Gemcitabine prodrugs and methods of making and using the same

Abstract

The application belongs to the field of antitumor drug preparation, and particularly relates to albumin binding and non-binding gemcitabine prodrugs, and a preparation method and application thereof. The acryloyl chloride gemcitabine prodrug is a compound formed by bridging gemcitabine and acryloyl chloride through an amide bond, and the two non-albumin binding prodrugs are compounds formed by bridging gemcitabine and 3-succinimidyl propionic acid / propionic acid through an amide bond. The ethylene group in the prodrug is a binding target for the free thiol group of the 34th cysteine on albumin, can be rapidly and specifically combined with albumin in the systemic circulation, and further forms an albumin prodrug complex. The complex can significantly improve the stability of gemcitabine in the systemic circulation, and prolong the systemic circulation time. The macromolecular prodrug strategy designed in the application can improve the delivery efficiency of chemotherapeutic drugs, improve the antitumor effect of chemotherapeutic drugs, and provides a new direction and new ideas for the delivery of chemotherapeutic drugs.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug formulations, specifically relating to albumin-bound and unbound gemcitabine prodrugs and their synthesis and application; gemcitabine prodrug solutions and their preparation methods and applications. Background Technology

[0002] In recent years, the incidence of malignant tumors has been increasing, seriously threatening human health. Chemotherapy is one of the most effective strategies in cancer treatment. Gemcitabine, as a pyrimidine nucleoside analogue antitumor drug, is widely used in the clinical treatment of various solid tumors. Gemcitabine hydrochloride for injection. It was approved by the FDA in May 1996 for the treatment of pancreatic cancer, and subsequently expanded to other types of cancer, such as non-small cell lung cancer and breast cancer. From 1996 to the present, Total sales reached $2.1 billion. However, gemcitabine is rapidly metabolized and degraded into biologically inactive metabolites by cytidine deaminase in the blood, with a plasma half-life of only 10-20 minutes. Furthermore, gemcitabine is a small-molecule hydrophilic drug, lacking specific distribution to tumor lesions and struggling to penetrate cell membranes via diffusion, relying instead on nucleoside transporters. Downregulation of these transporters leads to acquired resistance to gemcitabine, significantly limiting its clinical application. Frequent administration of gemcitabine (1000 mg / m²) has also resulted in side effects such as neutropenia, reversible hepatic transaminuria, nausea, and vomiting. Therefore, the development of a more promising gemcitabine drug delivery system is urgently needed.

[0003] Prodrugs are one of the hot topics in gemcitabine research. Currently, significant progress has been made in gemcitabine prodrug development. Their derivatization mainly involves conjugating various groups on the 4-NH2 and / or 5′-OH of gemcitabine to form amides or esters. The main objectives of synthesizing these prodrugs are: (1) to avoid degradation by cytidine deaminase, (2) to prolong intracellular release time, (3) for use in the absence of deoxycytidine kinase, and (4) for use in the absence of transporters. Although prodrugs have improved gemcitabine's disadvantages in drug delivery in one or more of these aspects, they still have shortcomings in targeting and accumulating in tumor lesions.

[0004] Albumin is the most abundant protein in blood plasma. Its primary physiological function is to transport many endogenous substances, including essential fatty acids, amino acids, and thyroid hormones. Furthermore, due to its long half-life of up to 19 days in plasma, albumin also serves as a carrier for binding or transporting large quantities of different exogenous drugs, thereby prolonging drug circulation time and improving their pharmacokinetic properties. In addition, albumin is an ideal carrier for chemotherapy drugs due to its EPR effect and albumin receptor-mediated tumor tissue targeting. Albumin-docetaxel nanoparticles were approved by the FDA in 2005 for the treatment of metastatic breast cancer, non-small cell lung cancer, and pancreatic cancer. Compared to traditional... It exhibits better tolerability, with the tolerable dose increasing from 175 mg / m2 to 260 mg / m2. By avoiding the use of Cremophor (polysorbate), which has sensitizing effects, this successfully demonstrates the high efficiency and low toxicity of albumin-based drug delivery systems, providing a new approach for improving the clinical application of chemotherapy drugs. Albumin surfaces contain various amino acid molecules that can be structurally modified, such as cysteine ​​and lysine. Cysteine ​​at position 34 of albumin is a specific active reaction site. INNO-206 is based on the Michael addition reaction between a prodrug containing a maleimide group and the free thiol group at Cys-34 of endogenous albumin under physiological conditions, forming an albumin-prodrug macromolecular complex. Chinese patent CN201810181464.5 also describes the excellent antitumor effects of maleimide-functionalized gemcitabine prodrugs.

