Hypoxic activated antibacterial and anticancer compound based on gemcitabine and application of hypoxic activated antibacterial and anticancer compound

Abstract

The invention discloses a gemcitabine-based hypoxic activated antibacterial and anticancer compound which releases an antibacterial drug LEV and an antitumor drug GEM under the action of nitroreductase after reaching a tumor hypoxic region; lEV kills bacteria in tumor cells, Gem inhibits growth of the tumor cells, and then good and effective antibacterial effect and anticancer effect are shown. The invention discloses an application of a gemcitabine-based hypoxia activated antibacterial and anticancer compound in preparation of a medicine for treating pancreatic cancer. The gemcitabine-based hypoxia activated antibacterial and anticancer compound has good lipid solubility, good hypoxia selectivity, no obvious toxicity to other tissues and good biological safety, and can be used for preparing drugs for treating pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a gemcitabine-based hypoxia-activated antibacterial and anticancer compound and application thereof. Background Art

[0002] Gemcitabine (GEM) is a first-line drug for the treatment of advanced pancreatic cancer, which benefits patients in clinical practice. However, GEM also has many defects: poor lipid solubility, poor transmembrane ability, and short half-life in vivo; GEM will be degraded by cytidine deaminase (CDA) in the blood, resulting in inactivation, resulting in reduced bioavailability. Frequent high-dose administration is required to ensure efficacy, leading to severe bone marrow suppression, hepatotoxicity, and nephrotoxicity, and drug resistance. In order to improve the metabolic stability of gemcitabine, circumvent GEM resistance, and improve efficacy, gemcitabine is chemically modified to block the deamination of deoxycytidine deaminase by protecting the function of amine, thereby enhancing the therapeutic benefits and reducing adverse reactions. For example, the prodrug valproic acid-GEM obtained by modifying the 4-NH2 of gemcitabine with valproic acid blocks the metabolism of free GEM. The prodrug releases GEM under the mediation of intracellular carboxylesterase 2 to prevent cancer cell proliferation. However, in Phase I clinical trials, valproic acid-GEM was suspended due to hepatotoxicity. Squalene-GEM conjugates have also been used to study the drug resistance and rapid inactivation of GEM, but the accumulation of squalene-GEM in gastric and intestinal tissues is significantly enhanced. The 4-NH2 of GEM was modified with 1,2-dithiolane, and then GEM was conjugated with 1,2-dithiolane through carbamate to synthesize the prodrug S-GEM. S-GEM can be activated by thioredoxin reductase in the body, releasing GEM to produce anti-tumor effects. The 5′-OH of GEM is esterified with fatty acids to reduce its hydrophilicity, overcoming the drug resistance of GEM caused by changes in nucleoside transporters. Trans-9-octadecenoic acid-GEM is formed by conjugating the 5′-OH of GEM with trans-9-octadecenoic acid, which can pass through the cell membrane by passive diffusion and then be converted into GEM in the cell by esterase. The uptake of trans-9-octadecenoic acid-GEM in cells has nothing to do with nucleoside transporters, which is beneficial to blocking the multidrug resistance mechanism; trans-9-octadecenoic acid-GEM is not a substrate for deaminase, which increases stability. However, the accumulation of squalene-GEM in gastric and intestinal tissues is significantly enhanced, while the absorption and accumulation in tumor tissues are slower. GEM phosphate prodrugs can also reduce the occurrence of drug resistance (Slusarczykm, et al. J Med Chem. 2014; 57(4): 1531-42.), but GEM phosphate prodrugs cannot prevent its metabolism by deoxycytidine deaminase, nor can they reduce its toxic side effects on non-tumor tissues; aminoacylation of gemcitabine phosphate prodrugs (WO2015 / 134334) can delay the metabolism of drugs by deoxycytidine deaminase, but still cannot reduce the toxic side effects of drugs on non-tumor tissues. In addition, cytidine deaminase (CDD L ) contribute to GEM inactivation and drug resistance. Although, GEM resistance can be reversed by systemic antibiotics, this treatment disrupts the balance of the intestinal microbiota.

