WO2018171665A1 - 氘代核苷酸类似物及其用途 - Google Patents

氘代核苷酸类似物及其用途 Download PDF

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WO2018171665A1
WO2018171665A1 PCT/CN2018/079990 CN2018079990W WO2018171665A1 WO 2018171665 A1 WO2018171665 A1 WO 2018171665A1 CN 2018079990 W CN2018079990 W CN 2018079990W WO 2018171665 A1 WO2018171665 A1 WO 2018171665A1
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deuterated
compound
nucleotide analog
group
analog according
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French (fr)
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谢永美
耿福能
魏于全
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Sichuan Good Doctor Panxi Pharmaceutical Co Ltd
Sichuan University
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Sichuan Good Doctor Panxi Pharmaceutical Co Ltd
Sichuan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/13—Dicarboxylic acids
    • C07C57/15—Fumaric acid

Definitions

  • the invention relates to deuterated nucleotide analogues and uses thereof, and belongs to the technical field of antiviral drugs.
  • Hepatitis B virus is a pathogen that seriously affects human health and is the culprit in causing chronic hepatitis B.
  • International research on antiviral drugs has made important progress, and some clinically effective antiviral drugs have been discovered, such as interferon, lamivudine, telbivudine, clafidine, entecavir, adefovir, and Norfovirtide, zidovudine, stavudine, nevirapine, indinavir and valaciclovir.
  • nucleoside antiviral drugs open-loop nucleoside compounds have low toxicity, good tolerance and broad-spectrum anti-DNA virus activity, and have strong killing effect on drug-resistant strains.
  • TDF is effective against a variety of viruses, including those resistant to nucleoside reverse transcriptase inhibitors.
  • Tenofovir is approved by the FDA in 2001 and 2008 for the treatment of HIV and HBV infection.
  • HCV hepatitis C virus
  • TDF can also cause viral resistance.
  • MT-2 in vitro human lymphoma cell line
  • tenofovir can cause acute renal failure, decreased bone density, Fanconi syndrome, proteinuria or tubular necrosis.
  • the first technical problem to be solved by the present invention is to provide a new antiviral drug for clinical use.
  • a novel antiviral drug of the present invention is a deuterated nucleotide analog
  • the deuterated nucleotide analog is a compound as shown in I or a pharmaceutically acceptable compound thereof Salt:
  • R 1 , R 3 and R 5 are independently hydrogen or deuterium
  • R 2 is halogen, amino, hydroxy, straight or branched or cyclic C 1 -C 6 alkylamino, straight or branched C 1 -C 6 alkoxy;
  • R 4 is an aryl or aralkyl group
  • R 6 is alkyl or haloalkyl
  • R 7 is alkyl or haloalkyl
  • X is O, S or Se
  • Y is O, S or NH
  • At least one of the groups R 1 , R 3 , R 5 , R 6 or R 7 contains ruthenium.
  • the R 7 is a C 3 alkyl group or a C 3 halogenated alkyl group.
  • the R 7 is isopropyl or deuterated isopropyl.
  • the R 7 is a haloalkyl group.
  • the R 6 is a methyl group or a deuterated methyl group.
  • the R 2 is an amino group, a linear or branched or cyclic C 1 -C 6 alkylamino group.
  • the R 4 is a phenyl group.
  • both X and Y are O.
  • the pharmaceutically acceptable salt is a hydrochloride, a sulfate, a fumarate, a succinate, a methanesulfonate or a sulfonate.
  • the pharmaceutically acceptable salt is a fumarate salt.
  • the deuterated nucleotide analog is one of the following compounds:
  • a second technical problem to be solved by the present invention is to provide an antiviral pharmaceutical composition.
  • the antiviral pharmaceutical composition comprises the above-described deuterated nucleotide analog or various crystal forms, hydrates or solvates of the deuterated nucleotide analog. .
  • a pharmaceutically acceptable excipient or an auxiliary ingredient may be added to the antiviral pharmaceutical composition.
  • a third technical problem to be solved by the present invention is to provide the use of the above-described deuterated nucleotide analog or antiviral pharmaceutical composition for the preparation of an antiviral drug.
  • the virus is hepatitis B virus or hepatitis C virus.
  • the deuterated nucleotide analog of the present invention has antiviral efficacy and provides a new choice for the development of antiviral drugs, and has great significance for anti-drug resistant viruses.
  • the deuterated nucleotide analog of the present invention has good stability in the liver, can enrich in the liver and slowly release the active ingredient.
  • the deuterated nucleotide analog of the present invention has low toxicity, particularly low nephrotoxicity and higher safety.
  • Figure 1 Stability of Compound 9 and GS-7340 in human plasma
  • Figure 2 Stability of Compound 9 and GS-7340 in human liver S9;
  • Figure 3 Compound 9 and GS-7340 in the liver as a function of time
  • FIG. 4 Tenofovir hydrolyzed by compound 11 and TAF in the liver over time
  • Figure 5 Evaluation of proliferation inhibitory activity of Compound 11 and TAF on HK-2 cells.
  • a novel antiviral drug of the present invention is a deuterated nucleotide analog
  • the deuterated nucleotide analog is a compound as shown in I or a pharmaceutically acceptable compound thereof Salt:
  • R 1 , R 3 and R 5 are independently hydrogen or deuterium
  • R 2 is halogen, amino, hydroxy, straight or branched or cyclic C 1 -C 6 alkylamino, straight or branched C 1 -C 6 alkoxy;
  • R 4 is an aryl or aralkyl group
  • R 6 is alkyl or haloalkyl
  • R 7 is alkyl or haloalkyl
  • X is O, S or Se
  • Y is O, S or NH
  • At least one of the groups R 1 , R 3 , R 5 , R 6 or R 7 contains ruthenium.
  • the R 7 is a C 3 alkyl group or a C 3 halogenated alkyl group.
  • the R 7 is isopropyl or deuterated isopropyl.
  • the R 7 is a haloalkyl group.
  • the R 6 is a methyl group or a deuterated methyl group.
  • the R 2 is an amino group, a linear or branched or cyclic C 1 -C 6 alkylamino group.