[0005] Besides maleimide groups, carbonyl reactive species (RCS), as the most reactive protein nucleophilic sites in plasma, can also undergo Michael addition with the free thiol group of Cys-34 in albumin. Carbonyl reactive species (RCS) mainly include α,β-unsaturated aldehydes and ketones, dialdehydes, ketaldehydes, etc., and possess direct cytotoxicity. They can also act as second messengers mediating oxidative stress and tissue damage, with acryloyl chloride being the most prominent class of carbonyl reactive species. Summary of the Invention

[0006] This invention synthesizes gemcitabine prodrugs that rapidly and specifically bind to endogenous albumin in vivo, using acryloyl chloride as a modifying structure, with 3-maleimide propionate gemcitabine prodrug as a positive control; and synthesizes albumin-unbound gemcitabine prodrugs using structurally similar propionic acid / 3-succinimide propionate as modifying structures. Acryloyl chloride gemcitabine prodrugs significantly improve the stability of the prodrug in systemic circulation, prolong the drug's circulation time, and improve the drug's pharmacokinetic behavior, allowing the drug to reach tumor lesions more effectively along with albumin, thereby enhancing antitumor activity. This invention provides a series of gemcitabine prodrugs with rapid binding to albumin in vivo, significantly prolonged drug half-life, and enhanced antitumor activity, along with their synthetic method and application in pharmaceutical formulations.

[0007] This invention provides, in one aspect, a prodrug of acryloyl chloride gemcitabine of general formula I and its pharmaceutically acceptable salts, solvates, polymorphs or isomers:

[0008]

[0009] Where X is (CH2)n or O(CH2)n, n = 1-10; preferably n = 2-6.

[0010] This invention also provides non-albumin-bound gemcitabine prodrugs of formula II or III, and pharmaceutically acceptable salts, solvates, polymorphs, or isomers thereof:

[0011]

[0012] Where X is (CH2)n or O(CH2)n, n = 1-10; preferably n = 2-6.

[0013] In the above technical solutions, further, the gemcitabine prodrugs represented by general formulas I, II, and III, and their pharmaceutically acceptable salts, solvates, polymorphs, or isomers, are selected from:

[0014]

[0015] Furthermore, in the above technical solution, the preparation method of the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salts, solvates, polymorphs, or isomers adopts the following steps:

[0016] The gemcitabine prodrug of Formula I or Formula II is prepared using the following steps:

[0017] An organic base, a fatty acid / acyl halide compound containing a double bond structure, or a saturated fatty acid / acyl halide compound without a double bond structure is dissolved in anhydrous DMF. Then, a DMF solution of gemcitabine is added, and the mixture is stirred under nitrogen protection to obtain a reaction solution. The DMF in the reaction solution is removed, and the product is separated by the preparation solution. The preparation solution is poured into ethyl acetate for extraction, and then the organic phases are combined and washed sequentially with saturated physiological saline. The organic phases are dried over anhydrous sodium sulfate, the solvent is recovered, and the purified product is obtained as a series I or II compound.

[0018] Preferably, the fatty acid or acyl halide containing a double bond structure used to prepare compound I is acryloyl chloride, and the saturated fatty acid or acyl halide used to prepare compound II is propionic acid;

[0019] Preferably, the organic base is one or more of triethylamine, N-methylmorpholine, diethylamine, and DIPEA, preferably DIPEA; the molar ratio is acryloyl chloride: DIPEA: gemcitabine = 1:(0.5-4):1;

[0020]

[0021] The gemcitabine prodrug of Formula III is prepared using the following steps:

[0022] The condensation catalyst and carboxylic acid were dissolved in anhydrous DMF. After activation in an ice bath, a DMF-diluted acid-binding organic base and gemcitabine solution were added sequentially. The mixture was stirred under nitrogen protection to obtain a reaction solution. The DMF in the reaction solution was removed, and the product was separated by the preparation solution. Post-processing yielded the III series compounds.

[0023]

[0024] Preferably, the carboxylic acid is a carboxylic acid containing a succinimide structural segment, and more preferably 3-succinimide propionic acid;

[0025] Preferably, the condensation catalyst is one or more of EDCI, HATU, HBTU, TBTU, CDI, DCC, DIC, and HOBt, with HATU being the most preferred; the organic base used is one or more of triethylamine, N-methylmorpholine, diethylamine, and DIPEA, with DIPEA being the most preferred; the molar ratio of 3-maleimide propionic acid: HATU: DIPEA: gemcitabine is 1:(1-4):(0.5-4):1.