[0003] In summary, there is an urgent need for the emergence of a new gemcitabine-based hypoxia-activated antibacterial and anticancer compound to solve the above-mentioned technical problems. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a gemcitabine-based hypoxia-activated antibacterial and anticancer compound, the chemical structure of the gemcitabine-based hypoxia-activated antibacterial and anticancer compound is as follows:

[0006]

[0007] After the gemcitabine-based hypoxia-activated antibacterial and anticancer compound reaches the hypoxic area of ​​the tumor, it releases the antibacterial drug LEV and the antitumor drug GEM under the action of nitroreductase; LEV kills the bacteria in the tumor cells, and Gem inhibits the growth of tumor cells, thereby showing good and effective antibacterial and anticancer effects.

[0008] Another object of the present invention is to use a hypoxia-activated antibacterial and anticancer compound based on gemcitabine in the preparation of a drug for treating pancreatic cancer.

[0009] Another object of the present invention is to provide a drug for treating pancreatic cancer, wherein the active ingredient is a hypoxia-activated antibacterial and anticancer compound based on gemcitabine.

[0010] The present invention has at least the following beneficial effects:

[0011] The hypoxia-activated antibacterial and anticancer compound based on gemcitabine provided by the present invention uses the hypoxia-responsive group p-nitrobenzyl to couple the antibacterial drug (such as levofloxacin LEV) and the anticancer drug GEM to obtain the hypoxia-activated antibacterial and anticancer compound (compound LEV-NO2-GEM). After reaching the hypoxic area of ​​the tumor, the compound LEV-NO2-GEM releases the antibacterial drug LEV and the antitumor drug GEM under the action of nitroreductase, thereby showing good effective antibacterial and anticancer effects.

[0012] In addition, gemcitabine-based hypoxia-activated antibacterial and anticancer compounds have good lipid solubility, good hypoxia selectivity, no obvious toxicity to other tissues, good biosafety, and can be used to prepare drugs for the treatment of pancreatic cancer.

[0013] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a preparation route of the p-nitrobenzyl ether-levofloxacin coupling compound LEV-NO2 in one embodiment of the present invention;

[0015] Figure 2 The synthetic route of the hypoxia activated antibacterial / anticancer prodrug LEV-NO2-GEM in one embodiment of the present invention;

[0016] Figure 3 This is a schematic diagram of the changes in tumor volume of mice in each group after administration of different drugs in one embodiment of the present invention;

[0017] Figure 4 This is a schematic diagram of the tumor mass of each group of mice after administration of different drugs in one embodiment of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0019] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0020] The present invention provides a hypoxia-activated antibacterial and anticancer compound based on gemcitabine, the chemical structure of which is shown below:

[0021]

[0022] The method for preparing the hypoxia-activated antibacterial and anticancer compound based on gemcitabine comprises the following steps:

[0023] (1) Preparation of p-nitrobenzyl ether-levofloxacin coupling compound (LEV-NO2) (preparation route as shown in Figure 1 shown):

[0024] Levofloxacin is dissolved in a solvent, and reacted under stirring at a suitable temperature for a certain time under the action of a condensing agent N,N'-dicyclohexylcarbodiimide (DCC) or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and a catalyst p-dimethylaminopyridine (DMAP); then 4-methyl-2,5-dihydroxymethylphenyl-4'-nitrobenzyl ether dissolved in a solvent is added dropwise, the temperature is raised to a suitable temperature, and the reaction is stirred in the dark; the reaction is cooled to room temperature, by-products are filtered out, the filtrate is concentrated, and purification is performed by chromatography or preparative liquid phase to obtain a p-nitrobenzyl ether-levofloxacin coupling compound LEV-NO2.

[0025] Wherein, the a solvent is one or more of DMSO, DMF, THF, MeCN and DCM.

[0026] The molar ratio of levofloxacin, the condensation agent and the catalyst is 1:1:1 to 1:20:20.

[0027] The mixing temperature of the levofloxacin, the condensation agent and the catalyst is 0 to 50° C., and the reaction is stirred for 0 to 2 hours.

[0028] The molar ratio of levofloxacin to 4-methyl-2,5-dihydroxymethylphenyl-4'-nitrobenzyl ether is 1:1-3, the temperature is 10-60° C., and the reaction is stirred in the dark for 0-24 hours.

[0029] The mixture is cooled to room temperature, by-products are filtered out, the filtrate is concentrated, and purified by chromatography or preparative liquid phase to obtain p-nitrobenzyl ether-levofloxacin coupling compound LEV-NO2.

