WO2017118355A1 - Nucleotide derivative of deuterated hcv ns5b inhibitor and use thereof - Google Patents

Nucleotide derivative of deuterated hcv ns5b inhibitor and use thereof Download PDF

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WO2017118355A1
WO2017118355A1 PCT/CN2016/113815 CN2016113815W WO2017118355A1 WO 2017118355 A1 WO2017118355 A1 WO 2017118355A1 CN 2016113815 W CN2016113815 W CN 2016113815W WO 2017118355 A1 WO2017118355 A1 WO 2017118355A1
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mixture
compound
hepatitis
virus
inhibitors
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PCT/CN2016/113815
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French (fr)
Chinese (zh)
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王喆
曾志宏
江荣珍
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上海长森药业有限公司
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Priority to CN201680077803.4A priority Critical patent/CN108473525A/en
Publication of WO2017118355A1 publication Critical patent/WO2017118355A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the invention belongs to the field of medicine, and particularly relates to a novel class of deuterated nucleoside (amino) phosphate compounds, compositions containing the same, and preparation of the compounds and medicaments thereof for HCV (Hepatitis C Virus) infectious diseases the use of.
  • HCV Hepatitis C Virus
  • Hepatitis C virus infection causes chronic liver diseases such as cirrhosis and liver cancer. Hepatitis C virus infection is one of the major infectious diseases. According to the World Health Organization, there are 170 million hepatitis C victims worldwide, and there are nearly 39 million hepatitis C infected people in China. Studies to date have shown that hepatitis C virus (HCV) is the leading cause of most non-A, non-B hepatitis. Hepatitis C virus is a flavivirus (F1aviviridae) positive single-stranded RNA virus. The standard protocol for the treatment of HCV was previously a combination of interferon or PEG interferon A and ribavirin. However, the side effects of interferon are obvious, and the side effects of interferon and ribavirin are combined to cause hemolysis, anemia and fatigue.
  • HCV hepatitis C virus
  • BMS BMS-986094 is a guanosine nucleotide phosphate prodrug for clinical treatment of hepatitis C.
  • the patient terminated his clinical trial after death due to heart failure.
  • Idenix's IDX19368 also stopped clinical trials. Therefore, there is still a need for effective compounds for the treatment and/or prevention of HCV infection.
  • R 1 H or D
  • R 2 H or D
  • R 3 H or D
  • R 4 H or D
  • R 5 CH 3 or CD 3
  • R 6 H or D
  • X -O Or -NH.
  • the compound is selected from the group consisting of:
  • the position of the ⁇ (D) generation in the compounds of the present application has at least 50% D, such as at least 90% D, 50-90% D.
  • the position of the ⁇ (D) generation in the compounds of the present application has at least 95% D.
  • the present application also provides an isomer mixture of the compounds described herein, wherein the two isomers of the mixture each comprise 50%, the mixture being as claimed in claim 5.
  • the application provides a compound of formula I, including the specific compounds listed, for use in the treatment or prevention of HCV infection.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a compound or a mixture of isomers of the present application, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present application further comprises one or more additional active ingredients.
  • the one or more additional active ingredients in the pharmaceutical composition of the present application are selected from the group consisting of (1) an immunomodulatory agent; (2) a hepatitis C virus protease (NS3) inhibitor; Hepatitis C virus NS4b inhibitor; (4) Hepatitis C virus NS5a inhibitor; (5) Hepatitis C virus polymerase (NS5b) inhibitor; (5) No Nucleosides and nucleoside derivatives belonging to (2)-(5); (6) Hepatitis B virus (HBV) inhibitors; (7) Human immunodeficiency virus (HIV) inhibitors; (8) Cancer drugs; 9) an anti-inflammatory drug; or (10) other compounds not belonging to the above (1) to (9).
  • the one or more additional active ingredients are selected from the group consisting of HCV NS3 protease inhibitors, HCV NS5A inhibitors, HCV NS5B polymerase inhibitors, interferons or interferon derivatives, ribavirin.
  • HCV NS3 protease inhibitors HCV NS5A inhibitors
  • HCV NS5B polymerase inhibitors interferons or interferon derivatives
  • ribavirin are selected from the group consisting of HCV NS3 protease inhibitors, HCV NS5A inhibitors, HCV NS5B polymerase inhibitors, interferons or interferon derivatives, ribavirin.
  • compositions of the present application include, but are not limited to, oral dosage forms and parenteral administration dosage forms.
  • the pharmaceutical composition may be an oral tablet, capsule, pill, powder, sustained release formulation, solution and suspension, sterile solution, suspension or emulsion for parenteral injection.
  • the pharmaceutical composition is in a unit dosage form suitable for single administration of precise dosages.
  • the amount of the compound ranges from about 0.001 mg/kg body weight/day to about 1000 mg/kg body weight/day. In other embodiments, the amount of the compound ranges from about 0.5 mg/kg body weight/day to about 50 mg/kg body weight/day. In some embodiments, the amount of the compound is from about 0.001 g/day to about 7 g/day.
  • the amount of the compound is from about 0.002 g/day to about 6 g/day. In other embodiments, the amount of the compound is from about 0.005 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.01 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.02 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.05 g/day to about 2.5 g/day. In other embodiments, the amount of the compound is from about 0.1 g/day to about 1 g/day. In other embodiments, a dose level below the lower limit of the above range may already be sufficient.
  • the compound is administered in a single dose, once a day. In other embodiments, the compound is administered in multiple doses more than once a day. In some embodiments, the compound is administered twice daily. In other embodiments, the compound is administered three times a day. In other embodiments, the compound is administered four times a day. In other embodiments, the compound is administered more than four times a day. In some embodiments, the individual to which the pharmaceutical composition is administered is a mammal. In other embodiments, the mammal is a human. In other embodiments, if the pharmaceutical composition comprises a plurality of active ingredients, the active ingredients may be combined in a single dosage form, and they may also be in separate dosage forms without mixing with one another.
  • the application provides the use of a compound or mixture of the present application in the manufacture of a medicament for the treatment or prevention of HCV infection.
  • the application provides the use of a compound or mixture of the present application in combination with one or more other active ingredients described herein for the manufacture of a medicament for the treatment or prevention of HCV infection.
  • the present application provides a method of treating or preventing an HCV infection, the method comprising administering an effective amount of a compound or mixture or pharmaceutical composition of the present application to a subject in need thereof.
  • the above method of treating or preventing an HCV infection comprises administering an effective amount of a compound or mixture of the present application in combination with one or more other active ingredients described herein to a subject in need thereof Tester.
  • the HCV infections described herein include chronic hepatitis C.
  • the terms "subject,” “patient,” or “individual” refer to an individual, including a mammal, and a non-mammal, having a disease, disorder, condition, or the like.
  • mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (eg, chimpanzees and other mites and monkeys); livestock, such as cattle, horses, sheep, goats, pigs; domesticated Animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs.
  • non-human mammals include, but are not limited to, birds and fish.
  • the mammal is a human.
  • treatment includes alleviating, alleviating or ameliorating the symptoms of a disease or condition, preventing other symptoms, ameliorating or preventing a potential metabolic cause of the symptoms, inhibiting the disease or condition, such as preventing the progression of the disease or condition, Ameliorating a disease or condition, ameliorating the disease or condition, alleviating the symptoms caused by the disease or condition, or terminating the symptoms of the disease or condition, and further, the term includes the purpose of prevention.
  • the term also includes obtaining a therapeutic effect and/or a preventive effect.
  • the therapeutic effect refers to curing or ameliorating the underlying disease to be treated.
  • the healing or amelioration of one or more physiological symptoms associated with a underlying disease is also a therapeutic effect, for example, although the patient may still be affected by the underlying disease, an improvement in the patient's condition is observed.
  • the composition can be administered to a patient at risk of developing a particular disease, or even if a diagnosis of the disease has not been made, the composition is administered to a patient who develops one or more physiological symptoms of the disease.
  • an "effective amount,” “therapeutically effective amount,” or “pharmaceutically effective amount,” as used herein, refers to at least one agent or compound that, after administration, is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent. The amount. The result can be a reduction and/or alleviation of signs, symptoms or causes, or any other desired change in the biological system.
  • an "effective amount” for treatment is an amount of a composition comprising a compound disclosed herein that is required to provide a significant conditional relief effect in the clinic.
  • An effective amount suitable for any individual case can be determined using techniques such as dose escalation testing.
  • administering refers to a method of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, duodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical and rectal administration.
  • parenteral injections including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion
  • topical and rectal administration topical and rectal administration.
  • the techniques of administration of the compounds and methods described herein are well known to those skilled in the art, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, those discussed in Pa.
  • the compounds and compositions discussed herein are administered orally.
  • pharmaceutically acceptable refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present application, and is relatively non-toxic, ie, the substance can be administered to an individual without causing undesirable organisms. The reaction or in an undesirable manner interacts with any of the components contained in the composition.
  • composition refers to a biologically active compound, optionally in admixture with at least one pharmaceutically acceptable chemical component, including but not limited to a carrier.
  • carrier refers to a relatively non-toxic chemical compound or agent that facilitates the introduction of a compound into a cell or tissue.
  • pharmaceutically acceptable salt refers to a salt that retains the biological effectiveness of the free acid and free base of the specified compound, and which has no adverse effects biologically or otherwise.
  • the compounds of the present application also include pharmaceutically acceptable salts.
  • a pharmaceutically acceptable salt refers to a form in which a base group in a parent compound is converted into a salt.
  • stereoisomer refers to an isomer produced by the different arrangement of atoms in a molecule in space.
  • the compounds of formula I contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereostructures and mixtures of Formula I, including racemic mixtures, are part of the current application.
  • the mixture of diastereomers can be separated into individual diastereomers, based on their different physicochemical properties, using well-known means, for example, resolution of the enantiomers can be carried out with appropriate optically active substances (eg chirality)
  • the alcohol or Mosher ⁇ s molyl chloride reaction is converted to a diastereomer which is separated and converted (e.g., hydrolyzed) to the corresponding single isomer.
  • Some of the compounds of Formula 1 may be atropisomers (e.g., substituted aryl groups) are also part of this application.
  • Enantiomers can also be separated using a chiral column.
  • the compounds of formula I may exist in different tautomeric forms, and such forms are embraced within the scope of the present application. For example, compounds in the form of keto-enol and imine-enamine.
  • the method for synthesizing the compounds in the present application includes, but is not limited to, the following reaction formulas and reaction steps:
  • EtOAc EtOAc
  • EtOAc eluted eluted (20ml)
  • the mixture was extracted, the aqueous phase and solid were combined, ethyl acetate was added, and 4N HCl aqueous solution was added dropwise with stirring to adjust pH ⁇ 2, and the organic phase was separated, and concentrated to give 1.19 g of a red foamy solid.
  • the compound 1f (93 mg, 0.261 mmol) was dissolved in 1.0 ml of THF, and a solution of t-BuMgCl (1M, 0.784 ml) in THF was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated, 0 to THF was added compound 11 (183mg, 0.418mmol) at the 5 °C (1.0 ml) solution, all the solid clear solution, warmed to room temperature stirred overnight, TLC tracking completion of the reaction was added saturated aqueous NH 4 Cl, EA, the organic phase was washed with The mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate and brine, dried over anhydrous magnesium sulfate, and filtered, and the filtrate was concentrated and then purified by column chromatography.
  • the eluent was n-heptane / ethyl
  • the compound 2c (130 mg, 0.47 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.41 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated.
  • the compound 3d (150 mg, 0.39 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.2 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated.
  • the compound 4e (150 mg, 0.39 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.2 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated.
  • Stable hepatitis C virus replicon GT1a and GT1b cell lines were transfected into Huh-7 cells by in vitro transcription of hepatitis C virus subgenomic GT1a (H77) and GT1b (Con1) replicon plasmid DNA into cells and selected by G418. And established.
  • the GT1a and GT1b stable cell lines were constructed by WuXi PharmaTech (Wuxi AppTech Huh7-GT1a (H77) and Huh7-GT1b (con1)).
  • Reagents or consumables DMEM medium, fetal bovine serum, streptomycin, MEM non-essential amino acids, glutamine, G418, 0.05% trypsin, DMSO, 96-well black cell culture plate, CellTiter-flour, Bright-Glo.
  • the compound was dissolved in DMSO to a 10 mmol/L concentrated stock solution and stored in a nitrogen cabinet. Gradient dilution of the compound: The compound was added to a 96-well plate with Echo, and each compound was subjected to a 1:3 serial dilution of 8 concentration points, double duplicate wells, and added to columns 3-10 of the 96-well plate. In column 11, only DMSO was added as an ineffective control well. The final volume of DMSO in each well was 607.5 nanoliters. See the layout below.
