A HDAC and CTSL dual-activated gemcitabine prodrug and its preparation method and application

Abstract

The present invention discloses a HDAC and CTSL dual-activated gemcitabine prodrug and its preparation method and application, wherein the structural formula is: #imgabs0# or #imgabs1# wherein R is selected from: H, tert-butyloxycarbonyl, 3-indoleethyl, 1-adamantanemethyl or octyl. Experimental results show that the multiple compounds have significant anti-tumor activity against HDAC and CTSL high-expressing strain A549, and generally lose anti-tumor activity against HDAC and CTSL low-expressing strain Caco-2, showing good selectivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleoside drugs. Specifically, the present invention relates to a gemcitabine prodrug, a preparation method thereof and an in vitro anti-tumor activity thereof, including the application of the gemcitabine prodrug in the anti-tumor field. The compound and the pharmaceutical composition thereof of the present invention are suitable for treating tumor diseases such as lung cancer and pancreatic cancer. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Gemcitabine (2′, 2′-difluoro-2′-deoxycytidine, dFdC) is a nucleoside analogue with the structural formula shown below. It is clinically used to treat a variety of cancers including pancreatic cancer, non-small cell lung cancer, breast cancer, etc.

[0004]

[0005] Gemcitabine enters cells through nucleoside transporters and is subsequently activated by phosphorylation by deoxycytidine kinase (dCK) to generate its 5′-monophosphate (dFdCMP), which is then phosphorylated by intracellular kinases to diphosphate (dFdCDP) and triphosphate (dFdCTP). dFdCTP competitively incorporates into DNA chains, causing chain termination, thereby inhibiting DNA synthesis; in addition, dFdCDP and dFdCTP reduce the amount of dCTP generated by inhibiting ribonucleic acid reductase, ultimately causing cell apoptosis. Gemcitabine is rapidly deaminated and converted into inactive 2′,2′-difluorouracil (dFdU) under the action of cytidine deaminase (CDA). Therefore, further modification of the amino group of gemcitabine is of great significance for improving the drug's selectivity for tumor cells, reducing toxicity, and improving bioavailability. Summary of the invention

[0006] Histone deacetylases (HDACs) are key enzymes involved in epigenetic regulation of histones and non-histone proteins. It has been well demonstrated in cancer biology that changes in HDACs expression may play a crucial role in tumor development. At the same time, cathepsin L (CTSL) also plays a key role in multiple stages of tumor progression and metastasis, and upregulation of CTSL is also considered a hallmark of metastatic cancer. Therefore, based on the increased expression of HDAC and CTSL in various tumor cells, gemcitabine prodrugs were designed, which were delivered to tumor cells and selectively released gemcitabine to exert anti-tumor effects.

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a HDAC and CTSL dual-activated gemcitabine prodrug and its preparation method and application. This series of prodrugs has anti-tumor activity.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a HDAC and CTSL dual-activated gemcitabine prodrug, the structural formula of which is:

[0010]

[0011] Wherein, R is selected from: H, tert-butyloxycarbonyl, 3-indoleethyl, 1-adamantylmethyl or octyl.

[0012] The representative compounds have the following structures, as shown in Table 1.

[0013] Table 1

[0014]

[0015] In a second aspect, the present invention provides a synthetic route of the HDAC and CTSL dual-activated gemcitabine prodrug as follows:

[0016]

[0017] In some embodiments, Boc-Lys(Ac)-OH, 4-methylmorpholine, 1-hydroxybenzotriazole and EDCI are added to a DMF solution of gemcitabine to obtain a mixed solution, which is stirred at 50-60° C. for 15-20 h in a nitrogen atmosphere, and then extracted, washed and chromatographed to obtain compounds C1 and C6;

[0018] In the mixed liquid, the concentration of Boc-Lys(Ac)-OH is 0.6-1.5 mol / L; the concentration of 4-methylmorpholine is 0.6-1.5 mol / L; the concentration of 1-hydroxybenzotriazole is 0.6-1.5 mol / L; and the concentration of EDCI is 0.6-1.5 mol / L.

