WO2023173708A1 - 三嗪类化合物或其可药用盐、异构体、药物组合物和用途 - Google Patents

三嗪类化合物或其可药用盐、异构体、药物组合物和用途 Download PDF

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WO2023173708A1
WO2023173708A1 PCT/CN2022/118629 CN2022118629W WO2023173708A1 WO 2023173708 A1 WO2023173708 A1 WO 2023173708A1 CN 2022118629 W CN2022118629 W CN 2022118629W WO 2023173708 A1 WO2023173708 A1 WO 2023173708A1
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compound
pharmaceutically acceptable
isomer
general formula
acceptable salt
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French (fr)
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蒋晟
肖易倍
郭炳华
张阔军
刘春河
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药康众拓(江苏)医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention belongs to the field of innovative medicinal chemistry and relates to a triazine compound, its preparation method, pharmaceutical composition and application.
  • Coronaviruses are a family of enveloped positive-strand RNA pathogenic viruses that cause acute and chronic diseases, including central nervous system disease, the common cold, lower respiratory tract infections, and diarrhea.
  • HCoV-229E and HCoV-OC43 are the first zoonotic strains discovered since 1995.
  • severe acute respiratory syndrome coronavirus now named SARS-CoV-1, caused the first global human coronavirus pandemic, causing progressive respiratory failure in more than 8,000 people and 916 deaths (case fatality rate 10 to 15 %).
  • zoonotic coronaviruses HCoV-NL64 and HCoV-HKU1 which were significantly less lethal, were discovered.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 is a highly pathogenic, large-scale zoonotic virus that belongs to the same family of coronaviruses as SARS-CoV-1 and MERS-CoV. These three viruses differ from several other coronaviruses, HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoVHKU1, in their ability to cause severe respiratory illness. Symptoms of SARS-CoV-2 infection range from asymptomatic illness to moderate and severe pneumonia, and life-threatening complications including hypoxic respiratory failure, acute respiratory distress syndrome, multisystem organ failure, and ultimately death.
  • Host cell ribosomes translate the open reading frames (ORFs) 1a and ORF1b of the viral genome into polyproteins pp1a and pp1b, respectively, encoding 16 nonstructural proteins (nsps), while the remaining ORFs encode structural proteins and accessory proteins.
  • ORFs open reading frames
  • PLpro papain-like cysteine protease
  • RTC replication-transcription complex
  • 3CLpro inhibitors include covalent peptidomimetic inhibitors and non-covalent small molecule inhibitors. Although peptoid covalent inhibitors have significant inhibitory activity against 3CLpro, covalent inhibitors have poor target selectivity, unpredictable toxic side effects, and poor metabolic stability. Non-covalent small molecule inhibitors are a better choice. However, currently reported non-covalent small molecule inhibitors are very scarce and suffer from problems such as single structure, weak enzyme inhibitory activity, and poor druggability.
  • the compound can significantly inhibit multiple SARS-CoV-2 mutant strains including ⁇ , ⁇ , ⁇ and Omicron, indicating its broad application potential as a therapeutic agent for the treatment of COVID-19.
  • S-217622 shows broad antiviral activity against a range of coronaviruses.
  • S-217622 has no inhibitory activity against host cell proteases such as caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin within the range of 100 ⁇ M, indicating that S-217622 has a high potency against coronavirus proteases. Selectivity. In addition, no safety issues such as hERG inhibition, mutagenicity/clastogenicity, and phototoxicity were found in in vitro studies of S-217622.
  • host cell proteases such as caspase-2, chymotrypsin, cathepsin B/D/G/L, and thrombin within the range of 100 ⁇ M
  • Deuterium is a stable form of non-radioactive isotope of hydrogen in nature. It is non-toxic and non-radioactive. The C-D bond is more stable than the C-H bond.
  • deuterium By replacing hydrogen in drug molecules with deuterium, it is possible to block the metabolic site, reduce the production of toxic metabolites, and extend the half-life of the drug in the body without affecting the pharmacological activity. Since 2000, deuteration strategies have been widely used in drug research. Deuterated S-217622 has better pharmacokinetic properties than S-217622, including increased drug blood concentration, extended half-life, and reduced single dose.
  • the technical problem to be solved by the present invention is to overcome the serious shortage of existing anti-coronavirus drugs and the lack of non-covalent high-efficiency 3CLpro small molecule inhibitors, and provide a triazine compound, its preparation method, pharmaceutical composition and application.
  • the triazine compound of the present invention is a non-covalent small molecule inhibitor of 3CLpro with significant activity, and has a good therapeutic effect on coronavirus infectious diseases.
  • the present invention solves the above technical problems through the following technical solutions.
  • the invention provides a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, with the following structure:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 or R 8 are independently selected from hydrogen or deuterium;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 or R 8 is deuterium.
  • a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is characterized in that, At least one of R 1 , R 2 , R 3 , R 4 , R 5 or R 6 is deuterium.
  • a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is characterized in that, At least one of R 1 , R 2 or R 3 is deuterium.
  • a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is characterized in that, At least one of R 4 , R 5 or R 6 is deuterium.
  • a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is characterized in that, At least one of R 7 or R 8 is deuterium.
  • a triazine compound having a structure represented by general formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, is characterized in that, R 7 and R 8 are both deuterium.
  • the compound having the structure represented by Formula I is any of the following compounds:
  • the invention provides a method for preparing a triazine compound having a structure represented by general formula I, or a pharmaceutically acceptable salt, isomer or metabolite thereof, which includes the following steps: in a solvent, under the action of a base , the compound represented by formula II and the compound represented by formula III are reacted as shown below;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 are as defined in claims 1 and 2.
  • the invention also provides the use of a triazine compound having a structure represented by the general formula I, or a pharmaceutically acceptable salt, isomer or metabolite thereof in the preparation of a 3C-like cysteine protease inhibitor. .
  • the present invention also provides the use of a triazine compound having a structure represented by general formula I, or a pharmaceutically acceptable salt, isomer or metabolite thereof in the preparation of a medicament for the treatment and/or prevention of viral infectious diseases. .
  • the viruses include but are not limited to Middle East syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), influenza A virus, influenza B virus, and new coronavirus Pneumonia (COVID-19), Spanish flu virus, arenavirus, bunyavirus, rabies virus, avian influenza virus, poliovirus, rhinovirus, adenovirus, Ebola virus, enterovirus, hepatitis A virus , hepatitis C virus, hepatitis E virus, enterovirus, HIV virus, echo virus, filovirus, measles virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, Newcastle disease virus, RS virus, blister stomatitis virus, mumps virus, dengue virus, coxsackie virus, rotavirus or tobacco mosaic virus.
