WO2023217279A1 - 一种吡啶衍生物、中间体、制备方法和应用 - Google Patents

一种吡啶衍生物、中间体、制备方法和应用 Download PDF

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WO2023217279A1
WO2023217279A1 PCT/CN2023/094017 CN2023094017W WO2023217279A1 WO 2023217279 A1 WO2023217279 A1 WO 2023217279A1 CN 2023094017 W CN2023094017 W CN 2023094017W WO 2023217279 A1 WO2023217279 A1 WO 2023217279A1
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formula
compound represented
substance
mycobacterium tuberculosis
compound
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PCT/CN2023/094017
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French (fr)
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李永国
王春娟
李磊
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广州嘉越医药科技有限公司
上海嘉坦医药科技有限公司
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Publication of WO2023217279A1 publication Critical patent/WO2023217279A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C07D213/127—Preparation from compounds containing pyridine rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C07D213/16—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom containing only one pyridine ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C07D213/16—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom containing only one pyridine ring
    • C07D213/18—Salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/62—Oxygen or sulfur atoms
    • C07D213/63—One oxygen atom
    • C07D213/64—One oxygen atom attached in position 2 or 6
    • 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 relates to a pyridine derivative, intermediate, preparation method and application.
  • Pulmonary tuberculosis refers to tuberculosis lesions that occur in lung tissue, trachea, bronchi and pleura. Tuberculosis is still a major infectious disease that endangers people's health today, and the tuberculosis epidemic situation remains severe. In 2019, approximately 10 million people suffered from tuberculosis (range: 8.9 million to 11 million), which is equivalent to an average of 130 people per 100,000 people. The annual incidence rate varies from place to place (5 to 500 people) per 100,000 people. The majority (approximately 90%) of people who develop TB are adults, with more men than women; among global TB patients, 58% are men over 15 years old, 34% are women, and 8% are children under 15 years of age.
  • Bedaquiline is a diarylquinoline derivative. It was approved for marketing in the United States in December 2012, in the European Union in March 2014, and in November 2016 in China. Bedaquiline (BDQ) was historically listed by the World Health Organization (WHO) as the preferred regimen for rifampicin-resistant tuberculosis (RR-TB) and multidrug-resistant tuberculosis (MDR-TB) in 2018.
  • WHO World Health Organization
  • the technical problem to be solved by the present invention is to provide a pyridine derivative and its application in order to overcome the drug resistance problem of tuberculosis diseases in the prior art.
  • the pyridine derivative of the present invention can inhibit the growth of sensitive Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis, especially bedaquiline-resistant strains, and has the advantages of longer elimination half-life in pharmacokinetics and high safety. .
  • the present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof;
  • the present invention also provides a compound represented by formula IX or a pharmaceutically acceptable salt thereof;
  • the present invention also provides a compound represented by formula IY or a pharmaceutically acceptable salt thereof;
  • the retention time of the compound represented by Formula I-Y under the following SFC conditions is 1.445 min;
  • the present invention also provides a compound represented by formula II,
  • R 1 is an amino protecting group.
  • R 1 is preferably -Bn, -Trt, -Dmb or -PMB, more preferably -Bn.
  • the present invention also provides a compound represented by formula II-X,
  • R 1 is the same as mentioned above.
  • the invention also provides a method for preparing a compound represented by formula I, which includes the following steps: in a solvent, the compound represented by formula II-X is subjected to a deprotection reaction in the presence of a deprotecting reagent, and then undergoes a chiral Resolve to obtain the compound represented by formula I;
  • R 1 is the same as mentioned above.
  • the method and conditions of the deprotection reaction may be conventional methods and conditions for such reactions in the art.
  • the solvent can be a conventional solvent for such reactions in the art, such as methylene chloride.
  • the amount of the solvent can be a conventional capacity for such reactions in the art.
  • the volume-to-mass ratio of the solvent to the compound represented by Formula II-X can be 3 mL/g to 6 mL/g. g, preferably 5mL/g, 4mL/g or 4.7mL/g.
  • the deprotecting reagent can be a conventional deprotecting reagent for such reactions in the art, such as 1-chloroethyl chloroformate and methanol.
  • the amount of the deprotecting reagent may be a conventional capacity for such reactions in the art.
  • the molar ratio of the deprotecting reagent to the compound represented by Formula II-X may be 1.1:1. ⁇ 1.8:1, preferably 1.4:1.
  • the temperature of the deprotection reaction may be a temperature conventional for such reactions in the art, such as room temperature.
  • the progress of the deprotection reaction can be monitored using conventional detection methods for such reactions in the art, such as TLC, HPLC, GC or NMR, preferably HPLC.
  • the deprotection reaction can also include the following post-processing steps: after monitoring the end of the deprotection reaction, quenching the reaction, adjusting the pH, extracting and separating, and the obtained organic phase is concentrated and dried, and then separated by column chromatography. , to obtain the compound represented by the formula I-X.
  • the present invention also provides a method for preparing a compound represented by formula I, which includes the following steps: in a solvent, the compound represented by formula II is subjected to a deprotection reaction as shown below in the presence of a deprotecting reagent, Obtain the compound represented by the formula I;
  • R 1 is the same as mentioned above.
  • the method and conditions of the deprotection reaction may be conventional methods and conditions for such reactions in the art.
  • the solvent can be a conventional solvent for such reactions in the art, such as methylene chloride.
  • the amount of the solvent can be a conventional capacity for this type of reaction in the art.
  • the volume-to-mass ratio of the solvent to the compound represented by Formula II can be 3 mL/g to 6 mL/g, Preferably it is 5mL/g, 4mL/g or 4.7mL/g.
  • the deprotecting reagent can be a conventional deprotecting reagent for such reactions in the art, such as 1-chloroethyl chloroformate and methanol.
  • the amount of the deprotecting reagent can be a conventional capacity for such reactions in the art.
  • the molar ratio of the deprotecting reagent to the compound represented by Formula II can be 1.1:1 to 1.8. :1, preferably 1.4:1.
  • the temperature of the deprotection reaction may be a temperature conventional for such reactions in the art, such as room temperature.
  • the progress of the deprotection reaction can be monitored using conventional detection methods for such reactions in the art, such as TLC, HPLC, GC or NMR, preferably HPLC.
  • the deprotection reaction can also include the following post-processing steps: after monitoring the end of the deprotection reaction, quenching the reaction, adjusting the pH, extracting and separating, and the obtained organic phase is concentrated and dried, and then separated by column chromatography. , that is, gain Compounds of formula I are described.
  • the present invention also provides a method for preparing a compound represented by formula II-X, which includes the following steps: in a solvent, the compound represented by formula III and the compound represented by formula IV are reacted as shown below to obtain the Compounds represented by formula II-X;
  • R 1 is the same as mentioned above.
  • the method and conditions of the above reaction can be conventional methods and conditions for such reactions in the art.
  • the solvent can be a conventional solvent for such reactions in the art, such as tetrahydrofuran.
  • the amount of the solvent can be a conventional capacity for this type of reaction in the art.
  • the volume-to-mass ratio of the solvent to the compound represented by Formula III can be 4 mL/g to 6 mL/g, Preferably it is 5mL/g.
  • the base may be a base conventional for such reactions in the art, such as n-butyllithium.
  • the amount of the base can be a conventional capacity for such reactions in the art.
  • the molar ratio of the base to the compound represented by Formula III can be 1.2:1 to 2:1, preferably is 1.65:1.
  • the temperature of the deprotection reaction can be a conventional temperature for such reactions in the art, such as -60°C to -80°C, preferably -72°C.
  • the progress of the above reaction can be monitored using conventional detection methods for such reactions in the art.
  • the post-processing step of the preparation method of the compound represented by formula II-X can be a conventional post-processing step for this type of reaction in the art, for example, the following steps are included: after monitoring the completion of the reaction, the resulting reaction liquid Add it to a saturated solution of NH 4 Cl, wash, extract and separate the layers. The obtained organic phase is washed with brine and then separated. The obtained organic phase is concentrated, dried and separated by column chromatography to obtain the compound represented by the formula II-X.
  • the present invention also provides the use of substance A or substance B in the preparation of drugs for inhibiting Mycobacterium tuberculosis;
  • the substance A is a compound represented by formula I or a pharmaceutically acceptable salt thereof;
  • the substance B is Compounds represented by formula I-X or pharmaceutically acceptable salts thereof.
  • the Mycobacterium tuberculosis is preferably sensitive Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis.
  • the susceptible Mycobacterium tuberculosis is preferably strain/ATCC 27830.
  • the drug-resistant Mycobacterium tuberculosis is preferably bedaquiline-resistant Mycobacterium tuberculosis; the bedaquiline-resistant Mycobacterium tuberculosis is preferably selected from BDQ-resistant strain 1, BDQ One or more of drug-resistant strain 2 and BDQ-resistant strain 3.
  • the medicament may include pharmaceutical excipients.
  • the substance A or substance B may be one of the active ingredients or the only active ingredient of the drug.
  • the substance A is preferably a compound represented by formula I.
  • the substance B is preferably a compound represented by formula I-X.
  • the administration method of the drug can be a conventional administration method in the art, such as oral administration.
  • the administration frequency of the drug can be a conventional administration frequency in the art, preferably once per week.
  • the present invention also provides an application of substance A or substance B in the preparation of medicine for treating Mycobacterium tuberculosis infection;
  • the substance A is a compound represented by formula I or a pharmaceutically acceptable salt thereof;
  • the substance B is a compound represented by formula I-X or a pharmaceutically acceptable salt thereof.
  • the Mycobacterium tuberculosis is preferably sensitive Mycobacterium tuberculosis or drug-resistant Mycobacterium tuberculosis.
  • the drug-resistant Mycobacterium tuberculosis is preferably strain/ATCC 27830.
  • the drug-resistant Mycobacterium tuberculosis is preferably bedaquiline-resistant Mycobacterium tuberculosis; the bedaquiline-resistant Mycobacterium tuberculosis is preferably selected from BDQ-resistant strain 1, BDQ One or more of drug-resistant strain 2 and BDQ-resistant strain 3.
  • the medicament may include pharmaceutical excipients.
  • the disease infected by Mycobacterium tuberculosis is preferably pulmonary tuberculosis.
