Gemcitabine prodrugs and medical uses thereof

Abstract

The application discloses a gemcitabine prodrug or a pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate thereof with a structure as shown in formula I, R 1 , R 2 are independently selected from H, C1-C6 alkyl or one or more hydrogens in C1-C6 alkyl can be replaced by R 3 , benzyl or benzyl substituted with R 4 on a benzene ring; 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 hydroxyl, halogen, cyano, nitro or methoxyl. The gemcitabine prodrug of the application can be activated by ROS, has stronger tumor targeting property, has ideal anti-tumor activity and is less affected by cytidine deaminase and nucleoside transporters. The application further discloses application of the gemcitabine prodrug or the pharmaceutically acceptable salt thereof in preparation of an anti-tumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a class of gemcitabine prodrugs and the medical use thereof in preparing anti-tumor drugs. BACKGROUND

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

[0003] Gemcitabine is a cytosine nucleoside analogue, which was approved by the FDA in 1996 and 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 shows many deficiencies: 1. Strong hydrophilicity, dependent on nucleoside transporters to enter cells, low transmembrane absorption efficiency; 2. Easily inactivated by cytidine deaminase (CDA) which is abundant in the liver and blood; 3. The down-regulation of deoxycytidine kinase in cancer cells and the up-regulation of efflux transporters lead to reduced drug efficacy. In addition, the distribution of gemcitabine in non-tumor tissues also leads to side effects such as myelosuppression, nephrotoxicity and gastrointestinal toxicity in clinical application. Prodrug strategy is an important way to optimize the anti-cancer activity of gemcitabine. LY2334737 is a valproylated gemcitabine, which improves the absorption of gemcitabine and achieves oral administration in clinical studies. NUC-1031 is a gemcitabine phosphoramidate prodrug developed by ProTide strategy, which does not require nucleoside transporters in the absorption process, can resist the metabolic action of cytidine deaminase, and does not depend on deoxycytidine kinase to exert efficacy, and is currently in clinical phase III study.

[0004] Hydrogen peroxide (H2O2) is the most representative oxidative substance among reactive oxygen species (ROS), which is most abundant and abnormally accumulated in tumor tissues. Existing reports have proved 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

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

[0006] The second purpose of the present application is to provide the medical use of the aforementioned gemcitabine prodrug.

[0007] In order to achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows:

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

[0009]

[0010] R 1 , R 2 are independently selected from: H, C1-C6 straight chain or branched alkyl, or one or more hydrogens in C1-C6 straight chain or branched alkyl can be substituted by R 3 , benzyl or benzene ring hydrogen substituted by R 4 ; 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 hydroxyl, halogen, cyano, nitro or methoxy.

[0012] Preferably, R 1 , R 2 are independently selected from: H, C1-C6 straight chain or branched alkyl, benzyl or benzene ring hydrogen substituted by R 4 ; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl;

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

[0014] More preferably, R 1 , R 2 are 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] Further preferably, R 1 , R 2independently selected from: H, C1-C4 straight chain or branched alkyl or benzyl; X is selected from: S or O; Y is selected from: O; Ar is selected from: phenyl; but not including: R 1 selected from: H, R 2 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] In particular, the gemcitabine prodrug is selected from any one of the following compounds:

[0018]

[0019]

[0020] In a second aspect, the present application provides use of the gemcitabine prodrug or its pharmaceutically acceptable salt, tautomer, mesomer, racemate, stereoisomer or solvate in the preparation of an antitumor drug.

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

[0022] In a fourth aspect, the present application provides use of the pharmaceutical composition in the preparation of an antitumor drug.

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

[0024] Preferably, the dosage form of the pharmaceutical composition is a tablet, pill, capsule, powder, granule, paste, emulsion, suspension, suppository, injection, inhalation, gel, microsphere, aerosol, lyophilized powder injection, etc.

[0025] Definitions

[0026] Unless otherwise indicated, the following terms used in the present application have the following meanings. A particular term should not be construed as indefinite or unclear if not specifically defined, but should be understood according to the ordinary meaning in the art. When a trade name appears in the text, it is intended to refer to its corresponding product or active ingredient thereof.