  • MERS-CoV Middle East syndrome-related coronavirus
  • SARS-CoV severe acute respiratory syndrome-related coronavirus
  • influenza A virus influenza B
  • the present invention also provides a pharmaceutical composition, which contains a triazine compound having a structure represented by general formula I, or a pharmaceutically acceptable salt, isomer or metabolite thereof, and a pharmaceutically acceptable carrier or Excipients.
  • the dosage of the triazine compound with the structure represented by the general formula I, or its pharmaceutically acceptable salt, isomer or metabolite is a therapeutically effective amount.
  • the present invention also provides the use of the above pharmaceutical composition in preparing 3C-like cysteine protease inhibitors.
  • the present invention also provides the use of the above pharmaceutical composition in preparing drugs for treating and/or preventing viral infectious diseases.
  • the viruses include but are not limited to Middle East syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome-related coronavirus (SARS-CoV), influenza A virus, influenza B virus, and new coronavirus Pneumonia (COVID-19), Spanish flu virus, arenavirus, bunyavirus, rabies virus, avian influenza virus, poliovirus, rhinovirus, adenovirus, Ebola virus, enterovirus, hepatitis A virus , hepatitis C virus, hepatitis E virus, enterovirus, HIV virus, echo virus, filovirus, measles virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, Newcastle disease virus, RS virus, blister stomatitis virus, mumps virus, dengue virus, coxsackie virus, rotavirus or tobacco mosaic virus.
  • MERS-CoV Middle East syndrome-related coronavirus
  • SARS-CoV severe acute respiratory syndrome-related coronavirus
  • influenza A virus influenza B
  • the pharmaceutical excipients can be those widely used in the field of pharmaceutical production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition. They can also provide a method to enable the active ingredients to dissolve at a desired rate after administration, or promote the activity of the composition after administration. Ingredients are absorbed effectively.
  • the pharmaceutical excipients may be inert fillers, or provide certain functions, such as stabilizing the overall pH value of the composition or preventing degradation of the active ingredients of the composition.
  • the pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, and anti-adhesion agents. Agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorings and sweeten
  • compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.
  • compositions of the present invention may be administered in any form, including injectable (intravenous), mucosal, oral (solid and liquid formulations), inhaled, ocular, rectal, topical or parenteral (infusion, injection, implant). Intravenous, subcutaneous, intravenous, intraarterial, intramuscular) administration.
  • the pharmaceutical composition of the present invention may also be in a controlled-release or delayed-release dosage form (eg, liposomes or microspheres).
  • solid oral dosage forms include, but are not limited to, powders, capsules, caplets, softgels, and tablets.
  • liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions.
  • topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations.
  • formulations for parenteral administration include, but are not limited to, injectable solutions, dry formulations which may be dissolved or suspended in a pharmaceutically acceptable carrier, injectable suspensions, and injectable emulsions.
  • suitable formulations of the pharmaceutical compositions include, but are not limited to, eye drops and other ophthalmic formulations; aerosols such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges agent.
  • salts refers to salts of compounds of the present invention prepared from compounds having specific substituents found in the present invention and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, phosphoric acid- Hydrogen salt, dihydrogen phosphate, sulfuric acid (forming sulfate or hydrogen sulfate), hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, etc.
  • organic acid salts also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid.
  • Certain specific compounds of the present invention contain basic and acidic functional groups, which can be Conversion to any base or acid addition salt.
  • the free form of the compound is regenerated by contacting the salt with a base or acid in a conventional manner and isolating the parent compound.
  • the free form of the compound and its various salt forms The difference lies in certain physical properties, such as solubility in polar solvents.
  • the "pharmaceutically acceptable salts" of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid groups or bases. In general, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
  • isomers refers to compounds that have the same chemical formula but different arrangements of atoms.
  • metabolite refers to the pharmaceutically active product produced by the metabolism of the compound represented by Formula I or its salt in the body. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Accordingly, the invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a period of time sufficient to obtain the metabolites thereof.
  • Metabolites are typically identified by preparing radiolabeled isotopes of the compounds of the invention and administering them parenterally to animals, such as rats, mice, guinea pigs, monkeys, at detectable doses (e.g., greater than about 0.5 mg/kg). , or human, allow sufficient time for metabolism to occur (typically about 30 seconds to 30 hours) and isolation of its transformation products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated by using antibodies capable of binding epitopes present in the metabolites). Metabolite structures are determined in a conventional manner, for example, by MS, LC/MS or NMR analysis.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C). All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the deuterated triazine compound of the present invention has good inhibitory activity against 3C-like cysteine protease.
  • the compound of the present invention can effectively increase the blood concentration, extend the half-life, and significantly reduce the single dose.
  • the compound of the present invention has good therapeutic effect on coronavirus infection.
  • Figure 1 shows the anti-infectious activities of Example 14 positive control groups and compound S11 in mouse infection models.
  • Example 12 SARS-CoV-2 virus 3C-like cysteine protease (3CLpro) enzyme inhibitory activity test experiment
  • the gene sequence of the full-length 3CLpro protein was constructed into the expression vector pET28a(+) vector and transferred into E. coli BL21 (DE3) competent cells. It was induced with Ni-NTA at a final concentration of 0.5mM IPTG at 25°C for 12 hours. Column purification. The purified protein was tested by SDS, and the part with a purity greater than 90% was used for further purification using the GE protein chromatography purification system AKTA Pure's Superdex 200 10/300 GL to obtain a protein with a purity greater than 95%. The protein concentration was measured using Nano Drop. Aliquot and quickly freeze in liquid nitrogen and store at -80°C.
  • the activity of SARS-CoV-2 3CLpro and the inhibitory activity of compounds against SARS-CoV-2 3CLpro were determined by fluorescence resonance energy transfer (FRET) technology.
  • the fluorescent substrate (Dabcyl-KTSAVLQ ⁇ SGFRKM-E(Edans)-NH 2 ) and Tris-HCl buffer (20mM Tris-HCl) with SARS-CoV-2 3CLpro cleavage site (arrow) were used in the assay. , 150mM NaCl, 10mM EDTA, pH 7.5). Compounds were dissolved in 100% DMSO.