  • HCV GT1a or GT1b cells were digested from T150 cell culture flasks, and the cell density was adjusted to 65,843 cells/ml with cell culture medium, and then the cell suspension was added to a 96-well plate to which the compound had been added. The well was added to 121.5 microliters. The final density of cells in 96-well plates was 8,000 cells/well, and the final concentration of DMSO in each well was 0.5%. No cells were added to the 100% effective control wells in column 2. A 96-well plate containing cells and compounds was placed in 5% CO 2 and cultured at 37 ° C for 3 days.
  • CellTiter-Flour was used to detect the cell viability of each well in a 96-well plate: 40 ⁇ l of CellTitter-flour was added to each well, and then cultured at 37 ° C, 5% CO 2 for 1 hour, and then read with EnVision. The fluorescence signal (excitation light 405 nm, emission light 515 nm) was detected. The anti-hepatitis C virus activity of the compounds was examined by Bright-Glo: the supernatant of the 96-well plate in which the activity of the cells was detected was removed, and then 100 ⁇ l of the prepared 1X Bright-Glo reagent was added to each well, and the chemiluminescence signal was detected within 5 minutes.
  • ZPE Mean value of DMSO ineffective control well signal
  • HPE 100% effective control well (cell-free control well) signal mean
  • RFU Relative Fluorescence Unit
  • the representative compound represented by the formula (I) of the present application exhibits an EC50 (50% inhibition rate for the HCV GT1a and GT1b subgene replicon inhibition test, and the sofosbuvir raw material drug is a reference compound) As shown in the table below.

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Abstract

Provided in the present application are a nucleotide derivative of a deuterated HCV (Hepatitis C Virus) NS5b inhibitor, or a pharmaceutically acceptable salt, stereoisomer, tauromer inhibitor thereof, and a use thereof in the preparation of drugs for treating or preventing HCV infections. The inhibitor is represented by general formula (I).

Description

氘代HCV NS5b抑制剂核苷酸衍生物及其用途Deuterated HCV NS5b inhibitor nucleotide derivative and use thereof 技术领域Technical field
本发明属于医药领域，具体涉及一类新的氘代核苷(氨基)磷酸酯化合物、含有该化合物的组合物以及所述化合物的制备及其在HCV(Hepatitis C Virus)感染性疾病的药物中的用途。The invention belongs to the field of medicine, and particularly relates to a novel class of deuterated nucleoside (amino) phosphate compounds, compositions containing the same, and preparation of the compounds and medicaments thereof for HCV (Hepatitis C Virus) infectious diseases the use of.
背景技术Background technique
丙型肝炎病毒感染造成慢性肝病，如肝硬化和肝癌。丙肝病毒感染是主要传染病之一。根据世界卫生组织统计，全球有1.7亿丙型肝炎受害者，我国将近有3900万丙肝感染者。至今的各项研究表明，丙型肝炎病毒(HCV)是引起大多数非甲非乙型肝炎的主要病原体。丙型肝炎病毒是一种黄病毒科(F1aviviridae)正单链RNA病毒。治疗HCV的标准方案以前是干扰素或PEG干扰素A与利巴韦林的联合用药。但干扰素的副作用明显，干扰素和利巴韦林联用的副作用更大，可导致溶血、贫血和疲乏等。Hepatitis C virus infection causes chronic liver diseases such as cirrhosis and liver cancer. Hepatitis C virus infection is one of the major infectious diseases. According to the World Health Organization, there are 170 million hepatitis C victims worldwide, and there are nearly 39 million hepatitis C infected people in China. Studies to date have shown that hepatitis C virus (HCV) is the leading cause of most non-A, non-B hepatitis. Hepatitis C virus is a flavivirus (F1aviviridae) positive single-stranded RNA virus. The standard protocol for the treatment of HCV was previously a combination of interferon or PEG interferon A and ribavirin. However, the side effects of interferon are obvious, and the side effects of interferon and ribavirin are combined to cause hemolysis, anemia and fatigue.
近年来，药物研发工作在丙型肝炎的治疗中取得不小进展。例如吉利德公司的索非布韦(Sofosbuvir)2013年12月6日在美国FDA获批，其用于HCV口服治疗方案的药物，在用于特定基因型(2型、3型)慢性丙型肝炎的治疗时，可消除对传统注射药物干扰素的需求。In recent years, drug research and development has made great progress in the treatment of hepatitis C. For example, Sofitosbuvir of Gilead was approved by the US FDA on December 6, 2013. Its drug for oral treatment of HCV is used for specific genotypes (type 2, type 3) chronic type C. The treatment of hepatitis eliminates the need for traditional injectable drug interferons.
尽管在丙型肝炎的治疗上已获得相当进展，但也出现了一些困难和挫折。例如，BMS的BMS-986094是一种鸟苷核苷酸磷酸酯前药，用于丙型肝炎的临床治疗。2012年8月，病人因心脏衰竭去世后终止了其临床试验。Idenix的IDX19368也停止了临床试验。因此，目前仍需要用于治疗和/或预防HCV感染的有效化合物。Despite considerable progress in the treatment of hepatitis C, there have been some difficulties and setbacks. For example, BMS BMS-986094 is a guanosine nucleotide phosphate prodrug for clinical treatment of hepatitis C. In August 2012, the patient terminated his clinical trial after death due to heart failure. Idenix's IDX19368 also stopped clinical trials. Therefore, there is still a need for effective compounds for the treatment and/or prevention of HCV infection.
发明内容Summary of the invention
本发明的目的在于提供一种用于治疗和/或预防HCV感染的由以下通式I表示的化合物及其药学可接受的盐、立体异构体、互变异构体。It is an object of the present invention to provide a compound represented by the following formula I and a pharmaceutically acceptable salt, stereoisomer, tautomer thereof for use in the treatment and/or prevention of HCV infection.
Figure PCTCN2016113815-appb-000001
Figure PCTCN2016113815-appb-000001
其中，R1＝H或D；R2＝H或D；R3＝H或D；R4＝H或D；R5＝CH3或CD3；R6＝H或D；X＝-O或-NH。Wherein R 1 =H or D; R 2 =H or D; R 3 =H or D; R 4 =H or D; R 5 =CH 3 or CD 3 ;R 6 =H or D;X=-O Or -NH.
根据本申请的一些具体实施方式，所述化合物选自以下的化合物：According to some embodiments of the present application, the compound is selected from the group consisting of:
Figure PCTCN2016113815-appb-000002
Figure PCTCN2016113815-appb-000002
Figure PCTCN2016113815-appb-000003
Figure PCTCN2016113815-appb-000003
Figure PCTCN2016113815-appb-000004
Figure PCTCN2016113815-appb-000004
根据本申请的一些实施方式，本申请的化合物中氘(D)代的位置具有至少50％的D，例如至少90％的D、50-90％的D。According to some embodiments of the present application, the position of the 氘(D) generation in the compounds of the present application has at least 50% D, such as at least 90% D, 50-90% D.
根据本申请的一些实施方式，本申请的化合物中氘(D)代的位置具有至少95％的D。According to some embodiments of the present application, the position of the 氘(D) generation in the compounds of the present application has at least 95% D.
根据本申请的一些方面，本申请还提供了本申请所述化合物的异构体混合物，其中所述混合物中的两种异构体各占50％，所述混合物如权利要求5所述。According to some aspects of the present application, the present application also provides an isomer mixture of the compounds described herein, wherein the two isomers of the mixture each comprise 50%, the mixture being as claimed in claim 5.
根据本申请的一个方面，本申请提供了式I化合物(包括所列出的具体化合物)，其用于治疗或预防HCV感染。According to one aspect of the present application, the application provides a compound of formula I, including the specific compounds listed, for use in the treatment or prevention of HCV infection.
根据本申请的又一个方面，本申请提供了一种药物组合物，其包含作为活性成分的本申请的化合物或异构体的混合物，以及药学上可接受的载体。According to still another aspect of the present application, the present application provides a pharmaceutical composition comprising as an active ingredient a compound or a mixture of isomers of the present application, and a pharmaceutically acceptable carrier.
根据本申请的一些实施方式，本申请的药物组合物进一步包括一种或多种其他的活性成分。According to some embodiments of the present application, the pharmaceutical composition of the present application further comprises one or more additional active ingredients.
根据本申请的一些实施方式，本申请的药物组合物中的一种或多种其他的活性成分选自(1)免疫调节剂；(2)丙型肝炎病毒蛋白酶(NS3)抑制剂；(3)丙型肝炎病毒NS4b抑制剂；(4)丙型肝炎病毒NS5a抑制剂；(5)丙型肝炎病毒聚合酶(NS5b)抑制剂；(5)不 属于(2)-(5)之核苷和核苷衍生物；(6)乙型肝炎病毒(HBV)抑制剂；(7)人类免疫缺损病毒(HIV)抑制剂；(8)癌症药物；(9)抗发炎药物；或(10)不属于上述(1)-(9)之其他化合物。例如，其中所述一种或多种其他的活性成分选自HCV NS3蛋白酶抑制剂、HCV NS5A抑制剂、HCV NS5B聚合酶抑制剂、干扰素或干扰素衍生物、利巴韦林。这些活性成分可以被混合在一个单一剂型中，也可以不相互混合而分别处于不同的剂型中。According to some embodiments of the present application, the one or more additional active ingredients in the pharmaceutical composition of the present application are selected from the group consisting of (1) an immunomodulatory agent; (2) a hepatitis C virus protease (NS3) inhibitor; Hepatitis C virus NS4b inhibitor; (4) Hepatitis C virus NS5a inhibitor; (5) Hepatitis C virus polymerase (NS5b) inhibitor; (5) No Nucleosides and nucleoside derivatives belonging to (2)-(5); (6) Hepatitis B virus (HBV) inhibitors; (7) Human immunodeficiency virus (HIV) inhibitors; (8) Cancer drugs; 9) an anti-inflammatory drug; or (10) other compounds not belonging to the above (1) to (9). For example, wherein the one or more additional active ingredients are selected from the group consisting of HCV NS3 protease inhibitors, HCV NS5A inhibitors, HCV NS5B polymerase inhibitors, interferons or interferon derivatives, ribavirin. These active ingredients may be mixed in a single dosage form or may be in separate dosage forms without being mixed with each other.
本申请的药物组合物包括但不限于口服剂型和胃肠外给药剂型。在一些实施方式中，所述药物组合物可以是口服的片剂、胶囊、丸剂、粉剂、缓释制剂、溶液和悬浮液，用于胃肠外注射的无菌溶液、悬浮液或乳液。在其它实施方式中，所述药物组合物为适合单次施予精确剂量的单位剂型。在其它实施方式中，所述化合物的量在约0.001mg/kg体重/天-约1000mg/kg体重/天的范围内。在其它实施方式中，所述化合物的量的范围为约0.5mg/kg体重/天-约50mg/kg体重/天。在一些实施方式中，所述化合物的量为约0.001g/天-约7g/天。在其它实施方式中，所述化合物的量为约0.002g/天-约6g/天。在其它实施方式中，所述化合物的量为约0.005g/天-约5g/天。在其它实施方式中，所述化合物的量为约0.01g/天-约5g/天。在其它实施方式中，所述化合物的量为约0.02g/天-约5g/天。在其它实施方式中，所述化合物的量为约0.05g/天-约2.5g/天。在其它实施方式中，所述化合物的量为约0.1g/天-约1g/天。在其它实施方式中，低于上述范围下限的剂量水平可能已经是足够的。在其它实施方式中，可能需要高于上述范围上限的剂量水平。在一些实施方式中，以单剂量施用所述化合物，每天一次。在其它实施方式中，以多剂量施用所述化合物，每天不只一次。在一些实施方式中，每天施用两次所述化合物。在其它实施方式中，每天施用三次所述化合物。在其它实施方式中，每天施用四次所述化合物。在其它实施方式中，每天施用四次以上的所述化合物。在一些实施方式中，所述药物组合物施用于的个体为哺乳动物。在其它实施方式中，所述哺乳动物是人。在其它实施方式中，如果药物组合物包含多种活性成分，这些活性成分可以被混合在一个单一剂型中，它们也可以不相互混合而分别处于不同的剂型中。Pharmaceutical compositions of the present application include, but are not limited to, oral dosage forms and parenteral administration dosage forms. In some embodiments, the pharmaceutical composition may be an oral tablet, capsule, pill, powder, sustained release formulation, solution and suspension, sterile solution, suspension or emulsion for parenteral injection. In other embodiments, the pharmaceutical composition is in a unit dosage form suitable for single administration of precise dosages. In other embodiments, the amount of the compound ranges from about 0.001 mg/kg body weight/day to about 1000 mg/kg body weight/day. In other embodiments, the amount of the compound ranges from about 0.5 mg/kg body weight/day to about 50 mg/kg body weight/day. In some embodiments, the amount of the compound is from about 0.001 g/day to about 7 g/day. In other embodiments, the amount of the compound is from about 0.002 g/day to about 6 g/day. In other embodiments, the amount of the compound is from about 0.005 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.01 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.02 g/day to about 5 g/day. In other embodiments, the amount of the compound is from about 0.05 g/day to about 2.5 g/day. In other embodiments, the amount of the compound is from about 0.1 g/day to about 1 g/day. In other embodiments, a dose level below the lower limit of the above range may already be sufficient. In other embodiments, dose levels above the upper limit of the above range may be required. In some embodiments, the compound is administered in a single dose, once a day. In other embodiments, the compound is administered in multiple doses more than once a day. In some embodiments, the compound is administered twice daily. In other embodiments, the compound is administered three times a day. In other embodiments, the compound is administered four times a day. In other embodiments, the compound is administered more than four times a day. In some embodiments, the individual to which the pharmaceutical composition is administered is a mammal. In other embodiments, the mammal is a human. In other embodiments, if the pharmaceutical composition comprises a plurality of active ingredients, the active ingredients may be combined in a single dosage form, and they may also be in separate dosage forms without mixing with one another.