[0027] The term "alkyl" refers to a group of formula C n H 2n+1The term "alkyl" refers to a straight or branched chain hydrocarbon group having 1 to 20 carbon atoms, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-decyl, and the like. The alkyl group can be substituted or unsubstituted. 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" in reference to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0030] As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned.

[0031] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof with a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of a compound of the present application to an organism.

[0032] The term "pharmaceutically acceptable excipient" refers to those excipients which do not cause significant irritation to an organism and do not abrogate the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, for example, carbohydrates, waxes, water soluble and / or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0033] The present application also includes isotope-labeled compounds of the present application which are the same as those described herein, but for the fact that one or more atoms are replaced by an atom 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 a compound of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as2H,3H,11C,13C,14C,13N,15N,15O,17O,18O,31P,32P,35S,18F,123I,125I, and36Cl, respectively.

[0034] The pharmaceutical composition of the present application can be prepared by combining a compound of the present application with a suitable pharmaceutically acceptable excipient, and can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms, such as tablets, pills, capsules, powders, granules, pastes, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols, and the like.

[0035] Typical routes of administration of the compounds of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof 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, dissolution methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

[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 invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, and the like for oral administration to a patient.

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

[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 examples of the present invention.

[0041] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on 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 transporters. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is the H NMR spectrum of compound 1.

[0045] Figure 2 Figure 2 is the result of H2O2 activation experiment; 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 studies are shown; A represents the effect of compound 1 on the tumor volume of nude mice; B represents the effect of compound 1 on the body weight of nude mice; and C represents the effect of compound 1 on the weight of nude mice tumors. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below by way of examples. In the present invention, the following examples are provided to better illustrate 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 flask. Dioxane-water (dioxane:water = 4:1, 25 mL) and di-tert-butyl dicarbonate (218 mg, 1 mmol) were added sequentially and reacted at room temperature for 48 hours. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate. The organic phases were combined, washed once with saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and Compound I-2 (white solid, 243 mg) was isolated by normal phase silica gel column chromatography (dichloromethane:methanol = 20:1 v / v) in a 67% yield. 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 two: N-(tert-butoxycarbonyl)-L-alanine (compound I-4, 6300 mg, 33 mmol), thiazolidine-2-one (compound I-3, 3100 mg, 30 mmol) and lithium chloride (2544 mg, 60 mmol) were placed in a two-neck flask, replaced with argon for 3 times, added THF at -30°C and stirred for 1 hour. Then added pivaloyl chloride (PivCl, 9.3 mL, 75 mmol) and triethylamine (7900 mg, 78 mmol) dropwise, continued to react at -30°C for 3 hours. After the reaction was completed, saturated aqueous ammonium chloride solution was added to the reaction solution, extracted with ethyl acetate three times, combined the organic phase, washed with saturated sodium carbonate three times, washed with saturated sodium chloride once, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, separated by normal phase silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 V / V) to obtain compound I-5 (white solid, 5822 mg), yield 71%. 1 H NMR (300 MHz, CDCl3) δ 5.25 (q, J = 7.5 Hz, 1H), 5.17 (s, 1H), 4.29-4.04 (m, 2H), 3.33 (td, J = 7.3, 4.0 Hz, 2H), 1.43 (s, 9H), 1.36 (d, J = 6.8 Hz, 3H).

[0054] Step three: (S)-(1-oxo-1-(2-oxothiazolidin-3-yl)propan-2-yl) tert-butyl carbamate (compound I-5, 4800 mg, 20 mmol) was placed in a single-neck flask, added hydrochloric acid-ethyl acetate (4M, 10 mL), and reacted at room temperature overnight. After the reaction was completed, the reaction solution was filtered to obtain compound I-6 (white solid, 2780 mg), yield 85%. 1 H NMR (300 MHz, DMSO) δ 8.57 (s, 3H), 4.68 (s, 1H), 4.11 (d, J = 7.6 Hz, 2H), 3.45 (d, J = 7.3 Hz, 2H), 1.39 (d, J = 6.9 Hz, 3H).

