Gemcitabine prodrug and medical application thereof

Abstract

The invention discloses a gemcitabine prodrug or pharmaceutically acceptable salt, tautomer, meracemate, racemate, stereoisomer or solvate of the gemcitabine prodrug, wherein the structure of the gemcitabine prodrug is shown as a formula I; R1 and R2 are independently selected from H, C1-C6 alkyl or C1-C6 alkyl, one or more hydrogen in the C1-C6 alkyl can be substituted by R3, and benzyl or benzyl of which hydrogen on a benzene ring is substituted by R4, or a pharmaceutically acceptable salt, tautomer, meracemate, racemate, stereoisomer or solvate of the gemcitabine prodrug, or pharmaceutically acceptable salt, tautomer, meracemate, racemate, stereoisomer or solvate thereof; x is selected from S or O; y is selected from S or O; ar is selected from phenyl, 1-naphthyl or 2-naphthyl; r3 is hydroxyl, halogen, C3-C6 cycloalkyl, phenyl or R4 substituted phenyl; and R4 is hydroxyl, halogen, cyano, nitro or methoxyl. The gemcitabine prodrug disclosed by the invention can be activated by ROS, has stronger tumor targeting property and ideal antitumor activity, and is less influenced by cytidine deaminase and nucleoside transporter. The invention also discloses an application of the gemcitabine prodrug or the pharmaceutically acceptable salt thereof in preparation of antitumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a class of gemcitabine prodrugs and their medical uses in preparing anti-tumor drugs. Background Art

[0002] Cancer has become the leading cause of death in the 21st century, and fighting cancer is a serious problem facing the world. Although a variety of anti-tumor drugs have been used in clinical practice, due to the complexity of tumor etiology, tumor drug resistance, and the toxic side effects of anti-tumor drugs, existing drugs still cannot meet clinical needs. It is of great significance to find new anti-tumor drugs with strong efficacy, strong targeting, and few toxic side effects.

[0003] Gemcitabine is a cytosine nucleoside analog that was approved for marketing by the US FDA in 1996. It is currently a first-line drug for the treatment of pancreatic cancer, breast cancer, non-small cell lung cancer and other cancers. However, gemcitabine also exhibits many shortcomings: 1. It is highly hydrophilic, relies on nucleoside transporters to enter the cell, and has low transmembrane absorption efficiency; 2. It is easily metabolically inactivated by cytidine deaminase (CDA) abundant in the liver and blood; 3. Downregulation of deoxycytidine kinase and upregulation of efflux transporters in cancer cells lead to reduced efficacy. In addition, the distribution of gemcitabine in non-tumor tissues also leads to side effects such as bone marrow suppression, renal toxicity and gastrointestinal toxicity in clinical applications. Prodrug strategy is an important way to optimize the anticancer activity of gemcitabine. LY2334737 is a valproyl-modified gemcitabine. As a classic amide prodrug, it improves the absorption of gemcitabine and achieves oral administration in clinical studies. NUC-1031 is a gemcitabine phosphamide prodrug developed using the ProTide strategy. Its absorption process does not require nucleoside transporters, can resist the metabolic effects of cytidine deaminase, and does not rely on deoxycytidine kinase to exert its efficacy. It is currently in Phase III clinical trials.

[0004] Hydrogen peroxide (H2O2) is the most representative oxidizing substance among reactive oxygen species (ROS), with the most abundant content and abnormal accumulation in tumor tissues. Existing reports have shown that H2O2-responsive prodrugs have ideal tumor targeting properties, but they are rarely used in the optimization and modification of gemcitabine. Based on the ProTide strategy, the inventors designed, synthesized and screened a large number of gemcitabine prodrugs in the early stage. Surprisingly, one class of prodrugs has excellent responsiveness to H2O2 and exhibits ideal pharmacological activity, which is expected to be further studied. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the first purpose of the present invention is to provide a novel gemcitabine prodrug, which can be specifically degraded in the presence of H2O2; the compound has ideal anti-tumor activity in vitro and in vivo, low toxicity to normal cells, and its efficacy is not easily affected by metabolic enzymes.

[0006] The second object of the present invention is to provide medical uses of the aforementioned gemcitabine prodrug.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a gemcitabine prodrug having a structural formula as shown in formula (I) or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof:

[0009]

[0010] R 1 , R 2 Independently selected from: H, C1-C6 straight chain or branched alkyl or one or more hydrogen atoms in the C1-C6 straight chain or branched alkyl may be replaced by R 3 Substitution, benzyl or hydrogen on the benzene ring is replaced by R 4 Substituted benzyl; X is selected from: S or O; Y is selected from: S or O; Ar is selected from: phenyl (Ph), 1-naphthyl or 2-naphthyl;

[0011] R 3 is hydroxyl, halogen, C3-C6 cycloalkyl, phenyl or R 4 Substituted phenyl; R 4 is hydroxy, halogen, cyano, nitro or methoxy.