根据本申请的一个方面，本申请提供了本申请的化合物或混合物在制备治疗或预防HCV感染的药物中的用途。According to one aspect of the present application, the application provides the use of a compound or mixture of the present application in the manufacture of a medicament for the treatment or prevention of HCV infection.
根据本申请的一个方面，本申请提供了本申请的化合物或混合物与本申请所述的一种或多种其他的活性成分联合用于制备治疗或预防HCV感染的药物中的应用。 According to one aspect of the present application, the application provides the use of a compound or mixture of the present application in combination with one or more other active ingredients described herein for the manufacture of a medicament for the treatment or prevention of HCV infection.
根据本申请的一个方面，本申请提供了一种治疗或预防HCV感染的方法，所述方法包括将有效量的本申请的化合物或混合物或药物组合物施用于有此需求的受试者。According to one aspect of the present application, the present application provides a method of treating or preventing an HCV infection, the method comprising administering an effective amount of a compound or mixture or pharmaceutical composition of the present application to a subject in need thereof.
根据本申请的一些实施方式，上述治疗或预防HCV感染的方法包括将有效量的本申请的化合物或混合物与本申请所述的一种或多种其他的活性成分联合施用于有此需求的受试者。According to some embodiments of the present application, the above method of treating or preventing an HCV infection comprises administering an effective amount of a compound or mixture of the present application in combination with one or more other active ingredients described herein to a subject in need thereof Tester.
根据本申请的一些实施方式，本申请所述的HCV感染包括慢性丙型肝炎。According to some embodiments of the present application, the HCV infections described herein include chronic hepatitis C.
术语定义Definition of Terms
某些药学术语本文所用的有关术语“受试者”、“患者”或“个体”是指患有疾病、病症或病况等的个体，包括哺乳动物和非哺乳动物。哺乳动物的实施例包括但不限于哺乳动物纲的任何成员：人，非人的灵长类动物(例如黑猩猩和其它猿类和猴)；家畜，例如牛、马、绵羊、山羊、猪；家养动物，例如兔、狗和猫；实验室动物，包括啮齿类动物，例如大鼠、小鼠和豚鼠等。非人哺乳动物的实施例包括但不限于鸟类和鱼类等。在本文提供的一个有关方法和组合物的实施方式中，所述哺乳动物为人。Certain pharmacy terms As used herein, the terms "subject," "patient," or "individual" refer to an individual, including a mammal, and a non-mammal, having a disease, disorder, condition, or the like. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (eg, chimpanzees and other mites and monkeys); livestock, such as cattle, horses, sheep, goats, pigs; domesticated Animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs. Examples of non-human mammals include, but are not limited to, birds and fish. In one embodiment of the methods and compositions provided herein, the mammal is a human.
本文所用的术语“治疗”和其它类似的同义词包括缓解、减轻或改善疾病或病症症状，预防其它症状，改善或预防导致症状的潜在代谢原因，抑制疾病或病症，例如阻止疾病或病症的发展，缓解疾病或病症，使疾病或病症好转，缓解由疾病或病症导致的症状，或者中止疾病或病症的症状，此外，该术语包含预防的目的。该术语还包括获得治疗效果和/或预防效果。所述治疗效果是指治愈或改善所治疗的潜在疾病。此外，对与潜在疾病相关的一种或多种生理症状的治愈或改善也是治疗效果，例如尽管患者可能仍然受到潜在疾病的影响，但观察到患者情况改善。就预防效果而言，可向具有患特定疾病风险的患者施用所述组合物，或者即便尚未做出疾病诊断，但向出现该疾病的一个或多个生理症状的患者施用所述组合物。The term "treatment" and other similar synonyms as used herein includes alleviating, alleviating or ameliorating the symptoms of a disease or condition, preventing other symptoms, ameliorating or preventing a potential metabolic cause of the symptoms, inhibiting the disease or condition, such as preventing the progression of the disease or condition, Ameliorating a disease or condition, ameliorating the disease or condition, alleviating the symptoms caused by the disease or condition, or terminating the symptoms of the disease or condition, and further, the term includes the purpose of prevention. The term also includes obtaining a therapeutic effect and/or a preventive effect. The therapeutic effect refers to curing or ameliorating the underlying disease to be treated. In addition, the healing or amelioration of one or more physiological symptoms associated with a underlying disease is also a therapeutic effect, for example, although the patient may still be affected by the underlying disease, an improvement in the patient's condition is observed. In terms of prophylactic effect, the composition can be administered to a patient at risk of developing a particular disease, or even if a diagnosis of the disease has not been made, the composition is administered to a patient who develops one or more physiological symptoms of the disease.
本文所使用术语“有效量”、“治疗有效量”或“药学有效量”是指服用后足以在某种程度上缓解所治疗的疾病或病症的一个或多个症状的至少一种药剂或化合物的量。其结果可以为迹象、症状或病因的消减和/或缓解，或生物***的任何其它所需变化。例如，用于治疗的“有效量”是在临床上提供显著的病症缓解效果所需的包含本文公开化合物的组合物的量。可使用诸如剂量递增试验的技术测定适合于任意个体病例中的有效量。 The term "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," as used herein, refers to at least one agent or compound that, after administration, is sufficient to alleviate one or more symptoms of the disease or condition being treated to some extent. The amount. The result can be a reduction and/or alleviation of signs, symptoms or causes, or any other desired change in the biological system. For example, an "effective amount" for treatment is an amount of a composition comprising a compound disclosed herein that is required to provide a significant conditional relief effect in the clinic. An effective amount suitable for any individual case can be determined using techniques such as dose escalation testing.
本文所用术语“服用”、“施用”、“给药”等是指能够将化合物或组合物递送到进行生物作用的所需位点的方法。这些方法包括但不限于口服途径、经十二指肠途径、胃肠外注射(包括静脉内、皮下、腹膜内、肌内、动脉内注射或输注)、外用和经直肠给药。本领域技术人员熟知可用于本文所述化合物和方法的施用技术，例如在Goodman and Gilman,The Pharmacological Basis of Therapeutics,current ed.；Pergamon；and Remington's,Pharmaceutical Sciences(current edition),Mack Publishing Co.,Easton,Pa中讨论的那些。在优选的实施方式中，本文讨论的化合物和组合物通过口服施用。The terms "administering," "administering," "administering," and the like, as used herein, refers to a method of delivering a compound or composition to a desired site for biological action. These methods include, but are not limited to, oral routes, duodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical and rectal administration. The techniques of administration of the compounds and methods described herein are well known to those skilled in the art, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, those discussed in Pa. In a preferred embodiment, the compounds and compositions discussed herein are administered orally.
本文针对制剂、组合物或成分所用术语“可接受的”是指对接受治疗的受试者的一般健康情况没有长期的有害影响。The term "acceptable" as used herein with respect to a formulation, composition or ingredient means that there is no long-term detrimental effect on the general health of the subject being treated.
本文所用术语“药学上可接受的”是指不影响本申请化合物的生物活性或性质的物质(如载体或稀释剂)，并且相对无毒，即该物质可施用于个体而不造成不良的生物反应或以不良方式与组合物中包含的任意组分相互作用。The term "pharmaceutically acceptable" as used herein, refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present application, and is relatively non-toxic, ie, the substance can be administered to an individual without causing undesirable organisms. The reaction or in an undesirable manner interacts with any of the components contained in the composition.
本文所用术语“药物组合物”是指任选地混合有至少一种药学上可接受的化学成分的生物活性化合物，所述药学上可接受的化学成分包括但不限于载体。本文所用术语“载体”是指相对无毒的化学化合物或试剂，其有助于将化合物引入到细胞或组织中。The term "pharmaceutical composition" as used herein, refers to a biologically active compound, optionally in admixture with at least one pharmaceutically acceptable chemical component, including but not limited to a carrier. The term "carrier," as used herein, refers to a relatively non-toxic chemical compound or agent that facilitates the introduction of a compound into a cell or tissue.
本文所用术语“药学上可接受的盐”是指保留了指定化合物的游离酸和游离碱的生物效力，并且在生物学或其它方面上没有不良作用的盐。本申请化合物还包括药学上可以接受的盐。药学上可接受的盐是指把母体化合物中的碱基基团转换成盐的形式。The term "pharmaceutically acceptable salt" as used herein, refers to a salt that retains the biological effectiveness of the free acid and free base of the specified compound, and which has no adverse effects biologically or otherwise. The compounds of the present application also include pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a form in which a base group in a parent compound is converted into a salt.
本文使用的“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体。式I化合物含有不对称或手性中心，因此，存在不同的立体异构形式。分子式I的所有立体结构和混合物一样，包括外消旋混合物，作为目前申请的一部分。非对映体混合物能够分离成单独的非对映体，基于它们不同的物理化学性质，采用众所周知的手段，例如，对映异构体的拆分可通过与适当的光学活性物质(例如手性醇或Mosher`s莫氏酰氯)反应转换为非对映异构体，将其分离并转化(如水解)为相对应的单一的异构体。式1中的一些化合物可能是阻转异构体(如取代芳基)也是本申请中的一部分。对映异构体也可利用手性色谱柱分离。式I中的化合物可能存在着不同的互变异构形式，这些形式都包含在本申请范围内。例如，酮-烯醇和亚胺-烯胺形式的化合物。 As used herein, "stereoisomer" refers to an isomer produced by the different arrangement of atoms in a molecule in space. The compounds of formula I contain asymmetric or chiral centers and, therefore, exist in different stereoisomeric forms. All stereostructures and mixtures of Formula I, including racemic mixtures, are part of the current application. The mixture of diastereomers can be separated into individual diastereomers, based on their different physicochemical properties, using well-known means, for example, resolution of the enantiomers can be carried out with appropriate optically active substances (eg chirality) The alcohol or Mosher`s molyl chloride reaction is converted to a diastereomer which is separated and converted (e.g., hydrolyzed) to the corresponding single isomer. Some of the compounds of Formula 1 may be atropisomers (e.g., substituted aryl groups) are also part of this application. Enantiomers can also be separated using a chiral column. The compounds of formula I may exist in different tautomeric forms, and such forms are embraced within the scope of the present application. For example, compounds in the form of keto-enol and imine-enamine.
具体实施方式detailed description
本申请的权利要求书特别陈述了本申请的新特征。在下文的陈述了利用本申请原理的示例性实施方式。通过参考以下内容可更好地理解本申请的特征和优点。The claims of the present application particularly set forth the novel features of the present application. Exemplary embodiments utilizing the principles of the present application are set forth below. The features and advantages of the present application will be better understood by reference to the following.