[0055] Step four: 3-(L-alaninyl)thiazolidine-2-one hydrochloride (compound I-6, 2100 mg, 10 mmol) and phenylphosphinic dichloride (2109 mg, 10 mmol) were placed in a two-necked flask, which was replaced with argon for 3 times, and then DCM (50 mL) was added. After the reaction solution was cooled to -50 °C, TEA (2780 μL, 20 mmol) was added dropwise and stirred for 30 min, then slowly warmed to room temperature and continued to react for 3 h. Five fluorophenol (1840 mg, 10 mmol) and DCM (10 mL) were added to the reaction solution, and TEA (1529 μL, 11 mmol) was added dropwise again. The reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was extracted with dichloromethane for three times, and the organic phase was combined. The organic phase was washed with saturated sodium bicarbonate for three times, saturated brine for one time, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the compound I-7 (white solid, 2800 mg) was obtained by separation with a yield of 58%. 1 H NMR (300 MHz, DMSO) δ 7.50-7.36 (m, 2H), 7.30-7.11 (m, 3H), 6.89 (ddd, J = 14.9, 10.4, 5.2 Hz, 1H), 5.08 (tqd, J = 9.9, 6.8, 2.6 Hz, 1H), 4.13-3.91 (m, 2H), 3.39 (ttt, J = 11.9, 6.5, 2.7 Hz, 2H), 1.25 (dd, J = 7.0, 3.3 Hz, 3H).

[0056] Step five: (2R,3R,5R)-tert-butyl 5-(4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)- 4,4-difluoro-2-(hydroxymethyl)tetrahydrofuran-3-yl carbonate (compound I-2, 180 mg, 0.5 mmol) was placed in a two-necked flask, which was replaced with argon for 3 times, and then THF (6 mL) was added. tert-Butyl magnesium chloride (1.7 M, 0.4 mL, 0.6 mmol) was added dropwise slowly under ice bath and stirred for 30 min, then phenyl N-((R)-1-oxo-1-(2-oxothiazolidin-3-yl)propan-2-yl)-P-(perfluorophenyl)phosphoramide (compound I-7, 480 mg, 1 mmol) was added and the reaction was carried out at room temperature overnight. After the reaction was completed, saturated aqueous ammonium chloride solution (6 mL) was added to quench, and the reaction solution was extracted with ethyl acetate for three times, and the organic phase was combined. The organic phase was washed with saturated brine for one time, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and compound I-8 (white solid, 215 mg) was obtained by separation with normal phase silica gel column chromatography (dichloromethane:methanol = 30:1 V / V) with a yield of 64%. 1H NMR (300 MHz, DMSO) δ 7.55 - 7.31 (m, 5H), 7.18 (q, J = 7.6 Hz, 3H), 6.31 - 6.03 (m, 2H), 5.79 (ddd, J = 11.3, 7.5, 2.5 Hz, 1H), 5.29 - 5.05 (m, 1H), 5.01 - 4.79 (m, 1H), 4.47 - 4.19 (m, 3H), 4.02 (p, J = 7.0 Hz, 2H), 3.45 - 3.33 (m, 2H), 1.45 (d, J = 2.0 Hz, 9H), 1.23 - 1.15 (m, 3H).

[0057] Step six: (2R,3R,5R)-5-(4-amino-2-oxo-1,2-dihydro-1,3-thiazin-3-yl)-4,4-difluoro-2-((((S)-1-oxo-1-(2-oxothiazolidin-3-yl)propan-2-yl)amino)(phenoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl butyl carbonate (compound I-8, 796 mg, 1.2 mmol) was placed in a single-neck flask, DCM (18 mL) was added, and trifluoroacetic acid (6 mL) was slowly added dropwise, and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was completed, it was neutralized with a saturated sodium bicarbonate solution, extracted with DCM, and the organic phases were combined and washed once with saturated sodium chloride, and the organic phase was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the product was separated by column chromatography on normal phase silica gel (dichloromethane:methanol = 15:1 V / V) to obtain compound 1 (white solid 492 mg) with a yield of 72%. The proton nuclear magnetic resonance spectrum of compound 1 is shown in Figure 1. Figure 1 . 1 H NMR (500 MHz, DMSO) δ 7.46 (d, J = 7.6 Hz, 1H), 7.44 (s, 1H), 7.41 - 7.34 (m, 3H), 7.23 - 7.13 (m, 3H), 6.39 (d, J = 6.4 Hz, 1H), 6.22 - 6.11 (m, 1H), 6.11 - 6.04 (m, 1H), 5.75 (d, J = 7.5 Hz, 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 = 6.9 Hz, 3H).