  • the R 4 is a phenyl group.
  • both X and Y are O.
  • the pharmaceutically acceptable salt is a hydrochloride, a sulfate, a fumarate, a succinate, a methanesulfonate or a sulfonate.
  • the pharmaceutically acceptable salt is a fumarate salt.
  • the deuterated nucleotide analog is one of the following compounds:
  • the antiviral pharmaceutical composition comprises the above-described deuterated nucleotide analog or various crystal forms, hydrates or solvates of the deuterated nucleotide analog. .
  • a pharmaceutically acceptable excipient or an auxiliary ingredient may be added to the antiviral pharmaceutical composition.
  • a third technical problem to be solved by the present invention is to provide the use of the above-described deuterated nucleotide analog or antiviral pharmaceutical composition for the preparation of an antiviral drug.
  • the virus is hepatitis B virus or hepatitis C virus.
  • Tenofovir (1) 1.44g (5mmol) was added to a 100ml round bottom flask, 40ml of anhydrous pyridine, 3ml of triphenyl phosphite (2), refluxed under nitrogen for 10 hours, completely detected by TLC. . Pyridine was distilled off under reduced pressure, and 5 ml of methanol was added thereto, and then a white solid was evaporated, filtered, and washed with 3 ml of methanol to give a white solid (3) (yield: 49.4%).
  • N-benzyloxy-L-alanine (4) (2.23 g, 10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), respectively. 5.75 g, 30 mmol), 4-dimethylaminopyridine (DMAP) (122 mg, 1 mmol), deuterated isopropanol (5) (511 mg, 7.5 mmol), triethylamine (4.04 g, 40 mmol) in a 200 ml flask. 100 ml of dichloromethane was added. The reaction was carried out at room temperature for 24 hours under a nitrogen atmosphere.
  • DMAP 4-dimethylaminopyridine
  • deuterated isopropanol (5) 511 mg, 7.5 mmol
  • triethylamine (4.04 g, 40 mmol
  • L-Alanine deuterated isopropyl ester 7 (1.97 g, 15 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane and triethylamine (30 mmol) were added. After stirring at room temperature for 30 minutes, the reaction was added dropwise. In the crude product of the previous step, stir at room temperature for 6 hours. The reaction was completed by TLC, and the mixture was washed with saturated aqueous sodium hydrogen sulfate and brine, and then evaporated. Separation and purification by silica gel column chromatography gave pale-yellow solid (8) 2.42 g, yield: 50.1%.
  • the chromatograph was prepared using a Waters SFC-80 model, and Compound 8 was resolved according to the following chromatographic conditions.
  • Peak time Percentage /% Asymmetry 13.397 100 1.219
  • Peak time Percentage /% Asymmetry 18.5925 100 1.3
  • Deuterated L-alanine 27 (4.7 g, 50 mmol) was taken in a 100 ml flask, and 30 ml of isopropanol was added. 1.5 ml of thionyl chloride was slowly added dropwise at 0 ° C, and reacted at room temperature for 5 hours. TLC (ninhydrin color development) was detected until the reaction was completed, and the solvent was evaporated under reduced pressure to give dec. MS: 136.09 [M+H] + .
  • the compound 28 (687 mg, 4 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane and triethylamine (5 mmol) were added thereto, and the mixture was stirred at room temperature for 30 minutes, and then the reaction liquid was added to the crude product of the previous step, and stirred at room temperature for 6 hours. .
  • the reaction was completed by TLC, and the mixture was washed with saturated aqueous sodium hydrogen sulfate and brine, and then evaporated. Separation and purification by silica gel column chromatography gave 107.3 mg of pale yellow solid 29, yield: 22%. MS: 488.29 [M+H] + .
  • Deuterated L-alanine 27 (4.66 g, 50 mmol) was taken in a 100 ml flask, and 30 ml of deuterated isopropanol was added. 1.5 ml of thionyl chloride was slowly added dropwise at 0 ° C, and reacted at room temperature for 5 hours. The reaction was completed by TLC (ninhydrin color development), and the solvent was evaporated under reduced pressure to give Compound 34. MS: 143.13 [M+H] + .
  • the compound 34 (712 mg, 4 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane and triethylamine (5 mmol) were added thereto, and the mixture was stirred at room temperature for 30 minutes, and then the reaction liquid was added to the crude product of the previous step, and stirred at room temperature for 6 hours. .
  • the reaction was completed by TLC, and the mixture was washed with saturated aqueous sodium hydrogen sulfate and brine, and then evaporated. Separation and purification by silica gel column chromatography gave 123.6 mg of pale yellow solid (yield: 25%). MS: 495.32 [M+H] + .
  • the compound 7 (553 mg, 4 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane and triethylamine (5 mmol) were added thereto. After stirring at room temperature for 30 minutes, the reaction liquid was added dropwise to the crude product of the previous step, and stirred at room temperature for 6 hours. . The reaction was completed by TLC, and the mixture was washed with saturated aqueous sodium hydrogen sulfate and brine, and then evaporated. Separation and purification by silica gel column chromatography gave 102 mg of pale yellow solid 40, yield: 21%. MS: 486.30 [M+H] + .
  • the compound 28 (687 mg, 4 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane and triethylamine (5 mmol) were added thereto. After stirring at room temperature for 30 minutes, the reaction liquid was added dropwise to the crude product of the previous step, and stirred at room temperature for 6 hours. . The reaction was completed by TLC, and the mixture was washed with saturated aqueous sodium hydrogen sulfate and brine, and then evaporated. Separation and purification by silica gel column chromatography gave 129.7 mg of pale yellow solid. MS: 481.29 [M + H] + .
  • the cytotoxicity of the compounds was examined by the MTT method.
  • the HepG2.2.15 cells in the logarithmic growth phase were inoculated into a 96-well culture plate, and the cell concentration was adjusted to 4 ⁇ 10 4 /ml in a DMEM medium containing 10% fetal bovine serum at a volume of 100 ⁇ l per well at 37 ° C. Incubate overnight under 5% CO 2 conditions. Each well was treated with different concentrations of test compound, and 3 replicate wells were set for each concentration.