  • Vero E6 cytotoxicity test The CCK8 method is used to detect the cytotoxicity of the test compound on mammalian Vero E6 cells. Vero E6 cells were added to the 96-well plate and cultured overnight. Then, the cells were incubated with different concentrations of test compounds for 48 h. Remove the culture medium in the well plate, replace it with fresh serum-free culture medium, add 10% CCK8 reagent, incubate at 37°C for 1 hour, and then use a microplate reader to detect the absorbance value at 450 nm.
  • the specific operations include the following steps: 1 Cell inoculation: Take Vero-E6 cells in the logarithmic growth phase, aspirate the culture medium, and digest the cells with trypsin. The cell count is: 1 ⁇ 106 cells/ml; take 4 ml of the above cells, add 6 ml of culture medium, prepare a cell suspension with a cell density of 4 ⁇ 105 cells/ml, and inoculate it into a 96-well plate, 100 ⁇ l per well, Cells 4 ⁇ 104.
  • Drug pretreatment of cells Change the cell culture medium to DMEM medium containing 2% FBS, add corresponding concentrations of drugs and DMSO, 100 ⁇ l per well, and then place it in a 37°C incubator for pretreatment for 1 hour.
  • 3Virus infection Take 0.3ml of virus, add 45ml of medium, mix, and dilute the virus to 100TCID50/0.05ml; discard the drug medium in the cell plate and vertically drop the virus diluent into the 96-well plate, and add the sample volume 50 ⁇ l/well, and add the corresponding drug culture medium (containing drugs of corresponding concentrations) at the same time, the sample volume is 50 ⁇ l/well, and mix; 4 Incubation: Mix the cell culture plate with the sample on a shaker and place it at 37 °C incubator, incubate for 1 hour.
  • the specific experimental results are shown in Table 2.
  • the compounds of the examples have low cytotoxicity, good inhibitory activity against SARS-CoV-2 virus infection, and good selectivity index.
  • mice Female BALB/c mice were first anesthetized by intraperitoneal injection of ketamine/xylazine (50mg/kg/5mg/kg), and then SARS-CoV-2 ⁇ strain (1 ⁇ 10 4 TCID 50 /mouse) was intranasally injected
  • the infection model was constructed by inoculation, and the mice in the negative control group were instilled with the same volume of physiological saline. After successful modeling, the animals were divided into a blank control group, an S-217622 positive control group and a drug administration group, with 6 animals in each group.
  • Compounds S-217622 and S11 were respectively suspended in 0.5% methylcellulose. They were orally administered once immediately after successful modeling and once 12 hours later.
  • S11 is administered at doses of 2 mg/kg, 8 mg/kg, 16 mg/kg and 32 mg/kg, and S-217622 is administered at 32 mg/kg. After 24 hours of virus infection, the mice were sacrificed and the virus titers in the lungs of the mice were observed.
  • Pharmacokinetic properties test in rat model Oral administration: Dissolve the compound in DMSO/0.5% methylcellulose (400cP) (1:4) system, and administer a dose of 2 ⁇ M/5ml/kg.
  • Intravenous administration: The compound was administered with DMSO/propylene glycol (v/v 1:1) at a dose of (1.0 ⁇ mol/mL/kg).
  • Pharmacokinetic properties test on beagle dogs Oral administration: The solvent is 0.5% methylcellulose (400cP), and the dosage is 3mg/2mL/kg.
  • Intravenous administration: The solvent is carbonate buffer (pH 9.0) of dimethylacetamide/ethanol/20% HP- ⁇ -CD (v:v:v 2:3:5). At 5min, 15min, 30min, 1h, 2h, 4h, 8h, 10h, 24h after administration, continuously collect blood from the fundus venous plexus and place it in an EP tube distributed with heparin, centrifuge at 8000rpm/min for 5min and then take the upper plasma, - Cryopreserve at 20°C for intravenous injection and wait for LC-MS/MS analysis. Based on the blood concentration-time data obtained from the test, WinNonlin software is used to calculate the pharmacokinetic parameters.