[0030] (2) Preparation of hypoxia-activated antibacterial and anticancer compounds (LEV-GEM-NO2) (preparation route: Figure 2 shown):

[0031] Take LEV-NO2 and dissolve it in solvent b. At an appropriate temperature, add diisopropylethylamine (DIPEA) and p-nitrophenyl chloroformate to react. After the reaction is completed, add GEM, heat to an appropriate temperature, and stir to react in the dark; cool to room temperature, wash with water 3 times, extract with DCM, collect the organic phase, dry, and remove the solvent by rotary evaporation to obtain a crude product. The crude product is purified by chromatography or preparative liquid phase to obtain a light yellow solid, namely, an oxygen-activated antibacterial and anticancer compound (LEV-GEM-NO2).

[0032] Wherein, the b solvent is one or more of DMSO, DMF, THF, MeCN and DCM.

[0033] The molar ratio of LEV-NO2, p-nitrophenyl chloroformate and catalyst DIPEA is 1:1:1 to 1:20:20.

[0034] The mixing temperature of the LEV-NO2, p-nitrophenyl chloroformate and catalyst DIPEA is 0-50°C, and the reaction is stirred for 0-24h.

[0035] The molar ratio of LEV-NO2 and GEM is 1:1-3, the temperature is 10-60°C, and the reaction is stirred in the dark for 0-48 hours.

[0036] The mixture is cooled to room temperature, by-products are filtered out, the filtrate is concentrated, and purification is performed by chromatography or preparative liquid phase to obtain a light yellow solid, namely, the hypoxia-activated antibacterial and anticancer compound LEV-GEM-NO2.

[0037] Example 1

[0038] Preparation of p-nitrobenzyl ether-levofloxacin coupling compound (LEV-NO2):

[0039] LEV (200 mg, 0.56 mmol), HATU (320 mg, 0.84 mmol) and TEA (156 μL, 1.12 mmol) were dissolved in 8 mL of acetonitrile, stirred and dissolved, reacted at room temperature for 30 min, and then p-nitrobenzyl ether (168 mg, 0.56 mmol) was added, and the reaction system was heated to 40 ° C for 12 h. After the reaction, the reaction system was cooled to room temperature, the solid was filtered out, the solvent in the filtrate was removed by rotary evaporation, and then separated and purified by column chromatography (eluent: V DCM :V MeOH =10:1) to obtain 117.5 mg of light yellow solid, namely p-nitrobenzyl ether-modified levofloxacin (LEV-NO2), with a yield of 65% and a melting point of 136.1-136.8°C. 1 H NMR (DMSO-d6, 600MHz, ppm): δ8.58 (s, 1H), 8.20 (d, J = 8.7Hz, 2H), 7.72 (d, J = 8.6Hz, 2H),7.42(d,J=12.4Hz,1H),7.29(d,J=14.6Hz,2H),5.26(d,J=12.5Hz,2H),5.19(s ,2H),4.72-4.69(m,1H),4.58(d,J=5.4Hz,2H),4.50(d,J=9.9Hz,1H),4.31(d,J=9. 3Hz, 1H), 3.40 (s, 4H), 2.94 (s, 3H), 2.59 (s, 4H), 2.31 (s, 3H), 1.37 (d, J = 6.8Hz, 3H). 13 C NMR(DMSO-d6,120MHz,ppm): δ171.79,165.39,156.31,154.69,152.75,147.29,147.01,146.07,140.98,135.84,133.73,1 30.26,130.16,129.34,128.46,124.25,123.93,109.00,104.29,74.88,68.66,61.93,58.37,54.19,49.01,21.08,18.17. MS analysis:Found:m / z 647.2504[M+H] + ;calcd for C 34 H 36 FN4O8:m / z647.2517[M+H] + .