[0012] Preferably, R 1 , R 2 Independently selected from: H, C1-C6 straight or branched alkyl, benzyl or hydrogen on the benzene ring is replaced by R 4 Substituted benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl;

[0013] R 4 is hydroxy, halogen, cyano, nitro or methoxy.

[0014] More preferably, R 1 , R 2 Independently selected from: H, C1-C6 straight chain or branched alkyl or benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl.

[0015] More preferably, R 1 , R 2Independently selected from: H, C1-C4 straight or branched alkyl or benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl; but does not include: R 1 Selected from: H, R 2 is selected from: isopropyl, tert-butyl, X is selected from: S, Y is selected from: O; Ar is selected from: phenyl.

[0016] Most preferably, R 1 , R 2 Independently selected from: H, CH3; X is selected from: S; Y is selected from: O; Ar is selected from: phenyl.

[0017] Specifically, the gemcitabine prodrug is selected from any one of the following compounds:

[0018]

[0019]

[0020] In a second aspect, the present invention provides the use of the gemcitabine prodrug or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof in the preparation of an anti-tumor drug.

[0021] In a third aspect, the present invention provides a pharmaceutical composition comprising the gemcitabine prodrug or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof, and a pharmaceutically acceptable excipient.

[0022] In a fourth aspect, the present invention provides use of the pharmaceutical composition in the preparation of anti-tumor drugs.

[0023] The dosage form of the pharmaceutical composition is solid, semi-solid, liquid or gaseous preparation.

[0024] Preferably, the dosage form of the pharmaceutical composition is tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, freeze-dried powder injections, etc.

[0025] definition

[0026] Unless otherwise specified, the following terms used in the present invention have the following meanings. A particular term should not be considered as uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commodity or its active ingredient.

[0027] The term "alkyl" refers to a group of the formula C n H 2n+1The alkyl group may be straight chain or branched. For example, the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl).

[0028] The term "halogen" refers to fluorine, chlorine, bromine, iodine.

[0029] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0030] As the pharmaceutically acceptable salt, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids and the like can be mentioned.

[0031] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present invention to an organism.

[0032] The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0033] The present invention also includes isotopically labeled compounds of the present invention that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2H, 3H, 11C, 13C, 14C, 13N, 15N, 15O, 17O, 18O, 31P, 32P, 35S, 18F, 123I, 125I, and 36Cl, etc., respectively.

[0034] The pharmaceutical composition of the present invention can be prepared by combining the compound of the present invention with suitable pharmaceutically acceptable excipients, for example, it can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0035] Typical routes of administration of the compounds of the invention or their pharmaceutically acceptable salts or their pharmaceutical compositions include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0036] The pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulating methods, making sugar-coated pills, grinding methods, emulsifying methods, freeze-drying methods, and the like.

[0037] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.

[0038] Solid oral compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into particles to obtain a tablet or dragee core. Suitable excipients include, but are not limited to, adhesives, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc.

[0039] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

[0040] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0041] The chemical reactions of the specific embodiments of the present invention are carried out in a suitable solvent, which must be suitable for the chemical changes of the present invention and the reagents and materials required. In order to obtain the compounds of the present invention, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The gemcitabine prodrug of the present invention can be activated by ROS, has stronger tumor targeting, has ideal anti-tumor activity, and has ideal safety; the gemcitabine prodrug is less affected by cytidine deaminase and nucleoside transporter. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound 1.

[0045] Figure 2 Figure 3 is the result of H2O2 activation experiment; wherein, Figure A is the activation result of gemcitabine prodrug (Compound 1-Compound 10) in H2O2, and Figure B is the stability result of gemcitabine prodrug (Compound 1-Compound 10) in PBS.

[0046] Figure 3 The effect of metabolic enzymes on the activity of compounds.

[0047] Figure 4 The results of in vivo antitumor activity study; wherein, A is the effect of compound 1 on the tumor volume of nude mice; B is the effect of compound 1 on the body weight of nude mice; C is the effect of compound 1 on the weight of nude mice tumor. DETAILED DESCRIPTION

[0048] The present invention is specifically described below by way of examples. In the present invention, the following examples are for better illustrating the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0049] Example 1

[0050] Synthesis of compound 1

[0051]

[0052] Step 1: At room temperature, gemcitabine hydrochloride (compound I-1, 300 mg, 1 mmol) and anhydrous sodium carbonate (530 mg, 5 mmol) were placed in a single-necked bottle, and dioxane-water (dioxane: water = 4: 1, 25 mL) and di-tert-butyl dicarbonate (218 mg, 1 mmol) were added in sequence, and the reaction was carried out at room temperature for 48 hours. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate, the organic phases were combined, the organic phases were washed once with saturated brine, and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and compound I-2 (white solid, 243 mg) was separated by normal phase silica gel column chromatography (dichloromethane: methanol = 20: 1 V / V), with a yield of 67%. 1 H NMR (300MHz, DMSO) δ7.65(d,J=7.5Hz,1H),7.43(d,J=5.4Hz,2H),6.22(t,J=9.3Hz,1H),5.82(d ,J=7.5Hz,1H),5.30-5.11(m,2H),4.15(dt,J=7.0,3.5Hz,1H),3.80-3.59(m,2H),1.46(s,9H).