  • the substance A or substance B may be one of the active ingredients or the only active ingredient of the drug.
  • the substance A is preferably a compound represented by formula I.
  • the substance B is preferably a compound represented by formula I-X.
  • the administration method of the drug can be a conventional administration method in the art, such as oral administration.
  • the administration frequency of the drug can be a conventional administration frequency in the art, preferably once per week.
  • the present invention also provides a pharmaceutical composition, which includes substance A or substance B, and pharmaceutical excipients; the substance A is a compound represented by formula I or a pharmaceutically acceptable salt thereof; the substance B is a compound of formula I The compound represented by I-X or a pharmaceutically acceptable salt thereof.
  • the compound represented by formula I-X is a chiral isomer mixture containing at least the compound represented by formula I.
  • the "room temperature” is 20°C-30°C, preferably 25°C.
  • pharmaceutically acceptable means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients.
  • the "patient” is preferably a mammal, more preferably a human.
  • pharmaceutically acceptable salts refers to salts of compounds of the present invention prepared with relatively non-toxic, pharmaceutically acceptable acids or bases.
  • base addition can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in pure solution or a suitable inert solvent.
  • a salt When compounds of the present invention contain relatively basic functional groups, acid addition can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in pure solution or a suitable inert solvent.
  • the pharmaceutically acceptable acids include inorganic acids and organic acids. When the compound of the present invention contains relatively acidic and relatively basic functional groups, it can be converted into a base addition salt or an acid addition salt.
  • pharmaceutical excipients refers to all substances included in pharmaceutical preparations, except active pharmaceutical ingredients, and is generally divided into two categories: excipients and additives. For details, please refer to the “Pharmacopoeia of the People's Republic of China (2020 Edition)” and Handbook of Pharmaceutical Excipients (Paul J Sheskey, Bruno C Hancock, Gary P Moss, David J Goldfarb, 2020, 9th Edition).
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the nitrogen position of an amino group.
  • Representative amino protecting groups include, but are not limited to: arylmethyl, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trityl (Trt) and 2.4-dimethoxybenzyl (Dmb).
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, embodiments of the present invention.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effects of the present invention are: (1) the pyridine derivatives of the present invention can inhibit the growth of sensitive Mycobacterium tuberculosis, drug-resistant Mycobacterium tuberculosis and bedaquiline-resistant strains;
  • the pyridine derivative of the present invention has a longer elimination half-life in pharmacokinetics
  • Comparative drug Compound II is provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.; the compound II is compound I-1 in Example 1 of PCT/CN2017/070835; isoniazid (INH), rifampicin (RFP), moxisha Star (MFX) and PA-824 were purchased from Sigma-Aidrich; bedaquiline (BDQ) was purchased from Shanghai Hanxiang Biotechnology Co., Ltd.;
  • strain/H37RV (ATCC 27294), strain/ATCC 30248, strain/ATCC 30031, strain/ATCC 30129, strain/ATCC 30050, strain/ATCC 30091, strain/ATCC 30144, strain/ATCC30180, strain/ATCC 30611, strain /ATCC 29065,strain/ATCC 30797,strain/ATCC 30390,strain/ATCC 29928,strain/ATCC 29925,strain/ATCC 30105,strain/ATCC 30586,strain/ATCC 28198,strain/ATCC 30367,strain/ATCC 30102,strain /ATCC 30149, strain /ATCC 30577 and strain/ATCC 27830 were provided by the National Tuberculosis Clinical Laboratory of Beijing Chest Hospital affiliated to Capital Medical University (Beijing Tuberculosis and Thoracic Oncology Institute), and the strains are preserved in the Drug Research Laboratory.
  • microplate Alamar Blue method (MABA) was determined with reference to Lu Yu, Wang Bin, Zheng Meiqin, etc., and research on the application of Alamar Blue and MTT to determine the minimum inhibitory concentration of anti-tuberculosis drugs.
  • Each clinically isolated strain was cultured in DifoTM Middlebrook 7H9 medium containing 10% OADC in a 5% CO 2 incubator at 37°C for 2-3 weeks until the logarithmic growth phase, and then frozen at -80°C.
  • DifoTM Middlebrook 7H9 medium containing 10% OADC in a 5% CO 2 incubator at 37°C for 2-3 weeks until the logarithmic growth phase, and then frozen at -80°C.
  • the cryopreservation solution of each clinical strain from the -80°C storage cabinet, dilute the concentration of each strain to 1*10 6 CFU/mL, and add 100 ⁇ L to the drug-containing wells and bacterial solution control wells on the 96-well plate.
  • the final volume of each well is 200 ⁇ L, and the final concentration of bacterial solution inoculation is 5*10 5 CFU/mL.
  • the MIC ranges of compound I and control drugs against five sensitive clinical strains of Mycobacterium tuberculosis were 0.142-0.273ug/ml; compound II: 0.073-0.107ug/ml; BDQ: 0.018-0.019ug/ml; INH: 0.029- 0.038ug/ml; RFP: 0.068-0.091ug/ml; MFX: 0.035-0.056ug/ml; PA-824: 0.077-0.288ug/ml; Activity and Compounds of Compound I against 5 sensitive clinical strains of Mycobacterium tuberculosis II, BDQ, INH, RFP, MFX, PA-824 are similar.
  • Compound I has good in vitro activity against clinical isolates of Mycobacterium tuberculosis, including sensitive strains and drug-resistant strains.
  • the activity is similar to compound II and BDQ, and there is no cross-resistance with other anti-tuberculosis drugs.
  • Postprandial administration Subjects will be admitted to the Phase I clinical trial ward the day before the corresponding trial, and their lives will be managed uniformly, and any food or drink other than a unified diet is prohibited. The subjects had dinner at around 18:00, and then had no food or water. At around 8:00 on the 1st day of single administration, the 1st day of multiple administrations, and the 4th to 14th days, the subjects ate a normal meal and completed the meal within 30 minutes; 30 minutes after the subjects started eating, the researchers Distribute the trial drug (active drug or placebo) and take it with 240ml of water. No water for 1 hour after taking the drug. Have lunch and dinner respectively 4h and 10h after taking the medicine.
  • Multiple doses 0h before the first dose (within 60 minutes before taking the drug), 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, 24h, 48h, 72h after the dose (before D4 dose), 96h (before D5 administration), 120h (before D6 administration), 144h (before D7 administration), 264h (before D12 administration); 0h before the last administration, 1h, 2h, 3h, 4h, 5h after the last administration , 6h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 264h blood collection.
  • the drug concentration detection in this trial is carried out by Shanghai Xihua Testing Technology Co., Ltd.
  • the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the concentration of compound II and compound I in human plasma.
  • the internal standard method was used for quantification.
  • the plasma sample dosage was 100 ⁇ L.
  • the lowest limit of quantitation (LLOQ) for the determination of the concentration of compound II in plasma. ) is 10.0ng/mL, and the detection range is 10.0 to 5000ng/mL; the lowest limit of quantification (LLOQ) of compound I is 0.100ng/mL, and the detection range is 0.100 to 50.0ng/mL.
  • the measurement of the test sample will be carried out. Quality control is performed in the measurement of biological samples to ensure the reliability of the established methods in practical applications. Prepare quality control samples of different concentrations to evaluate the analysis method.
  • the non-compartmental model was used to calculate the pharmacokinetic parameters of the plasma original drug compound II and compound I through Phoenix WinNonlin 8.1 software.
  • t1/2z terminal elimination half-life
  • ⁇ z elimination rate constant
  • Compound I has a longer human half-life than Compound II. After reaching a steady state, it can be administered once a week, making it convenient for patients to take medication and overcoming the heavy burden of patent medicines caused by the combined use of multiple drugs in tuberculosis patients.
  • Comparative drug Compound II was provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.; the compound II was compound I-1 of Example 1 of PCT/CN2017/070835; bedaquiline (BDQ) was purchased from Shanghai Hanxiang Biotechnology Co., Ltd.;
  • strain/H37RV strain/BDQ-resistant strain 1
  • strain/BDQ-resistant strain 2 strain/BDQ-resistant strain Drug strains 3 were provided by the National Tuberculosis Clinical Laboratory of Beijing Chest Hospital (Beijing Institute of Tuberculosis and Thoracic Tumors) affiliated to Capital Medical University, and the strains are preserved in the Drug Research Laboratory.
  • MABA microplate Alamar Blue method
  • microplate Alamar Blue method (MABA) was determined with reference to Lu Yu, Wang Bin, Zheng Meiqin, etc., and research on the application of Alamar Blue and MTT to determine the minimum inhibitory concentration of anti-tuberculosis drugs.
  • Compound I and the reference drug were accurately weighed.
  • Compound I, Compound II and bedaquiline (BDQ) were dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration storage solution (see Table 5).
  • Use 7H9 liquid The culture medium is diluted to the concentration of the application solution.
  • the final concentration of Compound I and the control drug in the culture medium is twice the concentration.
  • the final concentrations for the strains are: Compound I: 32 ⁇ 0.0156 ⁇ g/mL; Compound II: 32 ⁇ 0.0156 ⁇ g. /mL; BDQ: 10 ⁇ 0.005 ⁇ g/mL.
  • the BDQ-resistant strains induced by each laboratory were cultured in Difo TM Middlebrook 7H9 medium containing 10% OADC in a 5% CO 2 incubator at 37°C for 2-3 weeks to the logarithmic growth phase, and then frozen at -80°C. Take out the cryopreservation solution of each clinical strain from the -80°C storage cabinet, dilute the concentration of each strain to 1*10 6 CFU/mL, and add 100 ⁇ L to the drug-containing wells and bacterial solution control wells on the 96-well plate. The final volume of each well is 200 ⁇ L, and the final concentration of bacterial solution inoculation is 5*10 5 CFU/mL.
  • the fluorescence value of Alamar Blue in the 96-well culture plate was measured using a multifunctional microplate reader (Tecan infininte m200) at the wavelength (560nm, 590nm). The minimum inhibitory concentration of each drug was obtained by observing the color change and calculating the fluorescence value.