[0019] DMF is N,N-dimethylformamide; EDCI is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0020] Preferably, the extraction agent used in the extraction is ethyl acetate.

[0021] Preferably, the washing is to wash the organic layer with a sodium bicarbonate aqueous solution and a sodium chloride aqueous solution.

[0022] Preferably, compound C1 is dissolved in CF3COOH and diluted with dichloromethane (DCM), the concentration of C1 is 0.2-0.5 mol / L, the concentration of CF3COOH is 0.6-1.2 mol / L, stirred at 15-30°C for 1.5-2.5h, saturated sodium bicarbonate aqueous solution is slowly added dropwise until the solution is neutral, directly freeze-dried, and compounds C2 and C7 are obtained after chromatography. CF3COOH is used to remove the BOC group. It has been found through experiments that the use of saturated sodium bicarbonate aqueous solution as a neutralizing agent can remove excess acid, facilitate freeze-drying, and facilitate the stability of compounds C2 and C7.

[0023] Further preferably, a carboxylic acid containing different substituents is dissolved in DMF, 4-methylmorpholine, 1-hydroxybenzotriazole and EDCI are added thereto, and after stirring and mixing, compound C2 is added thereto to obtain a mixed solution, and stirring is continued for 1.5-2.5 hours; in the mixed solution, the concentration of the carboxylic acid containing different substituents is 0.08-0.15 mol / L, the concentration of 4-methylmorpholine is 0.08-0.15 mol / L; the concentration of 1-hydroxybenzotriazole is 0.08-0.15 mol / L; the concentration of EDCI is 0.08-0.15 mol / L; the concentration of C2 is 0.08-0.15 mol / L;

[0024] After the reaction is completed, extraction, washing, drying and column chromatography are performed to obtain compound C3, C4, C5, C8, C9 or C10.

[0025] More preferably, the carboxylic acid containing different substituents is 3-indoleacetic acid, 1-adamantanecarboxylic acid or octanoic acid.

[0026] In a third aspect, the present invention provides use of the HDAC and CTSL dual-activated gemcitabine prodrug in the preparation of anti-tumor drugs.

[0027] In a fourth aspect, the present invention provides an anti-tumor pharmaceutical composition, the active ingredient of which includes the gemcitabine prodrug.

[0028] In the so-called "pharmaceutical composition", the gemcitabine prodrug in the first aspect should be in a synergistic effective dose, and the drug dose is a technical content that can be routinely determined by those skilled in the art according to the different application purposes of the pharmaceutical composition. In addition, in addition to the gemcitabine prodrug in the first aspect, other ingredients with anti-tumor effects may also be included.

[0029] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0030] The present invention conducted an anti-tumor cell proliferation activity experiment on the above compounds. The experimental results showed that the multiple compounds had obvious anti-tumor activity against the HDAC and CTSL high-expressing strain A549, and generally lost anti-tumor activity against the HDAC and CTSL low-expressing strain Caco-2, showing good selectivity.

[0031] The preparation process of the gemcitabine prodrug of the present invention is shorter than that of existing nucleotide drugs, is suitable for industrial scale-up production, and has good application prospects. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0034] Example 1 Synthesis of Compounds

[0035] Prepared according to the following synthetic route

[0036]

[0037] (1) Synthesis of Compound C1

[0038]

