Gemcitabine prodrug as well as preparation method and application thereof

Abstract

The invention belongs to the field of anti-tumor medicine preparations, and particularly relates to albumin binding type and non-binding type gemcitabine prodrugs as well as a preparation method and application of the albumin binding type and non-binding type gemcitabine prodrugs. The acryloyl chloride gemcitabine prodrug is a compound formed by bridging gemcitabine and acryloyl chloride through an amido bond, and the two non-albumin combined prodrugs are compounds formed by bridging gemcitabine and 3-succinimide propionic acid / propionic acid through an amido bond. The vinyl group in the prodrug is a binding target head of free sulfydryl of cysteine at the 34th site on albumin, and can be rapidly and specifically bound with albumin in systemic circulation, so that an albumin prodrug compound is formed. The compound can significantly improve the stability of gemcitabine in systemic circulation and prolong the systemic circulation time of gemcitabine. According to the macromolecular prodrug strategy designed by the invention, the delivery efficiency of the chemotherapeutic drug can be improved, the anti-tumor effect of the chemotherapeutic drug is improved, and a new direction and a new idea are provided for the delivery of the chemotherapeutic drug.

Description

Technical Field

[0001] The invention belongs to the field of anti-tumor drug preparations, and specifically relates to albumin-bound and non-bound gemcitabine prodrugs and synthesis and application thereof; gemcitabine prodrug solution and preparation method and application thereof. Background Art

[0002] In recent years, the incidence of malignant tumors has been increasing, seriously threatening human health. Chemotherapy is one of the most effective strategies for cancer treatment. Gemcitabine, as a pyrimidine nucleoside analog 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. The sales of gemcitabine totaled $2.1 billion. However, since gemcitabine is rapidly metabolized and degraded into biologically inactive metabolites by cytidine deaminase in the blood, its plasma half-life is only 10-20 minutes. In addition, gemcitabine is a small molecule hydrophilic drug. It not only lacks specific distribution in tumor lesions, but also has difficulty entering the cell membrane by diffusion and can only rely on nucleoside transporters. Down-regulation of transporter expression can lead to acquired resistance to gemcitabine. The above deficiencies greatly limit its clinical application. Frequent administration of gemcitabine (1000 mg / m2) to patients has led to side effects such as neutropenia, reversible liver transaminuria, nausea and vomiting. Therefore, the development of a more promising gemcitabine drug delivery system is urgent.

[0003] Prodrugs are one of the hot topics in gemcitabine research. At present, great progress has been made in gemcitabine prodrugs. Its derivatization method is mainly to form amides or esters by conjugating various groups on the 4-NH2 and / or 5′-OH of gemcitabine. The main purposes of synthesizing these prodrugs are: (1) to avoid degradation by cytidine deaminase, (2) to prolong the release time in cells, (3) to be used in the absence of deoxycytidine kinase, and (4) to be used in the absence of transporters. Although prodrugs have improved the disadvantages of gemcitabine in the drug delivery process in one or more of the above aspects, they are still insufficient in targeting and accumulation in tumor lesions.

[0004] Albumin is the most abundant protein in plasma. Its physiological function is mainly to transport many endogenous substances, including essential fatty acids, amino acids, thyroxine, etc. In addition, since the half-life of albumin in plasma is as high as 19 days, another important function of albumin is to act as a carrier to bind or transport a large number of different types of exogenous drugs to prolong the circulation time of the drugs and improve their pharmacokinetic properties. In addition, albumin has become an ideal carrier for chemotherapeutic drugs because of the 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. It has better tolerance, and the tolerance dose is increased from 175mg / m2 to 260mg / m2. The use of Cremophor (polysorbate) with sensitizing effect is avoided, which successfully demonstrates that the albumin drug delivery system has the characteristics of high efficiency and low toxicity, and provides a new idea for improving the clinical application of chemotherapy drugs. There are a variety of amino acid molecules such as cysteine ​​and lysine on the surface of albumin that can be used for structural modification, among which cysteine ​​at position 34 of albumin is a specific active reaction site. INNO-206 is based on the fact that the prodrug containing maleimide group undergoes Michael addition reaction with the free thiol group of Cys-34 of endogenous albumin under physiological conditions to form an albumin-prodrug macromolecular complex. Chinese patent CN201810181464.5 also records that maleimide-functionalized gemcitabine prodrug has excellent anti-tumor effect.