  • Test products Compound I (Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) and Compound III (Wuhan WuXi AppTec New Drug Development Co., Ltd.);
  • Negative control group 0.3% DMSO (purchased from Sinopharm);
  • Negative control substance Measure the required volume of DMSO into the container as the storage solution
  • Test sample Under light-proof conditions, weigh an appropriate amount of Compound I and Compound III, add an appropriate amount of DMSO respectively, and prepare a 10 mmol/L storage solution. Put the prepared solution in a container and store it at room temperature in the dark;
  • Positive control substance Weigh an appropriate amount of haloperidol, add an appropriate amount of DMSO and dissolve it into a 1mmol/L stock solution, aliquot the prepared solution and store it at -20°C for later use.
  • the DMSO and Compound I group administration preparations were diluted with an appropriate amount of Tyrode's solution to the working solution of the required concentration.
  • the positive control group administration preparations were taken out from the -20°C refrigerator and thawed and diluted with an appropriate amount of Tyrode's solution to the required concentration.
  • Working fluid of required concentration The details are as follows in Table 7:
  • New Zealand white rabbits SPF grade; 40 pieces; 2.4-3.5kg, 3-5 months old; purchased from China Institute of Food and Drug Control.
  • test animal 3% sodium pentobarbital (approximately 45-60 mg/kg), ear vein injection, cut the abdomen, open the chest, expose the heart, and take out the heart.
  • Tyrode's solution (mmol): NaCl 137, KCl 4, MgSO 4 ⁇ 7H 2 O 1, NaH 2 PO 4 ⁇ 2H 2 O 0.4, NaHCO 3 12, CaCl 2 1.8, Glucose 5.6, PH 7.35-7.4, 95% O 2 +5% CO 2 mixed
  • the gas is saturated for more than 30 minutes;
  • Test working solution From low concentration to high concentration (0.3, 1, 3 and 10 ⁇ mol/L (Tyrode's solution)), add a higher concentration of the test working solution after the sample has been added for about 15 minutes and its effect reaches a steady state. .
  • DMSO Add the negative control working solution (0.3% DMSO (in Tyrode’s solution)) before adding the test product working solution. After the sample has been added for about 5 minutes and its effect reaches a steady state, a low-concentration test product working solution can be added.
  • Positive control working solution Select different tissues to be tested and add positive control working solution (1 ⁇ mol/L haloperidol (in Tyrode’s solution)).
  • RMP Resting potential
  • Vmax action potential 0 phase rise rate
  • APA action potential amplitude
  • APID action potential duration
  • RMP, APD, APA and Vmax are expressed as mean ⁇ standard deviation, and the percentage of data change before and after adding samples is calculated.
  • the average values of RMP, APD, APA and Vmax of each concentration group of the test article/control substance are corrected using the average values of RMP, APD, APA and Vmax of each tissue before adding the sample, and the RMP, APD, APA and Vmax of each concentration group are calculated.
  • the percentage change ( ⁇ %) the formula is as follows:
  • Test products compound I (provided by Shanghai Jiatan Pharmaceutical Technology Co., Ltd.) and compound III (provided by Wuhan WuXi AppTec New Drug Development Co., Ltd.);
  • Negative control substance DMSO (purchased from Sigma);
  • hCav1.2 cells were constructed by the laboratory of Beijing Aisiyipu Biotechnology Co., Ltd.
  • the voltage stimulation protocol for whole-cell patch clamp recording of hCav1.2 channel current is as follows: when whole-cell sealing is formed, the cell membrane voltage is clamped at -60mV. The clamping voltage is depolarized from -60mV to 0mV (the specific voltage is determined by the pilot IV) and maintained for 0.3 seconds. Repeat data collection every 20 seconds to observe the effect of the drug on the hCav1.2 channel current peak. Experimental data were collected by an EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software (software version: v2x73.2).
  • Capillary glass tubes (BF150-86-10, Sutter Instruments) were drawn into recording electrodes using a microelectrode drawing instrument (P97, Sutter Instruments).
  • the microelectrode manipulator (86PW420600, MCI Instruments) was operated under an inverted microscope (AE31E, Motic) to contact the recording electrode to the cells, and negative pressure suction was applied to form a G ⁇ seal. After the G ⁇ seal is formed, rapid capacitance compensation is performed, and then negative pressure is continued to be applied to break the cell membrane and form a whole-cell recording mode.