[0058] Example 2

[0059] Synthesis of compound 2

[0060]

[0061] The procedure of Example 1 was followed, replacing the thiazolidine-2-one in Example 1 with oxazolidine-2-one (870 mg, 10 mmol), and the remaining starting 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), to give compound 2 (white solid, 176 mg) as the final product. 1 H NMR (300 MHz, DMSO) δ 7.53 (br s, 1H), 7.46 (d, J = 6.5 Hz, 2H), 7.42 - 7.32 (m, 2H), 7.25 - 7.10 (m, 3H), 6.43 (d, J = 6.6 Hz, 1H), 6.21 - 6.05 (m, 2H), 5.75 (d, J = 7.7 Hz, 1H), 5.13 - 4.93 (m, 1H), 4.46 - 3.93 (m, 6H), 3.83 (t, J = 8.3 Hz, 2H), 1.22 (d, J = 7.1 Hz, 3H).

[0062] Example 3

[0063] Synthesis of compound 3

[0064]

[0065] The procedure of Example 1 was followed, replacing the (tert-butoxycarbonyl)-L-alanine in Example 1 with (tert-butoxycarbonyl)-D-alanine (1249 mg, 6.6 mmol), and the remaining starting 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), to give compound 3 (white solid, 250 mg) as the final product. 1 H NMR (300 MHz, DMSO) δ 7.53 (br s, 1H), 7.46 (d, J = 6.5 Hz, 2H), 7.42 - 7.32 (m, 2H), 7.25 - 7.10 (m, 3H), 6.43 (d, J = 6.6 Hz, 1H), 6.21 - 6.05 (m, 2H), 5.75 (d, J = 7.7 Hz, 1H), 5.13 - 4.93 (m, 1H), 4.46 - 3.93 (m, 6H), 3.83 (t, J = 8.3 Hz, 2H), 1.22 (d, J = 7.1 Hz, 3H).

[0066] Example 4

[0067] Synthesis of compound 4

[0068]

[0069] Referring to Example 1, (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with tert-butoxycarbonylglycine (1155 mg, 6.6 mmol), and the rest of the starting 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), and compound I-2 435 mg (1.2 mmol), to produce compound 4 (white solid, 256 mg). 1 H NMR (500 MHz, DMSO) δ 7.78 (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, (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with tert-butoxycarbonylglycine (1925 mg, 11 mmol), and thiazolidine-2-one was replaced with oxazolidine-2-one (870 mg, 10 mmol), and the rest of the starting materials were lithium chloride 848 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), and compound I-2 544 mg (1.5 mmol), to produce compound 5 (white solid, 160 mg). 1H NMR (300 MHz, DMSO) δ 7.61 (br s, 1H), 7.56 - 7.44 (m, 2H), 7.38 (t, J = 7.8 Hz, 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 = 7.8 Hz, 1H), 4.48 - 4.22 (m, 4H), 4.22 - 4.07 (m, 3H), 4.07 - 3.98 (m, 1H), 3.86 (t, J = 8.1 Hz, 2H).

[0074] Example 6

[0075] Synthesis of Compound 6

[0076]

[0077] Referring to Example 1, (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (S)-2-((tert-butoxycarbonyl)amino)butyric acid (2235 mg, 11 mmol), and the rest of the 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), to prepare compound 6 (white solid, 329 mg). 1 H NMR (300 MHz, DMSO) δ 7.61 (br s, 1H), 7.56 - 7.44 (m, 2H), 7.38 (t, J = 7.8 Hz, 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 = 7.8 Hz, 1H), 4.48 - 4.22 (m, 4H), 4.22 - 4.07 (m, 3H), 4.07 - 3.98 (m, 1H), 3.86 (t, J = 8.1 Hz, 2H).