[0040] Example 2

[0041] Preparation of p-nitrobenzyl ether-levofloxacin coupling compound (LEV-NO2):

[0042] LEV (200 mg, 0.56 mmol), HATU (320 mg, 0.84 mmol) and TEA (156 μL, 1.12 mmol) were dissolved in 8 mL DMF, stirred and dissolved, reacted at room temperature for 30 min, and then p-nitrobenzyl ether (168 mg, 0.56 mmol) was added, and the reaction system was heated to 40 ° C for 12 h. After the reaction, the reaction system was cooled to room temperature, the solid was filtered out, 20 mL DCM was added, and the brine was washed 3 times. The solvent in the filtrate was removed by rotary evaporation, and then separated and purified by column chromatography (eluent: V DCM :V MeOH =10:1) to obtain 126.5 mg of light yellow solid, namely p-nitrobenzyl ether-modified levofloxacin (LEV-NO2), with a yield of 70% and a melting point of 136.1-136.8°C. 1 H NMR (DMSO-d6, 600MHz, ppm): δ8.58 (s, 1H), 8.20 (d, J = 8.7Hz, 2H), 7.72 (d, J = 8.6Hz, 2H),7.42(d,J=12.4Hz,1H),7.29(d,J=14.6Hz,2H),5.26(d,J=12.5Hz,2H),5.19(s ,2H),4.72-4.69(m,1H),4.58(d,J=5.4Hz,2H),4.50(d,J=9.9Hz,1H),4.31(d,J=9. 3Hz, 1H), 3.40 (s, 4H), 2.94 (s, 3H), 2.59 (s, 4H), 2.31 (s, 3H), 1.37 (d, J = 6.8Hz, 3H). 13 C NMR(DMSO-d6,120MHz,ppm): δ171.79,165.39,156.31,154.69,152.75,147.29,147.01,146.07,140.98,135.84,133.73,1 30.26,130.16,129.34,128.46,124.25,123.93,109.00,104.29,74.88,68.66,61.93,58.37,54.19,49.01,21.08,18.17. MS analysis:Found:m / z 647.2504[M+H] + ;calcd for C 34 H36 FN4O8:m / z 647.2517[M+H] + .

[0043] Example 3

[0044] Preparation of p-nitrobenzyl ether-levofloxacin coupling compound (LEV-NO2):

[0045] LEV (200 mg, 0.56 mmol), HATU (320 mg, 0.84 mmol) and TEA (156 μL, 1.12 mmol) were dissolved in 8 mL DMSO, stirred and dissolved, reacted at room temperature for 30 min, and then p-nitrobenzyl ether (168 mg, 0.56 mmol) was added, and the reaction system was heated to 40 ° C for 12 h. After the reaction, the reaction system was cooled to room temperature, the solid was filtered out, 20 mL DCM was added, and the brine was washed 3 times. The solvent in the filtrate was removed by rotary evaporation, and then separated and purified by column chromatography (eluent: V DCM :V MeOH =10:1) to obtain 121.1 mg of light yellow solid, namely p-nitrobenzyl ether-modified levofloxacin (LEV-NO2), with a yield of 67% and a melting point of 136.1-136.8°C. 1 H NMR (DMSO-d6, 600MHz, ppm): δ8.58 (s, 1H), 8.20 (d, J = 8.7Hz, 2H), 7.72 (d, J = 8.6Hz, 2H),7.42(d,J=12.4Hz,1H),7.29(d,J=14.6Hz,2H),5.26(d,J=12.5Hz,2H),5.19(s ,2H),4.72-4.69(m,1H),4.58(d,J=5.4Hz,2H),4.50(d,J=9.9Hz,1H),4.31(d,J=9. 3Hz, 1H), 3.40 (s, 4H), 2.94 (s, 3H), 2.59 (s, 4H), 2.31 (s, 3H), 1.37 (d, J = 6.8Hz, 3H). 13C NMR(DMSO-d6,120MHz,ppm): δ171.79,165.39,156.31,154.69,152.75,147.29,147.01,146.07,140.98,135.84,133.73,1 30.26,130.16,129.34,128.46,124.25,123.93,109.00,104.29,74.88,68.66,61.93,58.37,54.19,49.01,21.08,18.17. MSanalysis:Found:m / z 647.2504[M+H] + ;calcd for C 34 H 36 FN4O8:m / z 647.2517[M+H] + .