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Abstract

本发明公开了具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，药物组合物和用途。本发明克服了现有抗冠状病毒药物结构单一、非共价类高效小分子抑制剂缺乏等缺陷，本发明提供的三嗪类类化合物对3C样半胱氨酸蛋白酶具有很好的抑制活性，具有优良的药代动力学性质，且对感染性疾病有良好的治疗作用。

Description

三嗪类化合物或其可药用盐、异构体、药物组合物和用途 技术领域
本发明属于创新药物化学领域，涉及一种三嗪类化合物、其制备方法、药物组合物及应用。
背景技术
冠状病毒(CoV)是一种包膜正链RNA致病病毒家族，可导致急性和慢性疾病，包括中枢神经系统疾病、普通感冒、下呼吸道感染和腹泻。HCoV-229E和HCoV-OC43是自1995年以来首次发现的人畜共患病毒株。2003年，严重急性呼吸综合征冠状病毒，现在命名为SARS-CoV-1，造成第一次全球人类冠状病毒大流行，导致8000多人进行性呼吸衰竭和916人死亡(病死率为10～15％)。在随后8年中，人们又发现了致命性明显降低的人畜共患病冠状病毒HCoV-NL64和HCoV-HKU1。2012年，类似SARs的中东呼吸综合征冠状病毒(MERS-CoV)被发现，该类病毒虽然具有较低的传播速率，但死亡率很高，自2012年出现至2021年2月2日，全球共有2567名确诊感染患者，死亡人数882人(死亡率34％)。2020年，由重症急性呼吸综合症冠状病毒-2(SARS-CoV-2)所引起的新型冠状病毒肺炎(COVID-19)目前正在全球蔓延，已经成为世界流行性疾病，给全球公共卫生防御和医疗系统带来严峻的挑战，给世界经济带来不确定因素。SARS-CoV-2是一种高致病性、大规模流行的人畜共患病毒，其与SARS-CoV-1和MERS-CoV同属于冠状病毒科。这三种病毒与其他几种冠状病毒HCoV-NL63、HCoV-229E、HCoV-OC43和HCoVHKU1不同，它们能够导致严重的呼吸道疾病。SARS-CoV-2感染的症状从无症状疾病到中度和重度肺炎，以及危及生命的并发症，包括低氧性呼吸衰竭、急性呼吸窘迫综合征、多系统器官衰竭，并最终出现死亡。更可怕的是，这种病毒不仅具有高度传染性，而且可以通过无症状感染者和那些处于症状期和症状前阶段的人进行传播。尽管目前全球已有多种不同的疫苗被批准上市或者获得紧急使用权，但是全球范围内有相当大的一部分群体由于自身的身体条件或者当地的医疗条件的限制而不能接种疫苗。另外，SARS-CoV-2病毒的S蛋白出现的疫苗逃逸突变对疫苗的有效性也提出了潜在的挑战，因而有效的抗新冠药物研发依然迫在眉睫。
冠状病毒进入宿主细胞后会被分解释放出核衣壳和病毒基因组。宿主细胞核糖体将病毒基因组的开放阅读框架(ORF)1a和ORF1b分别翻译成多聚蛋白pp1a和pp1b，用于编码16个非结构蛋白(nsps)，而其余的ORF编码结构蛋白和附属蛋白。3C样半胱氨酸蛋白酶(3CLpro)和木瓜样半胱氨酸蛋白酶(PLpro)催化PP裂解生成nsp2-16，进而形成复制-转录复合体(RTC)。这些蛋白酶活性缺失会导致病毒生命周期停止。并且，3CLpro的结构和功能在冠状病毒中高度保守。因此，3CLpro成为开发抗广谱冠状病毒药物的潜在有效靶点。目前报道的3CLpro抑制剂包括共价拟肽类抑制剂和非共价小分子抑制剂。拟肽类共价抑制剂虽然对3CLpro具有显著的抑制活性，但是共价抑制剂靶点选择性较差，存在不可预测的毒副作用以及代谢稳定性差等问题。非共价小分子抑制剂是更好的选择，然而目前报道的非共价小分子抑制剂非常匮乏，存在结构单一、酶抑制活性较弱、成药性较差等问题。
日本盐野义制药公司报道的非共价Mpro小分子抑制剂S-217662目前处于临床2/3期，该公司目前已经报道了部分积极临床研究结果。该化合物能够显著抑制包括α、β、γ和Omicron等多种SARS-CoV-2变异株，表明其作为治疗新冠的治疗剂具有广泛的应用潜力。而且S-217622对一系列冠状病毒显示出广泛的抗病毒活性。S-217622对caspase-2、胰凝乳蛋白酶、组织蛋白酶B/D/G/L、凝血酶等宿主细胞蛋白酶在100μM范围内均无抑制活性，说明S-217622对冠状病毒蛋白酶具有很高的选择性。此外，S-217622的体外研究中没有发现hERG抑制、致突变性/致裂性和光毒性等安全性问题。
氘为氢在自然界中的一种稳定形态的非放射性同位素，无毒、无放射性，C-D键比C-H键更加稳定。将药物分子中的氢用氘取代后，可能封闭代谢位点、减少有毒代谢物的生成、延长药物在体内的半衰期，同时不影响药理活性。自2000年以来，氘代策略便被广泛应用于药物的研究中。氘代的S-217622相对于S-217622具有更优良的药代动力学性质，包括药物血药浓度提高、半衰期延长、降低了单次给药剂量。
发明内容
本发明要解决的技术问题是克服现有抗抗新冠药物严重匮乏、非共价类高效 3CLpro小分子抑制剂缺乏，而提供了一种三嗪类化合物、其制备方法、药物组合物及应用。本发明的三嗪类化合物是一种活性显著的3CLpro非共价小分子抑制剂，且对冠状病毒感染性疾病具有较好的治疗作用。
本发明通过以下技术方案解决上述技术问题。
本发明提供了一种具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，结构如下：
Figure PCTCN2022118629-appb-000001
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7或R 8独立地选自氢或氘；
并且，R 1、R 2、R 3、R 4、R 5、R 6、R 7或R 8中至少有一个为氘。
在一些实施方案中，具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 1、R 2、R 3、R 4、R 5或R 6至少有一个为氘。
在一些实施方案中，具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 1、R 2或R 3至少有一个为氘。
在一些实施方案中，具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 4、R 5或R 6至少有一个为氘。
在一些实施方案中，具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 7或R 8至少有一个为氘。
在一些实施方案中，具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 7 和R 8均为氘。
在一些实施方案中，具有通式I所示结构的化合物为以下任一化合物：
Figure PCTCN2022118629-appb-000002
本发明提供一种具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物制备方法，其包括以下步骤：在溶剂中，在碱的作用下，将如式II所示化合物与如式III所示化合物进行如下所示的反应，即可；
Figure PCTCN2022118629-appb-000003
其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7和R 8定义如权利要求1和2所述。
本发明还提供了一种具有通式I所示结构的三嗪类类化合物、或其药学上可接受的盐、异构体或代谢产物在制备3C样半胱氨酸蛋白酶抑制剂中的用途。
本发明还提供了一种具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物在制备治疗和/或预防病毒感染性疾病药物中的用途。
进一步地，所述的病毒包括但不限于中东综合征相关冠状病毒(MERS-CoV)、严重急性呼吸综合征相关冠状病毒(SARS-CoV)、甲型流感病毒、乙型流感病毒、新型冠状病毒肺炎(COVID-19)、西班牙流感病毒、沙粒病毒、布尼亚病毒、狂犬病毒、禽流感病毒、骨髓灰质炎病毒、鼻病毒、腺病毒、埃博拉病毒、肠病毒、甲型肝炎病毒、丙型肝炎病毒、戊型肝炎病毒、肠病毒、HIV病毒、艾柯病毒、丝状病毒、麻疹病毒、黄热病病毒、日本脑炎病毒、西尼罗河病毒、新城病病毒、RS病毒、水泡性口炎病毒、流行性腮腺炎病毒、登革热病毒、柯萨奇病毒、轮状病毒或烟草花叶病毒。
本发明还提供了一种药物组合物，其含有具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物，及药学上可接受的载体或辅料。