  • TAF positive control group GS-7340 fumarate
  • the extracellular HBV-DNA copy number was quantitatively detected by fluorescent PCR, and the inhibitory effect of the test compound on extracellular HBV-DNA replication was evaluated.
  • the experimental steps are as follows:
  • the logarithmic growth phase of HepG2.2.15 cells was inoculated into a 24-well culture plate, and the cell concentration was adjusted to 4 ⁇ 10 4 /ml with DMEM medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 Incubate for 24 hours under conditions.
  • the cells were treated with a culture medium containing different concentrations of the test compound and the positive control TDF, respectively, and a blank control was set.
  • the obtained deuterated compounds have a good inhibitory effect on the secretion of HBV-DNA by HepG2.2.15 cells, especially the inhibition effect of compound 11 on HBV is significantly better than that of the control compound TAF, and the toxicity to HepG2.2.15 cells. Both are small (CC 50 >10 ⁇ M).
  • the stability of Compound 9 in human plasma and human liver S9 was examined while using GS-7340 as a control.
  • the concentration of the experimental drug in plasma was 2 ⁇ M, and the concentration of the experimental drug in liver S9 was 10 ⁇ M. Samples were taken at different time points after dosing before dosing.
  • the concentration of Compound 9 and GS-7340 in the sample was analyzed by LC-MS/MS method.
  • mice Seventy mice were used in the experiment and purchased from the Experimental Animal Center of Sichuan Provincial People's Hospital. Set 10 min, 20 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, a total of 12 blood sampling time points, 3 mice at each time point.
  • the administered animals were bled at the corresponding time after administration, and the liver was taken after sacrifice.
  • a whole blood sample was collected and not less than 0.3 mL was placed in a labeled centrifuge tube containing heparin sodium (0.5%) anticoagulant, centrifuged at 3000 rpm for 15 min at 4 ° C, and the supernatant plasma was centrifuged in a 100 ⁇ l centrifuge tube.
  • 400 ⁇ l of acetonitrile (HPLC) was added, placed on a shaker for 30 minutes, and centrifuged at 130,000 rpm for 15 minutes, and then the supernatant was taken and stored in a refrigerator until LC-MS/MS analysis.
  • liver tissue samples were weighed and pure water was added to prepare liver tissue homogenates. 200 ⁇ l of homogenate was added to 800 ⁇ l of acetonitrile, placed on a shaker for 30 minutes, and centrifuged at 130,000 rpm for 15 minutes, and then the supernatant was taken and stored in a refrigerator until LC-MS/MS analysis.
  • the concentration of the free form of Compound 11 in vivo (Compound 9) and the free form of TAF in vivo (GS-7340) and its metabolite tenofovir in mouse plasma and liver tissue were analyzed by LC-MS/MS method.
  • the lower limit of detection (LLOQ) of tenofovir in mouse plasma was 1.00 ng/mL
  • the upper limit of quantitation (ULOQ) was 10000 ng/mL.
  • Figure 4 shows that tenofovir concentration first increases and then decreases within the first 10 hours after intragastric administration, and the concentration of tenofovir from hydrolysis of compound 11 is significantly higher than the concentration of tenofovir from hydrolysis of TAF.
  • the above results indicate that Compound 11 has higher stability in the liver than TAF, and after absorption, more tenofovir is accumulated in the liver.
  • HK-2 cells were plated at a density of 1500/well/100 ⁇ L. After 24 h, 100 ⁇ L of compound 11 and TAF prepared in fresh medium were added to each well to give a final drug concentration of 0.39, 0.78, 1.56, 3.12, 6.25. , 12.5, 25, 50, 100, 200 ⁇ M. After 72 h of drug administration, the cell proliferation inhibitory activity was measured by MTT assay. The results are shown in Figure 5.
  • HK-2 cells were plated at a density of 1000/well/100 ⁇ L. After adhering, the supernatant was aspirated, and 100 ⁇ L of each of the compounds 11 and TAF prepared in fresh medium was added to each well at a concentration of 100 ⁇ M. After 0.25 h, 24 h, 48 h, and 72 h, the supernatant was aspirated and placed in a sterile EP tube and placed at -20 °C. Detection was performed according to the instructions of the NGAL ELISA test kit. The results are shown in Table 4.
  • NGAL is a secreted protein that is rarely expressed in the kidneys.
  • the damaged renal tubular epithelial cells induce the apoptosis of neutrophils infiltrating the tubulointerstitial by expressing NGAL to protect the kidney tissue from attack;
  • the expression of NGAL is up-regulated, and a large amount of secreted NGAL is taken up by early primitive renal tubular epithelial cells, which promotes iron transport and promotes the maturation of primitive renal epithelial cells.
