WO2018171665A1 - Deuterated nucleotide analog and use thereof - Google Patents

Deuterated nucleotide analog and use thereof 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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PCT/CN2018/079990
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French (fr)
Chinese (zh)
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谢永美
耿福能
魏于全
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四川好医生攀西药业有限责任公司
四川大学
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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

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  • 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

A deuterated nucleotide analog, which is a compound represented by formula I or a pharmaceutically acceptable salt thereof, wherein at least one group from among R1, R3, R5, R6 or R7 contains deuterium. A use of the deuterated nucleotide analog in the preparation of an antiviral drug for use in resisting the hepatitis B virus and hepatitis C virus.

Description

氘代核苷酸类似物及其用途Deuterated nucleotide analogues and uses thereof 技术领域Technical field
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。The invention relates to deuterated nucleotide analogues and uses thereof, and belongs to the technical field of antiviral drugs.
背景技术Background technique
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。Hepatitis B virus (HBV) 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. As an important family of 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. They play an important role in the field of antiviral therapy. Status, in which acyclic nucleotide antiviral drugs represented by tenofovir and adefovir dipivoxil are hot topics in recent years (Tang YB et al. Bioorganic & Medicinal Chemistry Letters, 2007, 17(22): 6350- 6353). Studies have shown that adefovir dipivoxil and tenofovir disoproxil fumarate (TDF) are effective against lamivudine-resistant strains, while tenofovir disoproxil inhibits DNA polymerase. 5 times the Defowe. In vitro, 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. In recent years, it has been found that tenofovir disoproxil has a good therapeutic effect on hepatitis C virus (HCV) infection (Tuma P, Vispo E et al. Enferm Infecc Microbiol Clin. 2008, 26 (Suppl 8): 31-37). Like other antiretroviral drugs, TDF can also cause viral resistance. Increasing the concentration of TDF in an in vitro human lymphoma cell line (MT-2) resulted in a virus strain that survived 2 μM TDF. In addition, tenofovir can cause acute renal failure, decreased bone density, Fanconi syndrome, proteinuria or tubular necrosis.
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。In order to reduce the side effects of tenofovir, Gilead further modified the structure of tenofovir to obtain a compound TAF (tenofovir alafenamide fumarate, tenofovir alafenamide fumarate, trade name: Vemlid) Below one tenth of TDF, it has a very high antiviral effect. However, researchers at Johns Hopkins Medical School found that Vemlid still causes kidney damage (Tessa et al, Medicine, 2017, 96 (36): e8046). In addition, Vemlidy also causes varying degrees of bone density (Kosh et al, Journal of Hepatology, 2018, 68(4): 672-681). Vemlidy's drug label carries a black box warning, suggesting that TAF has a risk of lactic acidosis, liver enlargement, and a sharp exacerbation of hepatitis B after treatment.
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。Therefore, the development of new antiviral drugs, especially more efficient, low-toxic antiviral drugs is of great significance.
发明内容Summary of the invention
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。The first technical problem to be solved by the present invention is to provide a new antiviral drug for clinical use.
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：In order to solve the above first technical problem, a novel antiviral drug of the present invention is a deuterated nucleotide analog, and the deuterated nucleotide analog is a compound as shown in I or a pharmaceutically acceptable compound thereof Salt:
Figure PCTCN2018079990-appb-000001
Figure PCTCN2018079990-appb-000001
其中among them
R 1、R 3和R 5独立地为氢或氘； R 1 , R 3 and R 5 are independently hydrogen or deuterium;
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 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为芳基或芳烷基； R 4 is an aryl or aralkyl group;
R 6为烷基或氘代烷基； R 6 is alkyl or haloalkyl;
R 7为烷基或氘代烷基； R 7 is alkyl or haloalkyl;
X为O、S或Se；Y为O、S或NH；X is O, S or Se; Y is O, S or NH;
所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。 At least one of the groups R 1 , R 3 , R 5 , R 6 or R 7 contains ruthenium.
优选的，所述R 7为C 3烷基或C 3氘代烷基。 Preferably, the R 7 is a C 3 alkyl group or a C 3 halogenated alkyl group.
优选的，所述R 7为异丙基或氘代异丙基。 Preferably, the R 7 is isopropyl or deuterated isopropyl.