在所述的药物组合物中，所述的如通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物用量为治疗有效量。
本发明还提供了上述药物组合物在制备3C样半胱氨酸蛋白酶抑制剂中的用途。
本发明还提供了上述药物组合物在制备治疗和/或预防病毒感染性疾病药物中的用途。
进一步地，所述的病毒包括但不限于中东综合征相关冠状病毒(MERS-CoV)、严重急性呼吸综合征相关冠状病毒(SARS-CoV)、甲型流感病毒、乙型流感病毒、新型冠状病毒肺炎(COVID-19)、西班牙流感病毒、沙粒病毒、布尼亚病毒、狂犬病毒、禽流感病毒、骨髓灰质炎病毒、鼻病毒、腺病毒、埃博拉病毒、肠病毒、甲型肝炎病毒、丙型肝炎病毒、戊型肝炎病毒、肠病毒、HIV病毒、艾柯病毒、丝状病毒、麻疹病毒、黄热病病毒、日本脑炎病毒、西尼罗河病毒、新城病病毒、RS病毒、水泡性口炎病毒、流行性腮腺炎病毒、登革热病毒、柯萨奇病毒、轮状病毒或烟草花叶病毒。
所述的药用辅料可为药物生产领域中广泛采用的那些辅料。辅料主要用于提供一个安全、稳定和功能性的药物组合物，还可以提供方法，使受试者接受给药后活性成分以所期望速率溶出，或促进受试者接受组合物给药后活性成分得到有效吸收。所述的药用辅料可以是惰性填充剂，或者提供某种功能，例如稳定该组合物的整体pH值或防止组合物活性成分的降解。所述的药用辅料可以包括下列辅料中的一种或多种：粘合剂、助悬剂、乳化剂、稀释剂、填充剂、成粒剂、胶粘剂、崩解剂、润滑剂、抗粘着剂、助流剂、润湿剂、胶凝剂、吸收延迟剂、溶解抑制剂、增强剂、吸附剂、缓冲剂、螯合剂、防腐剂、着色剂、矫味剂和甜味剂。
本发明的药物组合物可根据公开的内容使用本领域技术人员已知的任何方法来制备。例如，常规混合、溶解、造粒、乳化、磨细、包封、包埋或冻干工艺。
本发明所述的药物组合物可以任何形式给药，包括注射(静脉内)、粘膜、口服(固体和液体制剂)、吸入、眼部、直肠、局部或胃肠外(输注、注射、植入、皮下、静脉内、动脉内、肌内)给药。本发明的药物组合物还可以是控释或延迟释放剂型(例如脂质体或微球)。固体口服制剂的实例包括但不限于粉末、胶囊、囊片、软胶囊剂和片剂。口服或粘膜给药的液体制剂实例包括但不限于悬浮液、乳液、酏剂和溶液。局部用制剂的实例包括但不限于乳剂、凝胶剂、软膏剂、乳膏剂、贴剂、糊剂、泡沫剂、洗剂、滴剂或血清制剂。胃肠外给药的制剂实例包括但不限于注射用溶液、可以溶解或悬浮在药学上可接受载体中的干制剂、注射用悬浮液和注射用乳剂。所述的药物组合物的其它合适制剂的实例包括但不限于滴眼液和其他眼科制剂；气雾剂：如鼻腔喷雾剂或吸入剂；适于胃肠外给药的液体剂型；栓剂以及锭剂。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。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碱与这类化合物的游离体形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机氨或镁盐或类似的盐。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酸与这类化合物的游离体形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸 盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸(形成碳酸盐或碳酸氢盐)、磷酸(形成磷酸盐、磷酸一氢盐、磷酸二氢盐、硫酸(形成硫酸盐或硫酸氢盐)、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；有机酸盐还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。优选地，以常规方式使盐与碱或酸接触，再分离母体化合物，由此再生化合物的游离体形式。化合物的游离体形式与其各种盐的形式的不同之处在于某些物理性质，例如在极性溶剂中的溶解度不同。
本发明的“药学上可接受的盐”可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。一般地，优选醚、乙酸乙酯、乙醇、异丙醇或乙腈等非水介质。
术语“异构体”是指具有相同化学式而有不同的原子排列的化合物。
术语“代谢产物”是指式I所示化合物或其盐通过体内代谢产生的药学活性产物。这种产物可以从例如所给药的化合物的氧化、还原、水解、酰胺化、脱酰胺、酯化、脱酯、葡糖醛酸化、酶促裂解等产生。因此，本发明包括本发明的化合物的代谢产物，包括使本发明的化合物与哺乳动物接触足够得到其代谢产物的一段时间的方法而产生的化合物。
代谢产物的鉴定典型地通过制备本发明化合物的放射性标记的同位素、将其以可检测的剂量(例如，大于约0.5mg/kg)非肠道给予动物，例如大鼠、小鼠、豚鼠、猴、或人，允许充分的时间以发生代谢(典型地约30秒到30小时)和从尿、血液或其它生物样本分离其转化产物。这些产物容易分离，因为它们是被标记的(其它通过利用能够结合存在于代谢物中的抗原表位的抗体分离)。以常规的方式确定代谢物结构，例如，通过MS，LC/MS或NMR分析。通常，代谢物的分析是以与本领域技术人员公知的常规药物代谢研究相同的方法进行的。只要代谢物产物不是以其它方式在体内不能被发现，否则它们可用于本发明化合物的治疗剂量给药的检定测定法。本发明的化合物可以在一个或多个构成该化合物的 原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
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)本发明的氘代三嗪类化合物对3C样半胱氨酸蛋白酶具有很好的抑制活性。
(2)本发明化合物能够有效提高血药浓度、延长半衰期，并且显著降低了单次给药剂量。
(3)本发明化合物对冠状病毒感染具有较好的治疗作用。
附图说明
图1为实施例14种阳性对照组和化合物S11在小鼠感染模型中的抗感染活性。
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。
实施例1：化合物S1的合成
Figure PCTCN2022118629-appb-000004
步骤一：氘代化合物4a的合成
Figure PCTCN2022118629-appb-000005
(1)化合物1(37.2g，0.2mol)混悬于水(190ml)和浓盐酸(50ml，0.6mol)中，0℃下，将NaNO 2水溶液(13.80g,0.2mol,32.20mL)滴加至上述溶液中，然后搅拌30min，过滤，预冷的NaBF 4(24.20g,0.22mol)的水溶液(90ml)加入到上述滤液中，0℃下搅拌40min。停止搅拌，过滤，冷乙醇和乙醚洗涤滤饼，收集滤饼、干燥制得重氮盐(22.5g，0.097mol)。将重氮盐溶于CHCl 3(231mL)，将KOAc(15.15g,0.155mol)加入到上述溶液中，室温下搅拌，TLC监测，反应完全，停止搅拌。加水(200ml)淬灭反应，DCM萃取(100mL×3)，合并有机相，饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，浓缩，重结晶制得化合物2(25.49g，65％)。 1H NMR(300MHz,Chloroform-d)δ9.11(s,1H),8.73(d,J＝1.4Hz,1H),8.34(d,J＝1.4Hz,1H),7.77(s,1H)。
(2)将化合物2(19.6g,0.1mol)溶于无水DMF(300ml)中，0℃下，分批加入NaH(4.8g，0.12mol)，维持0℃搅拌30min，然后向上述混悬液中加入氘代碘甲烷(15.9g，0.11mol)，转移至室温，继续搅拌反应，TLC监测，直至原料反应完全。停止反应，将反应液置于0℃下，饱和氯化铵溶液(200ml)淬灭反应，DCM萃取(100ml×3)，合并有机相，饱和食盐水洗涤，无水Na 2SO 4 干燥，过滤，浓缩，柱层析纯化，制得化合物3(14.98g，70％)。 1H NMR(300MHz,Chloroform-d)δ8.75(d,J＝1.5Hz,1H),8.03(d,J＝1.5Hz,1H),7.80(s,1H)。
(3)将化合物3a(10g，0.047mol)溶于甲醇/四氢呋喃(100ml)中，加入甲酸铵(0.187mol，11.8g)和Pd/C(1g)，加热回流，TLC监测，直至原料反应完全。停止反应，硅藻土抽滤，滤液浓缩，加入DCM溶解(300ml)，水洗，饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，浓缩，柱层析分离纯化，制得化合物4(7.8g，90％)。 1H NMR(300MHz,Chloroform-d)δ7.93–7.85(m,2H),7.34(d,J＝1.5Hz,1H),5.75(s,2H).