  • Inhibition rate 1/[1+(IC 50 /c) h ];

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Abstract

提供了一种吡啶衍生物、中间体、制备方法和应用，具体为如式I所示的化合物或其药学上可接受的盐。所述的吡啶衍生物能够抑制敏感结核分枝杆菌、耐药结核分支杆菌，特别是贝达喹啉耐药菌株的生长，且具有在药代动力学中消除半衰期更长以及安全性更高的优势。

Description

一种吡啶衍生物、中间体、制备方法和应用
本申请要求申请日为2022/5/13的中国专利申请2022105241422的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种吡啶衍生物、中间体、制备方法和应用。
背景技术
肺结核(pulmonary tuberculosis，PTB)是指发生在肺组织、气管、支气管和胸膜的结核病变。结核病仍是当今危害人们健康的重大传染病，结核病疫情形势依然严峻。2019年，约有1000万人患结核病(范围为890万～1100万)，相当于平均每10万人有130人罹患结核病，每年各地发病率为(5～500人)/10万不等。大多数(约90％)罹患结核病的人是成年人，男性多于女性；在全球结核病患者中，年龄在15岁以上男性占58％，女性占34％，15岁以下儿童占8％。从地理上看，在2019年患结核病的人大部分在东南亚(44％)、非洲(25％)和西太平洋(18％)，另外所占比例较小的是地中海东部(8.2％)、美洲(2.9％)和欧洲(2.5％)。30个高结核病负担国家名单中的8个国家的结核病病例数占全球总数2/3：印度(26％)、印度尼西亚(8.5％)、中国(8.4％)、菲律宾(6.0％)、巴基斯坦(5.7％)、尼日利亚(4.4％)、孟加拉国(3.6％)和南非(3.6％)。30个高结核病负担国家中的其他22个国家的结核病病例数占全球总数的21％。2019年，有54个国家的结核病发病率较低(每10万人口每年少于10例)，主要分布在WHO美洲和欧洲区域以及东地中海的一些国家和西太平洋地区。
贝达喹啉是二芳基喹啉类衍生物，于2012年12月获批在美国上市，2014年3月获批在欧盟上市，并于2016年11月获批在国内上市。贝达喹啉(BDQ)在2018年历史上被世界卫生组织(WHO)列为耐利福平结核病(RR-TB)和耐多药结核病(MDR-TB)的首选方案。
申请号：201410335196.X的专利记载了吡啶衍生物及其作为抗分支杆菌的应用，用于治疗肺结核及多重耐药性肺结核。
传染性结核病的并发症引起多重耐药性结核病。世界范围内，4％的情况都与多重耐药性结核病有关。
耐多药结核病人群的比例在初步诊断中仍保持在3-4％左右，在治疗有经验的患者中约为18-21％。耐药结核病仍然是一种公共卫生威胁。目前漫长而复杂的治疗方案根本不足以遏制和战胜耐多药结核病感染，从而促使人们重新寻找更好的药物。
发明内容
本发明所要解决的技术问题是为了克服现有技术中结核类疾病的耐药性问题，而提供了一种吡啶衍生物和应用。本发明的吡啶衍生物能够抑制敏感结核分枝杆菌、耐药结核分支杆菌，特别是贝达喹啉耐药菌株的生长，且具有在药代动力学中消除半衰期更长以及安全性高的优势。
本发明提供一种如式I所示的化合物或其药学上可接受的盐；
本发明还提供一种如式I-X所示的化合物或其药学上可接受的盐；
本发明还提供一种如式I-Y所示的化合物或其药学上可接受的盐；
其中，所述的如式I-Y所示的化合物在下述SFC条件下的保留时间为1.445min；
色谱柱：ChiralpakAD-3.50A，4.6mm，内径3um；
流动相：A：CO2，B：EtOH(0.05％二乙醇胺)；
梯度：MPB(流动相B)在1.2分钟内从由5％至50％，保持1分钟，然后0.8分钟内由50％至5％；
流速：3.4mL/min；
柱温：35℃；
ABPR：1800psi。
本发明还提供一种如式II所示的化合物，
其中，R1为氨基保护基。
在一实施方案中，所述R1优选为-Bn、-Trt、-Dmb或-PMB，更优选为-Bn。
本发明还提供一种如式II-X所示的化合物，
其中，R1的定义同前所述。
本发明还提供一种如式I所示的化合物的制备方法，其包括以下步骤：溶剂中，所述式II-X所示的化合物在脱保护试剂的存在下进行脱保护反应，再经手性拆分，得所述式I所示的化合物；
其中，R1的定义同前所述。
在一实施方案中，所述脱保护反应的方法和条件可为本领域此类反应常规的方法和条件。
在一实施方案中，所述的溶剂可为本领域此类反应常规的溶剂，例如为二氯甲烷。
在一实施方案中，所述溶剂的用量可为本领域此类反应常规的容量，例如，所述溶剂与所述式II-X所示的化合物的体积质量比可为3mL/g～6mL/g，优选为5mL/g、4mL/g或4.7mL/g。
在一实施方案中，所述脱保护试剂可为本领域此类反应常规的脱保护试剂，例如为氯甲酸-1-氯乙酯和甲醇。
在一实施方案中，所述脱保护试剂的用量可为本领域此类反应常规的容量，例如，所述脱保护试剂与所述式II-X所示的化合物的摩尔比可为1.1:1～1.8:1，优选为1.4:1。
在一实施方案中，所述脱保护反应的温度可为本领域此类反应常规的温度，例如为室温。
在一实施方案中，所述脱保护反应的进程可采用本领域此类反应常规的检测方法进行监测，例如TLC、HPLC、GC或NMR，优选为HPLC。
在一实施方案中，所述脱保护反应还可包括以下后处理步骤：监测脱保护反应结束后，淬灭反应，调节PH后萃取和分液，所得有机相浓缩干燥后，经柱层析分离，即得所述式I-X所示的化合物。
本发明还提供一种如式I所示的化合物的制备方法，其包括以下步骤：溶剂中，所述式II所示的化合物在脱保护试剂的存在下，进行如下所示的脱保护反应，得所述式I所示的化合物；
其中，R1的定义同前所述。
在一实施方案中，所述脱保护反应的方法和条件可为本领域此类反应常规的方法和条件。
在一实施方案中，所述的溶剂可为本领域此类反应常规的溶剂，例如为二氯甲烷。
在一实施方案中，所述溶剂的用量可为本领域此类反应常规的容量，例如，所述溶剂与所述式II所示的化合物的体积质量比可为3mL/g～6mL/g，优选为5mL/g、4mL/g或4.7mL/g。
在一实施方案中，所述脱保护试剂可为本领域此类反应常规的脱保护试剂，例如为氯甲酸-1-氯乙酯和甲醇。
在一实施方案中，所述脱保护试剂的用量可为本领域此类反应常规的容量，例如，所述脱保护试剂与所述式II所示的化合物的摩尔比可为1.1:1～1.8：1，优选为1.4:1。
在一实施方案中，所述脱保护反应的温度可为本领域此类反应常规的温度，例如为室温。
在一实施方案中，所述脱保护反应的进程可采用本领域此类反应常规的检测方法进行监测，例如TLC、HPLC、GC或NMR，优选为HPLC。
在一实施方案中，所述脱保护反应还可包括以下后处理步骤：监测脱保护反应结束后，淬灭反应，调节PH后萃取和分液，所得有机相浓缩干燥后，经柱层析分离，即得所 述式I所示的化合物。
本发明还提供一种如式II-X所示的化合物的制备方法，其包括以下步骤：溶剂中，式III所示的化合物与式IV所示的化合物进行如下所示的反应，得所述式II-X所示的化合物；
其中，R1的定义同前所述。
在一实施方案中，上述反应的方法和条件可为本领域此类反应常规的方法和条件
在一实施方案中，所述的溶剂可为本领域此类反应常规的溶剂，例如为四氢呋喃。
在一实施方案中，所述溶剂的用量可为本领域此类反应常规的容量，例如，所述溶剂与所述式III所示的化合物的体积质量比可为4mL/g～6mL/g，优选为5mL/g。
在一实施方案中，所述碱可为本领域此类反应常规的碱，例如为正丁基锂。
在一实施方案中，所述碱的用量可为本领域此类反应常规的容量，例如，所述碱与所述式III所示的化合物的摩尔比可为1.2:1～2:1，优选为1.65:1。
在一实施方案中，所述脱保护反应的温度可为本领域此类反应常规的温度，例如为-60℃～-80℃，优选为-72℃。
在一实施方案中，上述反应的进程可采用本领域此类反应常规的检测方法进行监测。
在一实施方案中，所述式II-X所示的化合物的制备方法，其后处理步骤可为本领域该类反应常规的后处理步骤，例如包括以下步骤：监测反应结束后，所得反应液加入NH4Cl饱和溶液中，经洗涤、萃取和分液，所得有机相用盐水洗涤后分液，所得有机相浓缩干燥并柱层析分离，即得所述式II-X所示的化合物。
本发明还提供了一种物质A或物质B在制备抑制结核分枝杆菌的药物中的应用；所述物质A为式I所示的化合物或其药学上可接受的盐；所述物质B为式I-X所示的化合物或其药学上可接受的盐。
在一实施方案中，所述的结核分枝杆菌优选为敏感结核分枝杆菌或耐药结核分枝杆菌。
在一实施方案中，所述的敏感结核分枝杆菌优选为菌株/ATCC 27830。
在一实施方案中，所述的耐药结核分枝杆菌优选为贝达喹啉耐药结核分枝杆菌；所述贝达喹啉耐药结核分枝杆菌优选选自BDQ耐药菌株1、BDQ耐药菌株2和BDQ耐药菌株3中的一种或多种。
在一些实施方案中，所述的药物可包含药用辅料。
在一些实施方案中，所述的物质A或物质B可为所述药物的有效成分之一或者唯一有效成分。
在一些实施方案中，所述的物质A优选为式I所示的化合物。
在一些实施方案中，所述的物质B优选为式I-X所示的化合物。
在一些实施方案中，所述的药物的施用方式可为本领域常规的施用方式，例如为口服。
在一些实施方案中，所述的药物的施用频次可为本领域常规的施用频次，优选为1次/周。
本发明还提供了一种物质A或物质B在制备治疗结核分枝杆菌感染的药物中的应用；所述物质A为如式I所示的化合物或其药学上可接受的盐；所述物质B为如式I-X所示的化合物或其药学上可接受的盐。
在一实施方案中，所述的结核分枝杆菌优选为敏感结核分枝杆菌或耐药结核分枝杆菌。
在一实施方案中，所述的耐药结核分枝杆菌优选为菌株/ATCC 27830。
在一实施方案中，所述的耐药结核分枝杆菌优选为贝达喹啉耐药结核分枝杆菌；所述贝达喹啉耐药结核分枝杆菌优选选自BDQ耐药菌株1、BDQ耐药菌株2和BDQ耐药菌株3中的一种或多种。
在一些实施方案中，所述的药物可包含药用辅料。
在一些实施方案中，所述的结核分枝杆菌感染的疾病优选为肺结核。
在一些实施方案中，所述的物质A或物质B可为所述药物的有效成分之一或者唯一有效成分。
在一些实施方案中，所述的物质A优选为式I所示的化合物。
在一些实施方案中，所述的物质B优选为式I-X所示的化合物。
在一些实施方案中，所述的药物的施用方式可以为本领域常规的施用方式，例如为口服。
在一些实施方案中，所述的药物的施用频次可以为本领域常规的施用频次，优选为1次/周。
本发明还提供了一种药物组合物，其包括物质A或物质B，和药用辅料；所述物质A为式I所示的化合物或其药学上可接受的盐；所述物质B为式I-X所示的化合物或其药学上可接受的盐。
