[go: up one dir, main page]

WO2023030347A1 - 嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途 - Google Patents

嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途 Download PDF

Info

Publication number
WO2023030347A1
WO2023030347A1 PCT/CN2022/116003 CN2022116003W WO2023030347A1 WO 2023030347 A1 WO2023030347 A1 WO 2023030347A1 CN 2022116003 W CN2022116003 W CN 2022116003W WO 2023030347 A1 WO2023030347 A1 WO 2023030347A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
optionally
compound
substituted
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/116003
Other languages
English (en)
French (fr)
Inventor
常晓宇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of WO2023030347A1 publication Critical patent/WO2023030347A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A61K31/708—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid having oxo groups directly attached to the purine ring system, e.g. guanosine, guanylic acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Definitions

  • the invention relates to a synthesis process of purine nucleoside drugs and its application in the preparation of drugs for preventing or treating coronaviruses, including novel coronavirus (Covid-19) infectious diseases.
  • Coronavirus is a class of enveloped positive-sense single-stranded RNA viruses that spread widely among humans, other mammals, and birds, and can cause respiratory, intestinal, liver, and nervous system diseases. Seven CoVs are currently known to cause disease in humans, four of which, HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, circulate in humans and commonly cause common cold symptoms. The other three SARS-CoV, MERS-CoV and new coronavirus (COVID-19) all have serious hazards of fast onset, strong infectivity and high lethality.
  • the present invention provides the purposes of the compound represented by formula (I) or pharmaceutically acceptable salt thereof in the preparation of the medicament for preventing or treating coronavirus infectious disease;
  • X and Y are independently selected from: alkyl or substituted alkyl, halogen, -CN, -N 3 , -OH, -NH 2 , -NHR 3 , -NR 3 (R 3 ), -OR 3 or -SR 3 ;
  • R' and R are each independently selected from: -H, -COR 3 , -COOR 3 , -CONHR 3 or
  • Ar is selected from: aryl or substituted aryl, heteroaryl or substituted heteroaryl;
  • R and R are independently selected from: -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, or 1-3 haloalkyl;
  • R 1 and R 2 form a 3-6 membered ring saturated aliphatic ring
  • Each R is independently selected from -H, alkyl or substituted alkyl, alkynyl or substituted alkynyl, alkenyl or substituted alkenyl, 1-3 haloalkyl or substituted alkyl, aryl or substituted aryl, or Heteroaryl or substituted heteroaryl;
  • substituents in the "substituted alkyl”, “substituted aryl”, “substituted heteroaryl”, “substituted alkynyl” and “substituted alkenyl” are each independently selected from: alkyl, halogen, -CN, -N 3 , -OH, -NH 2 , -NHR 3 , -NR 3 (R 3 ), -OR 3 , -SR 3 ;
  • halogen atoms in the "halogen” and “haloalkyl” are selected from F, Cl, Br or I;
  • alkyl in the "alkyl” and “haloalkyl” is a C 1-20 straight chain or branched chain alkyl, optionally a C 1-10 straight chain or branched chain alkyl, optionally a C 1-6 Straight chain or branched chain alkyl; alternatives are: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, or isopentyl ;
  • the haloalkyl group is 2-chloroethyl, 2-fluoroethyl or trifluoroethyl;
  • alkynyl is C 2 -C 20 alkynyl; optional C 2 -C 10 alkynyl; optional acetylene;
  • alkenyl is C 2 -C 20 alkenyl; optionally C 2 -C 10 alkenyl; optionally vinyl;
  • aryl is a 6-10 membered aryl group; it can be phenyl or naphthyl;
  • heteroaryl is a 5-10 membered heteroaryl group containing at least one heteroatom selected from N, O or S in the ring.
  • X is selected from -H, -NH 2 , -NHR 3 , -NR 3 (R 3 ), -OR 3 or -SR 3 ;
  • Y is -NH 2 ;
  • R' is -H
  • R is -H or
  • R 1 and R 2 are independently selected from: -H or C 1-6 straight or branched chain alkyl
  • Each R 3 is independently C 1-6 straight chain or branched chain alkyl
  • Ar is selected from phenyl or substituted phenyl, naphthyl or substituted naphthyl;
  • the C 1-6 straight chain or branched chain alkyl is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-butyl Pentyl, or isopentyl.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is a compound of formula (II) or a pharmaceutically acceptable salt thereof,
  • X, Y, R' and R are as defined above.
  • the compound represented by formula (I) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof,
  • X is -H, -NH 2 , -NHMe, -N(Me) 2 , -N(Me)(Et), -N(Et) 2 , -OMe, -OEt , -O(iPr), -SMe, -SEt, or -S(iPr);
  • Ar is phenyl or naphthyl.
  • Another aspect of the present invention provides the compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound represented by the formula (I) or a pharmaceutically acceptable salt thereof is used for the preparation of Drugs for the prevention or treatment of coronavirus infectious diseases.
  • Another aspect of the present invention provides a method for preventing or treating coronavirus infectious diseases, which includes administering a therapeutically or preventively effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient in need.
  • the pharmaceutically acceptable salts include salts formed by compounds of formula (I) and inorganic acids or organic acids;
  • the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, hemisulfuric acid, nitric acid, phosphoric acid, or carbonic acid;
  • the organic acids include formic acid, ascorbic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, glucose acid, tartaric acid, glucuronic acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzoic acid, benzenesulfonic acid, p-bromobenzenesulfonic acid, glutamic acid, salicylic acid, or pamoic acid;
  • the pharmaceutically acceptable salt is the hydrochloride salt of the compound of formula (I);
  • the pharmaceutically acceptable salt is the hemisulfate salt of the compound of formula (I).