[0005] In addition to the maleimide group, carbonyl reactive species (RCS), as the most reactive protein nucleophilic site in plasma, can also undergo Michael addition with the free sulfhydryl group of Cys-34 of albumin. Carbonyl reactive species (RCS) mainly include α, β-unsaturated aldehydes and ketones, dialdehydes, ketoaldehydes, etc., which have direct cytotoxicity and can also act as second messengers to mediate oxidative stress and tissue damage. Among them, acryloyl chloride is the most important type of carbonyl reactive species. Summary of the invention

[0006] The present invention uses acryloyl chloride as a modified structure to synthesize a gemcitabine prodrug that can rapidly and specifically bind to endogenous albumin in vivo, and uses 3-maleimidopropionic acid gemcitabine prodrug as a positive control; uses structurally similar propionic acid / 3-succinimidylpropionic acid as a modified structure to synthesize albumin-unbound gemcitabine prodrug. Acryloyl chloride gemcitabine prodrug forms a complex with albumin in vivo, significantly improves the stability of the prodrug in systemic circulation, prolongs the drug's in vivo circulation time, improves the drug's in vivo pharmacokinetic behavior, and allows the drug to have more opportunities to follow albumin to reach tumor lesions, thereby improving anti-tumor activity. The present invention provides a series of gemcitabine prodrugs and a synthesis method that can rapidly bind to albumin in vivo, significantly prolong the drug half-life, and improve anti-tumor activity, as well as applications in pharmaceutical preparations.

[0007] In one aspect, the present invention provides a prodrug of acryloyl chloride gemcitabine and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof as shown in the general formula I:

[0008]

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

[0010] The present invention also provides a non-albumin-binding gemcitabine prodrug represented by general formula II or III and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof:

[0011]

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

[0013] In the above technical solution, further, the gemcitabine prodrug represented by general formula I, II, III and its pharmaceutically acceptable salt, solvate, polymorph or isomer is selected from:

[0014]

[0015] In the above technical solution, further, the preparation method of the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salt, solvate, polymorph or isomer is prepared by the following steps:

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

[0017] Dissolving 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 in anhydrous DMF, then adding a DMF solution of gemcitabine, stirring under nitrogen protection to obtain a reaction solution; removing DMF from the reaction solution, separating the product by using a preparation liquid phase, pouring the preparation liquid into ethyl acetate for extraction, then combining the organic phases, washing with saturated physiological saline in turn, drying the organic phase with anhydrous sodium sulfate, recovering the solvent, and purifying to obtain series I or II compounds;

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

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

[0020]

[0021] The gemcitabine prodrug of formula III is prepared by the following steps:

[0022] The condensation catalyst and carboxylic acid are dissolved in anhydrous DMF, activated in an ice bath, and then the acid-binding agent organic base and gemcitabine solution diluted in DMF are added in sequence, and stirred under nitrogen protection to obtain a reaction solution; the DMF in the reaction solution is removed, and the product is separated by using a preparative liquid phase, and a series III compound is obtained by post-treatment;

[0023]

[0024] Preferably, the carboxylic acid is a carboxylic acid containing a succinimide structural fragment, preferably 3-succinimidyl propionic acid;

[0025] Preferably, the condensation catalyst is one or more of EDCI, HATU, HBTU, TBTU, CDI, DCC, DIC, HOBt, preferably HATU; the organic base used is one or more of triethylamine, N-methylmorpholine, diethylamine, DIPEA, preferably DIPEA; in molar ratio, the 3-maleimidopropionic acid: HATU: DIPEA: gemcitabine = 1: (1-4): (0.5-4): 1.

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

[0027] In the above technical solution, further, the mass ratio of the gemcitabine prodrug to the 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 regulator, and the pH regulator is one or more combinations of citric acid, hydrochloric acid, phosphate buffer, and acetate buffer, preferably hydrochloric acid.

[0029] In the above technical solution, further, the preparation method of the aforementioned gemcitabine prodrug solution comprises the following steps:

[0030] (1) dissolving the 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 amount of amino acids in an appropriate amount of water for injection and adjust the pH to 3-5;

[0032] (3) mixing the organic solution containing gemcitabine prodrug and the amino acid solution evenly and vortexing for 10 minutes;

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

[0034] The third aspect of the present invention provides a pharmaceutical composition comprising the aforementioned gemcitabine prodrug and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof 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 salt, solvate, polymorph or isomer or the aforementioned pharmaceutical composition in the preparation of albumin-bound anti-tumor drugs; or in the preparation of drugs with enhanced anti-tumor activity.