[0026] A second aspect of the present invention provides a gemcitabine prodrug solution, the components of which include gemcitabine prodrug and amino acids; the pH value of the solution is 3-5.

[0027] In the above technical solution, the mass ratio of gemcitabine prodrug to amino acid is 1:1-1:40, preferably 1:10-1:30;

[0028] The amino acid is one or more combinations of L-arginine, L-lysine, and L-histidine, preferably L-arginine; the pH value is adjusted by a pH adjuster, which is one or more combinations of citric acid, hydrochloric acid, phosphate buffer, and acetate buffer, preferably hydrochloric acid.

[0029] Furthermore, in the above technical solution, the preparation method of the aforementioned gemcitabine prodrug solution includes the following steps:

[0030] (1) Dissolve the above gemcitabine prodrug in an organic solvent; wherein the organic solvent is one or more of DMSO, anhydrous ethanol, methanol, acetonitrile, and dichloromethane;

[0031] (2) Dissolve a certain mass of amino acids in an appropriate amount of water for injection and adjust the pH to 3-5;

[0032] (3) Mix the organic solution containing gemcitabine prodrug with the amino acid solution until homogeneous, and vortex for 10 min;

[0033] (4) Remove the organic solvent and add water for injection to the full amount according to the content of the prodrug solution.

[0034] A third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salts, solvates, polymorphs or isomers and a pharmaceutically acceptable carrier.

[0035] The fourth aspect of the present invention provides the use of the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salts, solvates, polymorphs or isomers, or the aforementioned pharmaceutical compositions in the preparation of albumin-bound antitumor drugs; or in the preparation of drugs with enhanced antitumor activity.

[0036] The fifth aspect of this invention provides the use of the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salts, solvates, polymorphs or isomers, or the aforementioned pharmaceutical compositions in the preparation of a long-circulating drug in vivo. The beneficial effects of this invention are:

[0037] This invention synthesizes a series of gemcitabine prodrugs and prepares gemcitabine prodrug solutions. The synthesis and preparation methods are simple and easy to implement. The albumin-bound gemcitabine prodrug is a compound composed of an albumin-binding group and gemcitabine bridging, wherein acryloyl chloride acts as the binding target for the free thiol group at the 34-position cysteine ​​of albumin, and the amide bond exhibits enzyme sensitivity. Experiments demonstrate that the gemcitabine prodrugs of this invention can rapidly and specifically bind to serum albumin, improving the stability of gemcitabine in systemic circulation, prolonging the systemic circulation time of gemcitabine, and enhancing antitumor activity. The macromolecular prodrug strategy designed in this invention can improve the delivery efficiency of chemotherapeutic drugs and enhance their antitumor effects, providing a new direction and approach for chemotherapeutic drug delivery. Attached Figure Description

[0038] Figure 1 For structural confirmation of the prodrug 1 of the present invention, A: 1H-NMR spectrum of prodrug 1, B: mass spectrum of prodrug 1.

[0039] Figure 2 For structural confirmation of the prodrug 2 of the present invention, A: 1H-NMR spectrum of prodrug 2, B: mass spectrum of prodrug 2.

[0040] Figure 3 For structural confirmation of the prodrug 3 of the present invention, A: 1H-NMR spectrum of prodrug 3, B: mass spectrum of prodrug 3.

[0041] Figure 4 This is the preparation of gemcitabine prodrug solution using L-arginine as a cosolvent, as described in Example 4 of the present invention.

[0042] Figure 5 This is an in vitro binding experiment of prodrug 1 with human serum albumin in Example 5 of the present invention.

[0043] Figure 6This is a cytotoxicity experiment of four gemcitabine prodrugs and two albumin-prodrug complexes in Example 6 of the present invention.

[0044] Figure 7 The degradation of the prodrugs in the plasma chemical stability of the four prodrugs in Example 7 of the present invention.

[0045] Figure 8 The formation of the parent drug in the plasma chemical stability of the four prodrugs in Example 7 of the present invention.

[0046] Figure 9 This relates to the generation of inactive metabolite dFdU in the plasma chemical stability of the four prodrugs in Example 7 of this invention.

[0047] Figure 10 The GEMs released during the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of the present invention.