  • the coronavirus infectious diseases include diseases caused by infecting humans or other animals;
  • the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV or COVID-19.
  • the dosage form of the drug is an immediate-release dosage form, a sustained-release dosage form, or a controlled-release dosage form;
  • the dosage form of the drug is tablet, capsule, aqueous or oily suspension, granule, emulsion, syrup, elixir, injection, or powder injection.
  • the present invention also provides a preparation method for the compound of formula (I) or a pharmaceutically acceptable salt thereof, comprising subjecting compound 5 to the dihydroxylation reaction of step e, then to the cyclic sulfoesterification reaction of step f, and then to the step g adopting triethylamine trihydrofluoride to open the ring, and then undergoing the post-hydrolysis cyclization reaction and benzoylation of step h to obtain the step of intermediate compound 9;
  • step e the following reagents and conditions are used: KMnO 4 , NaHCO 3 , acetone;
  • step f the following reagents and conditions are used: SOCl 2 , EtN 3 , NaClO;
  • step g the following reagents and conditions are used: Et 3 N ⁇ 3HF, Et 3 N, HCl;
  • step h the following reagents and conditions are used: BaCl, BzCl, DMAP, Et 3 N, CH 3 CN;
  • the method further includes: using intermediate compound 9 to obtain compound 11 through the reduction reaction of step i and the bromination reaction of step j in sequence, Then compound 11 with response to get Purine nucleosides, which are then obtained through an esterification step
  • step i the following reagents and conditions are used: LiAlH[OC(CH 3 ) 3 ] 3 , THF;
  • step j the following reagents and conditions are used: PPh 3 , CBr 4 , DCM;
  • the method also includes: reacting compound 1 and ethyl acrylate to obtain compound 2 through step a, then brominating step b, hydrolyzing step c, and fluorination reaction step d to obtain compound 5. :
  • step a the following reagents and conditions are used: triethylenediamine (DABCO);
  • step b the following reagents and conditions are used: NBS, DMS, DCM;
  • step c the following reagents and conditions are used: sodium acetate, reflux conditions;
  • step d the following reagents and conditions are used: perfluorobutylsulfonyl fluoride, Et 3 N ⁇ 3HF.
  • Dissolve compound 3 (118 g, 0.40 mol) obtained in the previous step in 1 L of ethanol, add 100 g of anhydrous sodium acetate, and heat to reflux until the reaction of the raw materials is complete. The insoluble matter was filtered off, the temperature of the reaction solution was lowered to 0° C., sodium ethoxide (8.3 g, 0.10 mol) was added, and the temperature was kept until the reaction of the raw materials was complete.
  • the crude compound 10 obtained in the previous step was dissolved in dichloromethane, and PPh 3 (8.0g, 30.7mmol) was added at -20°C. After stirring for 20 minutes, CBr 4 (11.0g, 33.18mmol) was added, and the temperature was raised to 0°C to continue the reaction After 2 hours, after monitoring the completion of the reaction, 30 mL of water was added to quench the reaction, extracted with dichloromethane, and dried over anhydrous sodium sulfate.
  • Potassium tert-butoxide (3.0g, 26.43mmol) was added in batches to a solution of 2-chloro-6-aminopurine (4.5g, 26.43mmol) in 30mL of tert-butanol at room temperature, and after stirring for 1 hour at 30°C, the mixture The solution was poured into the acetonitrile solution of compound 11 (4.0 g, 8.81 mmol) obtained in the previous step, and the temperature was gradually raised to 50 ° C. After 16 hours, TLC monitored that the reaction was complete.
  • CPE cytopathic effect
  • test product III (synthesized in this laboratory), this product is white to light yellow solid. Seal and store in a cool place.
  • Ribavirin injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., the batch number is 31712252, the specification is 100mg/ml, diluted to the required concentration when used, and stored in a refrigerator at 4°C.
  • H460 cells were inoculated in a 96-well plate at 1.5 ⁇ 104 cells/well, and after overnight culture, a maintenance solution containing the drug to be tested was added, and the drug to be tested was tested with 8 doses of three-fold dilution samples, and the culture was continued. After 3 days of administration, the toxicity of the drug to the cells under an inverted microscope (CPE method), and the half toxic concentration TC 50 was calculated by the Reed-Muench method, and the calculation formula was as follows:
  • A drug concentration with cumulative inhibitory rate ⁇ 50%
  • B cumulative inhibitory rate > 50% inhibitory rate
  • C cumulative inhibitory rate ⁇ 50% inhibitory rate
  • D log dilution factor
  • the experiment was carried out in the passaged H460 cells.
  • the cells were seeded in 96-well plates at 1.5 ⁇ 104 /well. After overnight culture, 100TCID 50 HCoV-OC43 virus liquid was used to infect the cells in the 96-well plates.
  • the drug to be tested was diluted with culture medium. Measuring at the same time as the infection, the drug to be tested was tested with three times diluted samples of 8 doses, each dose was set with 2 parallel wells, and the results were observed when the virus control group had lesions of 4+, recorded and analyzed by Reed-Muench
  • A drug concentration with cumulative inhibition rate ⁇ 50%
  • B inhibition rate with cumulative inhibition rate>50%
  • C inhibition rate with cumulative inhibition rate ⁇ 50%
  • D log dilution factor
  • the CC 50 of compound III determined by CPE method was >100 ⁇ M, and its IC 50 against HCoV-OC43 was 0.09 ⁇ M; the selection index SI was >1111; the positive control drug RBV half toxic concentration CC 50 was >0.36 ⁇ 0.06 ⁇ M, the IC50 against HCoV-OC43 is 0.015 ⁇ 0.01 ⁇ M, and the selection index SI is >24.