步骤二：化合物S1的合成
Figure PCTCN2022118629-appb-000006
(1)化合物6a的合成
将化合物5(100mg,0.436mmol)溶于乙腈(2ml)中，向上述溶液中加入碳酸钾(78.0mg,0.567mmol)和化合物12a(0.063mL,0.480mmol)，加热回流反应2h。冷却至室温，乙酸乙酯稀释反应液，抽滤，滤液浓缩，柱层析分离纯化制得化合物6a(151mg,93％)。 1H NMR(300MHz,Chloroform-d)δ1.33(3H,t,J＝7.4Hz),1.65(9H,s),3.15(2H,q,J＝7.4Hz),5.03(2H,s),6.91-7.01(2H,m).
(2)化合物7a的合成
向化合物6a(4.88g,13.08mmol)中加入TFA(9.8mL)，室温下搅拌反应过夜，浓缩，异丙醚打浆，抽滤，收集滤饼，干燥，制得化合物7a(4.01g,97％)。 1H NMR(400MHz,Chloroform-d)δ1.36(3H,t,J＝7.4Hz),3.20(2H,q,J＝7.4Hz),5.16(2H,s),6.97-7.08(2H,m),8.25(1H,br s)。
(3)化合物8a的合成
将化合物7a(2.50g,7.88mmol)溶于DMF(15ml)中，向上述溶液中加入溴乙酸甲酯(1.2ml,11.8mmol)，碳酸钾(3.27g,23.6mmol)和碘化钾(130mg，0.788mol)，加热至60℃，反应4h。冷却至室温，加入DCM稀释反应液，水洗， 饱和食盐水洗涤，无水Na 2SO4干燥，过滤，浓缩，柱层析分离纯化，制得化合物8a(2.51g，85％)。 1H NMR(300MHz,Chloroform-d)δ7.08(dtt,J＝7.9,5.0,1.0Hz,1H),6.94(td,J＝8.0,4.9Hz,1H),5.05(d,J＝0.9Hz,2H),4.55(s,2H),3.75(s,3H),2.53(s,3H).
(4)化合物9a的合成
将化合物8a(3.75g，10mmol)溶于甲醇(30ml)中，加入NaOH水溶液(2M，20mol，10ml)，室温下搅拌2h。将反应液转移至0℃下，稀盐酸调节pH至2～3，抽滤，收集滤饼，烘干，得到8a脱甲酯的产物。将8a脱甲酯产物溶于无水THF(20ml)中，-5℃下，向上述溶液中滴加氯甲酸异丁酯(1.6ml，12mmol)和三乙胺(2ml，15mmol)，搅拌30min。抽滤，滤液浓缩，加入无水THF(20ml)溶解，0℃下，加入氨/甲醇(7M，50mmol，7ml)，转移至室温搅拌1h。抽滤，滤液加入DCM稀释，水洗，饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，柱层析分离纯化，制得化合物9a(3.24g，90％)。 1H NMR(300MHz,Chloroform-d)δ7.16-7.22(m,1H),7.08(s,2H),6.94(td,J＝8.0,5.0Hz,1H),5.06(d,J＝1.1Hz,2H),4.56(s,2H),2.59(s,3H).
(5)化合物10a的合成
将化合物9a(3.6g，10mmol)混悬于DMF-DMA(1.5ml，11mmol)中，加热至95℃，加热30min后，冷却至室温，减压蒸掉DMF-DMA，加入1,2-二氯乙烷形成共沸物，以保证DMF-DMA除干净。然后将上述油状物溶于乙醇(7ml)中，无需纯化直接投入后面的反应。乙醇(30ml)和醋酸(7ml)的混合液在冰浴条件下冷却，向上述溶液中滴加水合肼(4ml，11mol)，滴加完毕后，将前面得到的中间体的乙醇溶液缓慢滴加至上述反应液中，滴加完成后，转移至室温，搅拌反应，TLC监测，直至原料反应完全，停止反应，减压蒸掉乙醇，加水稀释，过滤，水洗涤滤饼，收集滤饼，干燥，制得化合物10a(1.92g，50％)。 1H NMR(300MHz,Chloroform-d)δ9.11(s,1H),8.60(d,J＝4.6Hz,1H),7.12(dtt,J＝8.0,5.0,1.0Hz,1H),6.97(td,J＝8.1,5.0Hz,1H),5.06(d,J＝1.1Hz,2H),4.29(s,2H),2.53(s,3H).
(6)化合物11a的合成
将化合物10a(3.84g，10mmol)溶于DMF(20ml)中，加入碳酸钾(3.45g， 25mmol)，碘甲烷(7.5ml，12mmol)，升温至80℃，反应5h。冷却至室温，抽滤，DCM稀释反应液，水洗，饱和食盐水洗涤，无水硫酸钠干燥，过滤，浓缩，柱层析分离纯化，制得化合物11a。 1H NMR(300MHz,Chloroform-d)δ8.24(s,1H),7.12(dtt,J＝8.0,4.9,1.0Hz,1H),6.97(td,J＝8.1,5.0Hz,1H),5.06(d,J＝1.1Hz,2H),4.68(d,J＝12.4Hz,1H),4.29(d,J＝12.5Hz,1H),4.09(s,3H),2.57(s,3H).