[0053] Step 2: Place N-(tert-butyloxycarbonyl)-L-alanine (Compound I-4, 6300 mg, 33 mmol), thiazolidin-2-one (Compound I-3, 3100 mg, 30 mmol) and lithium chloride (2544 mg, 60 mmol) in a double-necked flask, replace with argon three times, add THF at -30 ° C and stir for 1 hour. Then add pivaloyl chloride (PivCl, 9.3 mL, 75 mmol) and triethylamine (7900 mg, 78 mmol) dropwise, and continue to react for 3 hours at -30 ° C. After the reaction is completed, add saturated aqueous ammonium chloride to the reaction solution, extract three times with ethyl acetate, combine the organic phases, wash the organic phases with saturated sodium carbonate three times, wash once with saturated sodium chloride, and dry over anhydrous sodium sulfate. Evaporate the solvent under reduced pressure, separate by normal phase silica gel column chromatography (petroleum ether: ethyl acetate = 1: 1 V / V), and obtain compound I-5 (white solid, 5822 mg), with a yield of 71%. 1 H NMR (300MHz, CDCl3) δ5.25(q,J=7.5Hz,1H),5.17(s,1H),4.29-4.04(m,2H),3.33(td,J=7.3,4.0Hz,2H),1.43(s,9H),1.36(d,J=6.8Hz,3H).

[0054] Step 3: Place (S)-(1-oxo-1-(2-oxothiazolidin-3-yl)propan-2-yl)carbamic acid tert-butyl ester (Compound I-5, 4800 mg, 20 mmol) in a single-necked bottle, add hydrochloric acid-ethyl acetate (4M, 10 mL), and react at room temperature overnight. After the reaction is completed, the reaction solution is filtered to obtain Compound I-6 (white solid, 2780 mg), with a yield of 85%. 1 H NMR (300MHz, DMSO) δ8.57 (s, 3H), 4.68 (s, 1H), 4.11 (d, J = 7.6Hz, 2H), 3.45 (d, J = 7.3Hz, 2H), 1.39 (d, J = 6.9Hz, 3H).

[0055] Step 4: 3-(L-alanyl)thiazolidine-2-one hydrochloride (compound I-6, 2100mg, 10mmol) and phenylphosphonium dichloride (2109mg, 10mmol) are placed in a double-necked bottle, argon is replaced 3 times, and DCM (50mL) is added. After the reaction solution is cooled to -50 ° C, TEA (2780μL, 20mmol) is added dropwise and stirred for 30 minutes, slowly warmed to room temperature and continued to react for 3 hours. Pentafluorophenol (1840mg, 10mmol) and DCM (10mL) are added to the reaction solution, and TEA (1529μL, 11mmol) is slowly added dropwise again, and the reaction is continued at room temperature overnight. After the reaction is completed, the reaction solution is extracted three times with dichloromethane, the organic phase is combined, the organic phase is washed three times with saturated sodium bicarbonate, washed once with saturated brine, and the organic phase is dried over anhydrous sodium sulfate. The solvent is evaporated under reduced pressure and separated to obtain compound I-7 (white solid, 2800mg), with a yield of 58%. 1 H NMR (300MHz, DMSO) δ7.50-7.36(m,2H),7.30-7.11(m,3H),6.89(ddd,J=14.9,10.4,5.2Hz,1H),5.08(tqd, J=9.9,6.8,2.6Hz,1H),4.13-3.91(m,2H),3.39(ttt,J=11.9,6.5,2.7Hz,2H),1.25(dd,J=7.0,3.3Hz,3H).