[0036] The fifth aspect of the present invention provides the use of the aforementioned gemcitabine prodrug and its pharmaceutically acceptable salt, solvate, polymorph or isomer or the aforementioned pharmaceutical composition in the preparation of a long-circulating drug in vivo. Beneficial effects of the present invention:

[0037] The present invention synthesizes a series of gemcitabine prodrugs and prepares gemcitabine prodrug solutions, and the synthesis method and preparation method are simple and easy; the albumin-bound gemcitabine prodrug is a compound composed of an albumin-binding group and a gemcitabine bridge, wherein acryloyl chloride is used as a target head for binding to the free thiol group of cysteine ​​at position 34 of albumin, and the amide bond is enzyme-sensitive. Experiments have shown that the gemcitabine prodrug of the present invention can quickly and specifically bind to serum albumin, improve the stability of gemcitabine in systemic circulation, prolong the systemic circulation time of gemcitabine, and improve the anti-tumor activity. The macromolecular prodrug strategy designed by the present invention can improve the delivery efficiency of chemotherapeutic drugs, improve the anti-tumor effect of chemotherapeutic drugs, and provide a new direction and new ideas for the delivery of chemotherapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 For the 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 To confirm the structure of prodrug 2 of the present invention, A: 1H-NMR spectrum of prodrug 2, B: mass spectrum of prodrug 2.

[0040] Figure 3 To confirm the structure of 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 the gemcitabine prodrug solution using L-arginine as a cosolvent according to Example 4 of the present invention.

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

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

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

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

[0046] Fig. 9 The formation of the inactive metabolite dFdU in the plasma chemical stability of the four prodrugs of Example 7 of the present invention.

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

[0048] Fig.11 It is the total GEM in the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of the present invention.

[0049] Fig.12 It is the inactive metabolite dFdU released in the in vivo pharmacokinetic experiments of the four prodrugs and two albumin-prodrug complexes of Example 8 of the present invention.

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

[0051] Fig.14 This is a graph showing the tumor growth curves of PANC02 tumor-bearing mice at a dosage of 10 mg / kg for the control group, four gemcitabine prodrugs and two albumin-prodrug complexes of Example 9 of the present invention.

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

[0053] Fig.16 This is a graph showing the weight changes of PANC02 tumor-bearing mice at a dosage of 10 mg / kg for the control group, four gemcitabine prodrugs and two albumin-prodrug complexes of Example 9 of the present invention.

[0054] Fig.17 This is a graph showing the tumor growth curves of PANC02 tumor-bearing mice at a dosage of 20 mg / kg for the control group, four gemcitabine prodrugs and two albumin-prodrug complexes of Example 9 of the present invention.

[0055] Fig.18 A graph showing the tumor-bearing rates of PANC02 tumor-bearing mice at a dose of 20 mg / kg for the control group, four gemcitabine prodrugs and two albumin-prodrug complexes of Example 9 of the present invention.

[0056] Fig.19 This is a graph showing the weight changes of PANC02 tumor-bearing mice at a dose of 20 mg / kg for the control group, four gemcitabine prodrugs and two albumin-prodrug complexes of Example 9 of the present invention.

[0057] The above gemcitabine and prodrugs 1, 2, 3, 4, and albumin prodrug complexes correspond to GEM, BG, n-BG, n-MG, MG, HSA-BG, and HSA-MG in the accompanying drawings, respectively. DETAILED DESCRIPTION

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

[0059] Example 1

[0060] Synthesis of albumin-binding gemcitabine prodrug (BG) using acryloyl chloride as the modification module.