(4)化合物S1的合成
0℃下，向化合物10a(300mg,0.727mmol)和4(172mg,0.946mmol)的四氢呋喃溶液中滴加LHMDS(1M，1.46mL,1.46mmol)，维持0℃搅拌3h，然后转移至室温搅拌40min。反应完成后，加入饱和氯化铵溶液(2ml)淬灭反应，乙酸乙酯萃取(2ml×3)，合并有机相，饱和食盐水洗涤，无水Na 2SO 4干燥，过滤，浓缩，柱层析纯化，制得化合物S1。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ3.94(3H,s),5.08(2H,s),5.36(2H,s),7.46(1H,m),7.48-7.62(2H,m),7.77(1H,s),8.42(1H,s),9.32(1H,s).MS(ESI,m/z):535(M ++1).
实施例2：化合物S2的合成
Figure PCTCN2022118629-appb-000007
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),4.18(3H,s),3.94(1H,s).MS(ESI,m/z):534(M ++1).
实施例3：化合物S3的合成
Figure PCTCN2022118629-appb-000008
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),4.18(3H,s),3.94(2H,s).MS(ESI,m/z):533(M ++1).
实施例4：化合物S4的合成
Figure PCTCN2022118629-appb-000009
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),3.94(3H,s).MS(ESI,m/z):535(M ++1).
实施例5：化合物S5的合成
Figure PCTCN2022118629-appb-000010
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),4.18(s,1H),3.94(3H,s).MS(ESI,m/z):534(M ++1).
实施例6：化合物S6的合成
Figure PCTCN2022118629-appb-000011
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),4.18(s,2H),3.94(3H,s).MS(ESI,m/z):533(M ++1).
实施例7：化合物S7的合成
Figure PCTCN2022118629-appb-000012
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.36(2H,s),5.08(2H,s),.MS(ESI,m/z):538(M ++1).
实施例8：化合物S8的合成
Figure PCTCN2022118629-appb-000013
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.08(2H,s),4.18(3H,s).MS(ESI,m/z):537(M ++1).
实施例9：化合物S9的合成
Figure PCTCN2022118629-appb-000014
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.08(2H,s),3.94(2H,s).MS(ESI,m/z):537(M ++1).
实施例10：化合物S10的合成
Figure PCTCN2022118629-appb-000015
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.08(2H,s),4.18(s,3H),3.94(3H,s).MS(ESI,m/z):534(M ++1).
实施例11：化合物S11的合成
Figure PCTCN2022118629-appb-000016
合成方法如实施例1，只需更换相应的原料即可。 1H NMR(300MHz,DMSO-d 6,DCl in D 2O)δ9.32(1H,s),8.42(1H,s),7.77(1H,s),7.46(1H,m),5.08(2H,s).MS(ESI,m/z):540(M ++1).
实施例12：SARS-CoV-2病毒3C样半胱氨酸蛋白酶(3CLpro)酶抑制活性测试实验
1. 3CLpro蛋白表达与纯化
将全长3CLpro蛋白的基因序列构建在表达载体pET28a(+)载体中并转入大肠杆菌BL21(DE3)感受态细胞，在终浓度0.5mM IPTG，25℃条件下诱导12小时后用Ni-NTA柱纯化。纯化得到的蛋白经SDS检测，纯度大于90％的部分用于进一步用GE蛋白质层析纯化系统AKTA Pure的Superdex 200 10/300 GL纯化，得到纯度大于95％的蛋白，使用Nano Drop测定蛋白浓度，分装并液氮速冻后放入-80℃保存。
2.SARS-CoV-2 3CLpro酶活筛选体系的建立与抑制剂抑制率及药物IC50的计算
通过荧光共振能量转移(FRET)技术测定SARS-CoV-2 3CLpro活性及化合物对SARS-CoV-2 3CLpro的抑制活性。测定试验中使用带有SARS-CoV-2 3CLpro切割位点(箭头所示)的荧光底物(Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH 2)和Tris-HCl缓冲液(20mM Tris-HCl，150mM NaCl，10mM EDTA，pH 7.5)。化合物由100％DMSO溶解。10μl化合物与40μl SARS-CoV-2 3CLpro(终浓度0.5μM，Tris-HCl缓冲液稀释)在25℃孵育10min，通过添加50μl荧光底物(终浓度20μM)引发反应。使用放射共振能量转移荧光分光光度计在340nm激发波长和490nm吸收波长下检测由于3CLpro催化的底物裂解而产生的Dabcyl荧光信号。SARS-CoV-2 3CLpro动力学常数(Vmax和Km)是通过将数据拟合到Michaelis Menten方程中得出的，V＝Vmax×[S]/(Km+[S])。然后根据公式kcat＝Vmax/[E]，计算kcat。使用Tris-HCl缓冲液将化合物按倍数稀释法进行梯度稀释，并使用上述相同终浓度的SARS-CoV-2 3CLpro和荧光底物体系进行测定。分别在存在和不存在目标化合物的情况下，确定3CLpro催化多肽底物水解的内在(V0i)和表观(Vappi，Kappi)催化参数的值。目标化合物与Mpro结合的表观抑制常数(Kappi)由Vappi对固定底物浓度([S])下抑制剂浓度([I])的依赖性根据方程Vappi＝Vapp×[I]/(Kappi+[I])得出。目标化合物与3CLpro结合的内在抑制常数(Ki)的值根据方程Kappi＝Ki×(1+[S]/Km)计算得出。化合物的抑制曲线由GraphPad Prism 8.0软件绘制并计算IC 50值。其结果如下表1 所示，实施例化合物对SARS-CoV-2病毒3CLpro具有较好的抑制活性，活性优于阳性药S-217622。
表1 SARS-CoV-2病毒3CLpro酶抑制活性
化合物编号 IC 50(μM) 化合物编号 IC 50(μM)
S1 0.018 S7 0.012
S2 0.018 S8 0.015
S3 0.015 S9 0.0145
S4 0.018 S10 0.020
S5 0.017 S11 0.010
S6 0.016 S-217622 0.025
实施例13：细胞毒性以及抗SARS-CoV-2病毒感染药效测试实验