[0056] Step 5: Place (2R, 3R, 5R)-5-(4-amino-2-oxopyrimidine-1(2H)-yl)-4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl carbonate tert-butyl ester (Compound I-2, 180 mg, 0.5 mmol) in a double-necked bottle, replace with argon three times, and add THF (6 mL). Slowly drop tert-butyl magnesium chloride (1.7 M, 0.4 mL, 0.6 mmol) under ice bath and stir for 30 minutes, then add phenyl N-((R)-1-oxo-1-(2-oxothiazolidine-3-yl)propane-2-yl)-P-(perfluorophenyl)phosphamide (Compound I-7, 480 mg, 1 mmol), and react at room temperature overnight. After the reaction is completed, saturated aqueous ammonium chloride solution (6 mL) is added to quench, the reaction solution is extracted three times with ethyl acetate, the organic phases are combined, the organic phases are washed once with saturated brine, and the organic phases are dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure and separated by normal phase silica gel column chromatography (dichloromethane:methanol=30:1 V / V) to obtain compound I-8 (white solid, 215 mg) with a yield of 64%. 1H NMR (300MHz, DMSO) δ7.55-7.31(m,5H),7.18(q,J=7.6Hz,3H),6.31-6.03(m,2H),5.79(ddd,J=11.3,7.5,2.5Hz,1H),5.29-5.05( m,1H),5.01-4.79(m,1H),4.47-4.19(m,3H),4.02(p,J=7.0Hz,2H),3.45-3.33(m,2H),1.45(d,J=2.0Hz,9H),1.23-1.15(m,3H).

[0057] Step 6: Place (2R,3R,5R)-5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((((S)-1-oxo-1-(2-oxothiazolidin-3-yl)propane-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-ylcarbonate tert-butyl ester (Compound I-8, 796 mg, 1.2 mmol) in a single-necked bottle, add DCM (18 mL), slowly add trifluoroacetic acid (6 mL) dropwise, and react at room temperature for 3 hours. After the reaction is completed, neutralize with saturated sodium bicarbonate solution, extract with DCM, combine the organic phases, wash the organic phases once with saturated sodium chloride, and dry the organic phases with anhydrous sodium sulfate. Evaporate the solvent under reduced pressure, separate by normal phase silica gel column chromatography (dichloromethane: methanol = 15: 1 V / V), and obtain compound 1 (white solid 492 mg), with a yield of 72%. The H NMR spectrum of compound 1 is shown in Figure 1 . 1 H NMR(500MHz,DMSO)δ7.46(d,J=7.6Hz,1H),7.44(s,1H),7.41-7.34(m,3H) ,7.23-7.13(m,3H),6.39(d,J=6.4Hz,1H),6.22-6.11(m,1H),6.11-6.04( m,1H),5.75(d,J=7.5Hz,1H),4.99-4.88(m,1H),4.33-4.26(m,1H),4.24- 4.11(m,2H),4.08-3.95(m,3H),3.44-3.26(m,2H),1.20(d,J=6.9Hz,3H).

[0058] Example 2

[0059] Synthesis of compound 2

[0060]

[0061] Referring to the method of Example 1, the thiazolidin-2-one in Example 1 was replaced with oxazolidin-2-one (870 mg, 10 mmol), and the remaining raw materials were: (tert-butoxycarbonyl)-L-alanine 2090 mg (11 mmol), lithium chloride 848 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), compound I-2 363 mg (1 mmol), and finally compound 2 (white solid, 176 mg) was obtained. 1 H NMR(300MHz,DMSO)δ7.53(br s,1H),7.46(d,J=6.5Hz,2H),7.42-7.32(m,2H),7.25-7.10(m,3H),6.43(d,J=6.6Hz,1H),6.21-6.05(m,2H ),5.75(d,J=7.7Hz,1H),5.13-4.93(m,1H),4.46-3.93(m,6H),3.83(t,J=8.3Hz,2H),1.22(d,J=7.1Hz,3H).

[0062] Example 3

[0063] Synthesis of compound 3

[0064]

[0065] Referring to the method of Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (tert-butoxycarbonyl)-D-alanine (1249 mg, 6.6 mmol), and the remaining raw materials were: thiazolidine-2-one 618 mg (6 mmol), lithium chloride 509 mg (15.6 mmol), pivaloyl chloride 1.95 mL (15 mmol), triethylamine 1578 mg (15.6 mmol), compound I-2 363 mg (1 mmol), and compound 3 (white solid, 250 mg) was obtained. 1 H NMR(300MHz,DMSO)δ7.51(br s,1H),7.48-7.33(m,4H),7.19(t,J=7.4Hz,3H),6.47(d,J=6.1Hz,1H),6.29-6.09(m,2H),5.77(d,J=7.5H z,1H),4.98-4.80(m,1H),4.35-4.14(m,2H),4.14-3.93(m,4H),3.35-3.21(m,2H),1.19(d,J=6.8Hz,3H).