尽管本文描述了本申请的优选实施方式，但是这些实施方式仅作为示例提供。应理解本文所述的本申请实施方式的变体也可用于实施本申请的技术方案。本领域普通技术人员应理解，可出现多种变体、变化和替换而不脱离本申请的范围。应理解本申请各个方面的保护范围由权利要求书决定，并且这些权利要求范围内的方法和结构以及其等价的方法和结构均在本申请权利要求书涵盖的范围之内。Although preferred embodiments of the present application are described herein, these embodiments are provided by way of example only. It should be understood that variations of the embodiments of the present application described herein may also be used to implement the technical solutions of the present application. It will be appreciated by those skilled in the art that various modifications, changes and substitutions may be made without departing from the scope of the application. It is to be understood that the scope of the invention is defined by the scope of the claims and the scope of the claims
合成路线synthetic route
本申请中化合物的合成方法，包括但不限于以下反应式和反应步骤：The method for synthesizing the compounds in the present application includes, but is not limited to, the following reaction formulas and reaction steps:
中间体1a合成路线：Intermediate 1a synthetic route:
Figure PCTCN2016113815-appb-000005
Figure PCTCN2016113815-appb-000005
化合物A的合成路线： Synthesis route of compound A:
Figure PCTCN2016113815-appb-000006
Figure PCTCN2016113815-appb-000006
化合物B的合成路线：Synthesis route of compound B:
Figure PCTCN2016113815-appb-000007
Figure PCTCN2016113815-appb-000007
化合物C的合成路线： Synthesis route of compound C:
Figure PCTCN2016113815-appb-000008
Figure PCTCN2016113815-appb-000008
化合物G的合成路线：Synthetic route of compound G:
Figure PCTCN2016113815-appb-000009
Figure PCTCN2016113815-appb-000009
化合物U的合成路线：Synthetic route of compound U:
Figure PCTCN2016113815-appb-000010
Figure PCTCN2016113815-appb-000010
化合物M的合成路线：Synthetic route of compound M:
Figure PCTCN2016113815-appb-000011
Figure PCTCN2016113815-appb-000011
化合物W的合成路线：Synthetic route of compound W:
Figure PCTCN2016113815-appb-000012
Figure PCTCN2016113815-appb-000012
实施例Example
实施例1：化合物A的制备Example 1: Preparation of Compound A
实施例1a：Example 1a:
Figure PCTCN2016113815-appb-000013
Figure PCTCN2016113815-appb-000013
将化合物10(84g,0.17mol)加入乙醇(1.2L)中溶解，分批加入NaBH4(25g,0.69mol)，加热至40～45℃反应4h，TLC跟踪反应完毕，冷却至0℃，加入丙酮(50ml)搅拌15min淬灭反应，过滤，滤液浓缩尽干，加入乙酸乙酯萃取，浓缩后，柱层析分离，淋洗液 为DCM/MeOH＝20:1～10:1，得到30.5g白色固体，收率71％。1HNMR(400MHz,d6-DMSO)：11.41(s,1H),8.13(d,1H,J＝8.3Hz),6.18(s,1H),5.89(d,1H,J＝5.9Hz),5.60(dd,1H,J＝8.2,2.0Hz),5.34(dd,1H,J＝4.3,3.5Hz),3.85(m,3H),3.62(dd,1H,J＝10.5,4.3Hz),1.40(s,3H)Compound 10 (84 g, 0.17 mol) was dissolved in ethanol (1.2 L), NaBH 4 (25 g, 0.69 mol) was added in portions, heated to 40-45 ° C for 4 h, TLC followed the reaction was completed, cooled to 0 ° C, added The acetone (50 ml) was stirred for 15 min to quench the reaction, filtered, and the filtrate was concentrated to dryness. ethyl acetate was evaporated, concentrated, and then purified by column chromatography. The eluent was DCM/MeOH = 20:1 to 10:1 to give 30.5 g. White solid, yield 71%. 1 H NMR (400 MHz, d6-DMSO): 11.41 (s, 1H), 8.13 (d, 1H, J = 8.3 Hz), 6.18 (s, 1H), 5.89 (d, 1H, J = 5.9 Hz), 5.60 ( Dd, 1H, J = 8.2, 2.0 Hz), 5.34 (dd, 1H, J = 4.3, 3.5 Hz), 3.85 (m, 3H), 3.62 (dd, 1H, J = 10.5, 4.3 Hz), 1.40 (s) , 3H)
实施例1b：Example 1b:
Figure PCTCN2016113815-appb-000014
Figure PCTCN2016113815-appb-000014
将化合物1a(26.4g,95mmol)溶于520ml吡啶，油泵减压蒸出260ml溶剂，冷却至室温加入DMAP(4.0g,33mmol)和DMTCl(120g,354mmol)，然后加热至40～45℃反应4～5h，TLC跟踪反应完全，减压浓缩吡啶尽干，加入DCM萃取，有机相浓缩后，柱层析得到53g白色固体化合物，收率96％。Compound 1a (26.4 g, 95 mmol) was dissolved in 520 ml of pyridine, and 650 ml of solvent was distilled off under reduced pressure, and cooled to room temperature. DMAP (4.0 g, 33 mmol) and DMTCl (120 g, 354 mmol) were added, and then heated to 40 to 45 ° C. ~5h, TLC followed the reaction completely, concentrated pyridine under reduced pressure, extracted with DCM, concentrated organic phase, and then purified by column chromatography to give 53 g of white solid compound.
1HNMR(400MHz,d6-DMSO)：11.47(s,1H),7.95(d,1H,J＝8.0Hz),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.20(s,1H),6.07(d,1H,J＝6.0Hz),4.95(d,1H,J＝7.6Hz),4.18(dd,1H,J＝6.4,3.2Hz),3.70(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.33(m,1H),1.45(s,3H). 1 HNMR (400MHz, d6-DMSO ): 11.47 (s, 1H), 7.95 (d, 1H, J = 8.0Hz), 7.34 (m, 4H), 7.21 (m, 5H), 6.88 (m, 4H), 6.20(s,1H),6.07(d,1H,J=6.0Hz), 4.95(d,1H,J=7.6Hz), 4.18(dd,1H,J=6.4,3.2Hz), 3.70(s,6H) ), 3.43 (dd, 1H, J = 3.6, 11.6 Hz), 3.33 (m, 1H), 1.45 (s, 3H).
实施例1c：Example 1c:
Figure PCTCN2016113815-appb-000015
Figure PCTCN2016113815-appb-000015
取化合物1b(6.24g)溶于60mlDCM，加入DMAP(2.9g)，冷却至0～5℃，滴加BzCl(1.8g)，加完升至室温反应2h，TLC跟踪反应完毕，加水洗涤，干燥后，浓缩溶剂，柱层析分离得到6.3g产物，收率86％。 Compound 1b (6.24g) was dissolved in 60ml DCM, DMAP (2.9g) was added, cooled to 0~5 °C, BzCl (1.8g) was added dropwise, and the reaction was allowed to rise to room temperature for 2h. The reaction was completed by TLC, washed with water and dried. Thereafter, the solvent was concentrated and subjected to column chromatography to give 6.3 g of product, yield 86%.
1HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.01(m,3H),7.72(t,1H,J＝7.8Hz),7.57(t,2H,J＝7.8Hz),7.29(d,2H,J＝8.0Hz),7.25(m,7H),6.75(m,4H),6.30(s,1H),5.78(d,1H,J＝8.0Hz),5.21(d,1H,J＝6.4Hz),4.43(d,1H,J＝8.8Hz),3.65(s,3H),3.63(s,3H),3.40(m,2H),1.55(s,3H). 1 H NMR (400 MHz, d6-DMSO): 11.52 (s, 1H), 8. s (m, 3H), 7.72 (t, 1H, J = 7.8 Hz), 7.57 (t, 2H, J = 7.8 Hz), 7.29 ( d, 2H, J = 8.0 Hz), 7.25 (m, 7H), 6.75 (m, 4H), 6.30 (s, 1H), 5.78 (d, 1H, J = 8.0 Hz), 5.21 (d, 1H, J) = 6.4 Hz), 4.43 (d, 1H, J = 8.8 Hz), 3.65 (s, 3H), 3.63 (s, 3H), 3.40 (m, 2H), 1.55 (s, 3H).
实施例1d：Example 1d:
Figure PCTCN2016113815-appb-000016
Figure PCTCN2016113815-appb-000016
将化合物1c(0.486g,0.71mmol)加入80％乙酸(5ml)中溶解，20～25℃反应4h，TLC跟踪反应完毕，加入水，乙酸乙酯萃取，有机相依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸镁干燥，过滤，滤液浓缩后，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1，得到0.25g白色泡沫状固体，收率93％。Compound 1c (0.486 g, 0.71 mmol) was dissolved in 80% acetic acid (5 ml), and reacted at 20-25 ° C for 4 h. The reaction was completed by TLC. Water was added and ethyl acetate was evaporated. The mixture was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered, and then filtered, and then evaporated. .
1HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.18(d,1H,J＝8.0Hz),8.00(d,2H,J＝8.4Hz),7.70(t,1H,J＝8.4Hz),7.54(t,2H,J＝8.4Hz),6.32(s,1H),5.73(d,1H,8.0Hz),5.49(m,2H),4.33(dt,1H,J＝2.0,8.4Hz),3.85(m,1H),3.63(m,1H),1.51(s,3H). 1 H NMR (400 MHz, d6-DMSO): 11.52 (s, 1H), 8.18 (d, 1H, J = 8.0 Hz), 8.00 (d, 2H, J = 8.4 Hz), 7.70 (t, 1H, J = 8.4) Hz), 7.54 (t, 2H, J = 8.4 Hz), 6.32 (s, 1H), 5.73 (d, 1H, 8.0 Hz), 5.49 (m, 2H), 4.33 (dt, 1H, J = 2.0, 8.4 Hz), 3.85 (m, 1H), 3.63 (m, 1H), 1.51 (s, 3H).
实施例1e：Example 1e:
Figure PCTCN2016113815-appb-000017
Figure PCTCN2016113815-appb-000017
将化合物1d(1.10g,2.88mmol)加入乙腈(3ml)和水(3ml)中，固体不能完全溶解，然后加入TEMPO(46mg,0.288mmol)和BAIB(2.04g,6.34mmol)，10℃反应18h，TLC跟踪反应基本完全，加入DCM(50ml)和饱和碳酸氢钠水溶液(50ml)，有大量固体(产物的钠盐)析出，过滤，固体用DCM洗涤，合并有机相，用饱和碳酸氢钠水溶液(20ml) 萃取，合并水相和固体，加入乙酸乙酯，搅拌下滴加4NHCl水溶液调pH<2，分出有机相，干燥浓缩后得到1.19g红色泡沫状固体，收率定量。Compound 1d (1.10g, 2.88mmol) was added to acetonitrile (3ml) and water (3ml), the solid was not completely dissolved, then TEMPO (46mg, 0.288mmol) and BAIB (2.04g, 6.34mmol) were added and reacted at 10 °C for 18h The reaction was essentially complete with TLC. EtOAc (EtOAc) (EtOAc) eluted eluted (20ml) The mixture was extracted, the aqueous phase and solid were combined, ethyl acetate was added, and 4N HCl aqueous solution was added dropwise with stirring to adjust pH <2, and the organic phase was separated, and concentrated to give 1.19 g of a red foamy solid.
1HNMR(400MHz,d6-DMSO)：13.0(br,1H),11.56(d,1H,J＝2.0Hz),8.00(m,3H),7.70(m,1H),7.57(t,2H,J＝8.0Hz),6.34(s,1H),5.78(dd,1H,J＝8.0,2.0Hz),5.72(br,1H),4.74(d,1H,J＝8.0Hz),1.55(s,3H). 1 HNMR (400MHz, d6-DMSO ): 13.0 (br, 1H), 11.56 (d, 1H, J = 2.0Hz), 8.00 (m, 3H), 7.70 (m, 1H), 7.57 (t, 2H, J = 8.0 Hz), 6.34 (s, 1H), 5.78 (dd, 1H, J = 8.0, 2.0 Hz), 5.72 (br, 1H), 4.74 (d, 1H, J = 8.0 Hz), 1.55 (s, 3H) ).
实施例1f：Example 1f:
Figure PCTCN2016113815-appb-000018
Figure PCTCN2016113815-appb-000018
将化合物1e(66mg,0.168mmol)溶于THF(1ml)中，0～5℃加入三乙胺(26mg,0.252mmol)，然后加入氯甲酸乙酯(22mg,0.202mmol)，15℃反应2h，TLC跟踪反应基本完全，过滤，滤液中加入NaBD4(21mg,0.504mmol)，15℃下滴加入D2O(0.5g,25mmol)，加完反应30min，TLC跟踪反应完全，加入乙酸乙酯，用水洗涤，分出有机相，干燥浓缩，柱层析分离得固体30mg，收率47％。The compound 1e (66 mg, 0.168 mmol) was dissolved in THF (1 ml), EtOAc (EtOAc (EtOAc) The reaction was completely completed by TLC. Filtration was carried out. NaBD 4 (21 mg, 0.504 mmol) was added to the filtrate. D 2 O (0.5 g, 25 mmol) was added dropwise at 15 ° C. After the reaction was completed for 30 min, the reaction was completed by TLC and ethyl acetate was added. After washing with water, the organic phase was separated, dried and concentrated, and then purified by column chromatography.
1HNMR(400MHz,d6-DMSO):11.54(s,1H),8.19(d,1H,J＝8.0Hz),8.00(d,2H,J＝7.2Hz),7.69(t,1H,J＝7.2Hz),7.55(t,2H,J＝7.2Hz),6.33(s,1H),5.73(d,1H,J＝8.0Hz),5.48(d,1H,J＝8.4Hz),5.47(s,1H),4.32(d,1H,J＝8.4Hz),1.51(s,3H). 1 H NMR (400 MHz, d6-DMSO): 11.54 (s, 1H), 8.19 (d, 1H, J = 8.0 Hz), 8.00 (d, 2H, J = 7.2 Hz), 7.69 (t, 1H, J = 7.2) Hz), 7.55 (t, 2H, J = 7.2 Hz), 6.33 (s, 1H), 5.73 (d, 1H, J = 8.0 Hz), 5.48 (d, 1H, J = 8.4 Hz), 5.47 (s, 1H), 4.32 (d, 1H, J = 8.4 Hz), 1.51 (s, 3H).