Vero E6细胞毒性测试：采用CCK8法检测待测化合物对哺乳动物Vero E6细胞中的细胞毒性。Vero E6细胞加入到96孔板中，培养过夜。然后，细胞与不同浓度的待测化合物共孵育48h。除掉孔板中的培养基，换成新鲜无血清的培养基，加入10％CCK8试剂，再在37℃孵育1h，随后采用酶标仪检测450nm处吸光度值。
筛选无细胞毒性或细胞毒性较小的化合物进行抗病毒感染的测试，具体操作包括以下步骤：①接种细胞：取处于对数生长期的Vero-E6细胞，吸出培养液，用胰酶消化细胞，细胞计数为：1×106个/ml；取上述细胞4ml，加入培养基6ml，制备得到细胞密度为4×105个/ml的细胞悬液，接种到96孔板中，每孔100μl，每孔细胞4×104个。②药物预处理细胞：将细胞培养基更换为含有2％FBS的DMEM培养基，加入相应浓度的药物和DMSO，每孔100μl，之后置于37℃培养箱，预处理1h。③病毒感染：取病毒0.3ml，加入45ml培养基，混匀，将病毒稀释至100TCID50/0.05ml；弃去细胞板中的药物培养基垂直悬滴病毒稀释液至96孔板内，加样体积50μl/孔，同时加入相应的药物培养基(含有相应浓度的药物)，加样体积50μl/孔，混匀；④孵育：将加好样的细胞培养板在震荡器上混匀，置于37℃培养箱，孵育1h。孵育结束后，吸去接种有细胞的病毒-血清混合液，加入相应浓度的药物和对照组DMSO，加样体积100μl/孔(100TCID50/孔)，置于37℃CO 2培养箱中培养48h；⑤收集上清液体检测病 毒RNA，用4％的多聚甲醛固定染色进行免疫荧光染色分析。
具体实验结果如表2所示，实施例化合物细胞毒性较小，对SARS-CoV-2病毒感染具有较好的抑制活性，且具有较好的选择指数。
表2待测化合物的细胞毒性和抗SARS-CoV-2病毒感染活性
Figure PCTCN2022118629-appb-000017
实施例14：化合物S11体内抗感染活性测试
雌性BALB/c小鼠，首先通过腹腔注射氯胺酮/甲苯噻嗪(50mg/kg/5mg/kg)使其麻醉，然后将SARS-CoV-2γ株(1×10 4TCID 50/只)通过鼻内接种构建感染模型，阴性对照组小鼠滴入相同体积的生理盐水。造模成功后，分为空白对照组、S-217622阳性对照组和给药组，每组6只。将化合物S-217622和S11分别混悬于0.5％甲基纤维素，造模成功后立即口服给药一次，12h后给药一次。S11的给药剂量为2mg/kg，8mg/kg，16mg/kg和32mg/kg，S-217622给药剂量为32mg/kg。病毒感染24h后，处死小鼠，观察小鼠肺部病毒滴度。
如图1所示，化合物S11给药两次后，相对于空白对照组，显著降低感染小鼠的肺匀浆中的病毒低毒，且呈剂量依赖性。阳性对照S-217622和化合物S11在16mg/kg和32mg/kg给药剂量下病毒滴度达到最低检出限。
实施例14：化合物S11的药代动力学性质测试
大鼠模型药代动力学性质测试：口服给药：将化合物用DMSO/0.5％甲基纤维素(400cP)(1:4)体系溶解，给药剂量2μM/5ml/kg。静脉给药：将化合物用DMSO/丙二醇(v/v＝1:1)，给药剂量(1.0μmol/mL/kg)。于给药后5min，15min，30min，1h，2h，4h，8h，10h，24h，从眼底静脉丛连续取血置于分布由肝素的EP管中，8000rpm/min离心5min后取上层血浆，-20℃冻存静脉注射给药，待LC-MS/MS分析，根据测试所得的血药浓度-时间数据，采用WinNonlin软件求算药代动力学参数。
比格犬药代动力学性质测试：口服给药：溶媒为0.5％甲基纤维素(400cP)，给药剂量3mg/2mL/kg。静脉给药：溶媒为二甲基乙酰胺/乙醇/20％HP-β-CD(v:v:v＝2:3:5)的碳酸盐缓冲液(pH 9.0)。于给药后5min，15min，30min，1h，2h，4h，8h，10h，24h，从眼底静脉丛连续取血置于分布由肝素的EP管中，8000rpm/min离心5min后取上层血浆，-20℃冻存静脉注射给药，待LC-MS/MS分析，根据测试所得的血药浓度-时间数据，采用WinNonlin软件求算药代动力学参数。
实验结果表明，化合物S11在大鼠中的口服生物利用度为97％，半衰期为5h；化合物S-217622在大鼠中的口服生物利用度为93％，半衰期为2.9h。化合物S11在比格犬中的口服生物利用度为77％，半衰期为30h；S-217622在比格犬中的口服生物利用度为69％，半衰期为28h。

Claims (10)

  1. 具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，结构如下：
    Figure PCTCN2022118629-appb-100001
    其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7或R 8独立地选自氢或氘；
    并且，R 1、R 2、R 3、R 4、R 5、R 6、R 7或R 8中至少有一个为氘。
  2. 根据权利要求1所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 1、R 2、R 3、R 4、R 5或R 6至少有一个为氘。
  3. 根据权利要求1和2所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 1、R 2或R 3至少有一个为氘。
  4. 根据权利要求1和2所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 4、R 5或R 6至少有一个为氘。
  5. 根据权利要求1所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 7或R 8至少有一个为氘。
  6. 根据权利要求1和5所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体、代谢产物、前药、溶剂合物或水合物，其特征在于，所述的R 7和R 8均为氘。
  7. 根据权利要求1-6所述的具有通式I所示结构的三嗪类化合物或其药学上可接受的盐、异构体或代谢产物，其特征在于，所述的式I所示化合物为以下任一化合物：
    Figure PCTCN2022118629-appb-100002
  8. 一种如权利要求1和2中所述的具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物制备方法，其特征在于，其包括以下步骤：在溶剂中，在碱的作用下，将如式II所示化合物与如式III所示化合物进行如下所示的反应，即可；
    Figure PCTCN2022118629-appb-100003
    其中，R 1、R 2、R 3、R 4、R 5、R 6、R 7和R 8定义如权利要求1和2所述。
  9. 一种药物组合物，其特征在于，所述药物组合物含有治疗有效量的一种或多种权利 要求1-7中任一所述的具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物，及药学上可接受的载体或辅料。
  10. 一种如权利要求1-7中任一所述的具有通式I所示结构的三嗪类化合物、或其药学上可接受的盐、异构体或代谢产物的用途，其特征在于，用于制备3C样半胱氨酸蛋白酶抑制剂或用于制备治疗和/或预防病毒感染性疾病的药物，优选用于制备3C样半胱氨酸蛋白酶抑制剂或用于制备治疗和/或预防病毒感染性疾病的药物。
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