如无特殊说明，本发明中，所述式I-X所示的化合物为至少包含式I所示化合物的手性异构体混合物。
如无特殊说明，本发明中，所述“室温”为20℃-30℃，优选为25℃。
如无特别说明，本发明所用术语具有如下含义：
术语“药学上可接受的”是指盐、溶剂、辅料等一般无毒、安全，并且适合于患者使用。所述的“患者”优选哺乳动物，更优选为人类。
术语“药学上可接受的盐”是指本发明化合物与相对无毒的、药学上可接受的酸或碱制备得到的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的碱与这类化合物的中性形式接触的方式获得碱加成盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的药学上可接受的酸与这类化合物的中性形式接触的方式获得酸加成盐。所述的药学上可接受的酸包括无机酸和有机酸。当本发明的化合物中含有相对酸性和相对碱性的官能团时，可以被转换成碱加成盐或酸加成盐。具体可参见Berge et al.,"Pharmaceutical Salts",Journal of Pharmaceutical Science 66:1-19(1977)、或、Handbook of Pharmaceutical Salts:Properties,Selection,and Use(P.Heinrich Stahl and Camille G.Wermuth,ed.,Wiley-VCH,2002)。
术语“药用辅料”是指除活性药物成分以外，包含在药物制剂中的所有物质，一般分为赋形剂和附加剂两大类。具体可参见《中华人民共和国药典(2020年版)》、Handbook of Pharmaceutical Excipients(Paul J Sheskey,Bruno C Hancock,Gary P Moss,David J Goldfarb,2020,9th Edition)。
术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB),9-芴基甲基(Fm)，三苯甲基(Trt)和2.4-二甲氧基苄基(Dmb)。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明采用下述缩略词：aq代表水；eq代表当量、等量；DCM代表二氯甲烷；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；Bn代表苄基；PMB代表对甲氧基苄基；Trt代表三苯甲基；和Dmb代表2.4-二甲氧基苄基。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：(1)本发明的吡啶衍生物能够抑制敏感结核分枝杆菌、耐药结核分枝杆菌以及贝达喹啉耐药菌株的生长；
(2)本发明的吡啶衍生物在药代动力学中消除半衰期更长；
(2)本发明的吡啶衍生物具有更高的安全性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
下述实施例中的SFC手性分离方法：
利用下面手性分离条件，获得对映异构体化合物Ⅰ；
色谱柱：ChiralpakAD-3.50A，4.6mm，内径3um；
流动相：A：CO2，B：EtOH(0.05％二乙醇胺)；
梯度：MPB在1.2分钟内从由5％至50％，保持1分钟，然后0.8分钟内由50％至5％；
流速：3.4mL/min；
柱温：35℃；
ABPR：1800psi。
对映异构体化合物Ⅰ的RT：1.445min。
实施例1：化合物I的制备方法
搅拌下，向500mL的反应瓶中依次加入IPA(100mL)，浓HCl(100mL)，α-乙酰基萘(55.0g)，苄基甲基胺(40g)，多聚甲醛(17.2g)。搅拌升温至回流，约为95℃。回流7.5h后，HPLC监控反应结束。浓缩至干，后加入乙酸乙酯，搅拌过夜。过滤，滤饼用乙酸乙酯洗涤至滤液无色。固体加入DCM。搅拌形成悬浊液后，用NaHCO3水溶液调节pH～9，分液。有机相干燥浓缩，得到淡黄色油状液体化合物3，共计75.0g，收率76％。1H NMR(400MHz,CDCl3)δ8.58(d,J＝8.5Hz,1H),7.94(d,J＝8.2Hz,1H),7.85(d,J＝7.9Hz,1H),7.80(d,J＝7.1Hz,1H),7.54(ddd,J＝14.9,14.5,7.3Hz,2H),7.47–7.41(m,1H),7.25–7.17(m,5H),3.50(s,2H),3.24(t,J＝7.1Hz,2H),2.91(t,J＝7.1Hz,2H),2.22(s,3H)。
向500mL反应瓶中加入THF(180mL)，TMP(24.90g)，搅拌降温至-72℃。开始控温滴加n-BuLi(12.46g),体系成乳白色悬浊液。控温反应约2h后，控温滴加化合物4 (36.4g)，滴加结束后，控温反应2h，监控反应，无明显原料。开始控温滴加3(42.78g)，控温反应三小时后，送样监控，反应结束。反应液加入到200mL的NH4Cl饱和溶液中，洗涤两次。有机相再用盐水洗一次，分液。有机相干燥浓缩后，拌样过柱得无色油状物化合物5。
向500mL反应瓶中加入5(50.0g)，DCM(250mL)，搅拌溶解后加入氯甲酸-1-氯乙酯(11.6g)，室温下搅拌3h，反应液浓缩至干，然后加入甲醇，继续室温搅拌反应2h。HPLC监控反应结束后，直接加入NaHCO3水溶液淬灭。至pH～9淬灭后加入DCM，洗涤分液。有机相干燥浓缩，固体拌样过柱纯化，得淡黄色固体，SFC手性拆分得到化合物I。RT：1.445，ee值：100％，纯度：98.93％，收率18％。1H NMR(400MHz,DMSO)δ8.61(d,J＝7.8Hz,1H),8.49(s,1H),8.41(s,1H),8.24(s,1H),7.92(d,J＝6.6Hz,2H),7.71(d,J＝8.1Hz,5H),7.62–7.50(m,4H),7.34(t,J＝7.5Hz,1H),7.17(d,J＝5.7Hz,2H),5.72(s,1H),4.14(s,4H),2.81(s,1H),2.32(s,1H),2.08(s,4H),1.99(s,1H)。
实施例2
搅拌下，向500mL的反应瓶中依次加入DCM(100mL)，通入甲基胺(31.4g)，降温至0度。分批加入三苯基氯甲烷(TrtCl，140g)。室温反应2小时，HPLC监控反应结束。碳酸钠水洗后，分液，浓缩得到油状液体化合物6，共计103.5g，收率75％。
搅拌下，向500mL的反应瓶中依次加入IPA(100mL)，浓HCl(100mL)，α-乙酰基萘(55.0g)，化合物6(90.1g)，多聚甲醛(17.2g)。搅拌升温至回流，约为95℃。回流7.5h后，HPLC监控反应结束。浓缩至干，后加入乙酸乙酯，搅拌过夜。过滤，滤饼用乙酸乙酯洗涤至滤液无色。固体加入DCM。搅拌形成悬浊液后，用NaHCO3水溶液调节pH～9，分液。有机相干燥浓缩，得到油状液体化合物3-1，共计115.6g，收率77％。 1H NMR(400MHz,CDCl3)δ8.53(d,J＝8.5Hz,1H),7.93(d,J＝8.2Hz,1H),7.80(d,J＝7.9Hz,1H),7.72(d,J＝7.1Hz,1H),7.51(ddd,J＝14.9,14.5,7.3Hz,2H),7.46-7.48(d,J＝7.5Hz,6H),7.45–7.41(m,1H),7.25–7.21(m,6H),7.18–7.14(t,J＝7.2Hz,14.4Hz,3H),3.27(t,J＝7.1Hz,2H),2.84(t,J＝7.1Hz,2H),2.24(s,3H)。
向500mL反应瓶中加入THF(180mL)，TMP(24.90g)，搅拌降温至-72℃。开始控温滴加n-BuLi(12.46g),体系成乳白色悬浊液。控温反应约2h后，控温滴加化合物4(36.41g)，滴加结束后，控温反应2h，监控反应，无明显原料。开始控温滴加3-1(64.24g)，控温反应三小时后，送样监控，反应结束。反应液加入到200mL的NH4Cl饱和溶液中，洗涤两次。有机相再用盐水洗一次，分液。有机相干燥浓缩后，拌样过柱得无色油状物化合物5-1。
向500mL反应瓶中加入5-1(62.4g)，DCM(250mL)，搅拌溶解后加入氯甲酸-1-氯乙酯(11.7g)，室温下搅拌3h，反应液浓缩至干，然后加入甲醇，继续室温搅拌反应2h。HPLC监控反应结束后，直接加入NaHCO3水溶液淬灭。至pH～9淬灭后加入DCM，洗涤分液。有机相干燥浓缩，固体拌样过柱纯化，得淡黄色固体，SFC手性拆分得到化合物I。RT：1.445，ee值：100％，纯度：98.94％，收率19％。1H NMR(400MHz,DMSO)δ8.61(d,J＝7.8Hz,1H),8.49(s,1H),8.41(s,1H),8.24(s,1H),7.92(d,J＝6.6Hz,2H),7.71(d,J＝8.1Hz,5H),7.62–7.50(m,4H),7.34(t,J＝7.5Hz,1H),7.17(d,J＝5.7Hz,2H),5.72(s,1H),4.14(s,4H),2.81(s,1H),2.32(s,1H),2.08(s,4H),1.99(s,1H)。
实施例3
搅拌下，向500mL的反应瓶中依次加入甲醇(100mL)，通入甲基胺(17.94g)，降温到20度，分批加入2,4-二甲氧基苯甲醛(80g)。室温反应2小时，分批加入硼氢化钠 (21.87g)，HPLC监控反应结束，用盐酸调节pH到弱酸性，浓缩后，用乙酸乙酯溶解，氯化钠水洗后，分液，浓缩得到化合物7，共计61.1g，收率70％。
搅拌下，向500mL的反应瓶中依次加入IPA(100mL)，浓HCl(100mL)，α-乙酰基萘(55.0g)，化合物7(58.6g)，多聚甲醛(17.2g)。搅拌升温至回流，约为95℃。回流7.5h后，HPLC监控反应结束。浓缩至干，后加入乙酸乙酯，搅拌过夜。过滤，滤饼用乙酸乙酯洗涤至滤液无色。固体加入DCM。搅拌形成悬浊液后，用NaHCO3水溶液调节pH～9，分液。有机相干燥浓缩，得到油状液体化合物3-2，共计89.2g，收率76％。1H NMR(400MHz,CDCl3)δ8.52(d,J＝8.5Hz,1H),7.91(d,J＝8.2Hz,1H),7.80(d,J＝7.9Hz,1H),7.72(d,J＝7.1Hz,1H),7.50(dd,J＝14.9,14.5,7.3Hz,2H),7.46–7.39(m,2H),6.43(d,J＝4.4Hz,1H),6.31(s,1H),3.81(s,6H),3.56(s,2H),3.23(t,J＝7.1Hz,2H),2.91(t,J＝7.1Hz,2H),2.26(s,3H)。