实施例1g：Example 1g:
Figure PCTCN2016113815-appb-000019
Figure PCTCN2016113815-appb-000019
将化合物1f(93mg,0.261mmol)加入1.0mlTHF中溶解，0～5℃滴加t-BuMgCl(1M,0.784ml)的THF溶液，滴完0～5℃反应30min，有大量固体析出，0～5℃下加入化合物11(183mg,0.418mmol)的THF(1.0ml)溶液，固体全部溶清，升至室温搅拌过夜，TLC跟踪反应完毕，加入NH4Cl饱和水溶液，EA萃取，有机相依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸镁干燥，过滤，滤液浓缩后，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1到1:2到0:1，得到95mg产物A，收率67％。The compound 1f (93 mg, 0.261 mmol) was dissolved in 1.0 ml of THF, and a solution of t-BuMgCl (1M, 0.784 ml) in THF was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated, 0 to THF was added compound 11 (183mg, 0.418mmol) at the 5 ℃ (1.0 ml) solution, all the solid clear solution, warmed to room temperature stirred overnight, TLC tracking completion of the reaction was added saturated aqueous NH 4 Cl, EA, the organic phase was washed with The mixture was washed with a saturated aqueous solution of sodium hydrogen carbonate and brine, dried over anhydrous magnesium sulfate, and filtered, and the filtrate was concentrated and then purified by column chromatography. The eluent was n-heptane / ethyl acetate = 1:1 to 1:2 to 0: 1. 95 mg of product A was obtained in a yield of 67%.
1HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.2Hz),7.36(t,2H,J＝7.9Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.37(s,1H),5.59(d,1H,J＝8.2Hz),4.94(m,1H),4.16(dd,1H,J＝9.4,2.3Hz),3.99(d,1H,J＝9.4Hz),3.90(dq,1H,J＝10.2,7.4Hz),1.51(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz). 1 H NMR (400 MHz, d4-MeOD): 7.73 (d, 1H, J = 8.2 Hz), 7.36 (t, 2H, J = 7.9 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J=7.9Hz), 6.37(s,1H), 5.59(d,1H,J=8.2Hz), 4.94(m,1H), 4.16(dd,1H,J=9.4,2.3Hz),3.99( d, 1H, J = 9.4 Hz), 3.90 (dq, 1H, J = 10.2, 7.4 Hz), 1.51 (s, 3H), 1.33 (dd, 3H, J = 7.1, 0.8 Hz), 1.20 (d, 3H) , J = 6.3 Hz), 1.19 (d, 3H, J = 5.9 Hz).
13CNMR(400MHz,d4-MeOD):172.91,172.86,164.19,150.80,150.68,150.61,129.50,124.87,119.90,119.85,101.65,80.17,80.09,76.57,68.77,50.23,50.21,21.50,20.55,20.46,19.24,19.18. 13 C NMR (400 MHz, d4-MeOD): 172.91, 172.86, 164.19, 150.80, 150.68, 150.61, 129.50, 124.87, 119.90, 119.85, 101.65, 80.17, 80.09, 76.57, 68.77, 50.23, 50.21, 21.50, 20.55, 20.46, 19.24, 19.18.
MS:正离子548[M+H]+，570[M+Na]+ MS: positive ion 548 [M+H] + , 570 [M+Na] +
负离子546[M-H]-，582[M+Cl]- Negative ion 546 [MH] - , 582 [M + Cl] -
实施例2：化合物B的制备Example 2: Preparation of Compound B
实施例2aExample 2a
Figure PCTCN2016113815-appb-000020
Figure PCTCN2016113815-appb-000020
将化合物1b(500mg,0.863mmol)和碳酸氢钠(363mg,4.32mmol)加入DCM(10ml)中，然后加入DMP(732mg,1.73mmol)，10～15℃反应3h，TLC跟踪反应基本完全，加入DCM(50ml)和饱和碳酸氢钠水溶液(30ml)，分出有机相，干燥浓缩后得到0.6g红色泡沫状固体直接用于下步反应。Compound 1b (500 mg, 0.863 mmol) and sodium hydrogencarbonate (363 mg, 4.32 mmol) were added to DCM (10 ml), then DMP (732 mg, 1.73 mmol) was added and reacted at 10-15 ° C for 3 h. DCM (50 ml) and saturated aqueous sodium bicarbonate (30 ml) were evaporated and evaporated.
实施例2b： Example 2b:
Figure PCTCN2016113815-appb-000021
Figure PCTCN2016113815-appb-000021
将化合物2a(600mg粗品,0.863mmol)溶于EtOD(6ml)中，10℃下加入NaBD4(83mg，1.99mmol)，反应2h，TLC跟踪反应基本完全，加入重水(1ml)淬灭反应，乙酸乙酯萃取，干燥浓缩，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝2：1到1：1，得到400mg白色固体，两步收率80％。Compound 2a (600 mg crude product, 0.863 mmol) was dissolved in EtOD (6 ml), NaBD 4 (83 mg, 1.99 mmol) was added at 10 ° C for 2 h, the reaction was completely complete by TLC, and the reaction was quenched by adding water (1 ml). The ethyl ester was extracted, dried and concentrated, and purified by column chromatography. The eluent was n-heptane / ethyl acetate = 2:1 to 1:1 to give 400 mg of white solid.
1HNMR(400MHz,d6-DMSO):11.49(s,1H),7.96(d,1H,J＝8.0Hz),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.21(s,1H),6.06(s,1H),4.94(d,1H,J＝7.6Hz),3.71(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.33(m,1H),1.45(s,3H). 1 HNMR (400MHz, d6-DMSO ): 11.49 (s, 1H), 7.96 (d, 1H, J = 8.0Hz), 7.34 (m, 4H), 7.21 (m, 5H), 6.88 (m, 4H), 6.21 (s, 1H), 6.06 (s, 1H), 4.94 (d, 1H, J = 7.6 Hz), 3.71 (s, 6H), 3.43 (dd, 1H, J = 3.6, 11.6 Hz), 3.33 (m) , 1H), 1.45 (s, 3H).
实施例2c：Example 2c:
Figure PCTCN2016113815-appb-000022
Figure PCTCN2016113815-appb-000022
将化合物2b(400mg，0.69mmol)溶于DCM(8ml)中，加入CF3COOH(1ml)，10～16℃反应3h，TLC跟踪反应基本完全，浓缩后柱层析分离，淋洗液为乙酸乙酯，得到固体156mg，收率82％。Compound 2b (400mg, 0.69mmol) was dissolved in DCM (8ml), added CF 3 COOH (1ml), reacted at 10-16 ° C for 3h, TLC followed the reaction was completely complete, concentrated and separated by column chromatography, the eluent was acetic acid Ethyl ester gave 156 mg of solid, yield 82%.
实施例2d：Example 2d:
Figure PCTCN2016113815-appb-000023
Figure PCTCN2016113815-appb-000023
将化合物2c(130mg，0.47mmol)加入1.5mlTHF中溶解，0～5℃滴加t-BuMgCl(1M)的THF溶液(1.41ml)，滴完0～5℃反应30min，有大量固体析出，0～5℃下加入化合物11(340mg，0.75mmol)的THF(1.5ml)溶液，固体全部溶清，升至室温搅拌过夜，TLC跟踪反应完毕，加入NH4Cl饱和水溶液，乙酸乙酯萃取，有机相依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸镁干燥，过滤，滤液浓缩后，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1到1:2到0:1，得到60mg化合物B。The compound 2c (130 mg, 0.47 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.41 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated. ~ THF (1.5ml) was added compound 11 (340mg, 0.75mmol) at 5 ℃ solution, all the solid clear solution, warmed to room temperature stirred overnight, TLC tracking completion of the reaction was added saturated aqueous NH 4 Cl, extracted with ethyl acetate, the organic The phases were washed successively with a saturated aqueous solution of sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, and filtered, and the filtrate was concentrated, and then separated by column chromatography. The eluent was n-heptane / ethyl acetate = 1:1 to 1:2 To 0:1, 60 mg of Compound B was obtained.
1HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.3Hz),7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),5.59(d,1H,J＝8.3Hz),4.94(m,1H),4.52(ddd,1H,J＝12.2,5.9,2.0Hz),4.38(ddd,1H,J＝11.8,5.5,3.2Hz),4.17(dd,1H,J＝4.7,2.3Hz),3.90(dq,1H,J＝10.2,7.0Hz),1.50(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz). 1 H NMR (400 MHz, d4-MeOD): 7.73 (d, 1H, J = 8.3 Hz), 7.36 (t, 2H, J = 8.2 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J=7.9Hz), 6.36(s,1H), 5.59(d,1H,J=8.3Hz), 4.94(m,1H), 4.52(ddd,1H,J=12.2,5.9,2.0Hz), 4.38 (ddd, 1H, J = 11.8, 5.5, 3.2 Hz), 4.17 (dd, 1H, J = 4.7, 2.3 Hz), 3.90 (dq, 1H, J = 10.2, 7.0 Hz), 1.50 (s, 3H) , 1.33 (dd, 3H, J = 7.1, 0.8 Hz), 1.20 (d, 3H, J = 6.3 Hz), 1.19 (d, 3H, J = 5.9 Hz).
13CNMR(400MHz,d4-MeOD):172.91,172.86,164.19,150.80,150.61,129.50,124.87,119.89,119.85,101.65,80.17,80.09,76.57,68.77,50.23,50.21,21.46,20.54,20.45,19.23,19.17. 13 C NMR (400 MHz, d4-MeOD): 172.91, 172.86, 164.19, 150.80, 150.61, 129.50, 124.87, 119.89, 119.85, 101.65, 80.17, 80.09, 76.57, 68.77, 50.23, 50.21, 21.46, 20.54, 20.45, 19.23, 19.17.
MS:正离子569[M+Na]+ MS: positive ion 569 [M+Na] +
负离子545[M-H]-，581[M+Cl]- Negative ion 545 [MH] - , 581 [M + Cl] -
实施例3：化合物C的制备Example 3: Preparation of Compound C
实施例3a：Example 3a:
Figure PCTCN2016113815-appb-000024
Figure PCTCN2016113815-appb-000024
将2b(2.66g,4.6mmol)溶于DM(26ml)，加入DMAP(1.12g,9.2mmol)，冷却至0～5℃，滴加BzCl(0.78g,5.6mmol),加完升至室温反应2h，TLC跟踪反应完毕，加水洗涤，干燥后，浓缩溶剂得3.4g粗品，直接用于下步反应。2b (2.66g, 4.6mmol) was dissolved in DM (26ml), DMAP (1.12g, 9.2mmol) was added, cooled to 0 to 5 ° C, BzCl (0.78g, 5.6mmol) was added dropwise, and the reaction was added to room temperature. 2h, TLC followed the completion of the reaction, washing with water, drying, and then concentrating the solvent to obtain 3.4 g of crude product, which was directly used for the next step.
实施例3b： Example 3b:
Figure PCTCN2016113815-appb-000025
Figure PCTCN2016113815-appb-000025
将3a(上步3.4g粗品，4.6mmol)加入DCM(30ml)中溶解，加入三氟乙酸(5ml)，20～25℃反应4h，TLC跟踪反应完毕，浓缩溶剂后，加入DCM，硅胶拌样，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1，得到1.0g白色泡沫状固体，两步收率57.2％。3a (upper step 3.4g of crude product, 4.6mmol) was dissolved in DCM (30ml), added trifluoroacetic acid (5ml), reacted at 20-25 ° C for 4h, TLC followed the reaction was completed, concentrated solvent, DCM, silica gel sample Separation by column chromatography, the eluent was n-heptane / ethyl acetate = 1:1 to give 1.0 g of white foamy solid.
实施例3c：Example 3c:
Figure PCTCN2016113815-appb-000026
Figure PCTCN2016113815-appb-000026
将3b(1.0g,2.88mmol)加入乙腈(3ml)和水(3ml)中，固体不能完全溶解，然后加入TEMPO(45mg,0.288mmol)和BAIB(2.04g,6.34mmol)，10℃反应18h，TLC跟踪反应基本完全，加入DCM(50ml)和饱和碳酸氢钠水溶液(50ml)，有大量固体(产物的钠盐)析出，过滤，固体用DM洗涤，合并有机相，用饱和碳酸氢钠水溶液(20ml)萃取，合并水相和固体，加入乙酸乙酯，搅拌下滴加4NHCl水溶液调pH<2，分出有机相，干燥浓缩后得到红色泡沫状固体，收率定量。3b (1.0g, 2.88mmol) was added to acetonitrile (3ml) and water (3ml), the solid was not completely dissolved, then TEMPO (45mg, 0.288mmol) and BAIB (2.04g, 6.34mmol) were added and reacted at 10 ° C for 18h. The reaction was essentially complete with TLC. EtOAc (EtOAc) (EtOAc)EtOAc. 20 ml) was extracted, and the combined organic phase and solid were added, ethyl acetate was added, and 4N aqueous HCl solution was added dropwise with stirring to adjust pH <2, and the organic phase was separated, and concentrated to give a red foamy solid.