  • the anti-HCoV-OC43 activity of the positive drug RBV was comparable to the results in the literature, indicating that the experimental system was established.
  • Example 3 Test of compound III anti-new coronavirus 2019-nCoV (COVID-19) activity and cytotoxicity in vitro
  • Virus strain 2019-nCoV (COVID-19);
  • Positive control drug remdesivir
  • Reagents DMEM medium (Gibco), fetal bovine serum (Gibco), double antibody, trypsin, MTT (Amresco), etc.;
  • Kit QIAamp viral RNA mini kit (52906, Qiagen), One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (RR096A TaKaRa)
  • MTT assay The toxicity of compound III to Huh7 cells was detected by MTT assay.
  • the full name of MTT is 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyltetrazoliumromide, which is a yellow dye.
  • MTT colorimetry is a method to detect cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in the cells, while dead cells do not have this function.
  • 10% SDS (dissolved in 0.01mol/L HCl solution) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 570nm with a multifunctional microplate reader, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number.
  • the survival rate of cells under the concentration of compound III can be calculated, thereby calculating the half-toxic concentration (CC 50 ) of compound III.
  • the antiviral activity was measured on the Huh7 cell model, and each experiment was set up with 3 replicate wells, which were repeated 3 times.
  • the One Step TB Green PrimeScript PLUS RT-PCR Kit (Perfect Real Time) kit was used for qRT-PCR detection, and the primers were RBD-qF1:5’-CAATGGTTTAACAGGCACAGG-3’; RBD-qR1:5’-CTCAAGTGTCTGTGGATCACG-3’.
  • the total volume of the reaction system is 20 ⁇ L: 10 ⁇ L 2X One Step TB Green RT-PCR Buffer 4, 1.2 ⁇ L TaKaRa Ex Taq HS Mix, 0.4 ⁇ L PrimeScript PLUS RTase Mix, 0.8 ⁇ L each of RBD-qF1 and RBD-qR1, 0.4 ⁇ L ROX Reference Dye (50X), 2 ⁇ L viral RNA, 4.4 ⁇ L RNase Free dH2O.
  • the reaction parameters were: reverse transcription at 42°C for 5min, pre-denaturation at 95°C for 10s, 40 cycles of PCR including denaturation at 95°C for 10s, annealing and extension at 60°C for 30s.
  • Test drug EC50 CC 50 Ti Compound III 0.08 ⁇ M >100 ⁇ M >1250 remdesivir 1.50 ⁇ M >45.0 ⁇ M >30.0
  • the concentration required for Compound III to inhibit 50% of the new coronavirus is 0.08 ⁇ M, while Redcivir requires 1.5 ⁇ M.
  • the activity of Compound III in inhibiting the new coronavirus is 18 times that of Redcivir, and the cytotoxicity is less .
  • compound III and its analogues have the effect of preventing or treating coronavirus infectious diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

式(I)所示的化合物或其药学上可接受的盐在制备预防或治疗冠状病毒感染疾病的药物中的用途。所述式(I)化合物在体外试验中表现出显著的抑制新冠病毒复制的活性，而且在测试的剂量范围没有明显的细胞毒性。

Description

嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途 技术领域