[0066] Example 4

[0067] Synthesis of compound 4

[0068]

[0069] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with tert-butoxycarbonylglycine (1155 mg, 6.6 mmol), and the remaining raw materials were: thiazolidine-2-one 619 mg (6 mmol), lithium chloride 509 mg (15.6 mmol), pivaloyl chloride 1.95 mL (15 mmol), triethylamine 1578 mg (15.6 mmol), compound I-2 435 mg (1.2 mmol), and compound 4 (white solid, 256 mg) was obtained. 1 H NMR(500MHz,DMSO)δ7.78(br s,1H),7.60(br s,1H),7.56-7.50(m,1H),7.40-7.34(m,2H),7.23-7.16(m,3H),6.51-6.36(m,1H),6.22-6.12(m,1H),5.88- 5.73(m,2H),4.43-4.33(m,1H),4.33-4.23(m,1H),4.21-4.09(m,3H),4.08-4.01(m,3H),3.42-3.35(m,2H).

[0070] Example 5

[0071] Synthesis of compound 5

[0072]

[0073] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with tert-butoxycarbonylglycine (1925 mg, 11 mmol), the thiazolidine-2-one was replaced with oxazolidin-2-one (870 mg, 10 mmol), and the remaining raw materials were: lithium chloride 848 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), compound I-2 was 544 mg (1.5 mmol), and compound 5 (white solid, 160 mg) was obtained. 1H NMR(300MHz,DMSO)δ7.61(br s,1H),7.56-7.44(m,2H),7.38(t,J=7.8Hz,2H),7.25-7.14(m,3H),6.54-6.42(m,1H),6.24-6.11(m,1H),5.97-5 .82(m,1H),5.78(t,J=7.8Hz,1H),4.48-4.22(m,4H),4.22-4.07(m,3H),4.07-3.98(m,1H),3.86(t,J=8.1Hz,2H).

[0074] Example 6

[0075] Synthesis of compound 6

[0076]

[0077] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (S)-2-((tert-butoxycarbonyl)amino)butyric acid (2235 mg, 11 mmol), and the remaining raw materials were: thiazolidine-2-one 1032 mg (10 mmol), lithium chloride 849 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), compound I-2 653 mg (1.8 mmol), and compound 6 (white solid, 329 mg) was obtained. 1 H NMR(300MHz,DMSO)δ7.59-7.30(m,5H),7.29-7.08(m,3H),6.58-6.33(m,1H),6.29-6.11(m,1H),6.11-5.95(m,1H),5.83-5.67(m,1H) ,4.90-4.46(m,1H),4.43-4.11(m,3H),4.11-3.88(m,3H),3.47-3.22(m,2H),1.73-1.50(m,1H),1.50-1.30(m,1H),0.91-0.68(m,3H).

[0078] Example 7

[0079] Synthesis of compound 7

[0080]

[0081] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (tert-butoxycarbonyl)-L-valine (3690 mg, 13.2 mmol), and the remaining raw materials were: thiazolidine-2-one 1238 mg (12 mmol), lithium chloride 1017 mg (24 mmol), pivaloyl chloride 3.7 mL (30 mmol), triethylamine 3157 mg (31.2 mmol), compound I-2 was 653 mg (1.8 mmol), and compound 7 (white solid, 90 mg) was obtained. 1 H NMR(500MHz,DMSO)δ7.50(br s,1H),7.48-7.44(m,1H),7.39-7.31(m,3H),7.18(d,J=7.8Hz,3H),6.46(d,J=6.3Hz,1H),6.17(t,J=8.3Hz,1H),5.86-5.74(m,2H),4. 95-4.80(m,1H),4.41-4.27(m,1H),4.27-3.90(m,5H),3.42-3.35(m,1H),3.32-3.24(m,1H),1.97-1.86(m,1H),0.76(d,J=6.6Hz,6H).

[0082] Example 8

[0083] Synthesis of compound 8

[0084]

[0085] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (3816 mg, 16.5 mmol), and the remaining raw materials were: thiazolidine-2-one 1547 mg (15 mmol), lithium chloride 1271 mg (304 mmol), pivaloyl chloride 4.6 mL (37.5 mmol), triethylamine 3946 mg (39 mmol), compound I-2 653 mg (1.8 mmol), and compound 8 (white solid, 400 mg) was obtained. 1H NMR(300MHz,DMSO)δ7.46(br s,1H),7.44-7.29(m,4H),7.22-7.12(m,3H),6.45(d,J=6.4Hz,1H),6.24-6.10(m,1H),5.88(t,J=11.7Hz,1H),5.81-5.66(m,1H),5.21-5. 03(m,1H),4.35-4.14(m,3H),4.14-4.02(m,1H),4.02-3.93(m,1H),3 .93-3.80(m,1H),3.42-3.27(m,1H),3.27-3.11(m,1H),0.87(s,9H).