优选的，所述R 7为氘代烷基。 Preferably, the R 7 is a haloalkyl group.
优选的，所述R 6为甲基或氘代甲基。 Preferably, the R 6 is a methyl group or a deuterated methyl group.
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。 Preferably, the R 2 is an amino group, a linear or branched or cyclic C 1 -C 6 alkylamino group.
优选的，所述R 4为苯基。 Preferably, the R 4 is a phenyl group.
优选的，所述X和Y均为O。Preferably, both X and Y are 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。Preferably, the pharmaceutically acceptable salt is a hydrochloride, a sulfate, a fumarate, a succinate, a methanesulfonate or a sulfonate.
更优选的，所述药学上可以接受的盐为富马酸盐。More preferably, the pharmaceutically acceptable salt is a fumarate salt.
优选的，所述氘代核苷酸类似物为下列化合物之一：Preferably, the deuterated nucleotide analog is one of the following compounds:
Figure PCTCN2018079990-appb-000002
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。A second technical problem to be solved by the present invention is to provide an antiviral pharmaceutical composition.
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。In order to solve the second technical problem of the present invention, the antiviral pharmaceutical composition comprises the above-described deuterated nucleotide analog or various crystal forms, hydrates or solvates of the deuterated nucleotide analog. .
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。A pharmaceutically acceptable excipient or an auxiliary ingredient may be added to the antiviral pharmaceutical composition.
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。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.
优选的，所述病毒为乙型肝炎病毒、丙型肝炎病毒。Preferably, the virus is hepatitis B virus or hepatitis C virus.
有益效果：Beneficial effects:
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。1. 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.
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。2. 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.
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。3. The deuterated nucleotide analog of the present invention has low toxicity, particularly low nephrotoxicity and higher safety.
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。4. The anti-viral effect of the deuterated nucleotide analogs of the present invention and salts thereof is very good.
具体实施方式detailed description 附图说明DRAWINGS
图1：化合物9和GS-7340在人血浆中的稳定性；Figure 1: Stability of Compound 9 and GS-7340 in human plasma;
图2：化合物9和GS-7340在人肝S9中的稳定性；Figure 2: Stability of Compound 9 and GS-7340 in human liver S9;
图3：肝脏中化合物9和GS-7340随时间变化情况；Figure 3: Compound 9 and GS-7340 in the liver as a function of time;
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；Figure 4: Tenofovir hydrolyzed by compound 11 and TAF in the liver over time;
图5：化合物11和TAF对HK-2细胞的增殖抑制活性评价。Figure 5: Evaluation of proliferation inhibitory activity of Compound 11 and TAF on HK-2 cells.
具体实施方式detailed description
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：In order to solve the above first technical problem, a novel antiviral drug of the present invention is a deuterated nucleotide analog, and the deuterated nucleotide analog is a compound as shown in I or a pharmaceutically acceptable compound thereof Salt:
Figure PCTCN2018079990-appb-000003
Figure PCTCN2018079990-appb-000003
其中among them
R 1、R 3和R 5独立地为氢或氘； R 1 , R 3 and R 5 are independently hydrogen or deuterium;
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 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为芳基或芳烷基； R 4 is an aryl or aralkyl group;
R 6为烷基或氘代烷基； R 6 is alkyl or haloalkyl;
R 7为烷基或氘代烷基； R 7 is alkyl or haloalkyl;
X为O、S或Se；Y为O、S或NH；X is O, S or Se; Y is O, S or NH;
所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。 At least one of the groups R 1 , R 3 , R 5 , R 6 or R 7 contains ruthenium.
优选的，所述R 7为C 3烷基或C 3氘代烷基。 Preferably, the R 7 is a C 3 alkyl group or a C 3 halogenated alkyl group.
优选的，所述R 7为异丙基或氘代异丙基。 Preferably, the R 7 is isopropyl or deuterated isopropyl.
优选的，所述R 7为氘代烷基。 Preferably, the R 7 is a haloalkyl group.
优选的，所述R 6为甲基或氘代甲基。 Preferably, the R 6 is a methyl group or a deuterated methyl group.
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。 Preferably, the R 2 is an amino group, a linear or branched or cyclic C 1 -C 6 alkylamino group.