[0048] Figure 11 The total GEM values ​​are obtained from the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of this invention.

[0049] Figure 12 This refers to the inactive metabolite dFdU released during the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of the present invention.

[0050] Figure 13 The total inactive metabolite dFdU released during the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of the present invention.

[0051] Figure 14 This is a tumor growth curve of PANC02-bearing mice at a dose of 10 mg / kg, representing the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes of Example 9 of the present invention.

[0052] Figure 15 This is a graph showing the tumor bearing rate of PANC02-bearing mice in the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes of Example 9 of the present invention at a dose of 10 mg / kg.

[0053] Figure 16 The graph shows the weight changes of PANC02 tumor-bearing mice in Example 9 of this invention, including the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes, at a dose of 10 mg / kg.

[0054] Figure 17 This is a tumor growth curve of PANC02-bearing mice at a dose of 20 mg / kg, representing the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes of Example 9 of the present invention.

[0055] Figure 18 The tumor bearing rate of PANC02-bearing mice in Example 9 of this invention, including the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes, at a dose of 20 mg / kg.

[0056] Figure 19 The graph shows the weight changes of PANC02 tumor-bearing mice in Example 9 of this invention, including the control group, four gemcitabine prodrugs, and two albumin-prodrug complexes, at a dose of 20 mg / kg.

[0057] The gemcitabine and prodrugs 1, 2, 3, 4, and albumin prodrug complexes mentioned above correspond to GEM, BG, n-BG, n-MG, MG, HSA-BG, and HSA-MG in the attached figure, respectively. Detailed Implementation

[0058] The present invention will be further illustrated below by way of examples. The synthesis of the prodrugs will be described using prodrugs 1, 2 and 3 as examples. The binding ability of the prodrug to albumin will be examined using prodrug 1 as an example. The preparation of the prodrug solution using L-arginine as a cosolvent will also be examined using prodrug 1 as an example. However, the invention is not limited to the scope of the examples described herein.

[0059] Example 1

[0060] Synthesis of albumin-bound gemcitabine prodrug (BG) modified with acryloyl chloride.

[0061] Under ice bath conditions, an appropriate amount of gemcitabine 263 mg (1 mmol) was dissolved in 5 mL of anhydrous DMF in a 100 mL round-bottom flask. After complete dissolution, 180 μL (2.4 mmol) of DIPEA (N,N-diisopropylethylamine) was added, followed by 90 μL (1 mmol) of acryloyl chloride diluted in 5 mL of DMF. Under nitrogen protection, the mixture was naturally heated and stirred at room temperature for 12 h. After the reaction was completed, the DMF was removed using an oil pump, and the product was separated by the preparation phase. The preparation phase was extracted with ethyl acetate, and the organic phases were combined and washed sequentially with saturated physiological saline. The organic phase was dried over anhydrous sodium sulfate, the solvent was recovered, and the gemcitabine prodrug BG was obtained after purification.

[0062] The synthesis route is shown in the following formula:

[0063]

[0064] The structure of the prodrug was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 1 As shown. The nuclear magnetic resonance spectroscopy analysis results are as follows: BG.

[0065] 1H NMR (600MHz, DMSO-d6) δ11.24(s,1H,16-NH),8.29(d,J=7.5Hz,1H,14-H),7.38(d,J =7.5Hz,1H,13-H),6.55(dd,J=17.0,10.2Hz,1H,22-H),6.41-6.35(m,2H,23a-Hand 17-OH),6.18(t,J=7.2Hz,1H,20-H),5.90(d,J=10.2Hz,1H,23b-H),5.32(s,1H,7-OH),4.21-4.16(m,1H, 19-H), 3.90 (dd, J=7.5, 3.8Hz, 1H, 1-H), 3.81 (d, J=12.7Hz, 1H, 6a-H), 3.66 (dd, J=12.8, 3.5Hz, 1H, 6b-H).

[0066] The mass spectrometry results are MS(ESI) m / z for C 12 H 13 F2N3O5[M+Na] + =340.2.

[0067] Example 2

[0068] Synthesis of albumin-unbound gemcitabine prodrug (n-BG) with propionic acid as a modification module.