[0086] Example 9

[0087] Synthesis of compound 9

[0088]

[0089] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (tert-butoxycarbonyl)-L-leucine (2544 mg, 11 mmol), and the remaining raw materials were: thiazolidine-2-one 1032 mg (10 mmol), lithium chloride 849 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), compound I-2 653 mg (1.8 mmol), and compound 9 (white solid, 210 mg) was obtained. 1 H NMR(300MHz,DMSO)δ7.52-7.40(m,3H),7.40-7.30(m,2H),7.24-7.09(m,3H ),6.50-6.38(m,1H),6.16(t,J=8.2Hz,1H),6.11-5.92(m,1H),5.77(dd,J= 9.4,7.5Hz,1H),5.04-4.83(m,1H),4.38-4.07(m,3H),4.07-3.87(m,3H),3 .48-3.22(m,2H),1.83-1.51(m,1H),1.46-1.18(m,2H),0.85-0.66(m,6H).

[0090] Example 10

[0091] Synthesis of compound 10

[0092]

[0093] Referring to Example 1, the (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (tert-butoxycarbonyl)-L-phenylalanine (3502 mg, 13.2 mmol), and the remaining raw materials were: thiazolidine-2-one 1238 mg (12 mmol), lithium chloride 1017 mg (24 mmol), pivaloyl chloride 3.7 mL (30 mmol), triethylamine 3157 mg (31.2 mmol), compound I-2 was 653 mg (1.8 mmol), and compound 10 (white solid, 72 mg) was obtained. 1 H NMR (500MHz, DMSO) δ7.40(d,J=7.8Hz,3H),7.31-7.21(m,9H),7.13(t,J=7.5Hz,1H),6.85(d,J=8.2Hz,2H),6.40(d,J=6.6Hz,1H),6. 25-6.11(m,2H),5.76(d,J=7.6Hz,1H),5.25-5.12(m,1H),4.09-3.98(m,4H),3.93(p,J=6.4Hz,1H),3.87(s,1H),3.44-3.36(m,2H).

[0094] Embodiment 11

[0095] H2O2 activation experiment of prodrug compounds

[0096] Test drugs: prodrug compounds (Compound 1-Compound 10); control drug: NUC-1031 (CAS: 840506-29-8); internal standard compound: sofosbuvir (SOF, CAS: 1190307-88-0).

[0097] Analytical instrument: LC-20A high performance liquid chromatograph; ZORBAX Eclipse XDB-C18 (4.6×150 mm, 5 μm).

[0098] Experimental steps:

[0099] 1. Dissolve the test drug in 100% DMSO to prepare a stock solution of 10 mg / mL; dilute the 10M H2O2 solution to a stock solution of 13.2 mM; dilute the sofosbuvir stock solution to 200 μg / mL with acetonitrile step by step to prepare an acetonitrile solution containing an internal standard.

[0100] 2.①PBS stability test: the mother solution of the compound was diluted to 400μg / mL with acetonitrile. 2mL of each solution was accurately measured and mixed with 2mL of PBS solution (pH=7.4) to obtain a working solution;②In the H2O2 stability test, the above PBS solution was replaced with H2O2 solution (PBS dilution), wherein the ratio of the prodrug concentration to the H2O2 concentration in the working solution was 1:20. Both groups of samples ① and ② were incubated in a 37℃ constant temperature water bath.

[0101] 3. Take 100 μL of sample at different time points and add it to 100 μL of acetonitrile (containing sofosbuvir internal standard) and shake thoroughly. After two centrifugations (12000 rpm × 5 min), take 60 μL of supernatant into the injection bottle. Use high performance liquid chromatography for stability testing and quantification of the product.

[0102] 4. Using sofosbuvir as the internal standard, the in vitro stability of the test compound was calculated by establishing a standard curve.

[0103] The experimental results are as follows Figure 2 As shown, Figure A shows H2O2 stability, and Figure B shows PBS stability. In H2O2 solution, the control drug NUC-1031 cannot be degraded, compounds 7 and 8 show a weak degradation trend, and the remaining compounds can be fully degraded. Compounds 1-10 all show good stability in PBS, indicating that the above compounds do not have non-specific degradation in the buffer. The above results show that the gemcitabine prodrug of the present invention can be activated by H2O2, suggesting that the gemcitabine prodrug can be metabolically activated in tumor tissues enriched in H2O2.

[0104] Example 12

[0105] Study on the antitumor activity of prodrugs in vitro

[0106] Test drugs: test compounds (Compound 1-Compound 10), control drug NUC-1031 (CAS: 840506-29-8).

[0107] Experimental materials: 96-well cell culture plates were purchased from Corning; CCK8 reagent was purchased from Biosai Biotechnology; DMSO was purchased from Sigma; tumor cell lines: human pancreatic adenocarcinoma cells (BxPC-3 cells and MiaPaCa-2 cells); normal cell lines: L02 human normal liver cells.

[0108] Experimental instrument: BioTek Instruments multifunctional enzyme label detector.