优选的，所述R 4为苯基。 Preferably, the R 4 is a phenyl group.
优选的，所述X和Y均为O。Preferably, both X and Y are 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。Preferably, the pharmaceutically acceptable salt is a hydrochloride, a sulfate, a fumarate, a succinate, a methanesulfonate or a sulfonate.
更优选的，所述药学上可以接受的盐为富马酸盐。More preferably, the pharmaceutically acceptable salt is a fumarate salt.
优选的，所述氘代核苷酸类似物为下列化合物之一：Preferably, the deuterated nucleotide analog is one of the following compounds:
Figure PCTCN2018079990-appb-000004
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。In order to solve the second technical problem of the present invention, the antiviral pharmaceutical composition comprises the above-described deuterated nucleotide analog or various crystal forms, hydrates or solvates of the deuterated nucleotide analog. .
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。A pharmaceutically acceptable excipient or an auxiliary ingredient may be added to the antiviral pharmaceutical composition.
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。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.
优选的，所述病毒为乙型肝炎病毒、丙型肝炎病毒。Preferably, the virus is hepatitis B virus or hepatitis C virus.
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。The embodiments of the present invention are further described in conjunction with the embodiments, and are not intended to limit the scope of the embodiments.
实施例1Example 1
化合物11和12的制备Preparation of compounds 11 and 12
Figure PCTCN2018079990-appb-000005
Figure PCTCN2018079990-appb-000005
化合物3的合成：Synthesis of Compound 3:
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] -。 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%). 1 H NMR (400MHz, D 2 O): 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 [MH] - .
化合物6的合成：Synthesis of Compound 6:
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] +。 Take 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. After completion of the reaction, the mixture was washed with water (100 ml × 3), and purified by silica gel column to obtain 1.47 g of N-benzyloxy-L-alanine isopropyl isopropyl ester (6) in a yield of 54%. 1 H 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：Synthesis of Compound 7:
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] +。 Take N-benzyloxy-L-alanine deuterated isopropyl ester (6) 1g, Pd / C200mg in a 200ml two-necked flask, add 50ml of methanol, replace with nitrogen three times, hydrogen replacement 3 times, room temperature reaction overnight . After the completion of the reaction, the mixture was filtered, and the solvent was evaporated under reduced pressure to give 415 mg of EtOAc (yield: 1 H NMR (400 MHz, 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：Synthesis of Compound 8:
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。Compound 3 (3.63 g, 10 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 2 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 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%. 1 H 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：Synthesis of compounds 9 and 10:
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。The chromatograph was prepared using a Waters SFC-80 model, and Compound 8 was resolved according to the following chromatographic conditions.
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。 Column: MS-OD 20×250 mm, 5 μm preparative column; column temperature: 35 ° C; mobile phase: CO 2 /IPA=80/20; flow rate: 40 ml/min; cycle time: 5.5 min; back pressure: 120 Bar; Wavelength: 260 nm; injection volume: 1 mL; two peaks were collected, and the mobile phase was removed under reduced pressure to obtain a target product. The amount of the solution was 4.2 g, and 1.2 g of the compound 9 and the compound 10 were respectively obtained. The optical purity analysis of Compound 9 and Compound 10 was carried out on a Waters UPC chromatograph. The results are shown in Tables 1 and 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 Column type: MS-OD 4.6×150mm, 5μm analytical column; instrument model: Waters UPC; sample solvent: MeOH; column: MS-OD; column size: 4.6×150mm mobile phase: CO 2 /IPA=90/10 Flow rate: 2.5 mL/min back pressure: 2000 Psi; pressure drop: 510 Psi column temperature: 45 ° C; detection wavelength: 260 nm; injection volume: 20 μL
表1 化合物9的色谱图数据Table 1 Chromatogram data of compound 9
出峰时间Peak time 百分含量/％Percentage /% 不对称度Asymmetry
13.39713.397 100100 1.2191.219
表2 化合物10的色谱图数据Table 2 Chromatogram data of compound 10
出峰时间Peak time 百分含量/％Percentage /% 不对称度Asymmetry
18.592518.5925 100100 1.31.3
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：The above results show that the obtained Compound 9 and Compound 10 have a very high optical purity. The nuclear magnetic resonance spectrum data is as follows:
化合物9：Compound 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] +。 1 H 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); 13 C NMR (100 MHz, CDCl 3 ): δ 173.59, 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：Compound 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] +。 1 H 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). 13 C NMR (100 MHz, CDCl 3 ): δ 173.35, 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：Synthesis of Compound 11:
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％。Compound 9 (4.83 g, 10 mmol) and fumaric acid (1.05 g, 9 mmol) were placed in a 200 ml flask, and 100 ml of acetonitrile was added thereto, and the mixture was heated to reflux until the solid was completely dissolved. The mixture was filtered while hot, and the filtrate was stirred at 5 ° C for 12 hours. A white precipitate precipitated, which was filtered and washed with EtOAc EtOAc. Drying gave 4.5 g of a white solid 11 in a yield of 83.3%.