[0069] Under ice bath conditions, an appropriate amount of gemcitabine 263 mg (1 mmol) was dissolved in 5 mL of anhydrous DMF in a 50 mL round-bottom flask. After complete dissolution, 180 μL (2.4 mmol) of DIPEA was added, followed by 88 μL (1 mmol) of propionic acid diluted in 5 mL of DMF. Under nitrogen protection, the mixture was naturally heated and stirred at room temperature for 12 h. After the reaction was complete, the DMF was removed by an oil pump, and the product was separated by the preparation solution. The preparation solution was extracted with ethyl acetate, and the organic phases were combined and washed sequentially with saturated physiological saline. The organic phase was dried over anhydrous sodium sulfate, the solvent was recovered, and the gemcitabine prodrug n-BG was obtained after purification.

[0070] The synthesis route is shown in the following formula:

[0071]

[0072] The structure of the prodrug was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 2 As shown. The nuclear magnetic resonance spectroscopy analysis results are as follows: n-BG.

[0073] 1H NMR (600MHz, DMSO-d6) δ10.97(s,1H,16-NH),8.23(d,J=7.6Hz,1H,14-H),7.28(d,J=7.6Hz,1H,1 3-H),6.32(s,1H,17-OH),6.17(t,J=7.4Hz,1H,20-H),5.30(t,J=5.5Hz,1H,7-OH),4.18(td,J=12 .6,8.3Hz,1H,19-H),3.88(dt,J=8.3,3.0Hz,1H,1-H),3.80(dt,J=12.8,3.6Hz,1H,6a-H),3.65( ddd,J=12.8,5.6,3.6Hz,1H,6b-H),2.42(q,J=7.5Hz,2H,22-CH2),1.02(t,J=7.5Hz,3H,23-CH3).

[0074] The mass spectrometry results are MS(ESI) m / z for C 12 H 15 F2N3O5[MH] - =318.07.

[0075] Example 3

[0076] Synthesis of albumin-unbound gemcitabine prodrug (n-MG) with 3-succinimide propionic acid as a modification module.

[0077] Under ice bath conditions, 100 mg (0.58 mmol) of 3-succinimide propionic acid was dissolved in 2 mL of DMF and placed in a 50 mL round-bottom flask. After complete dissolution, 440 mg of HATU (1.16 mmol) was added, and the mixture was activated in an ice bath for 10 min. Then, 100 μL of LIPEA (0.58 mmol) was added, and activation was continued for 2 h under nitrogen protection. After activation, 158 mg (0.58 mmol) of gemcitabine dissolved in 2 mL of DMF was added. The mixture was stirred at room temperature under nitrogen protection for 24 h. After the reaction was completed, anhydrous DMF was removed using an oil pump, and the product was separated using a preparative solution. The purified product was obtained as the albumin-unbound gemcitabine prodrug n-MG.

[0078] The synthesis route is shown in the following formula:

[0079]

[0080] The structure of the prodrug was determined using mass spectrometry and proton nuclear magnetic resonance spectroscopy, and the results are as follows: Figure 3 As shown. The nuclear magnetic resonance spectroscopy analysis results are as follows: n-MG.

[0081] 1H NMR (600MHz, DMSO-d6) δ11.09(s,1H,16-NH),8.25(d,J=7.7Hz,1H,14-H),7.21(d,J=7.6Hz,1H,13-H),6.37(s,1H,17-OH),6.17(t,J=7.3Hz,1H,20- H),5.34(t,J=5.6Hz,1H,7-OH),4.19(q,J=11.6Hz,1H,19-H),3.89(dt,J= 8.6,3.0Hz,1H,1-H),3.80(d,J=12.6Hz,1H,6a-H),3.69-3.57(m,3H,6b-H and 24-H), 2.70-2.63 (t, J=7.6Hz, 2H, 23-H), 2.61 (s, 4H, 27and 28-CH2).

[0082] The mass spectrometry results are MS(ESI) m / z for C 12 H 13 F2N3O5[MH] - =315.10749.

[0083] Example 4

[0084] Preparation of gemcitabine prodrug solution with L-arginine as a cosolvent

[0085] Accurately weigh prodrugs (BG, MG, n-BG, n-MG) and L-arginine (1:1-1:40) in different mass ratios. Add 50 μL of anhydrous ethanol and 0.5% DMSO (v / v) to the prodrugs and sonicate until completely dissolved. Add approximately 80% of the total volume of water for injection to the L-arginine and stir at room temperature until completely dissolved. Adjust the pH to 3-5 with hydrochloric acid solution, then add the organic solution containing the prodrugs and mix thoroughly, vortexing for 10 min. Remove ethanol by vacuum distillation, add water for injection to the prepared total volume, and filter to obtain an ethanol-free prodrug solution. Determine its concentration by HPLC.