[0078] Example 7

[0079] Synthesis of Compound 7

[0080]

[0081] Referring to Example 1, (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced with (tert-butoxycarbonyl)-L-valine (3690 mg, 13.2 mmol), and the rest of the starting 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-2 653 mg (1.8 mmol), to produce compound 7 (white solid, 90 mg). 1 H NMR (500 MHz, DMSO) δ 7.50 (br s, 1H), 7.48-7.44 (m, 1H), 7.39-7.31 (m, 3H), 7.18 (d, J = 7.8 Hz, 3H), 6.46 (d, J = 6.3 Hz, 1H), 6.17 (t, J = 8.3 Hz, 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 = 6.6 Hz, 6H).

[0082] Example 8

[0083] Synthesis of compound 8

[0084]

[0085] Referring to Example 1, (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 rest of the starting 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), to produce compound 8 (white solid, 400 mg). 1H NMR (300 MHz, DMSO) δ 7.46 (br s, 1H), 7.44 - 7.29 (m, 4H), 7.22 - 7.12 (m, 3H), 6.45 (d, J = 6.4 Hz, 1H), 6.24 - 6.10 (m, 1H), 5.88 (t, J = 11.7 Hz, 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, (tert-butoxycarbonyl)-L-leucine (2544 mg, 11 mmol) was used instead of (tert-butoxycarbonyl)-L-alanine in Example 1, and the other starting materials were: thiazolidin-2-one 1032 mg (10 mmol), lithium chloride 849 mg (20 mmol), pivaloyl chloride 3.1 mL (25 mmol), triethylamine 2630 mg (26 mmol), and compound I-2 653 mg (1.8 mmol), to produce Compound 9 (white solid, 210 mg). 1 H NMR (300 MHz, DMSO) δ 7.46 (br s, 1H), 7.44 - 7.29 (m, 4H), 7.22 - 7.12 (m, 3H), 6.45 (d, J = 6.4 Hz, 1H), 6.24 - 6.10 (m, 1H), 5.88 (t, J = 11.7 Hz, 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).

[0090] Example 10

[0091] Synthesis of Compound 10

[0092]

[0093] Referring to Example 1, (tert-butoxycarbonyl)-L-alanine in Example 1 was replaced by (tert-butoxycarbonyl)-L-phenylalanine (3502 mg, 13.2 mmol), and the rest of the 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-2 653 mg (1.8 mmol), to prepare compound 10 (white solid, 72 mg). 1 H NMR (500 MHz, DMSO) δ 7.40 (d, J = 7.8 Hz, 3H), 7.31-7.21 (m, 9H), 7.13 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 8.2 Hz, 2H), 6.40 (d, J = 6.6 Hz, 1H), 6.25-6.11 (m, 2H), 5.76 (d, J = 7.6 Hz, 1H), 5.25-5.12 (m, 1H), 4.09-3.98 (m, 4H), 3.93 (p, J = 6.4 Hz, 1H), 3.87 (s, 1H), 3.44-3.36 (m, 2H).

[0094] Example 11

[0095] H2O2 activation experiment of prodrug compound

[0096] Test drug: prodrug compound (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 type high performance liquid chromatograph; ZORBAX Eclipse XDB-C18 (4.6x150mm, 5μm).

[0098] Experimental procedure:

[0099] 1. The test drug was dissolved with 100% DMSO to prepare a stock solution of 10 mg / mL; the 10M H2O2 solution was diluted to a solution stock of 13.2 mM; the sofosbuvir stock solution was sequentially diluted with acetonitrile to 200μg / mL to prepare an internal standard acetonitrile solution.

[0100] 2. ① PBS stability experiment: the compound mother liquor was diluted with acetonitrile to 400 μg / mL. 2 mL of each solution was mixed with 2 mL of PBS solution (pH = 7.4) to obtain a working solution; ② In the H2O2 stability experiment, the above PBS solution was replaced with an H2O2 solution (PBS dilution), and the working solution had a prodrug concentration to H2O2 concentration ratio of 1:20. The above ① and ② two groups of samples were incubated in a 37°C constant temperature water bath.