向500mL反应瓶中加入THF(180mL)，TMP(24.90g)，搅拌降温至-72℃。开始控温滴加n-BuLi(12.46g),体系成乳白色悬浊液。控温反应约2h后，控温滴加化合物4(36.4g)，滴加结束后，控温反应2h，监控反应，无明显原料。开始控温滴加化合物3-2(51.3g)，控温反应三小时后，送样监控，反应结束。反应液加入到200mL的NH4Cl饱和溶液中，洗涤两次。有机相再用盐水洗一次，分液。有机相干燥浓缩后，拌样过柱得无色油状物化合物5-1。
向500mL反应瓶中加入化合物5-2(50.0g)，DCM(250mL)，搅拌溶解后加入氯甲酸-1-氯乙酯(11.6g)，室温下搅拌3h，反应液浓缩至干，然后加入甲醇，继续室温搅拌反应2h。HPLC监控反应结束后，直接加入NaHCO3水溶液淬灭。至pH～9淬灭后加入DCM，洗涤分液。有机相干燥浓缩，固体拌样过柱纯化，得淡黄色固体，SFC手性拆分得到化合物I。RT：1.445，ee值：100％，纯度：98.95％，收率17％。1H NMR(400MHz,DMSO)δ8.61(d,J＝7.8Hz,1H),8.49(s,1H),8.41(s,1H),8.24(s,1H),7.92(d,J＝6.6Hz,2H),7.71(d,J＝8.1Hz,5H),7.62–7.50(m,4H),7.34(t,J＝7.5Hz,1H),7.17(d,J＝5.7Hz,2H),5.72(s,1H),4.14(s,4H),2.81(s,1H),2.32(s,1H),2.08(s,4H),1.99(s,1H)。
实施例4
搅拌下，向500mL的反应瓶中依次加入IPA(100mL)，浓HCl(100mL)，α-乙酰 基萘(55.0g)，对甲氧基苄基甲基胺(46g)，多聚甲醛(17.2g)。搅拌升温至回流，约为95℃。回流7.5h后，HPLC监控反应结束。浓缩至干，后加入乙酸乙酯，搅拌过夜。过滤，滤饼用乙酸乙酯洗涤至滤液无色。固体加入DCM。搅拌形成悬浊液后，用NaHCO3水溶液调节pH～9，分液。有机相干燥浓缩，得到油状液体化合物3-3，共计83.4g，收率75％。1H NMR(400MHz,CDCl3)δ8.53(d,J＝8.5Hz,1H),7.93(d,J＝8.2Hz,1H),7.80(d,J＝7.9Hz,1H),7.72(d,J＝7.1Hz,1H),7.51(dd,J＝14.9,14.5,7.3Hz,2H),7.48–7.21(m,3H),6.45(d,J＝4.4Hz,2H),3.83(s,3H),3.56(s,2H),3.22(t,J＝7.1Hz,2H),2.94(t,J＝7.1Hz,2H),2.26(s,3H)。
向500mL反应瓶中加入THF(180mL)，TMP(24.90g)，搅拌降温至-72℃。开始控温滴加n-BuLi(12.46g),体系成乳白色悬浊液。控温反应约2h后，控温滴加化合物4(36.4g)，滴加结束后，控温反应2h，监控反应，无明显原料。开始控温滴加3-3(47.1g)，控温反应三小时后，送样监控，反应结束。反应液加入到200mL的NH4Cl饱和溶液中，洗涤两次。有机相再用盐水洗一次，分液。有机相干燥浓缩后，拌样过柱得无色油状物。
向500mL反应瓶中加入5(53g)，DCM(250mL)，搅拌溶解后加入氯甲酸-1-氯乙酯(11.6g)，室温下搅拌3h，反应液浓缩至干，然后加入甲醇，继续室温搅拌反应2h。HPLC监控反应结束后，直接加入NaHCO3水溶液淬灭。至pH～9淬灭后加入DCM，洗涤分液。有机相干燥浓缩，固体拌样过柱纯化，得淡黄色固体，SFC手性拆分得到化合物I。RT：1.445，ee值：100％，纯度：98.94％，收率19％。1H NMR(400MHz,DMSO)δ8.61(d,J＝7.8Hz,1H),8.49(s,1H),8.41(s,1H),8.24(s,1H),7.92(d,J＝6.6Hz,2H),7.71(d,J＝8.1Hz,5H),7.62–7.50(m,4H),7.34(t,J＝7.5Hz,1H),7.17(d,J＝5.7Hz,2H),5.72(s,1H),4.14(s,4H),2.81(s,1H),2.32(s,1H),2.08(s,4H),1.99(s,1H)。
实施例5
1.目的：测试化合物I对结核分枝杆菌临床分离菌株(包括药物敏感菌株及耐药菌株)的体外活性
2.实验材料：
对照药物：化合物Ⅱ由上海嘉坦医药科技有限公司提供；所述化合物Ⅱ为PCT/CN2017/070835实施例1中化合物I-1；异烟肼(INH)、利福平(RFP)、莫西沙星(MFX)及PA-824购于Sigma-Aidrich；贝达喹啉(BDQ)购于上海翰香生物科技有限公司；
菌株：菌株/H37RV(ATCC 27294)、菌株/ATCC 30248、菌株/ATCC 30031、菌株/ATCC 30129、菌株/ATCC 30050、菌株/ATCC 30091、菌株/ATCC 30144、菌株/ATCC30180、菌株/ATCC 30611、菌株/ATCC 29065、菌株/ATCC 30797、菌株/ATCC 30390、菌株/ATCC 29928、菌株/ATCC 29925、菌株/ATCC 30105、菌株/ATCC 30586、菌株/ATCC 28198、菌株/ATCC 30367、菌株/ATCC 30102、菌株/ATCC 30149、菌株/ATCC 30577、菌株/ATCC 27830均为首都医科大学附属北京胸腔医院(北京市结核病胸部肿瘤研究所)国家结核病临床实验室提供，药物研究室保存菌株。
培养基：7H9液体培养基(含10％OADC)，为BD公司产品。
3.实验方法
应用微孔板Alamar Blue法(MABA)测定化合物Ⅰ、化合物Ⅱ、贝达喹啉(BDQ)、异烟肼(INH)、利福平(RFP)、莫西沙星(MFX)及PA-824对5株结核分枝杆菌临床分离敏感菌株以及15株结核分支杆菌临床分离耐药菌株的最低抑菌浓度(Minimum Inhibitory Concentration,MIC)。
微孔板Alamar Blue法(MABA)测定参考陆宇，王彬，郑梅琴等，应用Alamar Blue和MTT测定抗结核药物最低抑菌浓度的研究.中国防痨杂质，2007，29(6)：499-501。
(1)化合物I与对照药物精准称量，异烟肼(INH)用灭菌蒸馏水溶解，化合物I、化合物II、利福平(RFP)、莫西沙星(MFX)、PA-824及贝达喹啉(BDQ)用二甲基亚砜(DMSO)溶解配置成高浓度储存液(见表1)，用7H9液体培养基稀释至应用液浓度，化合物I及对照药物在培养基中的终浓度为二倍浓度，对菌株的终浓度分别为：化合物I：32～0.0156μg/mL；化合物II：32～0.0156μg/mL；INH：40～0.0195μg/mL；RFP：40～0.0195μg/mL；MFX：20～0.0097μg/mL；PA-824：40～0.0195μg/mL；BDQ：10～0.005μg/mL。
表1化合物Ⅰ及对照药物配置表
(2)菌株培养与接种
各临床分离菌株在含10％OADC的DifoTM Middlebrook 7H9培养基中5％CO2培养箱37℃培养2-3周至对数生长期后，-80℃冻存。从-80℃存储柜中取出各临床菌株冻存液，将各菌株浓度稀释至1*106CFU/mL,吸取100μL依次加入到96孔板上的含药孔及菌液对照孔中，此时各孔终体积200μL，菌液接种终浓度5*105CFU/mL。
(3)培养与结果观察
将96孔板至于5％CO2培养箱37℃培养7天。7天后加Alamar Blue，在生物安全柜中避光操作，按照每孔Alamar Blue 20μL，20％吐温80 12.5μL的比例均匀混合后使用八通道移液器吸取32.5μL加入各孔。此时各孔色均为蓝色，继续放入5％CO2培养箱37℃培养24小时后观察颜色变化。应用多功能酶标仪(Tecan infininte m200)波长(560nm,590nm)测定96孔培养板中Alamar Blue荧光值，通过观察颜色变化及荧光值 计算，得到各药物最小抑菌浓度。
4.实验结果与结论
本实验中化合物I与对照药物对5株临床分离敏感菌株及15株临床分离耐药菌株的MIC数据见表2和表3。
表2 5株敏感结核分枝杆菌临床菌株的MIC结果(ug/ml)
化合物I及对照药物对5株敏感结核分枝杆菌临床菌株的MIC范围分别为0.142-0.273ug/ml；化合物II：0.073-0.107ug/ml；BDQ：0.018-0.019ug/ml；INH：0.029-0.038ug/ml；RFP：0.068-0.091ug/ml；MFX：0.035-0.056ug/ml；PA-824：0.077-0.288ug/ml；化合物I对5株敏感结核分枝杆菌临床菌株的活性与化合物II、BDQ、INH、RFP、MFX、PA-824类似。
表3对15株耐药结核分枝杆菌临床株的MIC结果(ug/ml)

化合物Ⅰ及对照药物对15株耐药结核分枝杆菌临床菌株的MIC结果显示，除了抑制菌株/ATCC 30144、菌株/ATCC 30586，化合物Ⅰ对其他13株耐药结核分枝杆菌临床菌株的抑菌活性与化合物Ⅱ、BDQ及PA-284接近，INH\RFP对15株耐药结核分枝杆菌临床菌株未显示出活性，MFX对15株耐药结核分枝杆菌临床菌株的部分菌株显示出活性。
结论：化合物I对结核分枝杆菌临床分离菌株，包括敏感菌株及耐药菌株均具有良好的体外活性，活性与化合物II、BDQ类似，且与其他抗结核药物不存在交叉耐药性。
实施例6
1.药物
化合物Ⅰ和化合物Ⅱ都由上海嘉坦医药科技有限公司提供；
2.给药方法
餐后给药(MAD)：受试者于相应试验的前一天入住I期临床试验病房，统一生活管理，禁止统一饮食以外的任何食物或饮料。受试者于18:00左右进食晚餐，其后禁食不禁水。单次给药第1天，多次给药第1天、第4至14天8:00左右，受试者进食普通餐，30min内进食完毕；受试者在开始进餐30min后，由研究人员发放试验用药(活性药或安慰剂)，用240ml水送服，服药后禁水1h。服药后4h、10h分别进食午餐和晚餐。
3.药代动力学评价
多次给药：于首次给药前0h(服药前60分钟内)、给药后1h、2h、3h、4h、5h、6h、8h、12h、24h、48h、72h(D4给药前)、96h(D5给药前)、120h(D6给药前)、144h(D7给药前)、264h(D12给药前)；末次给药前0h、给药后1h、2h、3h、4h、5h、6h、8h、12h、24h、48h、72h、96h、120h、144h、264h采血。
4.生物分析方法学验证和样本测试
本试验药物浓度检测由上海熙华检测技术有限公司负责。采用液相色谱-串联质谱(LC-MS/MS)法测定人血浆中化合物Ⅱ及化合物Ⅰ的浓度，内标法定量，血浆样品用量为100μL，血浆中化合物Ⅱ浓度测定的最低定量限(LLOQ)为10.0ng/mL，检测范围为10.0至5000ng/mL；化合物Ⅰ最低定量限(LLOQ)为0.100ng/mL，检测范围为0.100至50.0ng/mL。