实施例3d：Example 3d:
Figure PCTCN2016113815-appb-000027
Figure PCTCN2016113815-appb-000027
将化合物3c(508mg,1.28mmol)溶于THF(10ml)中，0～5℃加入三乙胺(194.2mg,1.92mmol)，然后加入氯甲酸乙酯(250mg,1.54mmol)，15℃反应2h，TLC跟踪反应基 本完全，过滤，滤液中加入NaBD4(160.7mg,3.84mmol)，15℃下滴加入D2O(2.0g,100mmol)，加完反应30min，TLC跟踪反应完全，加入乙酸乙酯，用水洗涤，分出有机相，干燥浓缩，柱层析分离得固体160mg，收率32.6％。Compound 3c (508 mg, 1.28 mmol) was dissolved in THF (10 mL), EtOAc (EtOAc (EtOAc,EtOAc) , TLC followed the reaction was almost complete, filtered, NaBD 4 (160.7mg, 3.84mmol) was added to the filtrate, D 2 O (2.0g, 100mmol) was added dropwise at 15 °C, the reaction was added for 30min, TLC followed the reaction completely, adding acetic acid The ester was washed with water, the organic phase was separated, dried and concentrated, and then purified by column chromatography.
1HNMR(400MHz,d4-CD3OD):7.29(d,1H,J＝8.0Hz),6.42(s,1H),5.72(d,1H,J＝8.0Hz),4.61(s,1H),1.56(s,3H). 1 HNMR (400MHz, d4-CD 3 OD): 7.29 (d, 1H, J = 8.0Hz), 6.42 (s, 1H), 5.72 (d, 1H, J = 8.0Hz), 4.61 (s, 1H), 1.56(s,3H).
实施例3e：Example 3e:
Figure PCTCN2016113815-appb-000028
Figure PCTCN2016113815-appb-000028
将化合物3d(150mg，0.39mmol)加入1.5mlTHF中溶解，0～5℃滴加t-BuMgCl(1M)的THF溶液(1.2ml)，滴完0～5℃反应30min，有大量固体析出，0～5℃下加入化合物11(354mg，0.78mmol)的THF(1.5ml)溶液，固体全部溶清，升至室温搅拌过夜，TLC跟踪反应完毕，加入NH4Cl饱和水溶液，乙酸乙酯萃取，有机相依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸镁干燥，过滤，滤液浓缩后，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1到1:2到0:1，得到40mg化合物C，收率19％。The compound 3d (150 mg, 0.39 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.2 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated. ~ THF (1.5ml) was added compound 11 (354mg, 0.78mmol) at 5 ℃ solution, all the solid clear solution, warmed to room temperature stirred overnight, TLC tracking completion of the reaction was added saturated aqueous NH 4 Cl, extracted with ethyl acetate, the organic The phases were washed successively with a saturated aqueous solution of sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, and filtered, and the filtrate was concentrated, and then separated by column chromatography. The eluent was n-heptane / ethyl acetate = 1:1 to 1:2 To 0:1, 40 mg of Compound C was obtained in a yield of 19%.
1HNMR(400MHz,d4-MeOD):7.73(d,1H,J＝8.3Hz),7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),5.59(d,1H,J＝8.0Hz),4.94(m,1H),4.16(d,1H,J＝2.3Hz),3.90(dq,1H,J＝10.2,7.0Hz),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz). 1 H NMR (400 MHz, d4-MeOD): 7.73 (d, 1H, J = 8.3 Hz), 7.36 (t, 2H, J = 8.2 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J=7.9Hz), 6.36(s,1H), 5.59(d,1H,J=8.0Hz), 4.94(m,1H), 4.16(d,1H,J=2.3Hz), 3.90(dq, 1H, J = 10.2, 7.0 Hz), 1.50 (s, 3H), 1.33 (d, 3H, J = 7.1 Hz), 1.20 (d, 3H, J = 6.3 Hz), 1.19 (d, 3H, J = 5.9) Hz).
MS:正离子571[M+Na]+ MS: positive ion 571 [M+Na] +
负离子547[M-H]-，583[M+Cl]- Negative ion 547 [MH] - , 583 [M + Cl] -
实施例4：化合物G的制备Example 4: Preparation of Compound G
实施例4a： Example 4a:
Figure PCTCN2016113815-appb-000029
Figure PCTCN2016113815-appb-000029
将化合物12(20g,9.5mmol)悬浮于重水(200ml)中，加入10％Pd/C(1.0g)，氮气置换3次后，用氢气置换3次，氢气1atm下加热回流96h，浓缩后得到粗品，直接进行下步反应。Compound 12 (20 g, 9.5 mmol) was suspended in heavy water (200 ml), 10% Pd/C (1.0 g) was added, and the mixture was replaced with nitrogen three times, then replaced with hydrogen three times, and heated under reflux for 96 h at 1 atm. For the crude product, proceed directly to the next step.
实施例4b：Example 4b:
Figure PCTCN2016113815-appb-000030
Figure PCTCN2016113815-appb-000030
将化合物4a(20g)粗品加入吡啶(200ml)中，减压蒸去溶剂，再次加入吡啶(200ml)，冷却至0～5℃，滴加BzCl(49.5g)，加完升至室温反应18h，减压蒸去溶剂，倒入含NaOH(92g)的1.17L溶液中，搅拌1h使产品溶解，过滤除去钯碳，滤液用浓盐酸调pH值为4.0左右，过滤，得到的固体用1.2L乙醇在回流温度下打浆30min，冷却至室温后过滤，固体烘干后得到20g产品。The crude product of compound 4a (20 g) was added to pyridine (200 ml), and the solvent was evaporated under reduced pressure, pyridine (200 ml) was added again, cooled to 0 to 5 ° C, BzCl (49.5 g) was added dropwise, and the mixture was allowed to react to room temperature for 18 h. The solvent was evaporated under reduced pressure, poured into a 1.17L solution containing NaOH (92 g), and the mixture was stirred for 1 hour to dissolve the product, and the palladium carbon was removed by filtration. The filtrate was adjusted to pH 4.0 with concentrated hydrochloric acid, and filtered to give a solid. It was beaten at reflux temperature for 30 min, cooled to room temperature and filtered, and dried to give 20 g of product.
1HNMR(400MHz,d6-DMSO):7.97(d,2H,J＝7.2Hz),7.58(t,1H,J＝7.2Hz),7.47(t,2H,J＝7.2Hz). 1 H NMR (400 MHz, d6-DMSO): 7.97 (d, 2H, J = 7.2 Hz), 7.58 (t, 1H, J = 7.2 Hz), 7.47 (t, 2H, J = 7.2 Hz).
实施例4c：Example 4c:
Figure PCTCN2016113815-appb-000031
Figure PCTCN2016113815-appb-000031
将4b(21g,0.097mol)加入氯苯(200ml)中，然后加入0.1g硫酸铵和HMDS(46.9g,0.29mol)，加热回流2h，固体全部溶解，冷却，油泵减压浓缩，再用氯苯带一次，除尽HMDS，残液加入氯苯(150ml)，滴加LW1006-7(21g,0.049mol)的氯苯(100ml) 溶液，再加入SnCl4(37.8g,0.145mol)，Ar保护下70℃反应20h，反应液中析出大量固体，DM萃取，NaHCO3洗涤，浓缩后得粗品，直接投入下步反应。4b (21g, 0.097mol) was added to chlorobenzene (200ml), then 0.1g ammonium sulfate and HMDS (46.9g, 0.29mol) were added, heated under reflux for 2h, all solids were dissolved, cooled, concentrated under reduced pressure, and then treated with chlorine. The benzene band was once removed, the HMDS was removed, the residue was added with chlorobenzene (150 ml), and a solution of LW1006-7 (21 g, 0.049 mol) in chlorobenzene (100 ml) was added dropwise, followed by the addition of SnCl 4 (37.8 g, 0.145 mol), Ar protection. After reacting at 70 ° C for 20 h, a large amount of solid was precipitated in the reaction liquid, extracted with DM, washed with NaHCO 3 and concentrated to give a crude product, which was directly poured into the next step.
实施例4d：Example 4d:
Figure PCTCN2016113815-appb-000032
Figure PCTCN2016113815-appb-000032
将4c粗品加入醋酸(300ml)和水(75ml)中，加热回流3h，TLC跟踪反应完毕，浓缩尽干，加入乙酸乙酯后再次浓缩，加入DM和硅胶，浓缩后柱层析得到16.9g泡沫状固体，两步收率71％。The crude product of 4c was added to acetic acid (300 ml) and water (75 ml), and the mixture was refluxed for 3h, and the mixture was refluxed for 3h. The reaction was completed by TLC, and concentrated to dryness. EtOAc was evaporated and then concentrated again. Solid, the yield in two steps was 71%.
实施例4e：Example 4e:
Figure PCTCN2016113815-appb-000033
Figure PCTCN2016113815-appb-000033
将化合物4d(8.4g,0.017mol)加入乙醇(120ml)中溶解，分批加入NaBH4(2.5g,0.069mol)，加热至40～45℃反应4h，TLC跟踪反应完毕，冷却至0℃，加入丙酮(5ml)搅拌15min淬灭反应，过滤，滤液浓缩尽干，加入乙酸乙酯萃取，浓缩后，柱层析分离，淋洗液为DCM/MeOH＝20:1～10:1，得到3.05g白色固体，收率71％。Compound 4d (8.4 g, 0.017 mol) was dissolved in ethanol (120 ml), NaBH 4 (2.5 g, 0.069 mol) was added in portions, and heated to 40-45 ° C for 4 h. The reaction was completed by TLC and cooled to 0 ° C. After adding acetone (5 ml) and stirring for 15 min, the reaction was quenched, filtered, and the filtrate was concentrated to dryness, ethyl acetate was evaporated, and concentrated, and then separated by column chromatography. The eluent was DCM/MeOH = 20:1 to 10:1 to obtain 3.05 g White solid in 71% yield.
1HNMR(400MHz,d6-DMSO)：11.41(s,1H),6.18(s,1H),5.93(d,1H,J＝5.9Hz),5.40(dd,1H,J＝4.3,3.5Hz),3.85(m,3H),3.61(dd,1H,J＝10.5,4.3Hz),1.39(s,3H) 1 HNMR (400MHz, d6-DMSO ): 11.41 (s, 1H), 6.18 (s, 1H), 5.93 (d, 1H, J = 5.9Hz), 5.40 (dd, 1H, J = 4.3,3.5Hz), 3.85 (m, 3H), 3.61 (dd, 1H, J = 10.5, 4.3 Hz), 1.39 (s, 3H)
实验4f： Experiment 4f:
Figure PCTCN2016113815-appb-000034
Figure PCTCN2016113815-appb-000034
将化合物4e(150mg，0.39mmol)加入1.5mlTHF中溶解，0～5℃滴加t-BuMgCl(1M)的THF溶液(1.2ml)，滴完0～5℃反应30min，有大量固体析出，0～5℃下加入化合物11(354mg，0.78mmol)的THF(1.5ml)溶液，固体全部溶清，升至室温搅拌过夜，TLC跟踪反应完毕，加入NH4Cl饱和水溶液，乙酸乙酯萃取，有机相依次用饱和碳酸氢钠水溶液和饱和食盐水洗涤，无水硫酸镁干燥，过滤，滤液浓缩后，柱层析分离，淋洗液为正庚烷/乙酸乙酯＝1:1到1:2到0:1，得到40mg化合物G，收率19％。The compound 4e (150 mg, 0.39 mmol) was dissolved in 1.5 ml of THF, and a solution of t-BuMgCl (1 M) in THF (1.2 ml) was added dropwise at 0 to 5 ° C, and the reaction was carried out at 0 to 5 ° C for 30 min, and a large amount of solid precipitated. ~ THF (1.5ml) was added compound 11 (354mg, 0.78mmol) at 5 ℃ solution, all the solid clear solution, warmed to room temperature stirred overnight, TLC tracking completion of the reaction was added saturated aqueous NH 4 Cl, extracted with ethyl acetate, the organic The phases were washed successively with a saturated aqueous solution of sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, and filtered, and the filtrate was concentrated, and then separated by column chromatography. The eluent was n-heptane / ethyl acetate = 1:1 to 1:2 To 0:1, 40 mg of compound G was obtained in a yield of 19%.