  • NGAL can attenuate apoptosis, suggesting that NGAL may have potential anti-apoptotic effects. Therefore, when HK-2 is necrotic or even apoptotic, NGAL is synthesized and secreted by renal tubular epithelial cells in order to play an anti-apoptotic effect;

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Abstract

氘代核苷酸类似物，为如式Ⅰ所示的化合物或其药学上可以接受的盐，所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。所述氘代核苷酸类似物在制备用于抗乙型肝炎病毒、丙型肝炎病毒的抗病毒药物中的用途。

Description

氘代核苷酸类似物及其用途 技术领域
本发明涉及氘代核苷酸类似物及其用途，属于抗病毒药物技术领域。
背景技术
乙型肝炎病毒(HBV)是一种严重影响人类健康的病原体，也是引发慢性乙型肝炎的元凶。国际上抗病毒药物的研究已经取得重要的进展，发现了一些临床有效的抗病毒药物，如干扰素、拉米夫定、替比夫定、克拉夫定、恩替卡韦、阿德福韦酯、替诺福韦酯、齐多夫定、司他夫定、奈韦拉平、茚地那韦和伐昔洛韦等。开环核苷类化合物作为核苷类抗病毒药物的重要一族，具有低毒、耐受性好和广谱抗DNA病毒活性，对耐药菌株有强烈的杀伤作用，在抗病毒治疗领域占有重要地位，其中以替诺福韦酯和阿德福韦酯为代表的无环核苷酸类抗病毒药是近年研究的热点(Tang YB等.Bioorganic&Medicinal Chemistry Letters,2007,17(22):6350-6353)。研究表明，阿德福韦二匹伏酯和替诺福韦酯富马酸盐(TDF)均对拉米夫定耐药株有效，而替诺福韦酯对DNA聚合酶的抑制常数是阿德福韦的5倍。在体外，TDF可有效对抗多种病毒，包括那些对核苷类逆转录酶抑制剂耐药的毒株。替诺福韦酯分别于2001年和2008年被FDA批准上市，用于治疗HIV和HBV感染，近年研究发现，替诺福韦酯对丙型肝炎病毒(HCV)感染也有良好的治疗作用(Tuma P,Vispo E等.Enferm Infecc Microbiol Clin.2008,26(Suppl 8):31-37)。与其他抗逆转录病毒药物一样，TDF也会引起病毒耐药。在体外人淋巴瘤细胞株(MT-2)中逐渐增加TDF浓度，会产生能在2μM TDF中存活的病毒株。此外，替诺福韦酯能引起急性肾功能衰竭、骨密度下降、Fanconi综合症、蛋白尿或肾小管坏死等。
为了降低替诺福韦酯的副作用，Gilead公司对替诺福韦的结构进一步修饰，获得一个化合物TAF(tenofovir alafenamide fumarate，替诺福韦艾拉酚胺富马酸盐，商品名：Vemlidy)在低于TDF十分之一剂量时，就具有非常高的抗病毒疗效。然而，约翰·霍普金斯医学院的研究人员发现，Vemlidy仍然会引起肾损伤(Tessa等，Medicine,2017,96(36):e8046)。此外，Vemlidy也会导致骨密度不同程度的下降(Kosh等，Journal of Hepatology,2018,68(4):672–681)。Vemlidy的药品标签中带有黑框警告，提示TAF具有乳酸中毒、肝脏肿大以及治疗后乙肝急剧加重的风险。
因而，开发新的抗病毒药物，特别是更加高效、低毒的抗病毒药物具有十分重要的意义。
发明内容
本发明要解决的第一个技术问题是为临床提供一种新的抗病毒药物。
为解决上述第一个技术问题，本发明的一种新的抗病毒药物为氘代核苷酸类似物，所述氘代核苷酸类似物为如Ⅰ所示的化合物或其药学上可以接受的盐：
Figure PCTCN2018079990-appb-000001
其中
R 1、R 3和R 5独立地为氢或氘；
R 2为卤素、氨基、羟基、直链或支链或环状C 1～C 6烷基氨基、直链或支链C 1～C 6烷氧基；
R 4为芳基或芳烷基；
R 6为烷基或氘代烷基；
R 7为烷基或氘代烷基；
X为O、S或Se；Y为O、S或NH；
所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。
优选的，所述R 7为C 3烷基或C 3氘代烷基。
优选的，所述R 7为异丙基或氘代异丙基。
优选的，所述R 7为氘代烷基。
优选的，所述R 6为甲基或氘代甲基。
优选的，所述R 2为氨基、直链或支链或环状C 1～C 6烷基氨基。
优选的，所述R 4为苯基。
优选的，所述X和Y均为O。
优选的，所述药学上可以接受的盐为盐酸盐、硫酸盐、富马酸盐、琥珀酸盐、甲磺酸盐或磺酸盐。
更优选的，所述药学上可以接受的盐为富马酸盐。
优选的，所述氘代核苷酸类似物为下列化合物之一：
Figure PCTCN2018079990-appb-000002
本发明要解决的第二个技术问题是提供一种抗病毒药物组合物。
为解决本发明的第二个技术问题，所述抗病毒药物组合物包含上述的氘代核苷酸类似物或所述氘代核苷酸类似物的各种晶型、水合物或溶剂合物。
所述抗病毒药物组合物中可以添加药学上常用的辅料或辅助性成分。
本发明要解决的第三个技术问题是提供上述氘代核苷酸类似物或抗病毒药物组合物在制备抗病毒药物中的用途。
优选的，所述病毒为乙型肝炎病毒、丙型肝炎病毒。
有益效果：
1、本发明的氘代核苷酸类似物具有抗病毒的药效，为抗病毒药物的开发提供新的选择，对于抗耐药病毒具有重大的意义。
2、本发明的氘代核苷酸类似物在肝中的稳定性好,能在肝脏富集并缓慢释放活性成分。
3、本发明的氘代核苷酸类似物毒性小，特别是肾毒性很低，安全性更高。
4、本发明的氘代核苷酸类似物及其盐的抗病毒效果非常好。
具体实施方式 附图说明
图1：化合物9和GS-7340在人血浆中的稳定性；
图2：化合物9和GS-7340在人肝S9中的稳定性；
图3：肝脏中化合物9和GS-7340随时间变化情况；
图4：化合物11和TAF水解的替诺福韦在肝脏中随时间变化情况；
图5：化合物11和TAF对HK-2细胞的增殖抑制活性评价。
具体实施方式
为解决上述第一个技术问题，本发明的一种新的抗病毒药物为氘代核苷酸类似物，所述氘代核苷酸类似物为如Ⅰ所示的化合物或其药学上可以接受的盐：
Figure PCTCN2018079990-appb-000003
其中
R 1、R 3和R 5独立地为氢或氘；
R 2为卤素、氨基、羟基、直链或支链或环状C 1～C 6烷基氨基、直链或支链C 1～C 6烷氧基；
R 4为芳基或芳烷基；
R 6为烷基或氘代烷基；
R 7为烷基或氘代烷基；
X为O、S或Se；Y为O、S或NH；
所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。
优选的，所述R 7为C 3烷基或C 3氘代烷基。
优选的，所述R 7为异丙基或氘代异丙基。