化合物12的合成：Synthesis of Compound 12:
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％。Compound 10 (4.83 g, 10 mmol) and fumaric acid (1.05 g, 9 mmol) were placed in a 200 ml flask, and 100 ml of acetonitrile was added thereto, and the mixture was heated to reflux until the solid was completely dissolved, and then filtered, and the filtrate was stirred at 5 ° C for 12 hours. A white precipitate precipitated, which was filtered and washed with EtOAc EtOAc. Drying gave 4.1 g of a white solid 12 in a yield of 75.9%.
实施例2Example 2
化合物17和18的合成Synthesis of Compounds 17 and 18
Figure PCTCN2018079990-appb-000006
Figure PCTCN2018079990-appb-000006
化合物13的合成：Synthesis of Compound 13:
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] -。 Compound 3 (363 mg, 1 mmol) was placed in a 50 ml sealed tube, 36 mg of 10% palladium carbon, 10 ml of heavy water was added, and the mixture was replaced with nitrogen three times, three times with hydrogen, and reacted at 125 ° C for 24 hours. The mixture was cooled to room temperature, filtered, and the solution was evaporated under reduced pressure. The solvent was evaporated to give a white solid, which was washed with a small amount of methanol and filtered to yield 25 g of product as white powder. MS: 364.11 [MH] - .
化合物14的合成：Synthesis of Compound 14:
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。Compound 13 (365 mg, 1 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 0.5 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid.
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] +。 Take L-alanine isopropyl ester hydrochloride (640 mg, 4 mmol) in a 50 ml round bottom flask, add 20 ml of dichloromethane, triethylamine (5 mmol), and stir at room temperature for 30 minutes. In one 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 71.8 mg of pale yellow solid 14 yield: 15%. 1 HNMR (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：Synthesis of compounds 15 and 16:
化合物15和16的合成步骤与化合物9和化合物10相同。The synthetic steps of the compounds 15 and 16 were the same as those of the compound 9 and the compound 10.
化合物17和18的合成：Synthesis of compounds 17 and 18:
化合物17和18的合成步骤与化合物11和化合物12相同。The synthetic steps of the compounds 17 and 18 were the same as those of the compound 11 and the compound 12.
实施例3Example 3
化合物25和26的合成：Synthesis of compounds 25 and 26:
Figure PCTCN2018079990-appb-000007
Figure PCTCN2018079990-appb-000007
化合物20的合成：Synthesis of Compound 20:
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] -。 Compound 1 (1.44 g, 5 mmol) was taken in a 100 ml round bottom flask, and then deuterated phenol 19 (990 mg, 10 mmol) and N-methylpyrrolidone 30 ml were added and heated to 85 °C. The mixture was refluxed with nitrogen for 10 hours, and the reaction was completed by TLC. Pyridine was distilled off under reduced pressure, and 0.8 ml of triethylamine was added. After the solid was completely dissolved, DCC (1.54 g, 7.5 mmol) was slowly added at 100 ° C for 16 hours. After cooling to room temperature, 20 ml of water was added, and the solid was filtered, and the filtrate was evaporated to dryness to give crystalljjjjjjjjjjjjjjjjjjjjjjj After 2 times, pH = 3 was adjusted with dilute hydrochloric acid, a white solid was precipitated, filtered, and the solid was washed with 3 ml of methanol to give compound 20. MS: 367.08 [MH] - .