[0039] Gemcitabine (500 mg, 1.90 mmol) was dissolved in DMF (3 mL), stirred at room temperature, and L-Boc-Lys(Ac)-OH (547.76 mg, 1.90 mmol), 4-methylmorpholine (213.50 μL, 1.90 mmol), 1-hydroxybenzotriazole (257.70 mg, 1.90 mmol), EDCI (364.18 mg, 1.90 mmol) were added. The mixture was reacted at 55 °C for 17 hours under N2 protection. After cooling to room temperature, the mixture was extracted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate aqueous solution and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Silica gel column chromatography (DCM: MeOH = 40: 1, 10: 1) gave 584 mg of a white solid, which was compound C1, with a yield of 57.8%. 1H NMR(400MHz, DMSO-d6)δ:8.27(d,J=9.27Hz,1H),7.83-7.80(m,1H),7.26(d,J=6.69 Hz,1H),7.13(d,J=6.69Hz,1H),6.38-6.37(m,1H),6.18(t,J=14.60Hz,1H),5.35(b r,1H),4.20-4.08(m,2H),3.91-3.88(m,1H),3.83-3.80(m,1H),3.67-3.61(m,1H), 3.17(s,1H),3.02-2.97(m,2H),1.78(s,3H),1.62-1.51(m,2H),1.37-1.28(m,13H). 13 CNMR(100MHz,DMSO-d6)δ:174.79,169.55,163.99,156.04,154.64,145.38,123.39(t,J=257Hz,C2′),96.41,95.06,84.62(t, J=31.0Hz, C1′),81.56,78.74,68.80(t,J=22.0Hz,C3′),59.20,55.77,55.35,49.06,31.13,29.20,28.62,23.52,23.03.HRMS calcd forC 22 H 34 F2N5O8[M+H] + 534.2375, found 534.2372.

[0040] (2) Synthesis of Compound C2

[0041]

[0042] Dissolve C1 (500 mg, 937.15 μmol) in CF3COOH (208.84 μL, 2.81 mmol), add 3 mL DCM to dilute, and stir at room temperature for 2 hours. Saturated sodium bicarbonate aqueous solution was slowly added dropwise to neutralize to pH = 7, and then directly freeze-dried. Silica gel column chromatography (DCM: MeOH = 10: 1) gave 304 mg of white solid, which was compound C2, with a yield of 74.8%. 1H NMR (400MHz, DMSO-d6) δ: 8.04 (d, J = 8.41Hz, 1H), 7.82-7.80 (m, 1H), 7.68 (d, J = 7. 15Hz,1H),7.52(s,1H),7.05(s,1H),6.14-6.10(m,1H),5.99(d,J=7.57Hz,1H),5. 50-4.46(m,1H),4.18-4.11(m,1H),3.81-3.75(m,2H),3.64-3.60(m,1H),3.01-2 .97(m,2H),1.77(s,3H),1.72-1.66(m,1H),1.62-1.54(m,1H),1.39-1.26(m,4H). 13 C NMR (100MHz, DMSO-d6) δ: 174.83, 169.49, 163.68, 155.08, 140.12, 123.59 (t, J = 256Hz, C2′), 95.96, 84.01 ( t,J=66.0Hz,C1′),80.94,69.09(t,J=22.0Hz,C3′),59.42,53.58,38.83,32.29,29.34,23.30,23.10.HRMS calcd forC 17 H 26 F2N5O6[M+H] + 434.1850, found 434.1841.

[0043] (3) Synthesis of Compound C3

[0044]

[0045] Dissolve 3-indoleacetic acid (24.25 mg, 138.44 μmol) in DMF (2 mL), add 4-methylmorpholine (15.56 μL, 138.44 μmol), 1-hydroxybenzotriazole (18.84 mg, 138.44 mmol), EDCI (26.54 mg, 138.44 mmol), stir for 20 min, add compound C2 (60 mg, 138.44 μmol), and continue stirring for 2 hours. Extract the mixture with ethyl acetate, wash the organic layer with saturated sodium bicarbonate aqueous solution and brine, dry with anhydrous sodium sulfate, and evaporate the solution under reduced pressure. Silica gel column chromatography (DCM: MeOH = 20: 1, 10: 1) to obtain 16.4 mg of white solid, which is compound C3, with a yield of 20.0%. 1H NMR(400MHz, DMSO-d6)δ:8.08(d,J=16.38Hz,1H),7.84(br,1H),7.65(d,J=7.37Hz,1H),7.53 -7.49(m,2H),7.38-7.27(m,2H),7.10(t,J=53.24Hz,2H),6.27-6.22(m,1H),6.02-5.98(m,1H ),5.39-5.27(m,1H),4.50-4.44(m,1H),4.18-4.10(m,1H),3.92(s,1H),3.73(d,J=12.69Hz, 1H),3.61-3.56(m,2H),3.00-2.99(m,2H),1.71(s,3H),1.62-1.51(m,2H),1.39-1.23(m,6H). 13 C NMR (100MHz, DMSO-d6) δ: 173.74, 170.88, 169.43, 163.71, 154.83, 140.63, 136.56, 130.13, 127.41, 124.62, 121.64, 119.64, 118.91 (t, J=256 Hz,C2′),111.96,106.56,96.16,84.70,79.02,70.59(t,J=14.0Hz,C3′ ),59.64,53.62,38.84,32.28,30.59,29.38,27.02,23.32,23.12.HRMS calcd for C 27 H 33 F2N6O7[M+H] + 591.2378, found 591.2376.