优选的，所述R 7为氘代烷基。
优选的，所述R 6为甲基或氘代甲基。
优选的，所述R 2为氨基、直链或支链或环状C 1～C 6烷基氨基。
优选的，所述R 4为苯基。
优选的，所述X和Y均为O。
优选的，所述药学上可以接受的盐为盐酸盐、硫酸盐、富马酸盐、琥珀酸盐、甲磺酸盐或磺酸盐。
更优选的，所述药学上可以接受的盐为富马酸盐。
优选的，所述氘代核苷酸类似物为下列化合物之一：
Figure PCTCN2018079990-appb-000004
为解决本发明的第二个技术问题，所述抗病毒药物组合物包含上述的氘代核苷酸类似物或所述氘代核苷酸类似物的各种晶型、水合物或溶剂合物。
所述抗病毒药物组合物中可以添加药学上常用的辅料或辅助性成分。
本发明要解决的第三个技术问题是提供上述氘代核苷酸类似物或抗病毒药物组合物在制备抗病毒药物中的用途。
优选的，所述病毒为乙型肝炎病毒、丙型肝炎病毒。
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所 述的实施例范围之中。
实施例1
化合物11和12的制备
Figure PCTCN2018079990-appb-000005
化合物3的合成：
取替诺福韦(Tenofovir,1)1.44g(5mmol)于100ml圆底烧瓶中，加入无水吡啶40ml、亚磷酸三苯酯(2)3ml，氮气保护下回流10小时，TLC检测至反应完全。减压蒸馏除去吡啶，加入5ml甲醇，冷却析出白色固体，过滤，用3ml甲醇洗涤，得0.9g白色固体(3)，收率：49.4％。 1H NMR(400MHz,D 2O):8.26(s,1H),8.17(s,1H),7.17(t,2H),7.05(d,1H),6.66(d,2H),4.36(d,1H),4.21(d,1H),4.02(d,1H),3.77(d,1H),3.50(d,1H),1.22(d,3H)；MS:362.35[M-H] -。
化合物6的合成：
分别取N-苄氧酰基-L-丙氨酸(4)(2.23g，10mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI)(5.75g，30mmol)、4-二甲氨基吡啶(DMAP)(122mg，1mmol)、氘代异 丙醇(5)(511mg，7.5mmol)、三乙胺(4.04g，40mmol)于200ml烧瓶中，加入100ml二氯甲烷。氮气保护下室温反应24小时。反应结束后水洗(100ml×3)，硅胶柱纯化得N-苄氧酰基-L-丙氨酸氘代异丙酯(6)1.47g，收率54％。 1H NMR(400MHz,CDCl 3)：7.50-7.32(m,5H),5.45(s,1H),5.15(s,2H),4.40–4.25(m,1H),1.30(d,3H)；MS:273.15[M+H] +。
化合物7的合成：
取N-苄氧酰基-L-丙氨酸氘代异丙酯(6)1g、Pd/C200mg于200ml两口瓶中，加入50ml甲醇，分别用氮气置换3次，氢气置换3次，室温反应过夜。反应结束后过滤，减压蒸馏除去溶剂得L-丙氨酸氘代异丙酯(7)415mg，产率81.7％。 1H NMR(400MHz,DMSO-d 6):4.20–4.03(m,1H),1.60(s,2H),1.15(d,3H)；MS:139.35[M+H] +。
化合物8的合成：
取化合物3(3.63g，10mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、2ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取L-丙氨酸氘代异丙酯7(1.97g，15mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(30mmol)，常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到淡黄色固体(8)2.42g，收率：50.1％。 1H NMR(400MHz,CDCl 3)：8.22(d,1H),8.02(d,1H),7.38–7.19(m,5H),6.58(s,2H),4.61(d,1H),4.42-4.28(m,1H),4.01-3.65(m,4H),3.58-3.40(m,1H),1.21-1.08(m,6H)；MS:484.15[M+H] +。
化合物9和10的合成：
用Waters公司SFC-80型制备色谱仪，根据以下的色谱条件，对化合物8进行拆分。
色谱柱：MS-OD 20×250mm,5μm制备柱；柱温：35℃；流动相：CO 2/IPA＝80/20；流速：40ml/min；循环时间：5.5min；背压：120Bar；检测波长：260nm；进样体积：1mL；收集两个峰，减压浓缩除去流动相后得到目标产品。投料量4.2g，分别得到1.2g化合物9和化合物10。在Waters公司UPC色谱仪上，对化合物9和化合物10进行光学纯度分析，结果见表1和表2。
色谱柱类型：MS-OD 4.6×150mm,5μm分析柱；仪器型号：Waters UPC；样品溶剂：MeOH；色谱柱：MS-OD；柱规格：4.6×150mm流动相：CO 2/IPA＝90/10；流速：2.5mL/min背压：2000Psi；压力降：510Psi柱温：45℃；检测波长：260nm；进样体积：20μL
表1 化合物9的色谱图数据
出峰时间 百分含量/％ 不对称度
13.397 100 1.219
表2 化合物10的色谱图数据
出峰时间 百分含量/％ 不对称度
18.5925 100 1.3
以上结果表明，所得化合物9和化合物10具有非常高的光学纯度。核磁共振氢谱数据如下：
化合物9：
1H NMR(400MHz,CDCl 3)：δ8.31(s,1H),8.00(s,1H),7.31-7.27(m,2H),7.17-7.11(m,3H),6.46(s,2H),4.45-4.41(m,1H)),4.20-4.15(m,1H)),4.08-3.95(m,4H),),3.74-3.69(m,1H),1.25-1.19(m,6H)； 13C NMR(100MHz,CDCl 3):δ173.59,173.54,155.76,152.90,150.24,150.14,150.05,141.72,129.63,124.89,120.75,120.70,119.16,76.70,76.57,68.45,65.15,63.60,49.66,48.23,21.00,20.95,16.64。MS:484.25[M+H] +。
化合物10：
1H NMR(400MHz,CDCl 3)：8.34(s,1H),8.00(s,1H),7.22-7.18(m,2H),7.09-7.00(m,3H),6.45(s,2H),4.41-4.32(m,2H)),4.14-4.05(m,2H)),3.95-3.89(m,2H),),3.71-3.65(m,1H),1.30-1.21(m,6H)。 13C NMR(100MHz,CDCl 3):δ173.35,173.30,155.84,152.97,150.12,150.08,150.04,141.60,129.65,124.87,120.47,120.38,120.33，119.27,76.35,76.22,68.48,65.28,63.74,49.95,48.39,21.54,21.50,16.48。MS:484.46[M+H] +。
