Example 1, and its killing effect on human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8 under both light and dark conditions. The light wavelength was 660 nm and the light energy density was 2.4 J·cm⁻¹. -2 Based on experimental data and Figure 4 It can be seen that under dark conditions, compared with the original drug chlorambucil, this prodrug has a stronger inhibitory effect on A2780 and ID8 cells, indicating that the bioavailability of chlorambucil is improved. Under light conditions, the drug exhibits strong phototoxicity against A2780 and ID8 cells, with a half-maximal inhibitory concentration (IC50) of 1 / 3. 50 The concentrations (μM and μM) were 0.39 μM and 0.60 μM, respectively (see Table 1). These results indicate that the β-galactose-modified chlorambucil-fluoroboropyrrole combination antitumor prodrug exhibits strong photodynamic and chemotherapeutic combined anticancer activity against A2780 and ID8, and can selectively kill cancer cells.

[0087] Table 1. IC50 of β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug on A2780 and ID8 cells. 50 value

[0088] <![CDATA[IC 50 (μM)]]> 0.39 0.60

[0089] Application Example 5

[0090] The reactive oxygen species (ROS) generation capacity of the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug (A) was investigated. ROS generated by photosensitizers during photodynamic therapy can directly kill tumor cells; therefore, the ability of photosensitizers to generate ROS within cells is particularly important. DCFH-DA (2,7-dichlorofluorescein diacetate) is an indicator for detecting intracellular ROS. It does not possess fluorescence itself. After entering the cell via diffusion, DCFH-DA is hydrolyzed into non-fluorescent DCFH by intracellular esterases. DCFH cannot permeate the cell membrane. After being rapidly oxidized by intracellular ROS, DCFH generates highly fluorescent DCF. The fluorescence of DCF can be detected to determine the strength of the compound's ability to generate ROS within cells.

[0091] Reactive oxygen species detection experiment:

[0092] Human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8, in good growth condition, were digested with trypsin (containing 0.25% EDTA) and prepared into 1×10⁻⁶ cells / day using DMEM medium (containing 10% fetal bovine serum). 5Cell suspension of cells / mL was evenly spread in 12-well plates and incubated overnight in an incubator. The plates were then removed, washed three times with PBS buffer, and each well was incubated with a cell culture medium (containing 0.9% DMSO and 0.1% Tween 80) containing a specific concentration (1 μM) of β-galactose-modified chlorambucil-fluoroboropyrrole combined with an antitumor prodrug. The plates were then co-incubated for 2 h. 1 μL of the DCFH-DA kit solution was diluted in 1 mL of culture medium and mixed thoroughly to prepare the DCFH-DA working solution. The plates were removed, washed three times with PBS buffer, and 1 mL of DCFH-DA working solution was added to each well. The plates were then incubated for another 1 h. The culture medium was aspirated from the wells, and the plates were washed twice with PBS. The culture medium was then replaced, and the plates were placed in a 660 nm LED light plate (20 mW / cm²). 2 After 2 min of illumination, the cells were washed three times with PBS, and then 1 mL of trypsin was added to each well for digestion. Fluorescence detection was performed using flow cytometry: the FITC channel (excitation wavelength 488 nm, emission wavelength 525 nm) was selected for detection, and the results are presented as a histogram. The horizontal axis represents the fluorescence intensity of intracellular DCF, and the vertical axis represents the number of cells.

[0093] This invention used flow cytometry to determine the fluorescence intensity of DCF produced in human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8 by the β-galactose-modified chlorambucil-fluoroboropyrrole combined antitumor prodrug prepared in Example 1. The excitation wavelength was 488 nm. Figure 5 ). Flow cytometry histograms and fluorescence intensity comparison diagrams of compound A prepared in Example 1 in A2780 and ID8 cells.

[0094] The experimental data show that both cell types produced high DCF fluorescence after light exposure. This phenomenon fully demonstrates that drug A can generate reactive oxygen species through photodynamic action, thereby killing cells.