化合物21的合成：Synthesis of Compound 21:
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] -。 Compound 20 (368 mg, 1 mmol) was placed in a 50 ml sealed tube, 36 mg of 10% palladium carbon, 10 ml of heavy water was added, the nitrogen was replaced three times, the hydrogen was replaced three times, and the reaction was carried out at 125 ° C for 24 hours. After cooling to room temperature, filtration, the solvent was evaporated under reduced pressure to give a white solid, which was washed with a small amount of methanol and filtered to afford 248.1 g of product as white powder. MS: 369.13 [MH] - .
化合物22的合成：Synthesis of Compound 22:
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。Compound 21 (370 mg, 1 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 0.5 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 Take L-alanine isopropyl ester hydrochloride (640 mg, 4 mmol) in a 50 ml round bottom flask, add 20 ml of dichloromethane, triethylamine (5 mmol), and stir at room temperature for 30 minutes, then add the reaction droplets to the above. In one step of the crude product, 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 91.9 mg of pale yellow solid (yield: 19%). MS: 484.26 [M + H] + .
化合物23和24的合成：Synthesis of compounds 23 and 24:
化合物23和24的合成步骤与化合物9和化合物10相同。The synthetic steps of Compounds 23 and 24 were the same as those of Compound 9 and Compound 10.
化合物25和26的合成：Synthesis of compounds 25 and 26:
化合物25和26的合成步骤与化合物11和化合物12相同。The synthetic steps of the compounds 25 and 26 were the same as those of the compound 11 and the compound 12.
实施例4Example 4
化合物32和33的合成：Synthesis of compounds 32 and 33:
Figure PCTCN2018079990-appb-000008
Figure PCTCN2018079990-appb-000008
化合物28的合成：Synthesis of Compound 28:
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] +。 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] + .
化合物29的合成：Synthesis of Compound 29:
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。Compound 21 (370 mg, 1 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 0.5 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 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] + .
化合物30和31的合成：Synthesis of Compounds 30 and 31:
化合物30和31的合成步骤与化合物9和化合物10相同。The synthetic steps of Compounds 30 and 31 were the same as those of Compound 9 and Compound 10.
化合物32和33的合成：Synthesis of compounds 32 and 33:
化合物32和33的制备步骤与化合物11和化合物12相同。The preparation steps of the compounds 32 and 33 were the same as those of the compound 11 and the compound 12.
实施例5Example 5
化合物38和39的合成：Synthesis of compounds 38 and 39:
Figure PCTCN2018079990-appb-000009
Figure PCTCN2018079990-appb-000009
化合物34的合成：Synthesis of Compound 34:
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] +。 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] + .
化合物35的合成：Synthesis of Compound 35:
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。Compound 21 (370 mg, 1 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 0.5 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 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] + .
化合物36和37的合成：Synthesis of compounds 36 and 37:
化合物36和37的合成步骤与化合物9和化合物10相同。The synthetic procedures for compounds 36 and 37 are the same as for compound 9 and compound 10.
化合物38和39的合成：Synthesis of compounds 38 and 39:
化合物38和39的合成步骤与化合物11和化合物12相同。The synthetic steps of Compounds 38 and 39 were the same as those of Compound 11 and Compound 12.
实施例6Example 6
化合物43和44的合成：Synthesis of compounds 43 and 44:
Figure PCTCN2018079990-appb-000010
Figure PCTCN2018079990-appb-000010
化合物40的合成：Synthesis of Compound 40:
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。Compound 13 (365 mg, 1 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 0.5 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 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] + .
化合物41和42的合成：Synthesis of compounds 41 and 42:
化合物41和42的合成步骤与化合物9和化合物10相同。The synthetic steps of Compounds 41 and 42 were the same as those of Compound 9 and Compound 10.
化合物43和44的合成：Synthesis of compounds 43 and 44:
化合物43和44的合成步骤与化合物11和化合物12相同。The synthetic steps of the compounds 43 and 44 were the same as those of the compound 11 and the compound 12.