化合物11的合成：
取化合物9(4.83g，10mmol)、富马酸(1.05g，9mmol)于200ml烧瓶中，加入乙腈100ml，加热回流至固体完全溶解，趁热过滤，滤液置于5℃搅拌12小时。析出白色沉淀，过滤，用乙腈40ml洗涤固体。干燥得4.5g白色固体11，收率83.3％。
化合物12的合成：
取化合物10(4.83g，10mmol)、富马酸(1.05g，9mmol)于200ml烧瓶中，加入乙腈100ml，加热回流至固体完全溶解，趁热过滤，滤液置于5℃搅拌12小时。析出白色沉淀，过滤，用乙腈40ml洗涤固体。干燥得4.1g白色固体12，收率75.9％。
实施例2
化合物17和18的合成
Figure PCTCN2018079990-appb-000006
化合物13的合成：
取化合物3(363mg，1mmol)于50ml封管中，加入36mg的10％钯碳、10ml重水，氮气置换三次，氢气置换三次，125℃反应24小时。冷至室温，过滤，取虑液减压蒸馏，除去溶剂得白色固体，加入少量甲醇超声洗涤，过滤得255.7mg白色粉末状产物13，收率70％。MS:364.11[M-H] -。
化合物14的合成：
取化合物13(365mg，1mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、0.5ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体，直接用于下一步反应。
取L-丙氨酸异丙酯盐酸盐(640mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(5mmol)，常温搅拌30分钟后，将反应液滴加到上一步产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到71.8mg淡黄色固体14，收率：15％。 1HNMR(400MHz,CDCl 3)：7.28-6.90(m,5H),6.32(s,2H),4.91(d,1H),4.33-4.18(m,1H),4.11-3.75(m,5H),3.68-3.50(m,1H),1.21-1.08(m,12H)；MS:479.26[M+H] +。
化合物15和16的合成：
化合物15和16的合成步骤与化合物9和化合物10相同。
化合物17和18的合成：
化合物17和18的合成步骤与化合物11和化合物12相同。
实施例3
化合物25和26的合成：
Figure PCTCN2018079990-appb-000007
化合物20的合成：
取化合物1(1.44g，5mmol)于100ml圆底烧瓶中，加入氘代苯酚19(990mg，10mmol)、N-甲基吡咯烷酮30ml，加热至85℃。加入氮气保护回流10小时，TLC检测至反应完全。减压蒸馏除去吡啶，加入0.8ml三乙胺。待固体完全溶解后，100℃下缓慢加入DCC(1.54g，7.5mmol)，反应16小时。冷至室温，加入20ml水，滤除固体，滤液经减压蒸馏除去溶剂，得泡沫状固体，加入10ml水，用氢氧化钠水溶液调节pH>11,过滤，滤液用乙酸乙酯(10ml)洗涤2次后，用稀盐酸调节pH＝3，析出白色固体，过滤，固体用3ml甲醇洗涤，得化合物20。MS:367.08[M-H] -。
化合物21的合成：
取化合物20(368mg，1mmol)于50ml封管中，加入36mg的10％钯碳、10ml重水，置换氮气三次，置换氢气三次，125℃反应24小时。冷至室温，过滤，取虑液减压蒸馏除去溶剂得白色固体，加入少量甲醇洗涤，过滤得248.1mg白色粉末状产物21，收率67％。MS:369.13[M-H] -。
化合物22的合成：
取化合物21(370mg，1mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、0.5ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取L-丙氨酸异丙酯盐酸盐(640mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、 三乙胺(5mmol)，常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到91.9mg淡黄色固体22，收率：19％。MS:484.26[M+H] +。
化合物23和24的合成：
化合物23和24的合成步骤与化合物9和化合物10相同。
化合物25和26的合成：
化合物25和26的合成步骤与化合物11和化合物12相同。
实施例4
化合物32和33的合成：
Figure PCTCN2018079990-appb-000008
化合物28的合成：
取氘代L-丙氨酸27(4.7g，50mmol)于100ml烧瓶，加入30ml异丙醇。于0℃下缓慢滴加二氯亚砜1.5ml，常温反应5小时。TLC(茚三酮显色)检测至反应完全，减压蒸馏除去溶剂，得氘代L-丙氨酸异丙酯盐酸盐28。MS:136.09[M+H] +。
化合物29的合成：
取化合物21(370mg，1mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、0.5ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取化合物28(687mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(5mmol)， 常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到107.3mg淡黄色固体29，收率：22％。MS:488.29[M+H] +。
化合物30和31的合成：
化合物30和31的合成步骤与化合物9和化合物10相同。
化合物32和33的合成：
化合物32和33的制备步骤与化合物11和化合物12相同。
实施例5
化合物38和39的合成：
Figure PCTCN2018079990-appb-000009
化合物34的合成：
取氘代L-丙氨酸27(4.66g，50mmol)于100ml烧瓶，加入30ml氘代异丙醇。于0℃下缓慢滴加二氯亚砜1.5ml，常温反应5小时。TLC(茚三酮显色)检测至反应完全，减压蒸馏除去溶剂，得化合物34。MS:143.13[M+H] +。
化合物35的合成：