1HNMR:1HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.52(m,1H,4.38(m,1H),4.18(dd,1H,J＝4.7,2.3Hz),4.00(m,1H),3.90(m,1H),1.50(s,3H),1.33(dd,3H,J＝7.1,0.8Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz). 1 H NMR: 1 H NMR (400 MHz, d4-MeOD): 7.36 (t, 2H, J = 8.2 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J = 7.9 Hz), 6.36 (s, 1H), 4.94 (m, 1H), 4.52 (m, 1H, 4.38 (m, 1H), 4.18 (dd, 1H, J = 4.7, 2.3 Hz), 4.00 (m, 1H), 3.90 (m) , 1H), 1.50 (s, 3H), 1.33 (dd, 3H, J = 7.1, 0.8 Hz), 1.20 (d, 3H, J = 6.3 Hz), 1.19 (d, 3H, J = 5.9 Hz).
MS：正离子570[M+Na]+ MS: positive ion 570 [M+Na] +
负离子546[M-H]-,582[M+Cl]- Negative ion 546 [MH] - , 582 [M + Cl] -
实施例5：化合物U的制备 Example 5: Preparation of Compound U
Figure PCTCN2016113815-appb-000035
Figure PCTCN2016113815-appb-000035
化合物U的合成参照实施例1中实施例1b到实施例1g的方法。The synthesis of Compound U was carried out in the same manner as in Example 1b to Example 1g of Example 1.
化合物5a:1HNMR(400MHz,d6-DMSO)：11.51(s,1H),7.34(m,4H),7.21(m,5H),6.88(m,4H),6.20(s,1H),6.10(d,1H,J＝6.0Hz),4.18(dd,1H,J＝6.0,9.6Hz),3.71(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),3.32(m,1H),1.45(s,3H).Compound 5a: 1 H NMR (400 MHz, d6-DMSO): 11.51 (s, 1H), 7.34 (m, 4H), 7.21 (m, 5H), 6.88 (m, 4H), 6.20 (s, 1H), 6.10 ( d, 1H, J = 6.0 Hz), 4.18 (dd, 1H, J = 6.0, 9.6 Hz), 3.71 (s, 6H), 3.43 (dd, 1H, J = 3.6, 11.6 Hz), 3.32 (m, 1H) ), 1.45 (s, 3H).
化合物5c:1HNMR(400MHz,d6-DMSO)：11.52(s,1H),8.00(d,2H,J＝8.0Hz),7.70(t,1H,J＝8.0Hz),7.55(t,2H,J＝8.0Hz),6.32(s,1H),5.49(d,2H,J＝8.4Hz),4.33(d,1H,J＝8.4Hz),3.86(m,1H),3.63(m,1H),1.51(s,3H).Compound 5c: 1 H NMR (400 MHz, d6-DMSO): 11.52 (s, 1H), 8.00 (d, 2H, J = 8.0 Hz), 7.70 (t, 1H, J = 8.0 Hz), 7.55 (t, 2H, J=8.0Hz), 6.32(s,1H), 5.49(d,2H,J=8.4Hz), 4.33(d,1H,J=8.4Hz),3.86(m,1H),3.63(m,1H) , 1.51 (s, 3H).
化合物U：1HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝7.9Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.16(dd,1H,J＝9.6,2.4Hz),3.99(d,1H,J＝9.6Hz),3.90(dq,1H,J＝10.2,7.4Hz),1.51(s,3H),1.33(d,3H,J＝7.2Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz). Compound U: 1 H NMR (400 MHz, d4-MeOD): 7.36 (t, 2H, J = 7.9 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J = 7.9 Hz), 6.36 (s, 1H), 4.94 (m, 1H), 4.16 (dd, 1H, J = 9.6, 2.4 Hz), 3.99 (d, 1H, J = 9.6 Hz), 3.90 (dq, 1H, J = 10.2, 7.4 Hz), 1.51 (s, 3H), 1.33 (d, 3H, J = 7.2 Hz), 1.20 (d, 3H, J = 6.3 Hz), 1.19 (d, 3H, J = 5.9 Hz).
MS：正离子572[M+Na]+ MS: positive ion 572 [M+Na] +
负离子548[M-H]-，584[M+Cl]- Negative ion 548 [MH] - , 584 [M + Cl] -
实施例6：化合物M的制备Example 6: Preparation of Compound M
Figure PCTCN2016113815-appb-000036
Figure PCTCN2016113815-appb-000036
化合物M的合成参照实施例2中实施例2a到实施例2d的方法。The synthesis of Compound M was carried out in the same manner as in Example 2a to Example 2d of Example 2.
化合物6b：1HNMR(400MHz,d6-DMSO):11.50(s,1H),7.33(m,4H),7.21(m,5H),6.89(m,4H),6.20(s,1H),6.07(s,1H),3.70(s,6H),3.43(dd,1H,J＝3.6,11.6Hz),1.45(s,3H).Compound 6b: 1 H NMR (400 MHz, d6-DMSO): 11.50 (s, 1H), 7.33 (m, 4H), 7.21 (m, 5H), 6.89 (m, 4H), 6.20 (s, 1H), 6.07 ( s, 1H), 3.70 (s, 6H), 3.43 (dd, 1H, J = 3.6, 11.6 Hz), 1.45 (s, 3H).
化合物M：1HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.4Hz),7.25(d,2H,J＝8.4Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.52(dd,1H,J＝12.2,5.9Hz),4.38(m,1H),4.16(d,1H,J＝2.0Hz),3.90(m,1H),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.0Hz),1.19(d,3H,J＝6.0Hz).Compound M: 1 H NMR (400 MHz, d4-MeOD): 7.36 (t, 2H, J = 8.4 Hz), 7.25 (d, 2H, J = 8.4 Hz), 7.19 (t, 1H, J = 7.9 Hz), 6.36 (s, 1H), 4.94 (m, 1H), 4.52 (dd, 1H, J = 12.2, 5.9 Hz), 4.38 (m, 1H), 4.16 (d, 1H, J = 2.0 Hz), 3.90 (m, 1H), 1.50 (s, 3H), 1.33 (d, 3H, J = 7.1 Hz), 1.20 (d, 3H, J = 6.0 Hz), 1.19 (d, 3H, J = 6.0 Hz).
MS：MS:
正离子549[M+1]+,571[M+Na]+ Positive ion 549[M+1] + , 571[M+Na] +
实施例7：化合物W的制备 Example 7: Preparation of Compound W
Figure PCTCN2016113815-appb-000037
Figure PCTCN2016113815-appb-000037
化合物W的合成参照实施例3中的实施例3a到实施例3eThe synthesis of the compound W is referred to the embodiment 3a to the embodiment 3e in the embodiment 3.
化合物W：1HNMR(400MHz,d4-MeOD):7.36(t,2H,J＝8.2Hz),7.25(d,2H,J＝7.9Hz),7.19(t,1H,J＝7.9Hz),6.36(s,1H),4.94(m,1H),4.16(d,1H,J＝2.3Hz),3.90(m,1H),1.50(s,3H),1.33(d,3H,J＝7.1Hz),1.20(d,3H,J＝6.3Hz),1.19(d,3H,J＝5.9Hz).Compound W: 1 H NMR (400 MHz, d4-MeOD): 7.36 (t, 2H, J = 8.2 Hz), 7.25 (d, 2H, J = 7.9 Hz), 7.19 (t, 1H, J = 7.9 Hz), 6.36 (s, 1H), 4.94 (m, 1H), 4.16 (d, 1H, J = 2.3 Hz), 3.90 (m, 1H), 1.50 (s, 3H), 1.33 (d, 3H, J = 7.1 Hz) , 1.20 (d, 3H, J = 6.3 Hz), 1.19 (d, 3H, J = 5.9 Hz).
MS：MS:
正离子573[M+Na]+ Positive ion 573[M+Na] +
负离子549[M-H]-，585[M+Cl]- Negative ion 549 [MH] - , 585 [M + Cl] -
生物活性测试实验Biological activity test
一、HCV GT1a和GT1b细胞学实验方法I. HCV GT1a and GT1b cytology experimental methods
GT1a和GT1b复制子细胞：GT1a and GT1b replicon cells:
稳转丙型肝炎病毒复制子GT1a和GT1b细胞系通过将丙型肝炎病毒亚基因组GT1a(H77)和GT1b(Con1)复制子质粒DNA体外转录为RNA后转入Huh-7细胞，并经G418选择而建立。Stable hepatitis C virus replicon GT1a and GT1b cell lines were transfected into Huh-7 cells by in vitro transcription of hepatitis C virus subgenomic GT1a (H77) and GT1b (Con1) replicon plasmid DNA into cells and selected by G418. And established.
实验材料： Experimental Materials:
GT1a和GT1b稳转细胞系，由药明康德构建(Wuxi AppTech Huh7-GT1a(H77)和Huh7-GT1b(con1))。The GT1a and GT1b stable cell lines were constructed by WuXi PharmaTech (Wuxi AppTech Huh7-GT1a (H77) and Huh7-GT1b (con1)).
试剂或耗材：DMEM培养液，胎牛血清，青链霉素，MEM非必需氨基酸，谷氨酰胺，G418，0.05％胰酶，DMSO，96孔黑色细胞培养版，CellTiter-flour，Bright-Glo。Reagents or consumables: DMEM medium, fetal bovine serum, streptomycin, MEM non-essential amino acids, glutamine, G418, 0.05% trypsin, DMSO, 96-well black cell culture plate, CellTiter-flour, Bright-Glo.
仪器：ViCell细胞计数仪(Beckman)，ECHO(Labcyte)，EnVision(PerkinElmer)。Instruments: ViCell Cell Counter (Beckman), ECHO (Labcyte), EnVision (PerkinElmer).
实验方法：experimental method:
将化合物用DMSO溶解成10毫摩尔/升浓储液，存放在氮气柜中。梯度稀释化合物：用Echo将化合物加入96孔板中，每个化合物进行1:3系列稀释8个浓度点，双复孔，加入到96孔板的第3-10列中。第11列则只加入DMSO做为无效作用对照孔。每个孔中的DMSO终体积为607.5纳升。见如下排版。The compound was dissolved in DMSO to a 10 mmol/L concentrated stock solution and stored in a nitrogen cabinet. Gradient dilution of the compound: The compound was added to a 96-well plate with Echo, and each compound was subjected to a 1:3 serial dilution of 8 concentration points, double duplicate wells, and added to columns 3-10 of the 96-well plate. In column 11, only DMSO was added as an ineffective control well. The final volume of DMSO in each well was 607.5 nanoliters. See the layout below.
Figure PCTCN2016113815-appb-000038
Figure PCTCN2016113815-appb-000038
细胞铺板：将HCV GT1a或GT1b细胞从T150细胞培养瓶中消化下来，并用细胞培养液将细胞密度调整为65,843细胞/毫升，然后将细胞悬液加入到已经加好化合物的96孔板中，每孔加入121.5微升。96孔板中细胞的终密度为8,000细胞/孔，每孔中DMSO的终浓度为0.5％.其中第2列100％有效对照孔中不加细胞。将含有细胞和化合物的96孔板放入5％CO2、37℃培养3天。培养3天后用，用CellTiter-Flour检测96孔板中每孔的细胞活性：每孔加入40微升CellTitter-flour，然后在37℃、5％CO2条件下培养1小时后，用EnVision读板检测荧光信号(激发光405nm，发射光515nm)。用Bright-Glo检测化合物的抗丙肝病毒活性：将检测完细胞活性的96孔板中上清去掉，然后每孔加入100微升配制好的1X Bright-Glo试剂，5分钟内检测化学发光信号。Cell plating: HCV GT1a or GT1b cells were digested from T150 cell culture flasks, and the cell density was adjusted to 65,843 cells/ml with cell culture medium, and then the cell suspension was added to a 96-well plate to which the compound had been added. The well was added to 121.5 microliters. The final density of cells in 96-well plates was 8,000 cells/well, and the final concentration of DMSO in each well was 0.5%. No cells were added to the 100% effective control wells in column 2. A 96-well plate containing cells and compounds was placed in 5% CO 2 and cultured at 37 ° C for 3 days. After 3 days of culture, CellTiter-Flour was used to detect the cell viability of each well in a 96-well plate: 40 μl of CellTitter-flour was added to each well, and then cultured at 37 ° C, 5% CO 2 for 1 hour, and then read with EnVision. The fluorescence signal (excitation light 405 nm, emission light 515 nm) was detected. The anti-hepatitis C virus activity of the compounds was examined by Bright-Glo: the supernatant of the 96-well plate in which the activity of the cells was detected was removed, and then 100 μl of the prepared 1X Bright-Glo reagent was added to each well, and the chemiluminescence signal was detected within 5 minutes.
数据分析 data analysis
细胞活力：将原始数据(RFU)用以下公式转换为每孔相对于DMSO对照组的细胞活力百分比％ViabilityCell viability: The raw data (RFU) was converted to the percentage of cell viability per well relative to the DMSO control group using the following formula.