[0046] (4) Synthesis of Compound C4

[0047]

[0048] 1-Adamantanecarboxylic acid (24.95 mg, 138.44 μmol) was dissolved in DMF (2 mL), 4-methylmorpholine (15.56 μL, 138.44 μmol), 1-hydroxybenzotriazole (18.84 mg, 138.44 mmol), EDCI (26.54 mg, 138.44 mmol) were added, and the mixture was stirred for 20 min before adding compound C3 (60 mg, 138.44 μmol), and stirring was continued for 2 hours. The mixture was extracted with ethyl acetate, and the organic layer was washed with saturated sodium bicarbonate aqueous solution and brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. Silica gel column chromatography (DCM: MeOH = 20: 1, 10: 1) gave 20.12 mg of a white solid, which was compound C4, with a yield of 24.4%. 1 H NMR(400MHz, DMSO-d6)δ:8.02(d,J=7.75Hz,1H),7.78-7.76(m,1H),7.62(d,J=7.75Hz,1H) ,7.49(s,1H),7.01(s,1H),6.23-6.20(m,1H),6.01(d,J=7.16Hz,1H),5.33-5.22(m,1H),4. 49-4.45(m,1H),4.14-4.12(m,1H),3.74-3.71(m,2H),3.61-3.57(m,2H),3.00-2.97(m,2H ),2.02-1.99(m,3H),1.93-1.84(m,4H),1.77(s,3H),1.71-1.57(m,6H),1.39-1.23(m,6H). 13 C NMR (100MHz, DMSO-d6) δ: 182.58, 175.52, 173.76, 169.45, 163.71, 154.83, 128.25 (t, J = 188Hz, C2′), 124.46, 119.31, 110.50, 96. 16,79.08,55.25,53.61,38.84,38.55,36.25,32.28,31.75,30.85,29.37,27.63,27.02,23.77,23.32,23.07,22.55,14.41.HRMS calcd for C 28 H 40 F2N5O7[M+H] + 596.2895, found 596.2895.

[0049] (5) Synthesis of Compound C5

[0050]