[0061] Under ice bath conditions, an appropriate amount of gemcitabine 263mg (1mmol) was dissolved in 5mL anhydrous DMF and placed in a 100mL eggplant-shaped bottle. After complete dissolution, DIPEA (N, N-diisopropylethylamine) 180μL (2.4mmol) was added, followed by 90μL (1mmol) of acryloyl chloride diluted with 5mL DMF. Under nitrogen protection, the temperature was naturally raised and stirred at room temperature for 12h. After the reaction was completed, DMF was removed with an oil pump, and the product was separated by using a preparation liquid phase. The preparation liquid was poured into ethyl acetate for extraction, and then the organic phases were combined, washed with saturated saline in turn, and the organic phase was dried over anhydrous sodium sulfate. The solvent was recovered and purified to obtain gemcitabine prodrug BG.

[0062] The synthetic route is shown below:

[0063]

[0064] Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the structure of the prodrug. Figure 1 The results of NMR spectrum analysis 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] 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-non-binding gemcitabine prodrug (n-BG) with propionic acid as the modification module.

[0069] Under ice bath conditions, an appropriate amount of gemcitabine 263mg (1mmol) was dissolved in 5mL anhydrous DMF and placed in a 50mL eggplant-shaped bottle. After complete dissolution, 180μL (2.4mmol) of DIPEA was added, followed by 88μL (1mmol) of propionic acid diluted with 5mL DMF. Under nitrogen protection, the temperature was naturally raised and stirred at room temperature for 12h. After the reaction was completed, the oil pump removed DMF, the product was separated by the preparation liquid phase, the preparation liquid was poured into ethyl acetate for extraction, and then the organic phases were combined, washed with saturated saline in turn, and the organic phase was dried with anhydrous sodium sulfate. The solvent was recovered and purified to obtain gemcitabine prodrug n-BG.

[0070] The synthetic route is shown below:

[0071]

[0072] Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the structure of the prodrug. Figure 2 The results of NMR spectrum analysis 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] Mass spectrometry results are MS (ESI) m / z for C 12 H 15 F2N3O5[MH] - =318.07.

[0075] Example 3

[0076] Synthesis of albumin-non-binding gemcitabine prodrug (n-MG) with 3-succinimidyl propionic acid as the modification module.

[0077] Under ice bath conditions, an appropriate amount of 3-succinimidyl propionic acid 100mg (0.58mmol) was dissolved in 2mL DMF and placed in a 50mL eggplant-shaped bottle. After complete dissolution, 440mg HATU (1.16mmol) was added. After ice bath activation for 10min, 100μLDIPEA (0.58mmol) was added and activation was continued for 2h under nitrogen protection. After activation, 158mg (0.58mmol) of gemcitabine dissolved in 2mL DMF was added. Under nitrogen protection, the mixture was stirred at room temperature for 24h. After the reaction was completed, anhydrous DMF was removed by an oil pump, and the product was separated by a preparative liquid phase, and the albumin-unbound gemcitabine prodrug n-MG was purified.

[0078] The synthetic route is shown below:

[0079]

[0080] Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the structure of the prodrug. Figure 3 The results of NMR spectrum analysis 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] 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 Using L-Arginine as Cosolvent

[0085] Accurately weigh different mass ratios of prodrugs (BG, MG, n-BG, n-MG) and L-arginine (1:1-1:40), add 50 μL of anhydrous ethanol and 0.5% DMSO (v / v) to the prodrug, and dissolve it completely by ultrasound. Add about 80% of the total amount of water for injection to L-arginine, stir it at room temperature to completely dissolve it, adjust the pH value to 3-5 with hydrochloric acid solution, then add the organic solution containing the prodrug and mix it evenly, and vortex for 10 minutes. Remove ethanol by vacuum distillation, add water for injection to the total amount of preparation, and filter to obtain a prodrug solution without ethanol. Determine its concentration by HPLC.

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

[0087] Example 5

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

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

[0090] In addition, a competitive binding experiment with albumin was conducted. HSA was pre-incubated with 3-maleimidopropionic acid in a shaker at 37°C for 1 hour. When the thiol groups in albumin were completely bound to 3-maleimidopropionic acid, BG solution was added and incubated for 30 minutes according to the binding experiment method. The incubation solution was filtered with a filter membrane and directly entered into HPLC to examine the binding of the prodrug to albumin after the free thiol groups in albumin were completely blocked.

[0091] In vitro binding assay results Figure 5 As shown in the figure, after 60 minutes of incubation, BG and albumin were almost completely bound. This experiment shows that BG can bind to albumin quickly. When human serum albumin was incubated with excess 3-maleimidopropionic acid in advance and then subjected to a binding experiment with BG, the BG peak area did not change significantly with time. This result shows that BG specifically binds to the free thiol group of cysteine ​​34 of albumin through the vinyl group, and there is almost no other binding mode.