为了保证建立的LC-MS/MS法测定血浆中化合物Ⅱ及化合物Ⅰ分析方法的可靠性，对方法进行了完整的方法学验证，该方法学验证结果符合ICH M10等相关指导原则的线性、准确度、精密度、选择性(干扰)、回收率、基质效应、稀释可靠性、批量样品检测过程的残留、批容量、稳定性等接受指标。
方法学验证符合要求后开展试验样本的测定。在测定生物样品中进行质量控制，以保证所建立的方法在实际应用中的可靠性。配制不同浓度的质控样品对分析方法进行考核。
5.药代动力学参数计算
根据每个受试者血药浓度，通过Phoenix WinNonlin 8.1软件采用非房室模型计算血浆原药化合物Ⅱ及化合物Ⅰ的药代动力学参数。
具体计算公式如下：t1/2z＝ln2/λz。
其中，t1/2z：终末端消除半衰期；λz：消除速率常数。使用线性回归方法计算的半对数药时曲线终末段的斜率的绝对值。
6.实验结果见表4
表4
从上述表格数据可以看出，化合物Ⅰ相对于化合物Ⅱ的人体半衰期很长。达到稳态后，可以每周给药一次，方便患者用药克服了因肺结核患者是多药并用导致成药负担很重的状况。
实施例7
1.目的：测试化合物Ⅰ对实验室诱导BDQ耐药菌株的体外活性
2.实验材料：
对照药物：化合物Ⅱ由上海嘉坦医药科技有限公司提供；所述化合物Ⅱ为PCT/CN2017/070835实施例1化合物I-1；贝达喹啉(BDQ)购于上海翰香生物科技有限公司；
菌株：菌株/H37RV、菌株/BDQ耐药菌株1、菌株/BDQ耐药菌株2、菌株/BDQ耐 药菌株3均为首都医科大学附属北京胸腔医院(北京市结核病胸部肿瘤研究所)国家结核病临床实验室提供，药物研究室保存菌株。
培养基：7H9液体培养基(含10％OADC)，为BD公司产品。
3.实验方法
应用微孔板Alamar Blue法(MABA)测定化合物Ⅰ、贝达喹啉、异烟肼(INH)、利福平(RFP)、对实验室诱导BDQ耐药菌株的最低抑菌浓度(Minimum Inhibitory Concentration,MIC)。
微孔板Alamar Blue法(MABA)测定参考陆宇，王彬，郑梅琴等，应用Alamar Blue和MTT测定抗结核药物最低抑菌浓度的研究.中国防痨杂质，2007，29(6)：499-501。
(1)化合物I与对照药物精准称量，化合物I、化合物II及贝达喹啉(BDQ)用二甲基亚砜(DMSO)溶解配置成高浓度储存液(见表5)，用7H9液体培养基稀释至应用液浓度，化合物I及对照药物在培养基中的终浓度为二倍浓度，对菌株的终浓度分别为：化合物I：32～0.0156μg/mL；化合物II：32～0.0156μg/mL；BDQ：10～0.005μg/mL。
表5化合物I及对照药物配置表
(2)菌株培养与接种
各实验室诱导BDQ耐药菌株在含10％OADC的DifoTM Middlebrook 7H9培养基中5％CO2培养箱37℃培养2-3周至对数生长期后，-80℃冻存。从-80℃存储柜中取出各临床菌株冻存液，将各菌株浓度稀释至1*106CFU/mL,吸取100μL依次加入到96孔板上的含药孔及菌液对照孔中，此时各孔终体积200μL，菌液接种终浓度5*105CFU/mL。
(3)培养与结果观察
将96孔板至于5％CO2培养箱37℃培养7天。7天后加Alamar Blue，在生物安全柜中避光操作，按照每孔Alamar Blue 20μL，20％吐温80 12.5μL的比例均匀混合后使用八通道移液器吸取32.5μL加入各孔。此时各孔色均为蓝色，继续放入5％CO2培养箱37℃培养24小时后观察颜色变化。应用多功能酶标仪(Tecan infininte m200)波长(560nm，590nm)测定96孔培养板中Alamar Blue荧光值，通过观察颜色变化及荧光值计算，得到各药物最小抑菌浓度。
4.实验结果与结论
本实验中化合物I与对照药物对实验室诱导BDQ耐药菌株的MIC数据见表6；
表6对实验室诱导BDQ耐药菌株的MIC结果(ug/ml)

化合物I及对照药物对实验室诱导BDQ耐药菌株的MIC结果显示，化合物I对BDQ耐药株1、BDQ耐药株2具有良好的体外活性，优于化合物Ⅱ、BDQ，且与其他抗结核药物不存在交叉耐药性。
实施例8
一、新西兰白兔浦肯野纤维研究化合物Ⅰ、化合物Ⅲ动作电位的影响
1.阳性对照品：氟哌啶醇(购于SIGMA)
供试品：化合物Ⅰ(上海嘉坦医药科技有限公司)和化合物Ⅲ(武汉药明康德新药开发有限公司)；
阴性对照组：0.3％DMSO(购于国药)；
2.给药制剂的配制和分析
(1)给药制剂储液配制方法
阴性对照品：量取所需体积的DMSO至容器中作为储液；
供试品：避光条件下，称取适量的化合物Ⅰ和化合物Ⅲ，分别加入适量的DMSO，配制成10mmol/L的储液，将配制好的溶液装于容器内，室温避光保存；
阳性对照品：称取适量的氟哌啶醇，加入适量的DMSO溶解成1mmol/L的储液，将配制好的溶液分装后-20℃保存备用。
(2)给药制剂工作液配制方法
动作电位测定前，DMSO和化合物Ⅰ组给药制剂分别用适量的Tyrode’s液稀释成所需浓度的工作液，阳性对照组给药制剂从-20℃冰箱取出融化后用适量的Tyrode’s液稀释成所需浓度的工作液。具体如下表7：
表7
3.实验方法
(1)实验动物
新西兰白兔，SPF级；40只；2.4～3.5kg，3～5月龄；购于中国食品药品检定研究院。
(2)离体组织制备
1)试验动物麻醉(3％戊巴比妥钠(约45～60mg/kg)，耳缘静脉注射)，剪开腹部，打开胸腔，暴露心脏，取出心脏。
2)将心脏迅速放置到经95％O2+5％CO2混合气体饱和的约37℃Tyrode’s液器皿中，轻轻挤压心脏，使心室内的剩余血液排出，其中Tyrode’s液(mmol)：NaCl 137，KCl 4，MgSO4·7H2O 1，NaH2PO4·2H2O 0.4，NaHCO3 12,CaCl2 1.8，Glucose 5.6，PH 7.35-7.4，95％O2+5％CO2混合气体饱和30min以上；
3)待心室内剩余血液排出后，将心脏迅速放置到经95％O2+5％CO2混合气体饱和的2-8℃高钾(27mM)Tyrode’s液器皿中，使心脏停博，其中高钾(27mM)Tyrode’s液(mmol)：NaCl 137，KCl 27，MgSO4·7H2O 1，NaH2PO4·2H2O 0.4，NaHCO3 12,CaCl21.8，Glucose 5.6，PH 7.35-7.4，95％O2+5％CO2混合气体饱和30min以上。
4)在显微镜下，小心分离出浦肯野纤维。
(3)加样
1)加样途径：使用WPI蠕动泵将工作液加至待测新西兰白兔浦肯野纤维所在的平皿中，使用Harvard微灌流槽保证蠕动泵灌流针出口温度为36±2℃。
2)加样组序和加样时间
供试品工作液：从低浓度到高浓度(0.3、1、3和10μmol/L(Tyrode’s液中))，待加样约15min其作用达稳态后加入更高浓度的供试品工作液。
DMSO：在加入供试品工作液前加入阴性对照品工作液(0.3％DMSO(Tyrode’s液中))，待加样约5min其作用达稳态后可加入低浓度的供试品工作液。
阳性对照品工作液：选择不同的待测组织加入阳性对照品工作液(1μmol/L氟哌啶醇(Tyrode’s液中))。
3)参数记录
静息电位(RMP)；动作电位0相上升速度(Vmax)；动作电位幅度(APA)；动作电位持续时间(APD)
4.数据处理和统计分析
(1)数据处理
通过Lab Chart 7.3.8软件完成实验数据分析。选取每个组织加样前后分别记录到的最后5个符合要求的动作电位进行分析。
RMP、APD、APA和Vmax均以均数±标准差表示，同时计算加样前后数据变化的百分比。供试品/对照品各浓度组RMP、APD、APA和Vmax的平均值采用每个组织加样前RMP、APD、APA和Vmax的平均值校正，计算出各个浓度组RMP、APD、APA和Vmax的变化百分比(△％)，公式如下：
RMP、APD、APA或Vmax的变化百分比(△％)＝[(A-B)/B]×100％
A：供试品/对照品各浓度组的RMP、APD、APA或Vmax
B：同一组织加样前的RMP、APD、APA或Vmax；
(2)统计分析
使用软件：SPSS Statistics 21。
RMP、APD、APA、Vmax及上述参数的变化百分比均进行统计分析，供试品各浓度组与阴性对照组相比时，均按照以下方法统计：①用Levene's检验检测数据的方差齐性，当方差齐(P>0.05)，则进行单因素方差分析(ANOVA)；当方差不齐(P≤0.05)，则Dunnett T3检验进行组间比较检验(0.05和0.01水平)。②当方差分析结果有统计学差异(P≤0.05)，则进一步用Dunnett t检验进行组间比较检验(0.05和0.01水平)；当方差分析结果无统计学差异(P>0.05)，则统计结束。
阳性对照组与DMSO相比时，用独立样本t检验。
5.实验结果
本试验条件下，给予0.3、1、3和10μmol/L的化合物Ⅰ后，新西兰白兔浦肯野纤维动作电位RMP、APA、Vmax、APD60和APD90的均值和变化百分比未见与给予供试品相关的明显变化；给予10μmol/L的化合物Ⅰ后，APD30的变化百分比降低，具体结果见表8；本试验条件下，给予0.3、1、3和10μmol/L的化合物Ⅲ后，新西兰白兔浦肯野纤维动作电位RMP、APA、Vmax、APD30、APD60和APD90的均值及RMP、APA、APD60和APD90的变化百分比无显著性变化，Vmax的变化百分比略有增加；给予10μmol/L的化合物Ⅲ后，新西兰白兔浦肯野纤维动作电位APD30的变化百分比降低，具体结果见表9。
表8化合物Ⅰ对新西兰白兔浦肯野纤维动作电位的影响

注：参数以均数±标准差表示；n：组织数；与0.3％DMSO组比较，P＞0.05。
表9化合物Ⅲ对新西兰白兔浦肯野纤维动作电位的影响

注：参数以均数±标准差表示；n：组织数；与0.3％DMSO组比较，P＞0.05。
6.实验结论：化合物Ⅰ影响新西兰白兔浦肯野纤维动作电位参数的浓度为10μmol/L。化合物Ⅲ影响新西兰白兔浦肯野纤维动作电位参数的浓度为0.3μmol/L。
二、化合物Ⅰ和化合物Ⅲ对hCav1.2通道的作用
1.阳性对照品：Nifedipine(购于Sigma)；
供试品：化合物Ⅰ(上海嘉坦医药科技有限公司提供)和化合物Ⅲ(武汉药明康德新药开发有限公司提供)；
所述化合物Ⅲ的结构式如下：
阴性对照品：DMSO(购于Sigma)；
2.给药制剂的配制和分析
(1)给药制剂储液配制方法
将称量出的化合物Ⅰ(10.07mg)和化合物Ⅲ(10.04mg)用DMSO配制成约29.941mM和29.969mM储液；
(2)给药制剂工作液配制方法
1)取待测样品储液用DMSO依次稀释为0.01mM，0.033mM，0.1mM，0.33mM和1mM；
2)取30μL上述各浓度待测液加入10mL的检测外液中稀释，配成0.03μM，0.1μM，0.3μM，1μM以及3μΜ液体。