本发明涉及嘌呤核苷类药物的合成工艺及其在制备预防或治疗冠状病毒，包括新冠病毒(Covid-19)感染性疾病的药物中的用途。
背景技术
冠状病毒(Coronavirus,CoV)是一类有包膜的正向单链RNA病毒，在人类、其他哺乳动物和鸟类中广泛传播，并可导致呼吸道、肠道、肝脏和神经系统等疾病。目前已知有七种CoV可导致人类疾病，其中四种HCoV-229E、HCoV-OC43、HCoV-NL63和HCoV-HKU1在人群中流行，并通常引起普通感冒症状。而其他三种SARS-CoV、MERS-CoV和新冠状病毒(COVID-19)都具有发病快、传染性强和致死率高的严重危害性。COVID-19自2019年底在全球爆发到2021年3月17日止，全球感染人数已达到1.17亿，死亡人数超过260万，成为全球公共卫生的巨大挑战。虽然有几款疫苗已经授予紧急使用权，但目前还没有有效的临床治疗药物，美国FDA批准紧急使用授权的瑞德西韦的临床疗效有限。因此，研发直接抗新冠病毒COVID-19的药物，十分迫切。
发明内容
本发明提供式(I)所示的化合物或其药学上可接受的盐在制备预防或治疗冠状病毒感染性疾病的药物中的用途；
Figure PCTCN2022116003-appb-000001
其中，
X和Y独立地选自：烷基或取代烷基、卤素、-CN、-N 3、-OH、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3或-SR 3；
R'和R各自独立地选自：-H、-COR 3、-COOR 3、-CONHR 3或
Figure PCTCN2022116003-appb-000002
Ar选自：芳基或取代芳基、杂芳基或取代杂芳基；
R 1和R 2独立地选自：-H、烷基或取代烷基、炔基或取代炔基、烯基或取代烯基、或1-3卤代烷基；
或R 1和R 2形成3-6元环饱和脂肪环；
各个R 3各自独立地选自-H、烷基或取代烷基、炔基或取代炔基、烯基或取代烯基、1-3卤代烷基或取代烷基、芳基或取代芳基、或者杂芳基或取代杂芳基；
所述“取代烷基”、“取代芳基”、“取代杂芳基”、“取代炔基”和“取代烯基”中的取代基各自独立的选自：烷基、卤素、-CN、-N 3、-OH、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3、-SR 3；
所述“卤素”和“卤代烷基”中的卤原子选自F、Cl、Br或I；
所述“烷基”和“卤代烷基”中的烷基为C 1-20直链或支链烷基，可选为C 1-10直链或支链烷基，可选为C 1-6直链或支链烷基；可选为：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、或异戊基；可选地，所述卤代烷基为2-氯乙基、2-氟乙基或三氟乙基；
所述“炔基”为C 2-C 20炔基；可选为C 2-C 10炔基；可选为乙炔；
所述“烯基”为C 2-C 20烯基；可选为C 2-C 10烯基；可选为乙烯；
所述“芳基”为6-10元芳基；可选为苯基或萘基；
所述“杂芳基”为环上含有选自N、O或S中至少一种杂原子的5-10元杂芳基。
可选地，式(I)中，
X选自-H、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3或-SR 3；
Y为-NH 2；
R'为-H；
R为-H或
Figure PCTCN2022116003-appb-000003
其中，
R 1和R 2独立地选自：-H或C 1-6直链或支链烷基；
各个R 3各自独立地为C 1-6直链或支链烷基；
Ar选自苯基或取代苯基，萘基或取代萘基；
可选地：所述C 1-6直链或支链烷基选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、或异戊基。
可选地，所述式(I)所示的化合物或其药学上可接受的盐为式(II)化合物或其药学上可接受的盐，
Figure PCTCN2022116003-appb-000004
其中，X、Y、R'和R如上定义。
可选地，式(I)所示的化合物或其药学上可接受的盐选自下列化合物或其药学上可接受的盐，
Figure PCTCN2022116003-appb-000005
式(I-a)和(I-b)中，X为-H、-NH 2、-NHMe、-N(Me) 2、-N(Me)(Et)、-N(Et) 2、-OMe、-OEt、-O(iPr)、-SMe、-SEt或-S(iPr)；
式(I-b)中，Ar为苯基或萘基。
本发明另一方面，提供上述式(I)所示的化合物或其药学上可接受的盐，其中，所述式(I)所示的化合物或其药学上可接受的盐用于制备用于预防或治疗冠状病毒感染性疾病的药物。
本发明另一方面，提供一种预防或治疗冠状病毒感染性疾病的方法，其中， 包括向有此需要的患者给予治疗或预防有效量的式(I)化合物或其药学上可接受的盐。
可选地，所述药学上可接受的盐包括式(I)化合物与无机酸或者有机酸形成的盐；
可选地，所述无机酸包括盐酸、氢溴酸、氢碘酸、硫酸、半硫酸、硝酸、磷酸、或碳酸；
可选地，所述有机酸包括甲酸、抗坏血酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、柠檬酸、枸橼酸、酒石酸、葡萄糖酸、酒石氢酸、葡萄糖醒酸、碳酸、苦味酸、甲磺酸、乙磺酸、对甲苯磺酸、苯甲酸、苯磺酸、对溴苯磺酸、谷氨酸、水杨酸、或双羟萘酸；
可选地，所述药学上可接受的盐为式(I)化合物的盐酸盐；
可选地，所述药学上可接受的盐为式(I)化合物的半硫酸盐。
可选地，所述冠状病毒感染性疾病包括感染人或其他动物所引起的疾病；
可选地，所述冠状病毒包括HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或COVID-19。
可选地，所述药物的剂型为速释剂型、缓释剂型、或控释剂型；
可选地，所述药物的剂型为片剂、胶囊剂、水性或油性混悬剂、颗粒剂、乳剂、糖浆剂、酏剂、注射液、或粉针剂。
本发明还提供所述式(I)化合物或其药学上可接受的盐的制备方法，包括将化合物5经过步骤e的双羟化反应，然后经步骤f的环磺酯化反应，然后经步骤g采用三乙胺三氢氟酸盐开环，然后经步骤h的水解后环化反应以及苯甲酰化，得到中间体化合物9的步骤；
Figure PCTCN2022116003-appb-000006
可选地，步骤e中，采用以下试剂和条件：KMnO 4，NaHCO 3，丙酮；
可选地，步骤f中，采用以下试剂和条件：SOCl 2，EtN 3，NaClO；
可选地，步骤g中，采用以下试剂和条件：Et 3N·3HF，Et 3N，HCl；