[0109] Experimental steps:

[0110] 1. Dissolve the test compound in 100% DMSO to prepare a 200 mM stock solution for storage;

[0111] 2. Digest and count the cells and prepare cell suspension (BxPC-3: 4.0×10 4 / mL, MiaPaCa-2: 5×10 4 / mL, L02: 3×10 4 / mL), add 100 μL of cell suspension to each well of a 96-well cell culture plate;

[0112] 3. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 24 hours;

[0113] 4. Dilute the test drug with culture medium to the required working solution concentration, and make the final DMSO concentration not exceed 0.1%. Add 100 μL of the corresponding drug-containing culture medium to each well of the 96-well plate, and set up a negative control group at the same time;

[0114] 5. Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 48 hours;

[0115] 6. Add 10 μL of CCK8 solution to each well and continue to culture in the incubator for 2-3 hours;

[0116] 7. Use a microplate reader to read the OD value of each well at 450nm;

[0117] 8. Calculate the inhibition rate of each group.

[0118] Table 1. In vitro antitumor activity (IC 50 , in μM)

[0119]

[0120]

[0121] The experimental results are shown in Table 1. All compounds have good anti-tumor activity against the selected tumor cell lines (BxPC-3 and MiaPaCa-2). Among them, the anti-proliferative activity of prodrugs 1, 3, and 4 against tumor cells is stronger than that of the positive drug NUC-1031. More noteworthy is that the cytotoxicity of the representative prodrugs of the present invention to normal cells (L02) is significantly lower than that to tumor cells. This suggests that the compounds of the present invention have good anti-tumor activity and have ideal safety for normal cells.

[0122] Embodiment 13

[0123] Effects of metabolic enzymes on the antitumor activity of prodrugs

[0124] Test drugs: Compound 1, control drug gemcitabine hydrochloride (Gem) (CAS: 122111-03-9).

[0125] The experimental materials and experimental instruments refer to Example 12. The metabolic enzyme inhibitors selected are: nucleoside transporter inhibitors dipyridamole (DPM) and S-(4-nitrobenzyl)-6-thioinosine (NBTI); cytidine deaminase inhibitor tetrahydrouridine (THU).

[0126] Experimental steps:

[0127] Bxpc-3 cells were cultured at 5 × 10 4 The cells were spotted at a density of 10 μM / mL in a 96-well plate and cultured for 12 hours. The inhibitor (final concentration of 10 or 20 μM) was pre-added to the 96-well plate and incubated for 30 minutes, followed by the addition of gemcitabine (1 μM) or compound 1 (2 μM). After incubation with the cells for 24 hours, CCK8 solution was added and continued to be cultured in the incubator for 1 hour. The OD value of each well was read at 450 nm using an enzyme reader and the inhibition rate was calculated.

[0128] Experimental results:

[0129] The experimental results are as follows Figure 3 As shown. Pre-administration of DPM and NBTI will lead to a decrease in nucleoside uptake, thereby reducing the anti-proliferative activity of Gem, indicating that the efficacy of Gem depends on nucleoside uptake; while compound 1 is insensitive to this treatment, indicating that the efficacy of compound 1 does not depend on nucleoside transporters and passively diffuses into the cell. Pre-administration of THU will lead to a decrease in cytidine deaminase activity, thereby increasing the anti-proliferative activity of Gem, indicating that the efficacy of Gem depends on cytidine deaminase; while compound 1 is insensitive to this treatment, indicating that the efficacy of compound 1 does not depend on cytidine deaminase and can effectively avoid the decrease in efficacy caused by cytidine deaminase.

[0130] Embodiment 14

[0131] Study on the antitumor activity of prodrugs in vivo

[0132] Test samples: Compound 1 and NUC-1031; the solvent was selected as CMC-Na solution with a concentration of 1 / 1000; the experimental groups were divided into a solvent group (Vehicle) and three dosing groups (Compound 1 at a dose of 0.15 or 0.3 mmol / kg, positive control drug NUC-1031 at a dose of 0.15 mmol / kg, Compound 1 or NUC-1031 was administered twice a week for 3 consecutive weeks, on days 1, 5, 8, 12, 15, 19, and 22).

[0133] Test animals: Balb / c nude mice, 6 weeks old, female; 8 mice in each group, 4 groups in total, 32 mice in total.

[0134] Experimental methods:

[0135] 1. Collect the cultured human pancreatic cancer cell suspension of Panc-1 at a concentration of 2×10 7 / mL, 0.1mL was inoculated subcutaneously in the right axilla of nude mice;

[0136] 2. The diameter of the transplanted tumor in nude mice was measured with a vernier caliper. The tumor grew to 100 mm. 3 At the same time, the mice in each group began to receive drugs by intraperitoneal injection, which was recorded as Day 1. After the experiment, the nude mice were killed and the tumors were surgically removed and weighed.