[0051] Dissolve octanoic acid (21.94 μL, 138.44 μmol) in DMF (2 mL), add 4-methylmorpholine (15.56 μL, 138.44 μmol), 1-hydroxybenzotriazole (18.84 mg, 138.44 mmol), EDCI (26.54 mg, 138.44 mmol), stir for 20 min, add compound C2 (60 mg, 138.44 μmol), and continue stirring for 2 hours. Extract the mixture with ethyl acetate, wash the organic layer with saturated sodium bicarbonate aqueous solution and brine, dry over anhydrous sodium sulfate, and evaporate the solution under reduced pressure. Silica gel column chromatography (DCM: MeOH = 20: 1, 10: 1) to obtain 18.6 mg of white solid, which is compound C5, with a yield of 24.0%. 1 H NMR(400MHz, DMSO-d6)δ:8.06(d,J=9.47Hz,1H),7.83-7.80(m,1H),7.64(d,J=8.52Hz,1H), 7.53(br,1H),7.06(br,1H),6.24-6.20(m,1H),6.01(d,J=6.63Hz,1H),5.36-5.25(m,1H),4 .49-4.44(m,1H),4.14-4.10(m,2H),3.76-3.72(m,1H),3.64-3.60(m,1H),3.02-2.97(m,2H ),2.46-2.42(m,2H),1.78(s,3H),1.72-1.54(m,4H),1.40-1.26(m,12H),0.87-0.84(m,3H). 13 C NMR (100MHz, DMSO-d6) δ: 173.71, 172.20, 169.40, 163.70, 154.82, 140.65, 122.23 (t, J = 251Hz, C2′), 96.14, 84.44, 79.00, 70. 28(t,J=34.0Hz,C3′),59.68,53.56,38.85,33.42,32.31,31.55,29.39,28.73,28.68,24.76,23.31,23.07,22.46,14.38.HRMS calcdfor C 25 H 40 F2N5O7[M+H] + 560.2895,found560.2890.

[0052] (6) Synthesis of Compound C6

[0053]

[0054] The operation was the same as in Example 1, except that the starting material was D-Boc-Lys(Ac)-OH. C6 was a white solid with a yield of 78%. 1 HNMR(400MHz,DMSO-d6)δ:8.27(d,J=8.00Hz,1H),7.82-7.80(m,1H),7.25(d ,J=8.00Hz,1H),7.14(d,J=8.00Hz,1H),6.38-6.36(m,1H),6.18(t,J=15.39H z,1H),5.35-5.32(m,1H),4.22-4.18(m,1H),3.91-3.80(m,2H),3.68-3.61(m ,1H),3.00-2.99(m,2H),1.78(s,3H),1.62-1.52(m,2H),1.38-1.29(m,13H). 13 C NMR (100MHz, DMSO-d6) δ: 174.83, 169.42, 163.38, 156.06, 154.61, 123.42 (t, J = 252Hz, C2′), 96 .41,84.87,81.53,78.74,68.82,59.25,55.80,38.74,31.08,29.22,28.63,23.55,23.07.HRMS calcd for C 22 H 34 F2N5O8[M+H] + 534.2375, found 534.2373.

[0055] (7) Synthesis of Compound C7

[0056]

[0057] The operation was the same as in Example 2, C7 was a white solid, and the yield was 66.7%. 1H NMR(400MHz, DMSO-d6)δ:8.01(d,J=7.48Hz,1H),7.85-7.82(m,1H),7.67(d,J=6.4 1Hz,1H),7.52(br,1H),7.04(br,1H),6.29-6.28(m,1H),6.13-6.09(m,1H),5.98( d,J=7.48Hz,1H),5.27-5.24(m,1H),4.48-4.43(m,2H),3.80-3.75(m,2H),3.65-3 .59(m,2H),3.03-2.98(m,2H),1.78(s,3H),1.71-1.57(m,2H),1.40-1.23(m,4H). 13 C NMR (100MHz, DMSO-d6) δ: 173.82, 169.51, 163.69, 155.06, 140.13, 123.59 (t, J = 273Hz, C2′), 95.9 6,83.21,80.92,69.13(t,J=22.0Hz,C3′),59.43,53.63,38.85,32.25,29.38,23.34,23.03.HRMS calcd for C 17 H 26 F2N5O6[M+H] + 434.1850, found 434.1844.