[0095] Application Example 6

[0096] The prepared β-galactose-modified chlorambucil-fluoroboropyrrole combined antitumor prodrug (A) was studied using live and dead cell staining fluorescence imaging. To visually observe the combined chemotherapy-photodynamic therapy effect, live and dead cells were stained with Calcein AM and PI double staining solutions. During photodynamic therapy, the ROS generated by the photosensitizer can directly kill tumor cells; therefore, the photosensitizer's ability to generate ROS within cells is particularly important. DCFH-DA (2,7-dichlorofluorescein diacetate) is an indicator for detecting intracellular ROS. It does not possess fluorescence itself. After entering the cell via diffusion, DCFH-DA is hydrolyzed into non-fluorescent DCFH by intracellular esterases. DCFH cannot permeate the cell membrane. After being rapidly oxidized by intracellular ROS, DCFH generates highly fluorescent DCF. The fluorescence of DCF can be used to determine the strength of the compound's ability to generate ROS within cells.

[0097] Dead and live cell staining experiment:

[0098] Human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8, in good growth condition, were digested with trypsin (containing 0.25% EDTA) and prepared into 1×10⁻⁶ cells / day using DMEM medium (containing 10% fetal bovine serum). 5 Cell suspension at a concentration of [number] cells / mL was evenly spread in 12-well plates and incubated overnight in an incubator. The plates were then removed, washed three times with PBS buffer, and each well was added to cell culture medium (containing 0.9% DMSO and 0.1% Tween 80) containing a specific concentration (0.5 μM) of β-galactose-modified chlorambucil-fluoroboropyrrole combined with an antitumor prodrug. The plates were co-cultured for 2 hours. The plates were then removed, washed three times with PBS buffer, and the culture medium was replaced. The plates were then placed under a 660 nm LED light (20 mW / cm²) lamp. 2 After 2 min of illumination, remove the cells and wash them three times with PBS, then continue incubation overnight. Accurately pipette 1 μL of calcium yellow-green methyl acetylate solution (Calcein-AM) and 1 μL of propidium iodide (PI) into 1 mL of DMEM, mix well to obtain the live cell detection solution. Add this detection solution to the wells and continue incubation for 1 h. Take pictures using a fluorescence microscope. (Calcein-AM fluorescence: excitation wavelength 488 nm, detection wavelength 510-570 nm; PI: excitation wavelength 543 nm, detection wavelength 550-620 nm).

[0099] This invention used fluorescence microscopy to determine the cell viability of the β-galactose-modified chlorambucil-fluboron dipyrrole combined with the antitumor prodrug prepared in Example 1 in human ovarian cancer cells A2780 and mouse ovarian cancer cells ID8. Figure 6 ).

[0100] Experimental data show that under dark conditions, compared with the original drug chlorambucil, this drug exhibits a stronger inhibitory effect on A2780 and ID8 cells, indicating improved bioavailability of chlorambucil. Under light conditions, the drug shows strong phototoxicity against A2780 and ID8 cells. These results indicate that the β-galactose-modified chlorambucil-fluoroboropyrrole combination antitumor prodrug exhibits strong photodynamic and chemotherapeutic combined anticancer activity against A2780 and ID8 cells, selectively killing cancer cells.

[0101] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug, characterized in that: The β-galactose-modified chlorambucil-fluoroboron dipyrrole co-antitumor prodrug is specifically a β-galactose-modified chlorambucil prodrug covalently linked via an esterification reaction, and its chemical structural formula is as follows:

2. A method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug as described in claim 1, characterized in that: Includes the following steps: (1) Using compounds Using these as starting materials, synthesize compounds (2) Then, using compound D synthesized in step (1) as the starting material and NaHCO3 as the base, a β-galactose-modified chlorambucil-fluoroboropyrrole co-antitumor prodrug was synthesized:

3. The method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug according to claim 2, characterized in that: The specific steps are as follows: (1) Compound 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-pyrrolidinylpyridine were dissolved in anhydrous dichloromethane in a molar ratio of 1:5:5 and stirred for 30 min in an ice bath; then, based on the molar amount of compound B, 1 equivalent of The solution was added to the reaction system in anhydrous dichloromethane, and the reaction was continued for 8 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 50:1 as the eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain a green powder compound. (2) Compound The compound was dissolved in a 1:4 (v / v) mixture of dichloromethane and methanol. 110 equivalents of sodium bicarbonate were added based on the molar amount of compound D, and the mixture was stirred for 4 hours. After the reaction was complete, 10 mL of ethyl acetate was added. The reaction solution was then washed three times with 20 mL of saturated brine each time. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using a 30:1 (v / v) mixture of dichloromethane and methanol as eluent. The solvent was removed by rotary evaporation under reduced pressure to obtain a green powdery β-galactose-modified chlorambucil-fluoroboron dipyrrole co-antitumor prodrug.