3)Nifedipine溶解于DMSO中配制成50mM储液，用二甲基亚砜(DMSO)将Nifedipine储液依次稀释为0.8μM，4μM，20μM，100μM以及500μM的溶液。
3.实验方法
(1)细胞系
1)采用了稳定表达hCav1.2通道的CHO细胞系，hCav1.2细胞由北京爱思益普生物科技股份有限公司实验室自行构建。
2)维持培养基：HAM’S F12以及20％FBS。
3)细胞传代：除去旧培养基并用PBS洗一次，然后加入1mL 0.25％trypsin-EDTA溶液，37℃孵育1.5分钟。当细胞从皿底脱离，加入5mL 37℃预热的完全培养基。
将细胞悬液用吸管轻轻吹打使聚集的细胞分离。将细胞悬液转移至无菌的离心管中，1000rmp/min离心5分钟收集细胞。扩增或维持培养，将细胞接种于6厘米细胞培养皿，每个细胞培养皿，接种细胞量为2.5*105cells(最终体积：10mL)。
(2)膜片钳检测
全细胞膜片钳记录hCav1.2通道电流的电压刺激方案如下：当形成全细胞封接后细胞膜电压钳制于-60mV。钳制电压由-60mV除极至0mV(具体电压由先导IV决定)维持0.3秒。每隔20秒重复采集数据，观察药物对hCav1.2通道电流峰值的作用。实验数据由EPC-10放大器(HEKA)进行采集并储存于PatchMaster(HEKA)软件中(软件版本：v2x73.2)。
用微电极拉制仪(P97，Sutter Instruments)将毛细玻璃管(BF150-86-10，Sutter Instruments)拉制成记录电极。在倒置显微镜(AE31E，Motic)下操纵微电极操纵仪(86PW420600，MCI Instruments)将记录电极接触到细胞上，给予负压抽吸，形成GΩ封接。形成GΩ封接后进行快速电容补偿，然后继续给予负压，吸破细胞膜，形成全细胞记录模式。
当全细胞记录的hCav1.2通道电流稳定后开始给药，每个药物浓度作用至5分钟(或者电流至稳定)后检测下一个浓度，每一个测试化合物检测多个浓度。将铺有细胞的盖玻片置于倒置显微镜镜头上方的记录浴槽中，测试化合物以及不含化合物的外液利用重力灌流的方法从低浓度到高浓度依次流经记录小室从而作用于细胞，在记录中利用真空泵进行液体交换。每一个细胞在不含化合物的外液中检测到的电流作为自己的对照组。
独立重复检测多个细胞。所有电生理实验在室温下进行。
4.数据分析
(1)将每一个药物浓度作用后的电流和空白对照电流标准化:Peak Current compound/Peak Current vehicle
(2)计算每一个药物浓度对应的抑制率:1-(Peak Current compound/Peak Current vehicle)
(3)对每一个浓度计算平均数和标准误，以上所有数值利用Microsoft Excel 2013计算获得。此外通过IGOR软件运用以下的方程计算每种化合物的半抑制浓度：
抑制率＝1/[1+(IC50/c)h]；
用以上方程对剂量依赖效应进行非线性拟合，其中c代表药物浓度，IC50为半抑制浓度，h代表希尔系数。曲线拟合以及IC50的计算利用IGOR软件完成(软件版本：6.0.1.0)。
5.结果
化合物Ⅰ和化合物Ⅲ对hCav1.2钙通道抑制作用，并通过拟合计算出样品对hCav1.2钙通道电流的半抑制浓度(IC50)，实验结果如下：
表10

阳性对照品的实验结果如下：
表11
表12化合物Ⅰ与化合物Ⅲ对不同离子通道的抑制作用
结论：在临床上，通常兔子浦肯野纤维测定AP浓度大于10μM表示没有心血管风险，hCav1.2钙通道的抑制作用大于3uM代表方法最高浓度，表示没有心血管风险；而化合物ⅢhCav1.2钙通道的抑制作用0.75和兔子浦肯野纤维测定AP浓度0.3表示有中高心血管风险。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不违背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (10)

  1. 一种如式I所示的化合物或其药学上可接受的盐；
  2. 一种如式I-X所示的化合物或其药学上可接受的盐；
    较佳地，所述的如式I-X所示的化合物在下述SFC条件下的保留时间为1.445min；
    色谱柱：ChiralpakAD-3.50A，4.6mm，内径3um；
    流动相：A：CO2，B：EtOH(0.05％二乙醇胺)；
    梯度：MPB在1.2分钟内从由5％至50％，保持1分钟，然后0.8分钟内由50％至5％；
    流速：3.4mL/min；
    柱温：35℃；
    ABPR：1800psi。
  3. 一种如式II所示的化合物，
    其中，R1为氨基保护基；优选为-Bn、-Trt、-Dmb或-PMB，更优选为-Bn。
  4. 一种如式II-X所示的化合物，
    其中，R1的定义如权利要求3所述。
  5. 一种式I所示化合物的制备方法，其特征在于，其包括以下步骤：溶剂中，所述式II-X所示的化合物在脱保护试剂的存在下进行脱保护反应，再经手性拆分，得所述式I所示的化合物；
    其中，R1的定义如权利要求3所述较佳地，满足以下条件中的一种或多种：
    1)所述的溶剂为二氯甲烷；
    2)所述溶剂与所述式II-X所示的化合物的体积质量比为3mL/g～6mL/g，优选为5mL/g、4mL/g或4.7mL/g；
    3)所述脱保护试剂为氯甲酸-1-氯乙酯和甲醇；
    4)所述脱保护试剂与所述式II-X所示的化合物的摩尔比为1.1:1～1.8:1，优选为1.4:1；
    5)所述脱保护反应的温度为室温；
    6)所述脱保护反应的进程采用TLC、HPLC、GC或NMR进行监测，优选为HPLC；
    7)所述脱保护反应包括以下后处理步骤：监测脱保护反应结束后，淬灭反应，调节PH后萃取和分液，所得有机相浓缩干燥后，经柱层析分离，即得所述式I-X所示的化合物。
  6. 一种如式I所示的化合物的制备方法，其包括以下步骤：溶剂中，所述式II所示的化合物在脱保护试剂的存在下，进行如下所示的脱保护反应，得所述式I所示的化合物；
    其中，R1的定义如权利要求3所述；
    较佳地，满足以下条件中的一种或多种：
    1)所述的溶剂为二氯甲烷；
    2)所述溶剂与所述式II所示的化合物的体积质量比为3mL/g～6mL/g，优选为5mL/g、4mL/g或4.7mL/g；
    3)所述脱保护试剂为氯甲酸-1-氯乙酯和甲醇；
    4)所述脱保护试剂与所述式II所示的化合物的摩尔比为1.1:1～1.8：1，优选为1.4:1；
    5)所述脱保护反应的温度为室温；
    6)所述脱保护反应的进程采用TLC、HPLC、GC或NMR进行监测，优选为HPLC；
    7)所述脱保护反应包括以下后处理步骤：监测脱保护反应结束后，淬灭反应，调节PH后萃取和分液，所得有机相浓缩干燥后，经柱层析分离，即得所述式I所示的化合物。
  7. 一种如式II-X所示的化合物的制备方法，其包括以下步骤：溶剂中，式III所示的化合物与式IV所示的化合物进行如下所示的反应，得所述式II-X所示的化合物；
    其中，R1的定义如权利要求3所述；较佳地，满足以下条件中的一种或多种：
    1)所述的溶剂为四氢呋喃；
    2)所述溶剂与所述式III所示的化合物的体积质量比为4mL/g～6mL/g，优选为5mL/g；
    3)所述碱与所述式III所示的化合物的摩尔比为1.2:1～2:1，优选为1.65:1；
    4)所述碱为正丁基锂；
    5)所述脱保护反应的温度为-60℃～-80℃，优选为-72℃；
    6)所述的制备方法包括以下后处理步骤：监测反应结束后，所得反应液加入NH4Cl饱和溶液中，经洗涤、萃取和分液，所得有机相用盐水洗涤后分液，所得有机相浓缩干燥并柱层析分离，即得所述式II-X所示的化合物。
  8. 一种物质A或物质B在制备抑制结核分枝杆菌的药物中的应用；所述物质A为如权利要求1所述的式I所示的化合物或其药学上可接受的盐；所述物质B为如权利要求2所述的式I-X所示的化合物或其药学上可接受的盐；较佳地，满足以下条件中的一种或多种：
    1)所述的结核分枝杆菌为敏感结核分枝杆菌或耐药结核分枝杆菌；所述敏感结核分枝杆菌优选为菌株/ATCC 27830；所述耐药结核分枝杆菌优选为贝达喹啉耐药 结核分枝杆菌；所述贝达喹啉耐药结核分枝杆菌优选选自BDQ耐药菌株1、BDQ耐药菌株2和BDQ耐药菌株3中的一种或多种；
    2)所述的药物包含药用辅料；
    3)所述的物质A或物质B为所述药物的有效成分之一或者唯一有效成分；
    4)所述的物质A为式I所示的化合物；
    5)所述的物质B为式I-X所示的化合物；
    6)所述的药物的施用方式为口服；
    7)所述的药物的施用频次为1次/周。
  9. 一种物质A或物质B在制备治疗结核分枝杆菌感染的药物中的应用；所述物质A为如权利要求1所述的式I所示的化合物或其药学上可接受的盐；所述物质B为如权利要求2所述的式I-X所示的化合物或其药学上可接受的盐；
    较佳地，满足以下条件中的一种或多种：
    1)所述的结核分枝杆菌为敏感结核分枝杆菌或耐药结核分枝杆菌；所述敏感结核分枝杆菌优选为菌株/ATCC 27830；所述耐药结核分枝杆菌优选为贝达喹啉耐药结核分枝杆菌；所述贝达喹啉耐药结核分枝杆菌优选选自BDQ耐药菌株1、BDQ耐药菌株2和BDQ耐药菌株3中的一种或多种；
    2)所述的药物包含药用辅料；
    3)所述的结核分枝杆菌感染的疾病为肺结核；
    4)所述的物质A或物质B为所述药物的有效成分之一或者唯一有效成分；
    5)所述的物质A为式I所示的化合物；
    6)所述的物质B为式I-X所示的化合物；
    7)所述的药物的施用方式为口服；
    8)所述的药物的施用频次为1次/周。
  10. 一种药物组合物，其包括物质A或物质B，和药用辅料；所述物质A为如权利要求1所述的式I所示的化合物或其药学上可接受的盐；所述物质B为如权利要求2所述的式I-X所示的化合物或其药学上可接受的盐。
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CN1671667A (zh) * 2002-07-25 2005-09-21 詹森药业有限公司 喹啉衍生物及其作为分枝杆菌抑制剂的应用
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WO2017121323A1 (zh) * 2016-01-13 2017-07-20 辰欣药业股份有限公司 一种吡啶衍生物类化合物的制备方法及其中间体和晶型

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