[0086] Experimental results are as follows Figure 4 As shown, the increase in prodrug solubility is not linearly related to the amount of L-arginine added. When the mass ratio of prodrug (BG, MG, n-BG, n-MG) to L-arginine is 1:15, 1:15, 1:20, and 1:20, the prodrug reaches saturation concentration.

[0087] Example 5

[0088] In vitro binding assay of prodrug 1 (BG) with human serum albumin

[0089] A 20% anhydrous ethanol solution of prodrug 1 (BG) was slowly added dropwise to an equal volume of human serum albumin solution at pH 7.4 phosphate. The final concentrations of the prodrug and albumin were 300 μM and 750 μM, respectively. The mixture was incubated in a shaker at 37°C. At 0 min, 5 min, 30 min, and 60 min, 100 μL of the incubation solution was taken for HPLC analysis to examine the binding strength of different prodrugs to albumin. The detection wavelength was 269 nm, and 20 μL was injected into the HPLC sample.

[0090] In addition, a competitive binding assay for albumin was performed. HSA and 3-maleimide propionic acid were pre-incubated in a shaker at 37°C for 1 hour. After the thiol groups in albumin were completely bound to 3-maleimide propionic acid, BG solution was added and incubated for 30 minutes according to the binding assay method. The incubation solution was filtered through a filter membrane and directly injected into HPLC to investigate the binding of the prodrug to albumin after the free thiol groups in albumin were completely blocked.

[0091] In vitro binding experiment results as follows Figure 5 As shown, after 60 minutes of incubation, BG almost completely bound to albumin. This experiment demonstrates that BG can rapidly bind to albumin. However, when human serum albumin was pre-incubated with an excess of 3-maleimide propionic acid before the binding experiment with BG, the peak area of ​​BG did not change significantly with time. This result indicates that BG specifically binds to the free sulfhydryl group of cysteine ​​at position 34 of albumin via its vinyl group, with almost no other binding mechanisms.

[0092] Example 6

[0093] Cytotoxicity assays of four prodrugs (BG, MG, n-BG, n-MG) with albumin-prodrug complexes HAS-BG and HAS-MG

[0094] 4T1 cells were seeded into 96-well plates (1000 cells per well) and incubated for 20 h. After cell attachment, fresh medium containing different concentrations of prodrug, albumin-prodrug complex, and gemcitabine was added to each well (200 μL per well, with 6 parallel wells for each concentration). After 48 h of incubation, the 96-well plates were removed, the old medium was discarded, and 135 μL of MTT solution diluted with fresh medium (100 μL fresh medium + 35 μL 5 mg / mL MTT solution) was added to each well. After incubation for 4 h, the medium was discarded, and the 96-well plates were inverted on filter paper to blot out any remaining liquid. 150 μL of LDMSO was added to each well, and the plates were shaken for 15 min to dissolve the blue-purple crystals. The absorbance was measured at 570 nm using a microplate reader.

[0095] The results are as follows Figure 6As shown, since prodrugs require a response activation process to exert their effects in cells, the cytotoxicity of gemcitabine prodrugs is generally weaker than that of gemcitabine solutions. Because the albumin-prodrug complex releases gemcitabine at a slower rate compared to the prodrug solution, the cytotoxicity of the albumin-prodrug complex is generally slightly lower than that of the corresponding gemcitabine prodrug.

[0096] The structural formula of the prodrug MG is as follows:

[0097]

[0098] Albumin-prodrug complexes HAS-BG and HAS-MG were obtained according to the method in Example 5. A 20% anhydrous ethanol solution of prodrug 1BG or prodrug MG was slowly added dropwise to an equal volume of human serum albumin at pH 7.4 phosphate solution. The final concentrations of the prodrug and albumin were 300 μM and 750 μM, respectively. The mixture was incubated in a shaker at 37°C for 60 min and 30 min, respectively, to obtain HSA-BG and HAS-MG.

[0099] Example 7

[0100] Plasma stability studies of four gemcitabine prodrugs (BG, MG, n-BG, n-MG) and two albumin-prodrug complexes, HAS-BG and HAS-MG.