[0058] (8) Synthesis of Compound C8

[0059]

[0060] The operation was the same as in Example 3, C8 was a white solid, and the yield was 20.0%. 1 H NMR(400MHz,DMSO-d6)δ:8.06(d,J=7.95Hz,1H),7.86-7.83(m,1H),7.52-7.49(m,2H) ,7.38-7.36(m,2H),7.27-7.26(m,1H),7.11-7.07(m,2H),7.00-6.96(m,1H),6.46-6.4 4(m,1H),6.16-6.11(m,1H),5.99(d,J=7.66Hz,1H),4.49-4.30(m,3H),4.03-4.01(m,1 H),3.82(s,2H),3.03-2.98(m,2H),1.78(s,3H),1.72-1.59(m,2H),1.41-1.24(m,6H). 13C NMR(100MHz,DMSO-d6)δ:173.74,171.85,169.44,163.70,154.89,136.56,130.13,127.50,124.61,121.59,119.01,118.89,111.93 ,107.16,96.31,77.78,70.52(t,J=24.0Hz,C3′),63.18,59.44,55.37,53.64,38.84,32.26,30.99,29.39,27.02,23.34,23.11.HRMS calcd for C 27 H 33 F2N6O7[M+H] + 591.2378, found 591.2368.

[0061] (9) Synthesis of Compound C9

[0062]

[0063] The operation was the same as in Example 4, C9 was a white solid, and the yield was 24.4%. 1 H NMR(400MHz, DMSO-d6)δ:8.10(d,J=8.18Hz,1H),7.85-7.83(m,2H),7.58-7.53(m,2H),7.37-7.27(m,2H),4.49-4.43(m,1H),4.36-4.33(m,1H),4 .03-3.98(m,2H),3.92-3.90(m,2H),3.05-3.00(m,2H),2.03-1.97(m,3H ),1.87-1.84(m,2H),1.78(s,3H),1.73-1.67(m,4H),1.39-1.24(m,12H). 13 C NMR (100MHz, DMSO-d6) δ: 176.66, 173.70, 169.45, 163.62, 154.83, 143.33, 128.13 (t, J = 188Hz, C2′), 124.09, 119.12, 110.77, 96. 22,85.66,55.37,53.64,38.84,36.37,35.30,32.27,31.75,30.86,29.38,27.73,27.02,23.33,23.07,22.56,16.01,14.41.HRMS calcdfor C 28 H 40 F2N5O7[M+H] +596.2895, found 596.2893.

[0064] (10) Synthesis of Compound C10

[0065]

[0066] The operation was the same as in Example 5, C10 was a white solid, and the yield was 24.0%. 1 H NMR(400MHz, DMSO-d6)δ:8.12-8.10(m,1H),7.87-7.84(m,1H),7.66(d,J=8.54Hz,1H),7.5 2-7.48(m,1H),7.05(br,1H),6.21-6.18(m,1H),6.13-6.11(m,1H),6.03-5.99(m,1H),5.3 4-5.31(m,1H),4.48-4.42(m,1H),3.81-3.73(m,1H),3.64-3.59(m,1H),3.02-2.97(m,2H) ,2.46-2.33(m,2H),1.78(s,3H),1.73-1.54(m,4H),1.40-1.24(m,12H),1.00-0.96(m,3H). 13 C NMR (100MHz, DMSO-d6) δ: 173.77, 172.14, 169.47, 163.85, 158.69, 154.85, 119.24 (t, J = 298Hz, C2′), 96.25, 85.07, 7 9.00,70.38,59.64,55.17,38.85,36.69,34.57,31.54,29.38,28.72,25.97,24.75,23.36,23.05,22.46,14.36.HRMS calcd for C 25 H 40 F2N5O7[M+H] + 560.2895,found560.2892.

[0067] Example 2

[0068] In vitro antitumor activity test of the compounds against HDAC and CTSL high-expressing tumor cell line A549 and low-expressing tumor cell line Caco-2

[0069] A549 cells were grown at 5 × 10 3 Caco-2 cells were seeded in 96-well plates at a density of 9×10 cells per well in DMEM 10% FBS medium. 3The cells were inoculated in a 96-well plate at a density of 100 cells / mL in DMEM 20% FBS medium. After 24 hours of growth, the culture medium was removed and the cells were treated with different concentrations of compounds, with gemcitabine as the control drug, and cultured in a 37°C constant temperature incubator for 72 hours. The culture medium containing 10% CCK8 was added and placed in the incubator for 0.5-2 hours. The absorbance value was measured at 450nm using an enzyme reader. The half effective concentration (EC50) of each compound was calculated using the software. 50 ), and the results are shown in Table 2.