实施例7Example 7
化合物48和49的合成：Synthesis of Compounds 48 and 49:
Figure PCTCN2018079990-appb-000011
Figure PCTCN2018079990-appb-000011
化合物45的合成：Synthesis of Compound 45:
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。Compound 3 (3.63 g, 10 mmol) was placed in a 50 ml round bottom flask, and 20 ml of dichloromethane, 0.5 ml of DMF, and 2 ml of oxalyl chloride were added, and the mixture was reacted at room temperature for 10 hours. The solvent was evaporated under reduced pressure to give a pale yellow solid, which was applied directly to the next step.
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] +。 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] + .
化合物46和47的合成：Synthesis of compounds 46 and 47:
化合物46和47的合成步骤与化合物9和化合物10相同。The synthetic procedures for compounds 46 and 47 are the same as for compound 9 and compound 10.
化合物48和49的合成：Synthesis of Compounds 48 and 49:
化合物48和49的合成步骤与化合物11和化合物12相同。The synthetic procedures for compounds 48 and 49 are the same as for compound 11 and compound 12.
试验例1Test example 1
体外抗HBV活性In vitro anti-HBV activity
(1)细胞毒性试验(1) Cytotoxicity test
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)。 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 μl 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. At the same time, the negative control group in which the inoculated cells were added to the culture medium and the non-inoculated cells were added to the blank control group of the medium alone and the positive control group GS-7340 fumarate (TAF), and the culture was continued for 72 hours. For MTT assay, half toxic concentration (CC 50) of cells is calculated.
(2)体外抗HBV活性(2) In vitro anti-HBV activity
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：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:
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。 a. 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.
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。b. 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.
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。c. Fresh medium containing different concentrations of test compound and positive control was replaced on the 3rd and 6th day after dosing.
d.收集培养液的上层清液，-20℃冷冻备用。d. Collect the supernatant of the culture solution and freeze at -20 °C for use.
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。 e. Quantitative PCR to detect the content of HBV-DNA, according to the kit instructions. Based on the detected HBV-DNA copy number of each sample, the inhibition of HBV-DNA replication by HepG2.2.15 was calculated for each compound, and the half effective concentration EC 50 of each compound was calculated by SPSS software.
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)。 From the results in Table 3, 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 μM).
表3 化合物对HBV DNA的抑制作用Table 3 Inhibition of HBV DNA by Compounds
Figure PCTCN2018079990-appb-000012
Figure PCTCN2018079990-appb-000012
Figure PCTCN2018079990-appb-000013
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Experimental Example 2 Stability of Compound 9 in Human Plasma and Human Liver S9
1.实验目的1. Experimental purpose
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。The purpose of this experiment was to investigate the stability of Compound 9 in Example 1 in human plasma, human liver S9, and to compare it with GS-7340.
2.材料与方法2. Materials and methods
2.1供试品信息2.1 Test information
Figure PCTCN2018079990-appb-000014
Figure PCTCN2018079990-appb-000014
2.2实验设计2.2 experimental design
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。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 μM, and the concentration of the experimental drug in liver S9 was 10 μM. Samples were taken at different time points after dosing before dosing.
2.3样品采集与制备2.3 sample collection and preparation
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。Take the appropriate amount of compound 9 or GS-7340 to make the experimental drug concentration 2μM; dilute the human liver S9 to the experimental concentration; and add the appropriate amount of compound 9 or GS-7340, so that the concentration of the experimental drug is 10μM, after mixing Add the A and B solutions to start the reaction. Before the addition, incubate the appropriate reaction solution for 5 min, 10 min, 20 min, 30 min, 40 min, 60 min, 80 min, 100 min, 120 min, 150 min, 180 min, 210 min, 240 min, and use 4 volumes of acetonitrile. (HPLC) The protein was quenched and precipitated, centrifuged at 130,000 rpm for 15 min, and the supernatant was then stored in a refrigerator until LC-MS/MS analysis.
2.3样品分析2.3 sample analysis
应用LC-MS/MS方法分析化合物9和GS-7340在样品中的浓度。The concentration of Compound 9 and GS-7340 in the sample was analyzed by LC-MS/MS method.
3.实验结果3. Experimental results
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。The stability of Compound 9 and GS-7340 in human plasma and human liver S9 is shown in Figures 1 and 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。The above results indicate that Compound 9 and GS-7340 are stable in human plasma, while in liver S9, Compound 9 is more stable than GS-7340.