可选地，步骤h中，采用以下试剂和条件：BaCl，BzCl，DMAP，Et 3N，CH 3CN；
可选地，该方法还包括：采用中间体化合物9依次经步骤i的还原反应及步 骤j的溴化反应得到化合物11，
Figure PCTCN2022116003-appb-000007
然后化合物11与
Figure PCTCN2022116003-appb-000008
反应得到
Figure PCTCN2022116003-appb-000009
嘌呤核苷，然后经过酯化的步骤获得
Figure PCTCN2022116003-appb-000010
可选地，步骤i中，采用以下试剂和条件：LiAlH[OC(CH 3) 3] 3，THF；
可选地，步骤j中，采用以下试剂和条件：PPh 3，CBr 4，DCM；
可选地，式(IV)反应得到式(VI)的反应步骤的试剂和条件：t-BuOK,t-BuOH/CH 3CN；
可选地，该方法还包括：将化合物1与丙烯酸乙酯经步骤a反应得到化合物2，再经步骤b的溴化，步骤c的水解反应，步骤d的氟化反应得到化合物5的反应步骤：
Figure PCTCN2022116003-appb-000011
Figure PCTCN2022116003-appb-000012
可选地，步骤a中，采用以下试剂和条件：三乙烯二胺(DABCO)；
可选地，步骤b中，采用以下试剂和条件：NBS，DMS，DCM；
可选地，步骤c中，采用以下试剂和条件：乙酸钠，回流条件；
可选地，步骤d中，采用以下试剂和条件：全氟丁基磺酰氟，Et 3N·3HF。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于示例性地对本发明进行说明，并不用于限制本发明。
实施例1.化合物的合成
1、化合物2的合成
Figure PCTCN2022116003-appb-000013
将R-丙酮缩甘油醛(化合物1，500g，3.85mol)溶于1500mL丙烯酸乙酯中，将DABCO(215g,1.93mol)加入上述溶液，置于0℃条件下反应12h，转到室温条件下继续反应6天。TLC检测反应结束后，减压浓缩至无馏出物(丙烯酸乙酯)，将残余物溶解于2L乙酸乙酯中，用0.5M稀盐酸洗涤有机相，至水相为无色，再用水，饱和食盐水依次洗涤。有机相用无水硫酸钠干燥。减压蒸出溶剂得淡黄色油状物(化合物2)(637g,2.77mol)，收率72％。 1H NMR(400MHz,CDCl 3)δ6.37-6.36(m,1H),5.97-5.96(m,1H),4.52-4.51(m,1H),4.36-4.29(m,1H),4.26-4.21(m,2H),3.93-3.92(m,2H),1.44(s,3H),1.35(s,3H),1.31(t,J＝7.2Hz,3H)ppm；
2、化合物3的合成
Figure PCTCN2022116003-appb-000014
0℃条件下，向NBS(124g,0.70mol)的DCM(1200mL)悬浮液中，滴加200mL二甲硫醚(47g,0.76mol)的DCM溶液。加毕后，继续搅拌30分钟，将200mL化合物2(146g,0.64mol)的DCM溶液滴加到上述所得反应液中，移至室温，TLC检测化合物2反应完全后，减压30℃蒸出DCM，残余物用500mL乙醚和500mL饱和食盐水萃取，有机相用0.5mol NaOH水溶液洗涤至有机相，水相无色，再用水和饱和食盐水依次洗涤。有机相用无水硫酸钠充分干燥。25-30℃减压浓缩蒸出乙醚得无色至淡黄色油状物(化合物3)(126g,0.56mol)，收率80％。 1H NMR(400MHz,CDCl 3)δ6.84(d,J＝8.4Hz,1H),4.96-4.90(m,1H),4.32-4.19(m,5H),3.74-3.70(m,1H),1.46(s,3H),1.42(s,3H),1.32(t,J＝6.8Hz,3H)ppm.
3、化合物4的合成
Figure PCTCN2022116003-appb-000015
将上步所得化合物3(118g,0.40mol)溶于1L乙醇中，加入100g无水乙酸钠，加热回流至原料反应完全。滤掉不溶物，将反应液降温到0℃，加入乙醇钠(8.3g,0.10mol)，保温至原料反应完全。加入0.3eq乙酸中和，减压蒸出乙醇，加入200mL水溶解，乙酸乙酯萃取至水相无产物，合并有机相依次用水、饱和碳酸氢钠溶液、食盐水洗涤后用无水硫酸钠干燥。减压浓缩，硅胶柱层析分离，石油醚：乙酸乙酯＝10:1-3:1,得淡黄色油状物(化合物4)(55.7g,0.24mol),收率60％。 1H NMR(400MHz,CDCl 3)δ6.80(d,J＝7.6Hz,1H),4.96(dd,J＝14.0,7.2Hz,1H),4.43-4.30(m,2H),4.27-4.17(m,3H),3.70-3.66(m,1H),1.45(s,3H),1.40(s,3H),1.32(t,J＝6.8Hz,3H)ppm；
4、化合物5的合成
Figure PCTCN2022116003-appb-000016
将化合物4(200g,0.87mol)、全氟丁基磺酰氟(525g,1.74mol)和Et 3N·3HF(210g,130mol)溶于1700mL无水四氢呋喃中，冰浴下滴加三乙胺(440g,4.35mol)，滴加过程控制反应液温度在20℃，加毕保温20-25℃，TLC监测至原料反应完全。加入2L石油醚搅拌5min，静置分层，收集上层有机相。下层棕色不溶油状物用石油醚/乙酸乙酯(10:1)混合液萃取，合并上层有机相，减压浓缩，硅胶柱层析分离，石油醚：乙酸乙酯＝80:1得淡黄色液体(化合物5)(121g,0.52mol)，收率60％。 1H NMR(400MHz,CDCl 3)δ＝7.02(ddd,J＝8,3.6,0.8Hz,1H),5.28(dd,J＝46.4,11.2Hz,1H),5.12(ddd,J＝48.4,11.6,1.2Hz,1H),5.02-4.96(m,1H),4.28-4.18(m,3H),3.71(dd,J＝8.4,7.2Hz,1H),1.47(s,3H),1.42(s,3H),1.32(t,J＝7.2Hz,3H)； 13C NMR(100MHz,CDCl 3)δ＝165.19(J＝3.8Hz),145.70(J＝4.6Hz),129.60(J＝15.4Hz),110.42,76.85(J＝163.5Hz),72.36,69.24(J＝3.4Hz),61.38,26.54,25.68,14.17.HRMS(ESI)m/z:[M+Na] +calcd for C 11H 17FNaO 4 255.1003,found 255.1006.