[0046] Example 4

[0047] Preparation of hypoxia-activated antibacterial and anticancer compounds (LEV-GEM-NO2):

[0048] LEV-NO2 (97 mg, 0.15 mmol) was dissolved in 4 mL DCM / DMSO (V DCM :V DMSO =1:1), DIPEA (131 μL, 0.75 mmol) and p-nitrophenyl chloroformate (121 mg, 0.6 mmol) were added at 0°C and reacted for 12 h. After the reaction, GEM (158 mg, 0.6 mmol) was added and the reaction system was heated to 40°C for 24 h. After the reaction, the reaction system was cooled to room temperature, water and DCM were added three times for extraction, the organic phase was collected, washed, dried, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated and purified by column chromatography (eluent: V DCM :V MeOH =18:1) to obtain 96 mg of a light yellow solid, namely, an anaerobic activated antibacterial and anticancer compound LEV-GEM-NO2, with a yield of 66% and a melting point of 164.1-164.6°C. 1H NMR (DMSO-d6, 600MHz, ppm): δ8.60(s,1H),8.22(d,J=7.7Hz,1H),8.19(d,J=8.5Hz,2H),7.74(d,J=8.5Hz,2H),7.43(s ,2H),7.38(d,J=42.7Hz,2H),7.06(d,J=7.6Hz,1H),6.32(d,J=6.4Hz,1H),6.15(t,J=7.3Hz,1H),5.29(d,J=6.7Hz,4H) ,5.24(s,2H),4.71(q,J=6.3Hz,1H),4.50(d,J=10.9Hz,1H),4.31(d,J=9.8Hz,1H),4.22-4.15(m,1H),3.89(d,J=8.4Hz ,1H),3.83-3.74(m,2H),3.68-3.63(m,1H),2.71(s,4H),2.44(s,4H),2.32(s,3H),1.37(d,J=6.7Hz,3H),1.23(s,3H). 13 C NMR(DMSO-d6,120MHz,ppm): δ171.84,165.46,163.75,156.38,154.76,154.40,154.05,153.38,147.9,147.10,145.86,135.29,132.3 4,131.85,130.18,129.40,128.42,124.26,123.89,108.85,104.32,95.25,75.50,68.61,62.88,61.68,59.27,54.22,20.91,181.16. MS analysis:Found:m / z 936.2996[M+H] + ;calcd forC 44 H 45 F3N7O 13 :m / z 936.3027[M+H] + .

[0049] Example 5

[0050] Preparation of hypoxia-activated antibacterial and anticancer compounds (LEV-GEM-NO2):

[0051] LEV-NO2 (97 mg, 0.15 mmol) was dissolved in 4 mL DCM / DMF (V DCM :V DMF=1:1), DIPEA (131 μL, 0.75 mmol) and p-nitrophenyl chloroformate (121 mg, 0.6 mmol) were added at 0°C and reacted for 12 h. After the reaction, GEM (158 mg, 0.6 mmol) was added and the reaction system was heated to 40°C for 24 h. After the reaction, the reaction system was cooled to room temperature, water and DCM were added three times for extraction, the organic phase was collected, washed, dried, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated and purified by column chromatography (eluent: V DCM :V MeOH =18:1) to obtain 90 mg of a light yellow solid, namely, an anaerobic activated antibacterial and anticancer compound LEV-GEM-NO2, with a yield of 62% and a melting point of 164.1-164.6°C. 1 H NMR (DMSO-d6, 600MHz, ppm): δ8.60(s,1H),8.22(d,J=7.7Hz,1H),8.19(d,J=8.5Hz,2H),7.74(d,J=8.5Hz,2H),7.43(s ,2H),7.38(d,J=42.7Hz,2H),7.06(d,J=7.6Hz,1H),6.32(d,J=6.4Hz,1H),6.15(t,J=7.3Hz,1H),5.29(d,J=6.7Hz,4H) ,5.24(s,2H),4.71(q,J=6.3Hz,1H),4.50(d,J=10.9Hz,1H),4.31(d,J=9.8Hz,1H),4.22-4.15(m,1H),3.89(d,J=8.4Hz ,1H),3.83-3.74(m,2H),3.68-3.63(m,1H),2.71(s,4H),2.44(s,4H),2.32(s,3H),1.37(d,J=6.7Hz,3H),1.23(s,3H). 13 C NMR(DMSO-d6,120MHz,ppm): δ171.84,165.46,163.75,156.38,154.76,154.40,154.05,153.38,147.9,147.10,145.86,135.29,132.3 4,131.85,130.18,129.40,128.42,124.26,123.89,108.85,104.32,95.25,75.50,68.61,62.88,61.68,59.27,54.22,20.91,181.16. MS analysis:Found:m / z 936.2996[M+H] + ;calcd forC44 H 45 F3N7O 13 :m / z 936.3027[M+H] + .