取化合物21(370mg，1mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、0.5ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取化合物34(712mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(5mmol)， 常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到123.6mg淡黄色固体35，收率：25％。MS:495.32[M+H] +。
化合物36和37的合成：
化合物36和37的合成步骤与化合物9和化合物10相同。
化合物38和39的合成：
化合物38和39的合成步骤与化合物11和化合物12相同。
实施例6
化合物43和44的合成：
Figure PCTCN2018079990-appb-000010
化合物40的合成：
取化合物13(365mg，1mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5mlDMF、0.5ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取化合物7(553mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(5mmol)，常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到102mg淡黄色固体40，收率：21％。MS:486.30[M+H] +。
化合物41和42的合成：
化合物41和42的合成步骤与化合物9和化合物10相同。
化合物43和44的合成：
化合物43和44的合成步骤与化合物11和化合物12相同。
实施例7
化合物48和49的合成：
Figure PCTCN2018079990-appb-000011
化合物45的合成：
取化合物3(3.63g，10mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、0.5ml DMF、2ml草酰氯，常温反应10小时。减压蒸馏除去溶剂得淡黄色固体粗产品，直接用于下一步反应。
取化合物28(687mg，4mmol)于50ml圆底烧瓶中，加入20ml二氯甲烷、三乙胺(5mmol)，常温搅拌30分钟后，将反应液滴加到上一步粗产品中，常温搅拌6小时。TLC检测至反应完全，反应液依次用饱和碳酸氢钠水溶液、饱和水盐水洗涤，取有机层，减压蒸馏出去溶剂得淡黄色固体。硅胶柱层析分离纯化得到129.7mg淡黄色固体45，收率：27％。MS:481.29[M+H] +。
化合物46和47的合成：
化合物46和47的合成步骤与化合物9和化合物10相同。
化合物48和49的合成：
化合物48和49的合成步骤与化合物11和化合物12相同。
试验例1
体外抗HBV活性
(1)细胞毒性试验
利用MTT法检测化合物的细胞毒性。将处于对数生长期的HepG2.2.15细胞接种到96孔培养板中，用含10％胎牛血清的DMEM培养液调节细胞浓度为4×10 4/ml，每孔体积100μl，于37℃， 5％CO 2条件下培养过夜。加入不同浓度的受试化合物对各孔细胞进行处理，每个浓度设3个复孔。实验同时设置接种细胞加入培养基的阴性对照组和未接种细胞仅加入培养基的空白对照组以及阳性对照组GS-7340富马酸盐(TAF)，继续培养72小时。进行MTT检测，计算细胞的半数毒性浓度(CC 50)。
(2)体外抗HBV活性
使用荧光PCR定量检测细胞外HBV-DNA拷贝数，评价待测化合物对细胞外HBV-DNA复制的抑制作用。实验步骤如下：
a.取对数生长期的HepG2.2.15细胞接种到24孔培养板中，用含10％胎牛血清的DMEM培养液调节细胞浓度为4×10 4/ml，于37℃，5％CO 2条件下培养24小时。
b.分别用含有不同浓度的待测化合物与阳性对照TDF的培养液对细胞进行处理，同时设置空白对照。
c.于加药后第3、6天更换新鲜含不同浓度待测化合物和阳性对照的培养基。
d.收集培养液的上层清液，-20℃冷冻备用。
e.荧光定量PCR检测HBV-DNA的含量，按试剂盒说明书进行操作。根据检测到的各个样本HBV-DNA拷贝数，计算各个化合物对HepG2.2.15细胞外HBV-DNA复制抑制作用，用SPSS软件计算各个化合物的半数有效浓度EC 50。
由表3中结果可知，所得氘代化合物对HepG2.2.15细胞分泌HBV-DNA具有良好的抑制作用，特别是化合物11对HBV的抑制效果显著优于对照化合物TAF，且对HepG2.2.15细胞的毒性均较小(CC 50>10μM)。
表3 化合物对HBV DNA的抑制作用
Figure PCTCN2018079990-appb-000012
Figure PCTCN2018079990-appb-000013
实验例2化合物9在人血浆、人肝S9中的稳定性研究
1.实验目的
本实验旨在考察实施例1中的化合物9在人血浆、人肝S9中的稳定性，并与GS-7340做对比。
2.材料与方法
2.1供试品信息
Figure PCTCN2018079990-appb-000014
2.2实验设计
考察化合物9在人血浆和人肝S9中的稳定性，同时用GS-7340做对照。血浆中实验药物浓度为2μM，肝S9中实验药物浓度为10μM。分别在加药前，加药后不同时间点取样处理检测。
2.3样品采集与制备
取新制的人血浆加入适量化合物9或GS-7340，使得实验药物浓度为2μM；取人肝S9稀释至实验浓度；并加入适量化合物9或GS-7340，使得实验药物浓度为10μM，混匀后加入A、B液开始反应，分别在加药前，孵育5min、10min、20min、30min、40min、60min、80min、100min、120min、150min、180min、210min、240min取适量反应液，用4倍体积乙腈(HPLC)淬灭并沉淀蛋白，130000rmp离心15min，然后取上清液保存在冰箱中直到进行LC-MS/MS分析。
2.3样品分析
应用LC-MS/MS方法分析化合物9和GS-7340在样品中的浓度。
3.实验结果
化合物9和GS-7340在人血浆和人肝S9中的稳定情况见图1和图2。
以上结果表明，化合物9和GS-7340在人血浆中的稳定相当，而在肝S9中的，化合物9的稳定性高于GS-7340。
试验例3肝脏和血液中的分布实验
1.实验目的
考察单次灌胃给予实施例1制备的化合物11和TAF在小鼠肝脏和血液中的分布情况。
2.材料与方法
2.1供试品信息
Figure PCTCN2018079990-appb-000015
2.2实验设计和动物准备
实验采用小鼠70只，购自四川省人民医院实验动物中心。设置10min、20min、30min、1h、2h、4h、6h、8h、12h、24h、48h，共12个采血取样时间点，每个时间点3只小鼠。
2.3制剂配制及给药
精确称量适量化合物11和TAF，置成浓度合适的生理盐水溶液。实验动物按照25mg/kg单次灌胃给予化合物11和TAF的生理盐水溶液。
2.4样品采集与制备