[0070] Table 2: In vitro anti-tumor activity test results

[0071]

[0072] Conclusion: From the above results, it can be seen that compounds C1, C2, C3, C6, and C7 all showed significant antitumor activity against HDAC and CTSL high-expressing strain A549 cells, and the antitumor activity was better than that of the positive control drug gemcitabine. In HDAC and CTSL low-expressing strains, the compounds showed poor antitumor cell activity. This shows that the compounds have enhanced antitumor activity and selectivity. Therefore, the example compounds can be used as preparations for antitumor drugs.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A HDAC and CTSL dual-activated gemcitabine prodrug, characterized in that: Its structural formula is: in, 2. The synthetic route of the HDAC and CTSL dual-activated gemcitabine prodrug according to claim 1, characterized in that: The steps include: Step a: Add Boc-Lys(Ac)-OH, 4-methylmorpholine, 1-hydroxybenzotriazole and EDCI to a DMF solution of gemcitabine to obtain a mixed liquid, stir and react at 50-60° C. for 15-20 h in a nitrogen atmosphere, extract, wash and chromatograph to obtain compounds C1 and C6; When the reactant is D-Boc-Lys(Ac)-OH, the product is C1; when the reactant is L-Boc-Lys(Ac)-OH, the reactant is C6; In the mixed liquid, the concentration of Boc-Lys(Ac)-OH is 0.6-1.5 mol / L; the concentration of 4-methylmorpholine is 0.6-1.5 mol / L; the concentration of 1-hydroxybenzotriazole is 0.6-1.5 mol / L; and the concentration of EDCI is 0.6-1.5 mol / L. Step b: Compound C1 was dissolved in CF3COOH and diluted with dichloromethane DCM. The concentration of C1 was 0.2-0.5 mol / L and the concentration of CF3COOH was 0.6-1.2 mol / L. The mixture was stirred at 15-30°C for 1.5-2.5 h. A saturated aqueous sodium bicarbonate solution was slowly added dropwise until the solution became neutral. The mixture was directly freeze-dried and chromatographed to obtain compounds C2 and C7. Step c: dissolving carboxylic acids containing different substituents in DMF, adding 4-methylmorpholine, 1-hydroxybenzotriazole and EDCI thereto, stirring and mixing, adding compound C2 thereto to obtain a mixed solution, and continuing stirring for 1.5-2.5 hours; in the mixed solution, the concentration of carboxylic acids containing different substituents is 0.08-0.15 mol / L, the concentration of 4-methylmorpholine is 0.08-0.15 mol / L; the concentration of 1-hydroxybenzotriazole is 0.08-0.15 mol / L; the concentration of EDCI is 0.08-0.15 mol / L; the concentration of C2 is 0.08-0.15 mol / L; After the reaction is completed, extraction, washing, drying and column chromatography are performed to obtain compound C3, C4, C5, C8, C9 or C10.

3. The synthetic route of the HDAC and CTSL dual-activated gemcitabine prodrug according to claim 2, characterized in that: The extraction agent used in the extraction is ethyl acetate.

4. The synthetic route of the HDAC and CTSL dual-activated gemcitabine prodrug according to claim 2, characterized in that: The washing is washing the organic layer with a saturated sodium bicarbonate aqueous solution and a sodium chloride aqueous solution.

5. The synthetic route of the HDAC and CTSL dual-activated gemcitabine prodrug according to claim 2, characterized in that: The carboxylic acid containing different substituents is 3-indoleacetic acid, 1-adamantanecarboxylic acid or octanoic acid.

6. Use of the HDAC and CTSL dual-activated gemcitabine prodrug according to claim 1 in the preparation of anti-tumor drugs.

7. An anti-tumor pharmaceutical composition, characterized in that: The active ingredients thereof include one, two or more of the gemcitabine prodrugs described in claim 1.

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