[0052] Example 6

[0053] Preparation of hypoxia-activated antibacterial and anticancer compounds (LEV-GEM-NO2):

[0054] LEV-NO2 (97 mg, 0.15 mmol) was dissolved in 4 mL THF / DMF (V THF :V DMF =1:1), DIPEA (131 μL, 0.75 mmol) and p-nitrophenyl chloroformate (121 mg, 0.6 mmol) were added at 0°C and reacted for 12 h. After the reaction, GEM (158 mg, 0.6 mmol) was added and the reaction system was heated to 40°C for 24 h. After the reaction, the reaction system was cooled to room temperature, water and DCM were added three times for extraction, the organic phase was collected, washed, dried, and the solvent was removed by rotary evaporation to obtain a crude product, which was separated and purified by column chromatography (eluent: V DCM :V MeOH =18:1) to obtain 100 mg of a light yellow solid, namely, an anaerobic activated antibacterial and anticancer compound LEV-GEM-NO2, with a yield of 68% and a melting point of 164.1-164.6°C. 1 H NMR (DMSO-d6, 600MHz, ppm): δ8.60(s,1H),8.22(d,J=7.7Hz,1H),8.19(d,J=8.5Hz,2H),7.74(d,J=8.5Hz,2H),7.43(s ,2H),7.38(d,J=42.7Hz,2H),7.06(d,J=7.6Hz,1H),6.32(d,J=6.4Hz,1H),6.15(t,J=7.3Hz,1H),5.29(d,J=6.7Hz,4H) ,5.24(s,2H),4.71(q,J=6.3Hz,1H),4.50(d,J=10.9Hz,1H),4.31(d,J=9.8Hz,1H),4.22-4.15(m,1H),3.89(d,J=8.4Hz ,1H),3.83-3.74(m,2H),3.68-3.63(m,1H),2.71(s,4H),2.44(s,4H),2.32(s,3H),1.37(d,J=6.7Hz,3H),1.23(s,3H). 13C NMR(DMSO-d6,120MHz,ppm): δ171.84,165.46,163.75,156.38,154.76,154.40,154.05,153.38,147.9,147.10,145.86,135.29,132.3 4,131.85,130.18,129.40,128.42,124.26,123.89,108.85,104.32,95.25,75.50,68.61,62.88,61.68,59.27,54.22,20.91,181.16. MS analysis:Found:m / z 936.2996[M+H] + ;calcd forC 44 H 45 F3N7O 13 :m / z 936.3027[M+H] + .

[0055] Example 7

[0056] The compound (prodrug) LEV-GEM-NO2 effectively improved the lipid solubility of GEM. The lipid-water partition coefficients of LEV, GEM and LEV-GEM-NO2 were measured by spectrophotometry. The results showed that the logP values ​​of LEV, GEM and LEV-GEM-NO2 were 0.20, -1.30 and 0.93, respectively. Compared with GEM, the logP value of LEV-GEM-NO2 increased, indicating that the lipid solubility of LEV-GEM-NO2 was enhanced, which effectively solved the problem of poor lipid solubility of GEM.

[0057] Example 8

[0058] Evaluation of the in vitro antibacterial activity of LEV-GEM-NO2. Using E. coli as a model, the antibacterial activity of LEV-GEM-NO2 prepared in Example 6 and its prototype compounds GEM, LEV, and GEM combined with LEV was evaluated. The E. coli bacterial solution was inoculated into a nutrient agar culture dish, spread evenly, and administered in the following manner: a non-drug group and a drug group (LEV-GEM-NO2, concentration 40 μmol / L) were set up, and GEM, LEV, and GEM combined with LEV were used as control groups. After culturing in a constant temperature shaker at 37°C for 20 hours, the drugs were taken out and the antibacterial effects of different drugs were measured. The results showed that GEM had no obvious inhibitory effect on E. coli; LEV had a strong killing effect on E. coli, reducing the bacterial survival rate to 30%;

[0059] LEV-GEM-NO2 and GEM combined with LEV have a significant inhibitory effect on the growth of E. coli, reducing the survival rate of bacteria to 20%, indicating that LEV-GEM-NO2 has a good and effective inhibitory effect on the growth of bacteria.