4. The method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug according to claim 2 or 3, characterized in that: The compound The synthesis method specifically includes the following steps: With compounds Using these as starting materials, synthesize compounds 5. The method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug according to claim 2 or 3, characterized in that: The compound The specific synthesis steps include: compound Dissolve in 150 mL of anhydrous toluene, as compound The molar quantity meter, then 2 equivalents Add the toluene to the reaction mixture and stir. Then add 1.2 mL piperidine, 1.0 mL glacial acetic acid, and anhydrous magnesium perchlorate, and reflux for 4 h. After the reaction is complete, remove most of the toluene using a rotary evaporator. The crude product is purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 50:1 as eluent, and the solvent is removed by rotary evaporation under reduced pressure to obtain a dark green solid compound.

6. The method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug according to claim 2 or 3, characterized in that: The compound The synthesis method specifically includes the following steps: (1) Using compounds Synthesizing compounds from starting materials (2) Using compounds Synthesizing compounds from starting materials (3) Using compounds Synthesizing compounds from starting materials (4) Using compounds Using these as starting materials, synthesize compounds 7. The method for preparing the β-galactose-modified chlorambucil-fluboron dipyrrole prodrug according to claim 2 or 3, characterized in that: The compound The specific synthesis steps include: (1) Under ice bath conditions, Dissolve in 20 mL of anhydrous dichloromethane solution, with Using a molar ratio, 7 equivalents of hydrobromoacetic acid solution were slowly added dropwise to the reaction system, and the mixture was stirred rapidly until homogeneous. The reaction was carried out in the dark for 3 hours. After the reaction was completed, the mixture was washed three times with 30 mL of ice-cold saturated sodium bicarbonate solution, and then washed three times with 30 mL of ice water. The organic phase was dried with anhydrous sodium sulfate and obtained as a transparent gel by rotary evaporation under reduced pressure. (2) Mix cesium carbonate with a mixed solvent of N,N-dimethylformamide / tetrahydrofuran at a volume ratio of 1:9 until homogeneous. Add dropwise a solution containing 0.25 equivalents of cesium carbonate. A mixed solvent of N,N-dimethylformamide / tetrahydrofuran with a volume ratio of 1:9 was added dropwise, followed by the addition of 0.25 equivalents of... A mixture of N,N-dimethylformamide and tetrahydrofuran (volume ratio 1:9) was used as a solvent, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, 50 mL of ethyl acetate was added and stirred for 10 min. The reaction solution was then washed three times with 50 mL of saturated brine each time. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate (volume ratio 4:3) as eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound. (3) Dissolve completely in 5 mL of anhydrous tetrahydrofuran, Using a molar ratio, 4 equivalents of a tetrahydrofuran solution of lithium tri-tert-butyloxyaluminum hydride (LiAlH(OtBu)3) was added dropwise to the mixed solution under ice bath conditions. After reacting for 3 hours, 5 mL of saturated ammonium chloride aqueous solution and 10 mL of ethyl acetate were added to the mixture, and stirring was continued for 1 hour. After the reaction was completed, 10 mL of a prepared saturated potassium hydrogen tartrate aqueous solution was added, and the mixture was extracted three times with 20 mL of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powder as the compound. (4) 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-pyrrolidinylpyridine were dissolved in anhydrous dichloromethane at a molar ratio of 5:10:16 and stirred in an ice bath for 30 min; The molar quantity is 1.25 equivalent. The solution was added to the reaction system in 2 mL of anhydrous dichloromethane, and the reaction was continued for 8 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol at a volume ratio of 100:1 as eluent, and the solvent was removed by rotary evaporation under reduced pressure to obtain a white powder compound.

8. The use of the β-galactose-modified chlorambucil-fluboron dipyrrole combined antitumor prodrug as described in claim 1 in the preparation of antitumor drugs.

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