[0137] Observation indicators:

[0138] 1. Body weight: Record the animal weight on Day 1, 5, 8, 12, 15, 19, and 22;

[0139] 2. Tumor volume: Tumor volume was recorded on Day 1, 5, 8, 12, 15, 19, and 22;

[0140] Tumor volume (TV; TV = 1 / 2 × a × b 2 ; where a and b represent length and width respectively) and relative tumor volume (RTV; RTV=V t / V1; V1 is the tumor volume measured on day 1, V t is the tumor volume at each measurement).

[0141] 3. Evaluation indicators of anti-tumor activity:

[0142] Relative tumor proliferation rate T / C (%)

[0143] T / C(%)=T RTV / C RTV × 100%, where T RTV RTV, C RTV The solvent group RTV.

[0144] Tumor growth inhibition rate TI (%)

[0145] TI (%) = (average tumor weight of vehicle group - average tumor weight of drug administration group) / average tumor weight of vehicle group × 100%.

[0146] 4. Statistical analysis: The mean was expressed as X±SD, and the t-test was used for inter-group analysis. SPSS (Staffstical Package for the Social Science) 17.0 was used for statistical analysis of the results.

[0147] Table 2. In vivo antitumor activity of compounds

[0148]

[0149] The experimental results are as follows Figure 4 As shown in Table 2, the tumor growth inhibition rates (TI) of NUC-1031 (0.15mmol / kg), low-dose compound 1 (0.15mmol / kg) and high-dose compound 1 (0.3mmol / kg) on ​​day 22 were 48.75%, 55.89% and 63.46%, respectively. Compared with the vehicle group, compound 1 was able to effectively reduce tumor weight ( Figure 4 A, C), and reduced tumor proliferation rate (Table 2). At the same dose, compound 1 was slightly more effective than NUC-1031, and showed better antitumor activity at high doses ( Figure 4 A). During the efficacy evaluation, compound 1 had no significant effect on the body weight of mice ( Figure 4 B), and no mouse death occurred; no obvious pathological changes were found in the mouse organs (heart, liver, kidney, lung and intestine) through pathological section examination. The above experiments show that the compound has good safety.

[0150] Embodiment 15

[0151] tablet

[0152] Compound 1 (50 g), hydroxypropylmethylcellulose E (150 g), starch (200 g), appropriate amount of povidone K30 and magnesium stearate (1 g) were mixed, granulated and tableted.

[0153] In addition, according to the conventional preparation method of the Pharmacopoeia 2015 edition, Compound 1 and Compound 2 can be added with different pharmaceutical excipients to prepare capsules, powders, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches, etc.

Claims

1. A gemcitabine prodrug having a structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof: in, R 1 , R 2 Independently selected from: H, C1-C6 straight chain or branched alkyl or one or more hydrogen atoms in the C1-C6 straight chain or branched alkyl may be replaced by R 3 Substitution, benzyl or hydrogen on the benzene ring is replaced by R 4 Substituted benzyl; X is selected from: S or O; Y is selected from: S or O; Ar is selected from: phenyl, 1-naphthyl or 2-naphthyl; R 3 is hydroxyl, halogen, C3-C6 cycloalkyl, phenyl or R 4 Substituted phenyl; R 4 is hydroxy, halogen, cyano, nitro or methoxy.

2. The gemcitabine prodrug according to claim 1, characterized in that: R 1 , R 2 Independently selected from: H, C1-C6 straight or branched alkyl, benzyl or hydrogen on the benzene ring is replaced by R 4 Substituted benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl; R 4 is hydroxy, halogen, cyano, nitro or methoxy.

3. The gemcitabine prodrug according to claim 2, characterized in that: R 1 , R 2 Independently selected from: H, C1-C6 straight chain or branched alkyl or benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl.

4. The gemcitabine prodrug according to claim 1, characterized in that: R 1 , R 2 Independently selected from: H, C1-C4 straight or branched alkyl or benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl; but does not include: R 1 Selected from: H, R 2 is selected from: isopropyl, tert-butyl, X is selected from: S, Y is selected from: O; Ar is selected from: phenyl.

5. A gemcitabine prodrug having a structural formula as shown in Formula I or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof: in, R 1 , R 2 Independently selected from: H, CH3; X is selected from: S; Y is selected from: O; Ar is selected from: phenyl.

6. A gemcitabine prodrug selected from any one of the following compounds or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof:

7. Use of the gemcitabine prodrug according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof in the preparation of an anti-tumor drug.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the gemcitabine prodrug according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof, and a pharmaceutically acceptable excipient.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of anti-tumor drugs.

10. The pharmaceutical composition according to claim 8, characterized in that: The dosage form of the pharmaceutical composition is a solid, semi-solid, liquid or gaseous preparation, preferably a tablet, pill, capsule, powder, granule, ointment, emulsion, suspension, suppository, injection, inhalant, gel, microsphere, aerosol, lyophilized powder injection.

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