[0101] 3. 100 μL of the sample was taken at different time points and added to 100 μL of acetonitrile (containing sofosbuvir internal standard) and mixed well. After two centrifugations (12000 rpm x 5 min), 60 μL of the supernatant was taken into a sample bottle. High performance liquid chromatography was used for stability detection and product quantification.

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

[0103] The experimental results are shown in Figure 2 Fig. A is the H2O2 stability, and Fig. B is the PBS stability. In the H2O2 solution, the control drug NUC-1031 cannot be degraded, and compounds 7 and 8 show a weak degradation trend, and the rest of the compounds can be fully degraded. Compounds 1-10 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 prodrugs of the application can be activated by H2O2, suggesting that the gemcitabine prodrugs can be metabolically activated in H2O2-rich tumor tissues.

[0104] Example 12

[0105] In vitro anti-tumor activity of prodrugs

[0106] Test drugs: test compounds (compounds 1-10), 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 Bosheng Biological; 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 instruments: BioTek Instruments multifunctional enzyme marker detector.

[0109] Experimental steps:

[0110] 1. The test compound was dissolved with 100% DMSO to prepare a 200 mM mother liquor for storage;

[0111] 2. Digest the cells, count, and configure cell suspensions (BxPC-3: 4.0 x 10 4 4 4 6. Add 10 μL CCK8 solution to each well and continue to incubate in the incubator for 2-3 hours.

[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 desired working solution concentration, and make the final concentration of DMSO not more than 0.1%, and add 100 μL of the corresponding drug-containing culture medium to each well of the 96-well plate, while setting up a negative control group.

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

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

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

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

[0118] Table 1. In vitro anti-tumor activity (IC 50 , unit: μM) of compounds

[0119]

[0120]

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

[0122] Example 13

[0123] Effect of metabolic enzymes on anti-tumor activity of prodrugs

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

[0125] The experimental materials and instruments were the same as those in Example 12. The metabolic enzyme inhibitors selected were: nucleoside transporter inhibitors dipyridamole (DPM) and S-(4-nitrobenzyl)-6-thioinosine (NBTI); and cytidine deaminase inhibitor tetrahydrouridine (THU).

[0126] Experimental steps:

[0127] Bxpc-3 cells were cultured at 5 × 10 4 Cells were plated at a density of 100 μM / mL in a 96-well plate and incubated for 12 hours. Inhibitors (final concentration of 10 or 20 μM) were pre-incubated in the 96-well plate 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 the cells were incubated in the incubator for another hour. The OD value of each well was read at 450 nm using a microplate 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 leads to decreased nucleoside uptake, thereby reducing Gem's antiproliferative activity, indicating that Gem's efficacy depends on nucleoside uptake. However, compound 1 is insensitive to this treatment, indicating that compound 1's efficacy is independent of nucleoside transporters and passively diffuses into the cell. Pre-administration of THU leads to decreased cytidine deaminase activity, thereby increasing Gem's antiproliferative activity, indicating that Gem's efficacy depends on cytidine deaminase. However, compound 1 is insensitive to this treatment, indicating that compound 1's efficacy is independent of cytidine deaminase and can effectively circumvent the efficacy reduction caused by cytidine deaminase.

[0130] Example 14

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

[0132] Test samples: Compound 1 and NUC-1031; the solvent was a CMC-Na solution with a concentration of 1 / 1000; the experimental groups were a vehicle 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] Experimental 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 culture of human pancreatic cancer cell Panc-1 cell suspension, the concentration is 2x10 7 6 mice per group, 0.1 mL per mouse, subcutaneously inoculate in the right armpit of nude mice;

[0136] 2. Measure the diameter of the transplanted tumor of nude mice with vernier caliper, when the tumor grows to 100 mm 3 3, the animals are randomly divided into groups. At the same time, each group of mice starts intraperitoneal injection of drug, and is recorded as Day 1 (Day 1); after the end of the experiment, the nude mice are immediately sacrificed, and the tumor mass is surgically stripped and weighed.