5、化合物6的合成
Figure PCTCN2022116003-appb-000017
将化合物5(81g,0.35mol)、NaHCO 3(88g,1.05mol)和乙二醇(87g,1.40mol)溶于1500mL丙酮。0℃下分批量加入KMnO4(66g,0.42mol)，维持反应液中温度不超过5℃,加毕保温30min。缓慢加入300mL饱和的亚硫酸氢钠水溶液淬灭反应，室温搅拌2h至棕色退去。抽滤，丙酮洗涤滤饼。减压蒸出滤液中丙酮，乙酸乙酯萃取3-4次，合并有机相。依次用水，食盐水洗涤有机相，无水硫酸钠干燥，浓缩蒸干。加入甲苯150mL蒸干，用100mL甲苯、500mL正庚烷加热溶解，冷却析出白色固体，抽滤得白色固体(化合物6)(40g,0.45mol)(纯度在75％-80％)，收率43％。 1H NMR(CDCl 3,400MHz):δ4.87–4.56(m,2H),4.41–4.33(m,1H),4.21–4.08(m,4H),4.06–4.00(m,1H),3.82(t,J＝7.6Hz,1H),2.89(d,J＝9.2Hz,1H),1.36–1.31(m,9H)ppm； 13C NMR(CDCl 3,100MHz):δ171.5(d,J C-F＝3.8Hz),109.7,86.5(d,J C-F＝172.8Hz),78.5(d,J C-F＝17.9Hz),74.1,73.9(d,J C-F＝5.7Hz),67.0,62.8,26.4,25.1,14.0ppm；HRMS(ESI)m/z:[M+Na] +calcd for C 11H 19FNaO 6 289.1058,found 289.1060.
6、化合物7的合成
Figure PCTCN2022116003-appb-000018
将化合物6(8.0g,30mmol)溶于15mL乙腈和60mL乙酸异丙酯的混合溶液中，加入三乙胺(7.28g,72mmol)，缓慢滴加SOCl 2(4.12g,34.6mmol)，加料过程中保持反应体系温度不超过20℃，加料完毕后保温30min。反应结束后，加入30mL水搅拌后静置，弃去水相，有机相依次用30mL水和30mL饱和NaHCO 3洗涤。向洗涤后的有机相中加入15mL乙腈和5.04g NaHCO 3固体，将所得混合液降温至10℃，缓慢加入有效氯为8-10％的NaClO溶液60mL(约为2倍量)，加毕移至室温反应2h。反应结束后，静置弃去水相，有机相用30mL饱和Na 2SO 3水溶液洗涤两次，用无水硫酸钠干燥。加入0.5mL三乙胺作为稳定剂，减压蒸出溶剂后，硅胶柱层析分离,石油醚：乙酸乙酯＝6:1，得无色油状物(化合物7)(8g,24.3mmol)，收率为81％。 1H NMR(CDCl 3,400MHz):δ5.14–5.04(m, 1H),5.03–4.92(m,2H),4.59–4.55(m,1H),4.36(d,J＝7.2Hz,2H),4.21(dd,J＝10.0,6.0Hz,1H),4.10(dd,J＝10.0,2.8Hz,1H),1.42(s,3H),1.38–1.35(m,6H)ppm； 13C NMR(CDCl 3,100MHz):δ164.7(d,J C-F＝7.4Hz),111.6,88.4(d,J C-F＝17.8Hz),82.5(d,J C-F＝1.8Hz),81.3(d,J C-F＝179.1Hz),71.7(d,J C-F＝2.7Hz),67.2,64.1,26.8,24.9,14.0ppm；HRMS(ESI)m/z:[M+H] +calcd for C 11H 17FNaO 8S 351.0520,found 351.0519.
7、化合物9的合成
Figure PCTCN2022116003-appb-000019
将化合物7(8g,24.4mmol)、Et 3N·3HF(1.96g,12.2mmol)和三乙胺(2.96g,29.3mmol)的混合物加热至90℃反应3-4h，冷却至室温后加入浓盐酸4mL，加毕90℃反应30min得到化合物8。化合物8未经处理直接加入30mL饱和BaCl 2溶液，90℃下继续反应4h，加入200mL正丙醇共沸蒸出溶剂，蒸干后加入150mL甲苯蒸干带水两次。将所得混合物用60mL乙腈溶解，依次加入苯基酰氯(10.29g,73.2mmol)、DMAP(298mg,2.44mmol)和三乙胺(7.41g,73.2mmol)，控制温度不超过45℃。反应结束后，加入30mL水淬灭反应，静置弃去水相，有机相依次用饱和NaHCO 3水溶液和饱和食盐水洗涤，无水硫酸钠干燥。减压浓缩蒸出溶剂，硅胶柱层析分离，石油醚/乙酸乙酯＝5:1，得白色固体化合物9(3.8g,9.76mmol)，收率为40％。 1H NMR(CDCl 3,400MHz):δ8.08–8.99(m,4H),7.66–7.55(m,2H),7.50–7.40(m,4H),5.88(ddd,J＝13.2,7.2,2.0Hz,1H),5.09–5.05(m,1H),4.95–4.92(m,1H),4.83–4.78(m,2H),4.62(dd,J＝12.8,4.8Hz,1H)ppm； 13C NMR(CDCl 3,100MHz):δ167.1(d,J C-F＝17.9Hz),165.8,165.2,134.3,133.6,130.2,129.8,128.9,128.8,128.6,127.8,89.1(dd,J C-F＝194.1,24.2Hz),79.0(dd,J C-F＝174.4,35,2Hz),78.2,68.0(dd,J C-F＝14.3,2.1Hz),62.2ppm；HRMS(ESI)m/z:[M+H] +calcd for C 20H 17F 2O 6 391.0988,found 391.0987.