试验例3肝脏和血液中的分布实验Test Example 3 Distribution experiment in liver and blood
1.实验目的1. Experimental purpose
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。The distribution of Compound 11 and TAF prepared in Example 1 in the liver and blood of mice was examined by single gavage.
2.材料与方法2. Materials and methods
2.1供试品信息2.1 Test information
Figure PCTCN2018079990-appb-000015
Figure PCTCN2018079990-appb-000015
2.2实验设计和动物准备2.2 Experimental design and animal preparation
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。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.
2.3制剂配制及给药2.3 formulation preparation and administration
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。An appropriate amount of Compound 11 and TAF were accurately weighed and placed in a physiological saline solution of a suitable concentration. The experimental animals were administered a physiological saline solution of Compound 11 and TAF in a single gavage at 25 mg/kg.
2.4样品采集与制备2.4 sample collection and preparation
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。The administered animals were bled at the corresponding time after administration, and the liver was taken after sacrifice. At each time point, 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 μl centrifuge tube. 400 μl 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 μl of homogenate was added to 800 μl 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.
2.5样品分析2.5 sample analysis
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。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. In this experiment, the lower limit of detection (LLOQ) of tenofovir in mouse plasma was 1.00 ng/mL, and the upper limit of quantitation (ULOQ) was 10000 ng/mL.
3.实验结果3. Experimental results
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。By mass spectrometry, it was found that in the blood samples from 10 minutes to 48 hours after intragastric administration of Compound 11 and TAF, the free form of Compound 11 in vivo (Compound 9) and the free form of TAF in vivo (GS-7340) and their The tenofovir content of the hydrolysate was below the detection limit. The changes in the free form of Compound 11 in vivo (Compound 9) and the free form of TAF in vivo (GS-7340) and its hydrolysate tenofovir in the liver are shown in Figures 3 and 4. Figure 3 shows that during the first hour, the concentration of Compound 9 gradually decreased with time and then fell below the detection limit. The concentration of GS-7340 in the liver has been below the detection limit. 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.
试验例4肾毒性研究Test Example 4 Nephrotoxicity study
1.实验目的1. Experimental purpose
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。The effects of Compound 11 and TAF prepared in Example 1 on human renal tubular epithelial cell HK-2 neutrophil gelatinase-associated lipocalin (NGAL) were examined.
2.材料与方法2. Materials and methods
2.1供试品信息2.1 Test information
Figure PCTCN2018079990-appb-000016
Figure PCTCN2018079990-appb-000016
2.2实验设计2.2 experimental design
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。First, we evaluated the ability of Compound 11 and TAF to inhibit HK-2 cell proliferation, and then examined the effect of compounds on the secretion of NGAL from HK-2 cells at a non-cytotoxic concentration.
(1)抑制细胞增殖活性评价(1) Evaluation of inhibition of cell proliferation activity
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。HK-2 cells were plated at a density of 1500/well/100 μL. After 24 h, 100 μL 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 μM. After 72 h of drug administration, the cell proliferation inhibitory activity was measured by MTT assay. The results are shown in Figure 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。According to Figure 5, Compound 11 and TAF were below 200 μM and showed no cytotoxicity within 72 hours.
(2)对HK-2分泌NGAL的影响(2) Impact on HK-2 secretion of NGAL
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。HK-2 cells were plated at a density of 1000/well/100 μL. After adhering, the supernatant was aspirated, and 100 μL of each of the compounds 11 and TAF prepared in fresh medium was added to each well at a concentration of 100 μM. 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.
表4 化合物11对HK-2细胞分泌NGAL的影响Table 4 Effect of Compound 11 on the secretion of NGAL from HK-2 cells
Figure PCTCN2018079990-appb-000017
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。As can be seen from Table 4, the level of NGAL secreted by Compound 11-treated HK-2 cells was not affected as the duration of drug action was prolonged, whereas the level of NGAL secreted by TAF-treated cells increased with time.
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；NGAL is a secreted protein that is rarely expressed in the kidneys. However, when the renal tubules are stimulated to be damaged, the damaged renal tubular epithelial cells induce the apoptosis of neutrophils infiltrating the tubulointerstitial by expressing NGAL to protect the kidney tissue from attack; In contrast, when renal tubular epithelial cells are damaged, 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. Studies have found that 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;
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。The above studies show that Compound 11 is safer than TAF at the cellular level.