[0092] Example 6

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

[0094] 4T1 cells were inoculated in 96-well plates (1000 cells per well) and incubated in an incubator for 20 hours. After they adhered to the wall, fresh culture medium containing different concentrations of prodrug, albumin-prodrug complex and gemcitabine was added, 200 μL per well, and 6 parallel wells were set for each concentration. After incubation for 48 hours, the 96-well plate was removed, the old culture medium was poured out, and 135 μL of MTT solution diluted with fresh culture medium was added to each well (100 μL fresh culture medium + 35 μL 5 mg / mL MTT solution). After incubation in the incubator for 4 hours, the culture medium was discarded, the 96-well plate was turned upside down on the filter paper to fully absorb the residual liquid, and 150 μL DMSO was added to each well and oscillated on an oscillator for 15 minutes to dissolve the blue-purple crystals. The absorbance value was measured at 570 nm using an ELISA reader.

[0095] The results are as follows Figure 6As shown in the figure, since the prodrug needs to undergo a response activation process to work in cells, the cytotoxicity of gemcitabine prodrugs is weaker than that of gemcitabine solutions. Since the release rate of gemcitabine from albumin-prodrug complexes is slower than that from prodrug solutions, the cytotoxicity of albumin-prodrug complexes is slightly lower than that of the corresponding gemcitabine prodrugs.

[0096] Among them, the structural formula of the prodrug MG is as follows:

[0097]

[0098] The albumin-prodrug complexes HAS-BG and HAS-MG were obtained according to the method of Example 5. A 20% anhydrous ethanol solution of prodrug 1BG or prodrug MG was slowly added dropwise to an equal volume of pH 7.4 phosphate solution of human serum albumin, and the final concentrations of prodrug and albumin were 300 μM and 750 μM, respectively. The mixed solution was placed in a shaking incubator 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, HAS-MG

[0101] The prodrug / albumin-prodrug was uniformly mixed with fresh rat plasma (1:10, v / v) at 37°C for 72 h to explore the in vitro release trend of GEM. The release rate of GEM in plasma was detected by HPLC at a predetermined time. The results are shown in Figure 7 As shown in the figure, due to the presence of a large amount of albumin in plasma, BG and MG can quickly bind to albumin in plasma after incubation with plasma and have good plasma chemical stability. As the incubation time increases, they slowly degrade. The release amount of GEM from the two complexes within 12 hours is less than 7% ( Figure 8 ), which provides a guarantee for the in vivo pharmacokinetic behavior of the drug. Fig. 9 It shows that the release trend of the generated dFdU is consistent with that of GEM. The more GEM is released, the more dFdU is metabolized. Moreover, the dFdU produced by BG and MG is significantly less than that of n-BG and n-MG. In addition, when the free thiol groups on the albumin in the plasma are occupied by premature binding, BG and MG cannot bind to them and are quickly cleared within 12 hours. This further illustrates that the binding of BG and MG to the free thiol group at position 34 of cysteine ​​is highly specific and the process is rapid. The above results show that the use of albumin binding strategy can effectively reduce the degradation of gemcitabine by CDA, significantly improve the stability of the prodrug in the systemic circulation, and avoid the off-target effects caused by premature degradation of the prodrug.

[0102] Example 8

[0103] Male Sprague-Dawley rats (200-230 g) 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 injected with GEM, prodrugs (BG, MG, n-BG, n-MG) and albumin prodrug complexes (HSA-BG, HSA-MG) via the tail vein at a dose equivalent to 6 mg / kg GEM. After administration, blood samples were collected at each predetermined time point. Plasma samples were then obtained by centrifugation and stored in a refrigerator at -80°C. The concentrations of prodrugs, dFdU and GEM were determined by UPLC-MS / MS. Fig.10 As shown in the figure, due to the short half-life of GEM, the GEM solution was rapidly cleared from the blood, with AUC and t1 / 2 of 54.137±21.276mg / L*h and 2.277±0.692h, respectively. In contrast, the total GEM content of the BG group and the MG group was much higher than that of the GEM group, 7 times and 45 times, respectively, and the half-life was extended by 8 times relative to the GEM group. In the BG group, GEM in the form of albumin-prodrug complex accounted for 80% of the total amount, indicating that most of the GEM can be protected by albumin in the systemic circulation and avoid CDA degradation. This may be attributed to the good affinity of BG for albumin. This property allows BG to bind to albumin faster in the systemic circulation, thereby better utilizing the protection provided by the albumin carrier and the advantages of prolonged circulation time. In contrast, the n-BG group and the n-MG group were rapidly cleared in the systemic circulation due to their inability to covalently bind to albumin. However, the total GEM content measured by the HSA-MG group and HSA-BG group, which were covalently bound to albumin in vitro, was inconsistent with the trend of the BG group and MG group, which were endogenously bound to albumin. This may be because MG underwent ring-opening hydrolysis in the PBS environment during in vitro incubation, and its ability to bind to albumin was weakened, thus failing to show better pharmacokinetic results.