[0101] The prodrug / albumin-prodrug mixture was homogenized with fresh rat plasma (1:10, v / v) at 37°C for 72 h to investigate the in vitro release trend of GEM. The release rate of GEM in plasma was detected by HPLC at predetermined time points. Results are as follows: Figure 7 As shown, due to the presence of a large amount of albumin in plasma, BG and MG can rapidly bind to albumin in plasma after incubation, exhibiting good plasma chemical stability. However, they degrade slowly with prolonged incubation time, and the release of GEM from the two complexes within 12 hours is less than 7%. Figure 8 This provides a guarantee for the pharmacokinetic behavior of drugs in vivo. Figure 9 The results showed that the generated dFdU followed the same release trend as GEM. The more GEM released, the more was metabolized into dFdU. Furthermore, BG and MG produced significantly less dFdU than n-BG and n-MG. In addition, when the free sulfhydryl groups on albumin in plasma were prematurely bound and occupied, BG and MG could not bind to them and were rapidly cleared within 12 hours. This further demonstrates that the binding of BG and MG to the free sulfhydryl group at position 34 of cysteine ​​is highly specific and the process is rapid. These results indicate that the albumin binding strategy can effectively reduce the degradation of gemcitabine by CDA, significantly improve the stability of the prodrug in systemic circulation, and avoid off-target effects caused by premature prodrug degradation.

[0102] Example 8

[0103] Male Sprague-Dawley rats (200-230g) were randomly divided into 7 groups (n=6 per group) to study the pharmacokinetic properties of the prodrug and its albumin prodrug complex.

[0104] SD rats were administered GEM, prodrugs (BG, MG, n-BG, n-MG), and albumin-prodrug complexes (HSA-BG, HSA-MG) via tail vein injection at doses equivalent to 6 mg / kg GEM. Blood samples were collected at predetermined time points after administration. Plasma samples were then obtained by centrifugation and stored at -80°C. The concentrations of prodrugs, dFdU, and GEM were determined by UPLC-MS / MS. Figure 10 As shown, due to its short half-life, GEM solution was rapidly cleared from the blood, with AUC and t1 / 2 of 54.137±21.276 mg / L*h and 2.277±0.692 h, respectively. In contrast, the total GEM content in the BG and MG groups was significantly higher than that in the GEM group, being 7 times and 45 times higher, respectively, with a half-life 8 times longer. In the BG group, 80% of the GEM was in the form of an albumin-prodrug complex, indicating that most of the GEM was protected by albumin in systemic circulation, avoiding CDA degradation. This may be attributed to the high affinity of BG for albumin. This characteristic allows BG to bind to albumin more quickly in systemic circulation, thus better utilizing the protection and extended circulation time provided by the albumin carrier. In contrast, the n-BG and n-MG groups were rapidly cleared from systemic circulation because they could not covalently bind to albumin. The total GEM content measured by in vitro covalently binding albumin in the HSA-MG and HSA-BG groups showed an inconsistent trend with that in the BG and MG groups that endogenously bound albumin. This may be because MG undergoes ring-opening hydrolysis in the PBS environment during in vitro incubation, which weakens its binding ability to albumin, thus failing to show better pharmacokinetic results.

[0105] Example 9

[0106] Antitumor pharmacodynamic experiments of four prodrugs and two albumin-prodrug complexes

[0107] PANC02 cells (2 × 10⁶ cells / mL) resuspended in 100 μL PBS were subcutaneously injected into the right dorsal region of male C57BL / 6 mice. When the tumor volume reached 100 mm³, the tumor-bearing mice were randomly divided into groups (n=6 per group): a negative control group (saline), a gemcitabine solution group, four gemcitabine prodrug solutions (MG, BG, n-MG, n-BG), and two albumin-prodrug complexes (HSA-MG, HSA-BG). Administered the drugs every three days for a total of four times at doses of 10 mg / kg and 20 mg / kg. The tumor volume (long axis * short axis * short axis / 2) and body weight of the tumor-bearing mice were measured and recorded daily, and curves showing the changes in tumor volume and body weight over time were plotted. After the experiment, the mice were sacrificed, the tumor tissue was dissected, and the tumor bearing rate (tumor weight / mouse body weight * 100%) was calculated.