8、化合物10的合成
Figure PCTCN2022116003-appb-000020
称取化合物9(10g,25.6mmol)溶于100mL无水THF中，-20℃下滴加31mL 1M的(t-BuO) 3AlH的THF溶液，维持温度反应4h，TLC检测有原料剩余，补加7.4mL 1M的(t-BuO) 3AlH的THF溶液，继续反应2h，加入100mL水淬灭反应，静置弃去水相，加压蒸出大部分四氢呋喃，加入150mL乙酸乙酯溶解，用饱和食盐水洗涤，有机相无水硫酸钠干燥，加压浓缩至干，得无色油状物，将所得油状物置于45℃中8h，得白色固体化合物10，不经进一步处理，直接用于下一步。
9、化合物11的合成
Figure PCTCN2022116003-appb-000021
将上步所得化合物10粗品溶于二氯甲烷，-20℃下加入PPh 3(8.0g,30.7mmol)，搅拌20分钟后，加入CBr 4(11.0g,33.18mmol)，升温至0℃继续反应2小时，监测反应结束后，加入30mL水淬灭反应，二氯甲烷萃取，无水硫酸钠干燥。减压浓缩蒸出溶剂，硅胶柱层析分离，石油醚/乙酸乙酯＝5:1，得白色固体化合物11(6.16g,13.57mmol)，两步总收率为53％。 1H NMR(CDCl 3,400MHz):δ8.12(d,J＝7.6Hz,2H),8.00(d,J＝8.0Hz,2H),7.64–7.57(m,2H),7.50–7.43(m,4H),6.64(s,1H),5.32–5.29(m,1H),4.91(dt,J＝6.0,3.2Hz,1H),4.87–4.71(m,2H),4.69–4.51(m,2H)ppm； 13C NMR(CDCl 3,100MHz):δ166.0,165.7,134.1,133.7,130.2,129.7,129.2,128.8,128.5,95.0(dd,J C-F＝214.5,17.8Hz),87.1(dd,J C-F＝23.6,6.3Hz),81.5(d,J C-F＝1.3Hz),81.3(dd,J C-F＝181.9,27.5Hz),68.5(dd,J C-F＝14.9,5.0Hz),62.1ppm；HRMS(ESI)m/z:[M+H] +calcd for C 20H 18BrF 2O 5 455.0300,found 455.0302.
10、化合物12的合成
Figure PCTCN2022116003-appb-000022
室温下将叔丁醇钾(3.0g,26.43mmol)分批加入2-氯-6-氨基嘌呤(4.5g,26.43mmol)的30mL叔丁醇溶液，30℃下搅拌1小时后，将上述混合液倒入上一步得到的化合物11(4.0g,8.81mmol)的乙腈溶液中，逐渐升温至50℃，16小时后，TLC监测反应完全，降至室温后加水淬灭反应，乙酸乙酯萃取(50mL x 3)，合并有机相，无水硫酸钠干燥，柱层析色谱,石油醚/乙酸乙酯＝5:1，分离得到白 色固体β构型化合物12(2.2g,4.14mmol)，产率47％。 1H NMR(CDCl 3,400MHz):δ7.96–7.93(m,2H),7.89–7.87(m,2H),7.84(s,1H),7.58–7.54(m,1H),7.47–7.43(m,1H),7.40–7.36(m,2H),7.28–7.24(m,2H,overlapped with the peak of chloroform),6.67(dd,J＝22.0,8.8Hz,1H),6.26(d,J＝18.0Hz,1H),5.60(s,2H),5.21–5.16(m,1H),4.86(dt,J＝10.8,5.6Hz,1H),4.78–4.44(m,3H)ppm； 13C NMR(CDCl 3,100MHz):δ166.4,165.7,159.2,152.5,152.4,141.8,134.0,133.4,130.0,129.7,129.1,128.6,128.4,128.0,126.0,100.7(dd,J C-F＝190.3,17.4Hz),89.5(dd,J C-F＝39.6,1.8Hz),81.9(dd,J C-F＝174.6,24.8Hz),78.2,71.4(dd,J C-F＝15.6,5.2Hz),62.9ppm；HRMS(ESI)m/z:[M+H] +calcd for C 25H 21ClF 2N 5O 5 544.1194,found 544.1197.
11、化合物13的合成
Figure PCTCN2022116003-appb-000023
称取化合物12(1.5g,2.76mmol)溶于甲醇中，加入3mL33％的甲胺醇溶液，85℃反应16小时，反应结束后，直接蒸干，硅胶柱层析分离，二氯甲烷：甲醇＝15:1，得白色固体化合物13(740mg,2.23mmol)，收率81％。 1H NMR(CD 3OD,400MHz):δ7.92(s,1H),6.23(d,J＝18.4Hz,1H),4.75(dd,J＝23.6,9.2Hz,2H),4.60(dt,J＝47.6,11.2Hz,1H),4.44–4.23(m,1H),4.09–4.00(m,2H),3.86(dd,J＝12.4,2.8Hz,1H),2.99(s,3H)ppm； 13C NMR(CD 3OD,100MHz):δ161.9,157.2,151.0,137.8,114.9,101.6(dd,J C-F＝184.9,17.0Hz),89.3(d,J C-F＝39.5Hz),83.6,82.0(dd,J C-F＝173.5,27.6Hz),69.2(dd,J C-F＝17.1,4.7Hz),60.9,27.5ppm；HRMS(ESI)m/z:[M+H] +calcd for C 12H 17F 2N 6O 3 331.1325,found 331.1328.