Figure PCTCN2016113815-appb-000039
Figure PCTCN2016113815-appb-000039
化合物抗丙肝病毒活性：将原始数据(RLU)用以下公式转换为每孔相对于100％有效对照组的抗丙肝病毒活性百分比inhibition％Compound anti-hepatitis C virus activity: The raw data (RLU) was converted to the percentage of anti-hepatitis C virus activity per well relative to the 100% effective control group using the following formula:
Figure PCTCN2016113815-appb-000040
Figure PCTCN2016113815-appb-000040
CPD：化合物孔的信号值CPD: signal value of compound pore
ZPE：DMSO无效作用对照孔信号平均值ZPE: Mean value of DMSO ineffective control well signal
HPE：100％有效对照孔(无细胞对照孔)信号平均值HPE: 100% effective control well (cell-free control well) signal mean
RFU：相对荧光单位RFU: Relative Fluorescence Unit
RLU：相对化学发光单位RLU: Relative chemiluminescence unit
用GraphPad Prism 5软件中的Sigmoidal dose-response(variable slope)程序分析化合物对HCV GT1a或GT1b稳转细胞株的EC50值和CC50值。Analysis of HCV GT1a 50 value of the compound or GT1b stably transfected cell lines with EC 50 values of CC and GraphPad Prism 5 software Sigmoidal dose-response (variable slope) program.
二、HCV GT1a的活性和GTA1b的活性2. Activity of HCV GT1a and activity of GTA1b
根据上述方法测定，本申请式(I)所示的代表性化合物表现出的对于HCV GT1a和GT1b亚基因复制子抑制试验的EC50(50％抑制率,索非布韦原料药为参比化合物)如下表所示。According to the above method, the representative compound represented by the formula (I) of the present application exhibits an EC50 (50% inhibition rate for the HCV GT1a and GT1b subgene replicon inhibition test, and the sofosbuvir raw material drug is a reference compound) As shown in the table below.
表1:部分化合物抑制丙型肝炎病毒的活性测试结果Table 1: Results of test results for inhibition of hepatitis C virus by some compounds
Figure PCTCN2016113815-appb-000041
Figure PCTCN2016113815-appb-000041
此外，表1中的化合物都表现出大于1000的选择性指数(CC50/EC50)。 Furthermore, the compounds in Table 1 all exhibited a selectivity index (CC 50 /EC 50 ) of greater than 1000.

Claims (10)

  1. 如式I表示的化合物或其药学可接受的盐、立体异构体、互变异构体：A compound represented by formula I or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof:
    Figure PCTCN2016113815-appb-100001
    Figure PCTCN2016113815-appb-100001
    其中，among them,
    R1＝H或D；R 1 =H or D;
    R2＝H或D；R 2 = H or D;
    R3＝H或D；R 3 = H or D;
    R4＝H或D；R 4 = H or D;
    R5＝CH3或CD3；R 5 =CH 3 or CD 3 ;
    R6＝H或D；和R 6 =H or D; and
    X＝-O或-NH。X = -O or -NH.
  2. 根据权利要求1所述的化合物，其选自以下的化合物：A compound according to claim 1 which is selected from the group consisting of:
    Figure PCTCN2016113815-appb-100002
    Figure PCTCN2016113815-appb-100002
    Figure PCTCN2016113815-appb-100003
    Figure PCTCN2016113815-appb-100003
    Figure PCTCN2016113815-appb-100004
    Figure PCTCN2016113815-appb-100004
  3. 根据权利要求1或2所述的化合物，其中氘代的位置具有至少50％的D。A compound according to claim 1 or 2 wherein the deuterated position has at least 50% D.
  4. 根据权利要求3所述的化合物，其中氘代的位置具有至少90或至少95％的D。 The compound of claim 3 wherein the deuterated position has a D of at least 90 or at least 95%.
  5. 权利要求2所述的化合物的异构体混合物，其中所述混合物中的两种化合物各占50％：The isomer mixture of the compound of claim 2 wherein the two compounds in the mixture each comprise 50%:
    Figure PCTCN2016113815-appb-100005
    的混合物；
    Figure PCTCN2016113815-appb-100005
    mixture;
    Figure PCTCN2016113815-appb-100006
    的混合物；
    Figure PCTCN2016113815-appb-100006
    mixture;
    Figure PCTCN2016113815-appb-100007
    的混合物；
    Figure PCTCN2016113815-appb-100007
    mixture;
    Figure PCTCN2016113815-appb-100008
    的混合物；
    Figure PCTCN2016113815-appb-100008
    mixture;
    Figure PCTCN2016113815-appb-100009
    的混合物；
    Figure PCTCN2016113815-appb-100009
    mixture;
    Figure PCTCN2016113815-appb-100010
    的混合物；
    Figure PCTCN2016113815-appb-100010
    mixture;
    Figure PCTCN2016113815-appb-100011
    的混合物；
    Figure PCTCN2016113815-appb-100011
    mixture;
    Figure PCTCN2016113815-appb-100012
    的混合物；
    Figure PCTCN2016113815-appb-100012
    mixture;
    Figure PCTCN2016113815-appb-100013
    的混合物；
    Figure PCTCN2016113815-appb-100013
    mixture;
    Figure PCTCN2016113815-appb-100014
    的混合物；
    Figure PCTCN2016113815-appb-100014
    mixture;
    Figure PCTCN2016113815-appb-100015
    的混合物；
    Figure PCTCN2016113815-appb-100015
    mixture;
    Figure PCTCN2016113815-appb-100016
    的混合物；
    Figure PCTCN2016113815-appb-100016
    mixture;
    Figure PCTCN2016113815-appb-100017
    的混合物；
    Figure PCTCN2016113815-appb-100017
    mixture;
    Figure PCTCN2016113815-appb-100018
    的混合物；
    Figure PCTCN2016113815-appb-100018
    mixture;
    Figure PCTCN2016113815-appb-100019
    的混合物；
    Figure PCTCN2016113815-appb-100019
    mixture;
    Figure PCTCN2016113815-appb-100020
    的混合物；
    Figure PCTCN2016113815-appb-100020
    mixture;
    Figure PCTCN2016113815-appb-100021
    的混合物；
    Figure PCTCN2016113815-appb-100021
    mixture;
    Figure PCTCN2016113815-appb-100022
    的混合物；
    Figure PCTCN2016113815-appb-100022
    mixture;
    Figure PCTCN2016113815-appb-100023
    的混合物；
    Figure PCTCN2016113815-appb-100023
    mixture;
    Figure PCTCN2016113815-appb-100024
    的混合物；
    Figure PCTCN2016113815-appb-100024
    mixture;
    Figure PCTCN2016113815-appb-100025
    的混合物；
    Figure PCTCN2016113815-appb-100025
    mixture;
    Figure PCTCN2016113815-appb-100026
    的混合物；
    Figure PCTCN2016113815-appb-100026
    mixture;
    Figure PCTCN2016113815-appb-100027
    的混合物；
    Figure PCTCN2016113815-appb-100027
    mixture;
    Figure PCTCN2016113815-appb-100028
    的混合物；
    Figure PCTCN2016113815-appb-100028
    mixture;
    Figure PCTCN2016113815-appb-100029
    的混合物；
    Figure PCTCN2016113815-appb-100029
    mixture;
    Figure PCTCN2016113815-appb-100030
    的混合物；
    Figure PCTCN2016113815-appb-100030
    mixture;
    Figure PCTCN2016113815-appb-100031
    的混合物；
    Figure PCTCN2016113815-appb-100031
    mixture;
    Figure PCTCN2016113815-appb-100032
    的混合物；
    Figure PCTCN2016113815-appb-100032
    mixture;
    Figure PCTCN2016113815-appb-100033
    的混合物；
    Figure PCTCN2016113815-appb-100033
    mixture;
    Figure PCTCN2016113815-appb-100034
    的混合物；
    Figure PCTCN2016113815-appb-100034
    mixture;
    Figure PCTCN2016113815-appb-100035
    的混合物；
    Figure PCTCN2016113815-appb-100035
    mixture;
    Figure PCTCN2016113815-appb-100036
    的混合物；
    Figure PCTCN2016113815-appb-100036
    mixture;
    Figure PCTCN2016113815-appb-100037
    的混合物；
    Figure PCTCN2016113815-appb-100037
    mixture;
    Figure PCTCN2016113815-appb-100038
    的混合物；
    Figure PCTCN2016113815-appb-100038
    mixture;
    Figure PCTCN2016113815-appb-100039
    的混合物；
    Figure PCTCN2016113815-appb-100039
    mixture;
    Figure PCTCN2016113815-appb-100040
    的混合物；
    Figure PCTCN2016113815-appb-100040
    mixture;
    Figure PCTCN2016113815-appb-100041
    的混合物；
    Figure PCTCN2016113815-appb-100041
    mixture;
    Figure PCTCN2016113815-appb-100042
    的混合物；
    Figure PCTCN2016113815-appb-100042
    mixture;
    Figure PCTCN2016113815-appb-100043
    的混合物；
    Figure PCTCN2016113815-appb-100043
    mixture;
    Figure PCTCN2016113815-appb-100044
    的混合物；
    Figure PCTCN2016113815-appb-100044
    mixture;
    Figure PCTCN2016113815-appb-100045
    的混合物；
    Figure PCTCN2016113815-appb-100045
    mixture;
    Figure PCTCN2016113815-appb-100046
    的混合物；
    Figure PCTCN2016113815-appb-100046
    mixture;
    Figure PCTCN2016113815-appb-100047
    的混合物；
    Figure PCTCN2016113815-appb-100047
    mixture;
    Figure PCTCN2016113815-appb-100048
    的混合物；
    Figure PCTCN2016113815-appb-100048
    mixture;
    Figure PCTCN2016113815-appb-100049
    的混合物；
    Figure PCTCN2016113815-appb-100049
    mixture;
    Figure PCTCN2016113815-appb-100050
    的混合物。
    Figure PCTCN2016113815-appb-100050
    mixture.
  6. 一种药物组合物，其包含作为活性成分的权利要求1-4中任一项的化合物或权利要求5中的混合物，以及药学上可接受的载体。A pharmaceutical composition comprising as an active ingredient a compound according to any one of claims 1 to 4 or a mixture according to claim 5, and a pharmaceutically acceptable carrier.
  7. 根据权利要求6所述的药物组合物，其中所述组合物进一步包括一种或多种其他的活性成分，所述其他的活性成分选自(1)免疫调节剂；(2)丙型肝炎病毒蛋白酶抑制剂(NS3，NS3/4a)；(3)丙型肝炎病毒NS4b抑制剂,(4)丙型肝炎病毒NS5a抑制剂,(5)丙型肝炎病毒聚合酶抑制剂；(6)不属于(2)-(3)之核苷和核苷衍生物；(7)乙型肝炎病毒(HBV)抑制剂；(7)人类免疫缺损病毒(HIV)抑制剂；(9)癌症药物；(10)抗发炎药物；或(11)不属于上述(1)-(10)之其他化合物。The pharmaceutical composition according to claim 6, wherein said composition further comprises one or more other active ingredients selected from the group consisting of (1) an immunomodulatory agent; (2) a hepatitis C virus Protease inhibitors (NS3, NS3/4a); (3) Hepatitis C virus NS4b inhibitors, (4) Hepatitis C virus NS5a inhibitors, (5) Hepatitis C virus polymerase inhibitors; (6) Not part of (2)-(3) nucleosides and nucleoside derivatives; (7) Hepatitis B virus (HBV) inhibitors; (7) Human immunodeficiency virus (HIV) inhibitors; (9) Cancer drugs; (10) An anti-inflammatory drug; or (11) other compounds not belonging to the above (1) to (10).
  8. 根据权利要求7所述的药物组合物，其中所述一种或多种其他的活性成分选自HCV NS3(NS3/4a)蛋白酶抑制剂、HCV NS4b抑制剂、HCV NS5A抑制剂、HCV NS5B聚合酶抑制剂、干扰素或干扰素衍生物、或利巴韦林。The pharmaceutical composition according to claim 7, wherein the one or more other active ingredients are selected from the group consisting of HCV NS3 (NS3/4a) protease inhibitors, HCV NS4b inhibitors, HCV NS5A inhibitors, HCV NS5B polymerase Inhibitor, interferon or interferon derivative, or ribavirin.
  9. 权利要求1-4中任意一项所述的化合物或权利要求5所述的混合物在制备治疗或预防HCV感染的药物中的应用。Use of a compound according to any one of claims 1 to 4 or a mixture according to claim 5 for the preparation of a medicament for the treatment or prevention of HCV infection.
  10. 权利要求1-4中任意一项所述的化合物或权利要求5所述的混合物与权利要求7或8所述的一种或多种其他的活性成分联合用于制备治疗或预防HCV感染的药物中的应用。 A compound according to any one of claims 1 to 4 or a mixture according to claim 5 in combination with one or more other active ingredients according to claim 7 or 8 for the preparation of a medicament for the treatment or prevention of HCV infection Application in .
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