给药动物在给药后相应时间采血，处死后取肝脏。每个时间点采集全血样品不少于0.3mL放入贴有标签的含有肝素钠(0.5％)抗凝剂的离心管中，4℃，3000rpm离心15min，取上清血浆100μl离心管中，加入乙腈(HPLC)400μl，放置于摇床30分钟后130000rpm离心15分钟，然后取上清液保存在冰箱中直到进行LC-MS/MS分析。称取肝组织样品，加入纯水，制备肝组织匀浆。取200μl匀浆加入800μl乙腈，放置于摇床30分钟后130000rpm离心15分钟，然后取上清液保存在冰箱中直到进行LC-MS/MS分析。
2.5样品分析
应用LC-MS/MS方法分析化合物11在体内的游离形式(化合物9)和TAF在体内的游离形式(GS-7340)及其代谢物替诺福韦在小鼠血浆及肝组织中的浓度。本实验中，替诺福韦在小鼠血浆中的检测定量下限(LLOQ)为1.00ng/mL，定量上限(ULOQ)为10000ng/mL。
3.实验结果
通过质谱检测，发现化合物11和TAF灌胃给药后，10分钟到48小时的血样中，化合物11在体内的游离形式(化合物9)和TAF在体内的游离形式(GS-7340)及它们的水解产物替诺福韦含量低于检测限。在肝脏中化合物11在体内的游离形式(化合物9)和TAF在体内的游离形式(GS-7340)及其水解产物替诺福韦的随时间的变化情况见图3和图4。图3表明，在前1小时内，化合物9的浓度随时间增加逐渐减小，之后低于检测限。而GS-7340在肝中的浓度一直低于检测限。图4表明灌胃给药后的前10小时内，替诺福韦浓度先增加后减小，且来自化合物11水解的替诺福韦浓度显著高于来自TAF水解的替诺福韦浓度。以上结果表明，化合物11在肝脏中比TAF具有更高的稳定性，且吸收后，有更多的替诺福韦聚集在肝脏。
试验例4肾毒性研究
1.实验目的
考察实施例1制备的化合物11和TAF对人肾小管上皮细胞HK-2中性粒细胞明胶酶相关脂质运载蛋白(NGAL)的影响。
2.材料与方法
2.1供试品信息
Figure PCTCN2018079990-appb-000016
2.2实验设计
首先，我们对化合物11和TAF抑制HK-2细胞增殖的能力进行评价，然后在无细胞毒的浓度下考察化合物对HK-2细胞分泌NGAL的影响。
(1)抑制细胞增殖活性评价
HK-2细胞以1500/孔/100μL的密度铺96孔板，24h后，每孔各加100μL用新鲜培养基配制的化合物11和TAF，使最终药物浓度为0.39,0.78,1.56,3.12,6.25，12.5,25,50,100,200μM。加药72h后，MTT法检测细胞增殖抑制活性。结果见图5。
根据图5，化合物11和TAF在200μM以下，72小时内未显示出细胞毒性。
(2)对HK-2分泌NGAL的影响
HK-2细胞以1000/孔/100μL的密度铺96孔板，贴壁后，吸出上清液，每孔各加100μL用新鲜培养基配制的化合物11和TAF，浓度为100μM。加药0.25h、24h、48h、72h后，吸出上清液置于无菌EP管中，置于-20℃。根据NGAL的ELISA检测试剂盒的说明书进行检测。结果见表4。
表4 化合物11对HK-2细胞分泌NGAL的影响
Figure PCTCN2018079990-appb-000017
由表4可知，随着药物作用时间的延长，化合物11处理的HK-2细胞分泌NGAL的水平并没有受到影响，而用TAF处理的细胞，分泌NGAL的水平随着时间延长而增大。
NGAL是一种分泌蛋白，一般情况下在肾脏中的表达量极少。然而当肾小管受到刺激出现损伤时，受损的肾小管上皮细胞通过表达NGAL以此诱导大量浸润于肾小管间质中的中性粒细胞发生凋亡，以保护肾组织免受攻击；另一方面，当肾小管上皮细胞发生损伤时，NGAL的表达出现上调，大量分泌的NGAL被早期的原始肾小管上皮细胞摄取，在介导铁转运的同时促进原始肾上皮细胞的成熟。有研究发现NGAL能够减轻细胞凋亡，提示NGAL可能具有潜在的抗凋亡作用。因此，HK-2发生坏死甚至凋亡时，NGAL为了起到抗凋亡作用，被肾小管上皮细胞大量合成并分泌；
以上研究表明，在细胞水平上，化合物11比TAF具有更高的安全性。

Claims (13)

  1. 氘代核苷酸类似物，其特征在于，所述氘代核苷酸类似物为如Ⅰ所示的化合物或其药学上可以接受的盐：
    Figure PCTCN2018079990-appb-100001
    其中，R 1、R 3和R 5独立地为氢或氘；
    R 2为卤素、氨基、羟基、直链或支链或环状C 1～C 6烷基氨基、直链或支链C 1～C 6烷氧基；
    R 4为芳基或芳烷基；
    R 6为烷基或氘代烷基；
    R 7为烷基或氘代烷基；
    X为O、S或Se；Y为O、S或NH；
    所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。
  2. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述R 7为C 3烷基或C 3氘代烷基。
  3. 根据权利要求2所述的氘代核苷酸类似物，其特征在于，所述R 7为异丙基或氘代异丙基。
  4. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述R 7为氘代烷基。
  5. 根据权利要求2所述的氘代核苷酸类似物，其特征在于，所述R 6为甲基或氘代甲基。
  6. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述R 2为氨基、直链或支链或环状C 1～C 6烷基氨基。
  7. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述R 4为苯基。
  8. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述X和Y均为O。
  9. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述药学上可以接受的盐为盐酸盐、硫酸盐、富马酸盐、琥珀酸盐、甲磺酸盐或磺酸盐。
  10. 根据权利要求9所述的氘代核苷酸类似物，其特征在于，所述药学上可以接受的盐为富马酸盐。
  11. 根据权利要求1所述的氘代核苷酸类似物，其特征在于，所述氘代核苷酸类似物为下列化合物之一：
    Figure PCTCN2018079990-appb-100002
  12. 抗病毒药物组合物，其特征在于，所述抗病毒药物组合物包含权利要求1～11任一项所述的氘代核苷酸类似物或所述氘代核苷酸类似物的各种晶型、水合物或溶剂合物。
  13. 如权利要求1～11任一项所述的氘代核苷酸类似物或权利要求12所述的抗病毒药物组合物在制备抗病毒药物中的用途，其所述病毒优选为乙型肝炎病毒、丙型肝炎病毒。
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CN105669751A (zh) * 2015-03-05 2016-06-15 洛阳聚慧医药科技有限公司 非环核苷酸磷酰胺类化合物及其盐的制备以及在抗病毒方面的应用

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