[0108] The results showed that, in the low-dose group, the gemcitabine solution group exhibited a significant tumor-suppressive effect compared to the saline group. In contrast, the in vivo albumin-bound prodrug solutions (MG, BG) further improved the anti-tumor effect and delayed tumor growth compared to the free gemcitabine group. In vitro albumin-bound HSA-BG and HSA-MG showed strong tumor-suppressive effects compared to the gemcitabine solution group, with tumor inhibition rates of 42.3% and 40.6%, respectively. Notably, n-MG and n-BG showed the worst tumor-suppressive effects due to their inability to bind to albumin in systemic circulation, but still possessed some tumor-suppressive efficacy. In the high-dose group, all mice died on day nine after increasing the MG dosage, indicating a potential safety risk associated with high-dose MG. This suggests that drug development should fully consider the efficacy and safety of drugs, not just their therapeutic effects. Furthermore, BG did not significantly improve the anti-tumor effect compared to free gemcitabine, but its safety was improved compared to the MG group. While the antitumor effects of n-BG and n-MG were relatively weak, they still exhibited some antitumor activity. The above experimental results indicate that n-BG and n-MG, due to their inability to bind to albumin in systemic circulation, exhibited poor stability in systemic circulation, with no significant increase in circulation time compared to the GEM group. The released free GEM was easily degraded into inactive metabolites dFdU by cytidine deaminase in plasma, leading to poor efficacy. In contrast, BG and MG, which can rapidly and selectively covalently bind to endogenous albumin via the Michael addition reaction, as well as the in vitro prepared albumin-prodrug complexes HSA-BG and HSA-MG, showed significantly reduced degradation by cytidine deaminase under the protection of albumin. This prevented premature release of GEM from the albumin-prodrug complexes, improved their metabolic enzyme stability and bioavailability, and prolonged systemic circulation time. Therefore, the measured in vivo GEM content was much higher than that of n-BG and n-MG, demonstrating better pharmacodynamic results.

[0109] Overall, the novel gemcitabine prodrug BG of this invention has a superior antitumor effect compared to the parent drug, and has low toxicity. Although the novel gemcitabine prodrugs n-BG and n-MG have relatively weaker antitumor effects than the parent drug, they have lower toxicity. In addition, the antitumor effect of prodrug BG is significantly improved after binding to albumin.

[0110] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Gemcitabine prodrug and its pharmaceutically acceptable salt, characterized in that, The compound is: 。 2. The method for preparing the gemcitabine prodrug and its pharmaceutically acceptable salt according to claim 1, characterized in that, Prepared using the following steps: The gemcitabine prodrug of Formula 1 is prepared using the following steps: An organic base and acryloyl chloride were dissolved in anhydrous DMF, and then a gemcitabine DMF solution was added. The mixture was stirred under nitrogen protection to obtain a reaction solution. The DMF in the reaction solution was removed, and the product was separated by the preparation solution. The preparation solution was poured into ethyl acetate for extraction, and then the organic phases were combined and washed sequentially with saturated physiological saline. The organic phases were dried with anhydrous sodium sulfate, the solvent was recovered, and the compound was purified to obtain compound 1. The organic base is DIPEA; the molar ratio of acryloyl chloride: DIPEA: gemcitabine is 1:(0.5-4):

1.

3. A gemcitabine prodrug solution, characterized in that, The solution comprises gemcitabine prodrug and amino acids as described in claim 1; the pH of the solution is 3-5.

4. The gemcitabine prodrug solution according to claim 3, characterized in that, The mass ratio of gemcitabine prodrug to amino acid as described in claim 1 is 1:1 to 1:40; The amino acid is one or more combinations of L-arginine, L-lysine, and L-histidine; the pH value is adjusted by a pH adjuster, which is one or more combinations of citric acid, hydrochloric acid, phosphate buffer, and acetate buffer.

5. The gemcitabine prodrug solution according to claim 3, characterized in that, The preparation of the gemcitabine prodrug solution includes the following steps: (1) Dissolve the gemcitabine prodrug as described in claim 1 in an organic solvent; wherein the organic solvent is one or more combinations of DMSO, anhydrous ethanol, methanol, acetonitrile, and dichloromethane; (2) Dissolve a certain mass of amino acids in an appropriate amount of water for injection and adjust the pH to 3-5; (3) Mix the organic solution containing gemcitabine prodrug with the amino acid solution until homogeneous, and vortex for 10 min; (4) Remove the organic solvent and add water for injection to the full amount according to the content of the prodrug solution.

6. A pharmaceutical composition, characterized in that, It includes the gemcitabine prodrug of claim 1 and its pharmaceutically acceptable salt.

7. The use of the gemcitabine prodrug of claim 1 and its pharmaceutically acceptable salt or the pharmaceutical composition of claim 6 in the preparation of an albumin-bound antitumor drug; or in the preparation of a drug with enhanced antitumor activity.

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