[0060] Example 9

[0061] Evaluation of the anticancer effect of LEV-GEM-NO2 in vitro. Using mouse pancreatic cancer cells PANC02 as a model, the LEV-GEM-NO2 prepared in Example 6 and its prototype compounds GEM, LEV, and GEM combined with LEV were evaluated for cytotoxicity using the CCK method under hypoxic conditions (94% N2, 1% O2, 5% CO2, 37°C) and normoxic conditions (75% N2, 20% O2, 5% CO2, 37°C). At the same time, HUVEC human umbilical vein endothelial cells were used to determine the toxicity of normal cells. Cells in the logarithmic growth phase were inoculated with 2 to 10 × 10 3 The cells were plated on a 96-well plate and grown for 24 hours. The drugs were administered as follows: a drug-free group and a drug-added group (LEV-GEM-NO2, concentration 4-64 μmol / L) were set up, and GEM, LEV and GEM combined with LEV were used as control groups. After culturing for 48 and 72 hours, 10 μL of CCK-8 solution was added, and the OD value at 450 nm was measured after incubation for 2 hours. The present invention has the following beneficial effects: LEV does not affect the viability of PANC02 and HUVEC cells under either normoxic or hypoxic conditions, GEM and GEM combined with LEV have similar effects on the activity of PANC02 and HUVEC cells, and the ability to inhibit cell proliferation increases with the increase of incubation time and drug concentration; LEV-GEM-NO2 shows good hypoxia selectivity: under normoxic conditions, LEV-GEM-NO2 does not affect the viability of PANC02 and HUVEC cells; under hypoxic conditions, LEV-GEM-NO2 seriously affects the viability of PANC02 cells, and the hypoxic cytotoxicity ratio (HCR: ratio of hypoxic cytotoxicity to normoxic cytotoxicity) reaches 9.2. The results show that LEV-GEM-NO2 exhibits good hypoxia selectivity, and is only activated under hypoxic conditions, effectively inhibiting the growth of tumor cells.

[0062] Example 10

[0063] Evaluation of the in vivo antibacterial and anticancer effects of LEV-GEM-NO2. Using E. coli-infected tumors (pancreatic cancer cells PANC02) as a model, the in vivo anticancer effects of LEV-GEM-NO2 prepared in Example 6 and its prototype compounds GEM, LEV, and GEM combined with LEV were evaluated. A non-drug group and a drug-added group (LEV-GEM-NO2, the dosage was 40 mg / kg) were set up, and GEM, LEV, and GEM combined with LEV were used as control groups. The drugs were administered once every 2 days for a total of 6 times. The nude mice were sacrificed 72 hours after the last dose. The results are shown in Figure 3 (Changes in tumor volume after administration of different drugs) and Figure 4 (Tumor mass after administration of different drugs). Compared with the Control group, the inhibition rates of LEV, GEM, and GEM combined with LEV were 16.5%, 62.3%, and 71.2%, respectively. The LEV-GEM-NO2 group showed excellent tumor inhibition, with an inhibition rate of 95.6%. During the treatment, the weight of mice did not decrease significantly. After the tumor tissue was homogenized and spread on LB solid culture medium for observation, the colonies of the Control group and GEM group were significantly more than those of the LEV group, GEM combined with LEV group, and LEV-GEM-NO2 group, indicating that LEV-GEM-NO2 can effectively release the antibacterial drug LEV in mice and kill bacteria colonized in tumor tissues. Blood biochemistry analysis was performed on the eyeball blood of mice in each group. Compared with the Control group, no significant changes occurred in the LEV-GEM-NO2 group, indicating that LEV-GEM-NO2 had no obvious toxicity to the heart, liver, and kidneys of mice, and had good biosafety.

[0064] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A hypoxia-activated antibacterial and anticancer compound based on gemcitabine, characterized in that: The chemical structure of the gemcitabine-based hypoxia-activated antibacterial and anticancer compound is shown below:

2. Use of the gemcitabine-based hypoxia-activated antibacterial and anticancer compound as claimed in claim 1 in the preparation of a drug for treating pancreatic cancer.

3. A drug for treating pancreatic cancer, characterized in that: The active ingredient is the gemcitabine-based hypoxia-activated antibacterial and anticancer compound as claimed in claim 1.

Images