[0105] Example 9

[0106] Antitumor pharmacodynamics 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 (6 in each group), and the negative control saline group, gemcitabine solution group, four gemcitabine prodrug solutions (MG, BG, n-MG, n-BG) and two albumin-prodrug complexes (HSA-MG, HSA-BG) were set up respectively. The drug was administered once every three days for a total of 4 times, with a dose of 10 mg / kg and 20 mg / kg. The tumor volume (long diameter*short diameter*short diameter / 2) and body weight of tumor-bearing mice were measured and recorded every day, and the curves of tumor volume and body weight over time were drawn. After the experiment, the mice were killed, the tumor tissue was peeled off, and the tumor-bearing rate (tumor weight / mouse weight*100%) was calculated by weighing.

[0108] The results showed that in the low-dose group, the gemcitabine solution group showed significant tumor inhibition compared with the saline group. In contrast, the prodrug solution group (MG, BG) covalently bound to albumin in vivo further improved the anti-tumor effect and delayed tumor growth compared with the free gemcitabine group. HSA-BG and HSA-MG covalently bound to albumin in vitro showed strong tumor inhibition compared with the gemcitabine solution group, with tumor inhibition rates of 42.3% and 40.6%, respectively. It is worth noting that n-MG and n-BG showed the worst tumor inhibition effect due to their inability to bind to albumin in the systemic circulation, but still had a certain tumor inhibition effect. In the high-dose group, after the MG increased the dosage, all mice died on the ninth day, indicating that there may be safety risks in the high-dose MG group. This suggests that we need to fully consider the efficacy and safety of drugs in drug development, not just their therapeutic effects. In addition, BG did not significantly improve the anti-tumor effect compared with free gemcitabine, but its safety was improved compared with the MG group. Although the anti-tumor effects of n-BG and n-MG are relatively weak, they still have certain anti-tumor effects. The above experimental results show that n-BG and n-MG cannot bind to albumin in the systemic circulation, have poor stability in the systemic circulation, and the systemic circulation time is not significantly improved compared with the GEM group. The released free GEM is easily degraded into inactive metabolites dFdU by cytidine deaminase in plasma, resulting in poor efficacy results. BG and MG, which can be quickly and highly selectively covalently bound to endogenous albumin through the Michael addition reaction, and the albumin-prodrug complexes HSA-BG and HSA-MG prepared in vitro, greatly reduce the degradation by cytidine deaminase under the protection of albumin, avoid the early release of GEM in the albumin-prodrug complex, improve its metabolic enzyme stability and bioavailability, and prolong the systemic circulation time. Therefore, the measured GEM content in the body is much higher than that of n-BG and n-MG, showing better pharmacodynamic results.

[0109] In general, the new gemcitabine prodrug BG of the present invention has an anti-tumor effect that is superior to that of the mother drug and has low toxicity. Although the anti-tumor effect of the new gemcitabine prodrugs n-BG and n-MG is relatively weaker than that of the mother drug, their toxicity is lower than that of the mother drug. In addition, after the prodrug BG is combined with albumin, its anti-tumor effect is significantly improved.

[0110] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. Gemcitabine acryloyl chloride prodrugs and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof as shown in general formula I: in, X is (CH2)n, O(CH2)n, n=1-10; preferably n=2-6.

2. Non-albumin-binding gemcitabine prodrugs represented by general formula II or III and pharmaceutically acceptable salts, solvates, polymorphs or isomers thereof: in, X is (CH2)n, O(CH2)n, n=1-10; preferably n=2-6.