[0137] Observation index:

[0138] 1. Body weight: record the body weight of animals once on Day 1, 5, 8, 12, 15, 19, 22;

[0139] 2. Tumor volume: record the tumor volume once on Day 1, 5, 8, 12, 15, 19, 22;

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

[0141] 3. Evaluation index of antitumor activity:

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

[0143] T / C (%) = T RTV / C RTV x 100%, where T RTV is the RTV of the drug administration group, and C RTV is the RTV of the solvent group.

[0144] Tumor growth inhibition rate TI (%)

[0145] TI (%) = (average tumor weight of the solvent group - average tumor weight of the drug administration group) / average tumor weight of the solvent group x 100%.

[0146] 4. Statistical processing: mean is represented by X ± SD, t test is used for statistical processing between groups, and SPSS (Staffstical Package for the Social Science) 17.0 is used for statistical analysis of the results.

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

[0148]

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

[0150] Example 15

[0151] Tablet

[0152] Compound 1 (50 g), hydroxypropyl methyl cellulose E (150 g), starch (200 g), polyvinylpyrrolidone K30 (appropriate amount) and magnesium stearate (1 g) were mixed, granulated and tableted.

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

Claims

1. A gemcitabine prodrug or a pharmaceutically acceptable salt thereof having a structure as shown in Formula I: wherein R 1 , R 2 is independently selected from the group consisting of: H, C1-C6 straight or branched chain alkyl or one or more hydrogens in C1-C6 straight or branched chain alkyl can be replaced by R 3 , benzyl or benzyl in which the hydrogens on the phenyl ring are replaced by R 4 ; X is selected from the group consisting of: S or O; Y is selected from the group consisting of: S or O; Ar is selected from the group consisting of: phenyl, 1-naphthyl or 2-naphthyl; R 3 is hydroxy, halogen, C3-C6cycloalkyl, phenyl or R 4 substituted phenyl; R 4 is hydroxy, halogen, cyano, nitro or methoxy.

2. The gemcitabine prodrug of claim 1, wherein: R 1 , R 2 is independently selected from the group consisting of: H, C1-C6 straight chain or branched alkyl, benzyl or benzyl in which a hydrogen on the phenyl ring is replaced by R 4 ; X is selected from the group consisting of: S or O; Y is selected from the group consisting of: O; Ar is selected from the group consisting of: phenyl; R 4 is hydroxy, halogen, cyano, nitro or methoxy.

3. The gemcitabine prodrug of claim 2, wherein: R 1 , R 2 is 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 of claim 1, wherein: R 1 , R 2 is independently selected from the group consisting of: H, C1-C4 straight chain or branched alkyl or benzyl; X is selected from the group consisting of: S or O; Y is selected from the group consisting of: O; Ar is selected from the group consisting of: phenyl; but not including: R 1 is selected from the group consisting of: H, R 2 is selected from the group consisting of: isopropyl, t-butyl, X is selected from the group consisting of: S, Y is selected from the group consisting of: O; Ar is selected from the group consisting of: phenyl.

5. A gemcitabine prodrug or a pharmaceutically acceptable salt thereof having a structure as shown in Formula I: wherein R 1 , R 2 is independently selected from the group consisting of: H, CH3; X is selected from the group consisting of: S; Y is selected from the group consisting of: O; Ar is selected from the group consisting of: phenyl.

6. A gemcitabine prodrug or a pharmaceutically acceptable salt thereof selected from any one of the following compounds:

7. Use of the gemcitabine prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-6 in the preparation of an antitumor drug, wherein the tumor is pancreatic cancer, breast cancer, non-small cell lung cancer.

8. A pharmaceutical composition, characterized by: The pharmaceutical composition comprises the gemcitabine prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1-6, and a pharmaceutically acceptable excipient.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of an antitumor drug, wherein the tumor is pancreatic cancer, breast cancer, non-small cell lung cancer.

10. The pharmaceutical composition of claim 8, wherein: The dosage form of the pharmaceutical composition is a solid, semi-solid, liquid or gaseous preparation.

11. The pharmaceutical composition of claim 10, wherein: The dosage form of the pharmaceutical composition is a tablet, a pill, a capsule, a powder, a granule, a paste, an emulsion, a suspension, a suppository, an injection, an inhaler, a gel, a microsphere, an aerosol, a freeze-dried powder injection.

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