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：A deuterated nucleotide analog, characterized in that the deuterated nucleotide analog is a compound as shown in I or a pharmaceutically acceptable salt thereof:
    Figure PCTCN2018079990-appb-100001
    Figure PCTCN2018079990-appb-100001
    其中，R 1、R 3和R 5独立地为氢或氘； Wherein R 1 , R 3 and R 5 are independently hydrogen or deuterium;
    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 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为芳基或芳烷基； R 4 is an aryl or aralkyl group;
    R 6为烷基或氘代烷基； R 6 is alkyl or haloalkyl;
    R 7为烷基或氘代烷基； R 7 is alkyl or haloalkyl;
    X为O、S或Se；Y为O、S或NH；X is O, S or Se; Y is O, S or NH;
    所述R 1、R 3、R 5、R 6或R 7中至少一个基团含有氘。 At least one of the groups R 1 , R 3 , R 5 , R 6 or R 7 contains ruthenium.
  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。 The deuterated nucleotide analog according to claim 1, wherein the R 7 is a C 3 alkyl group or a C 3 deuterated alkyl group.
  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。 The deuterated nucleotide analog according to claim 2, wherein the R 7 is isopropyl or deuterated isopropyl.
  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。 The deuterated nucleotide analog according to claim 1, wherein R 7 is a haloalkyl group.
  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。 The deuterated nucleotide analog according to claim 2, wherein the R 6 is a methyl group or a deuterated methyl group.
  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。 The deuterated nucleotide analog according to claim 1, wherein the R 2 is an amino group, a linear or branched or cyclic C 1 -C 6 alkylamino group.
  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。 The deuterated nucleotide analog according to claim 1, wherein the R 4 is a phenyl group.
  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。The deuterated nucleotide analog according to claim 1, wherein both X and Y are 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。The deuterated nucleotide analog according to claim 1, wherein the pharmaceutically acceptable salt is a hydrochloride, a sulfate, a fumarate, a succinate, a methanesulfonate or a sulfonate. Acid salt.
  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。The deuterated nucleotide analog according to claim 9, wherein the pharmaceutically acceptable salt is a fumarate.
  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：The deuterated nucleotide analog according to claim 1, wherein the deuterated nucleotide analog is one of the following compounds:
    Figure PCTCN2018079990-appb-100002
    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。An antiviral pharmaceutical composition comprising the deuterated nucleotide analog of any one of claims 1 to 11 or various crystals of the deuterated nucleotide analog Type, hydrate or solvate.
  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。The use of the deuterated nucleotide analog according to any one of claims 1 to 11 or the antiviral pharmaceutical composition according to claim 12 for the preparation of an antiviral drug, wherein the virus is preferably hepatitis B virus Hepatitis C virus.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113121600A (en) * 2021-03-31 2021-07-16 澳门科技大学 Remdesivir dimer, preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104710477A (en) * 2013-12-16 2015-06-17 安徽贝克联合制药有限公司 Tenofovir cyclic phosphonate compound and pharmaceutically acceptable salt thereof, and preparation methods and applications thereof
CN105669751A (en) * 2015-03-05 2016-06-15 洛阳聚慧医药科技有限公司 Preparation of non-cyclic nucleotide phosphoamides and salts thereof and application of non-cyclic nucleotide phosphoamides and salts thereof in aspect of antivirus

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017148290A1 (en) * 2016-03-01 2017-09-08 深圳市塔吉瑞生物医药有限公司 Substituted adenine compound and pharmaceutical composition thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104710477A (en) * 2013-12-16 2015-06-17 安徽贝克联合制药有限公司 Tenofovir cyclic phosphonate compound and pharmaceutically acceptable salt thereof, and preparation methods and applications thereof
CN105669751A (en) * 2015-03-05 2016-06-15 洛阳聚慧医药科技有限公司 Preparation of non-cyclic nucleotide phosphoamides and salts thereof and application of non-cyclic nucleotide phosphoamides and salts thereof in aspect of antivirus

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113121600A (en) * 2021-03-31 2021-07-16 澳门科技大学 Remdesivir dimer, preparation method and application thereof

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