3. The gemcitabine prodrug represented by the general formula I, II, III as claimed in claim 1 or 2, and its pharmaceutically acceptable salt, solvate, polymorph or isomer, selected from:

4. The method for preparing the gemcitabine prodrug and its pharmaceutically acceptable salt, solvate, polymorph or isomer according to claim 1 or 2, characterized in that: Prepared by the following steps: The gemcitabine prodrug of Formula I or Formula II is prepared by the following steps: Dissolving 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 in anhydrous DMF, then adding a DMF solution of gemcitabine, stirring under nitrogen protection to obtain a reaction solution; removing DMF from the reaction solution, separating the product by using a preparation liquid phase, pouring the preparation liquid into ethyl acetate for extraction, then combining the organic phases, washing with saturated physiological saline in turn, drying the organic phase with anhydrous sodium sulfate, recovering the solvent, and purifying to obtain series I or II compounds; Preferably, the fatty acid or acyl halide compound containing a double bond structure used to prepare the compound of formula I is acryloyl chloride, and the saturated fatty acid or acyl halide compound used to prepare the compound of formula II is propionic acid; Preferably, the organic base is one or more of triethylamine, N-methylmorpholine, diethylamine, and DIPEA, preferably DIPEA; in molar ratio, the acryloyl chloride:DIPEA:gemcitabine=1:(0.5-4):1; The gemcitabine prodrug of formula III is prepared by the following steps: The condensation catalyst and carboxylic acid are dissolved in anhydrous DMF, activated in an ice bath, and then the acid-binding agent organic base and gemcitabine solution diluted in DMF are added in sequence, and stirred under nitrogen protection to obtain a reaction solution; the DMF in the reaction solution is removed, and the product is separated by using a preparative liquid phase, and a series III compound is obtained by post-treatment; Preferably, the carboxylic acid is a carboxylic acid containing a succinimide structural fragment, preferably 3-succinimidyl propionic acid; Preferably, the condensation catalyst is one or more of EDCI, HATU, HBTU, TBTU, CDI, DCC, DIC, HOBt, preferably HATU; the organic base used is one or more of triethylamine, N-methylmorpholine, diethylamine, DIPEA, preferably DIPEA; in molar ratio, the 3-maleimidopropionic acid: HATU: DIPEA: gemcitabine = 1: (1-4): (0.5-4):

1.

5. A gemcitabine prodrug solution, characterized in that: The components of the solution include gemcitabine prodrug and amino acid; the pH value of the solution is 3-5.

6. The solution according to claim 5, characterized in that The mass ratio of the gemcitabine prodrug to the amino acid is 1:1-1:40, preferably 1:10-1:30; 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 regulator, and the pH regulator is one or more combinations of citric acid, hydrochloric acid, phosphate buffer, and acetate buffer, preferably hydrochloric acid.

7. A method for preparing the gemcitabine prodrug solution as claimed in claim 5, characterized in that: The steps include: (1) dissolving the gemcitabine prodrug in an organic solvent; wherein the organic solvent is one or more of DMSO, anhydrous ethanol, methanol, acetonitrile, and dichloromethane; (2) Dissolve a certain amount of amino acids in an appropriate amount of water for injection and adjust the pH to 3-5; (3) mixing the organic solution containing gemcitabine prodrug and the amino acid solution evenly and vortexing for 10 minutes; (4) Remove the organic solvent and add water for injection to the full amount according to the content of the prodrug solution.

8. A pharmaceutical composition comprising the gemcitabine prodrug according to any one of claims 1 to 3 and a pharmaceutically acceptable salt, solvate, polymorph or isomer thereof and a pharmaceutically acceptable carrier.

9. Use of the gemcitabine prodrug according to any one of claims 1 to 3 and its pharmaceutically acceptable salt, solvate, polymorph or isomer or the pharmaceutical composition according to claim 8 in the preparation of albumin-bound anti-tumor drugs; or in the preparation of drugs with enhanced anti-tumor activity.

10. Use of the gemcitabine prodrug according to any one of claims 1 to 3 and its pharmaceutically acceptable salt, solvate, polymorph or isomer or the pharmaceutical composition according to claim 8 in the preparation of a long-circulating drug in vivo.

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