12、化合Ⅲ的合成
Figure PCTCN2022116003-appb-000024
氮气保护下，称取化合物13(50mg,0.15mmol)溶于THF，0℃，缓慢滴加270μL 1M的叔丁基氯化镁溶液，保温15分钟后将反应体系移至室温15分钟， 再将体系移至0℃后，加入N-[(S)-(2,3,4,5,6-五氟苯氧基)苯氧基磷酰基]-L-丙氨酸异丙酯(100mg,0.22mmol)，保温30分钟后移至室温继续反应18小时，监测反应基本反应完全，加入饱和氯化铵淬灭反应，乙酸乙酯萃取(50mL x 3)，合并有机相，无水硫酸钠干燥，柱层析色谱分离，二氯甲烷：甲醇＝15:1-10:1,得到白色固体化合物Ⅲ(50mg,0.087mmol)，收率为58％。 1H NMR(CD 3OD,400MHz):δ7.77,7.76(ss,1H),7.28(t,J＝7.6Hz,2H),7.23–7.17(m,2H),7.12(t,J＝7.2Hz,1H),6.23(d,J＝18.8Hz,1H),4.97–4.89(m,1H),4.84–4.80(m,1H),4.72–4.40(m,4H),4.23–4.21(m,1H),3.90–3.80(m,1H),2.99(s,3H),1.25–1.22(m,3H),1.13–1.10(m,6H)ppm； 13C NMR(CD 3OD,100MHz):δ174.4(d,J C-P＝5.4Hz),162.1,157.2,152.1(d,J C-P＝6.8Hz),138.0,130.8,130.7,126.1,121.4(d,J C-P＝4.8Hz),121.3(d,J C-P＝4.8Hz),115.1,101.5(dd,J C-F＝184.8,16.7Hz),89.3(d,J C-F＝40.3Hz),82.3(dd,J C-F＝173.5,26.4Hz),81.9(d,J C-F＝8.1Hz),70.8(dd,J C-F＝17.0,4.7Hz),70.12,70.08,67.3(d,J C-P＝4.4Hz),51.6(d,J C-P＝1.5Hz),21.9,21.8,20.5(d,J C-P＝6.4Hz)ppm；HRMS(ESI)m/z:[M+H] +calcd for C 24H 33F 2N 7O 7P 600.2142,found 600.2141.
实施例2.化合物Ⅲ体外抗冠病毒活性和细胞毒性的测试
(一)实验目的
为研究化合物在体外抗冠状病毒的药效，拟用细胞病变效应(CPE)实验测定其对H460细胞的细胞毒性(CC 50)，以及对冠状病毒HCoV-OC43的半数抑制浓度(IC 50)。采用利巴韦林(RBV)作为阳性对照药物。
(二)实验材料与试剂
1.供试品名称：化合物Ⅲ(本实验室合成)，本品为白色至淡黄色固体。密封，在阴凉处保存。
2.阳性对照药
阳性对照药：利巴韦林注射液购自天津金耀集团湖北天药药业股份有限公司，批号为31712252，规格为100mg/ml，用时稀释至所需浓度，4℃冰箱保存。
3.细胞:传代人肺癌细胞H460细胞均为本室传代保存，在含10％胎牛血清(inactivated fetal bovine serum)和1％双抗(青霉素和链霉素)的DMEM培养基中，37℃，5％CO 2培养箱中培养。2-3天传代一次。
4.毒株:HCoV-OC43于HCT-8细胞中传代，保存于-80℃冰箱。
5.试剂:DMEM液体培养基、胎牛血清(fetal bovine serum)、青霉素和链霉素溶液(penicillin-streptomycin)、PBS(pH＝7.4)和0.25％Trypsin-EDTA均购自 Invitrogen公司。
6.实验用品及仪器:细胞培养瓶、96孔培养板和移液管为美国Corning公司产品；二氧化碳孵箱(Model 3111)为美国Thermo公司产品；生物安全柜为美国NUAIRE公司产品；倒置显微镜，奥林巴斯公司产品；真空泵为INTEGRA Biosciences公司产品；12道移液器和单道移液器为Eppendorf产品。
(三)实验步骤
1.细胞培养
在长满H460细胞的培养瓶内加0.25％Trypsin-EDTA 3ml，37℃消化1～2分钟，弃消化液，加培养液吹打，1:3传代，2-3天传代一次，种板时配制成每毫升15万个细胞，接种96孔细胞培养板，每孔0.1ml，37℃，5％CO 2培养过夜，细胞长成单层后进行实验。
2.CPE法测定药物对细胞毒性
H460细胞按1.5×10 4个/孔接种于96孔板中，过夜培养后加入含待测药物的维持液，待测药物以三倍稀释8个剂量的样品进行实验，继续培养。给药3天后倒置显微镜下药物对细胞的毒性(CPE法)，并用Reed-Muench法计算半数有毒浓度TC 50，计算公式如下：
Figure PCTCN2022116003-appb-000025
其中：A＝累积抑制率<50％的药物浓度，B＝累积抑制率>50％的抑制率，C＝累积抑制率<50％的抑制率，D＝log稀释倍数
3.抗HCoV-OC43的活性测定(CPE法)
实验在传代H460细胞中进行，细胞以1.5×10 4个/孔接种于96孔板中，过夜培养后将100TCID 50HCoV-OC43病毒液感染96孔板内细胞，待测药物用培养液稀释，于感染同时给药进行测定，待测药物以三倍稀释8个剂量的样品进行实验，每个剂量设2个平行孔，待病毒对照组病变达4+号时观察结果，记录并用Reed-Muench法计算药物对病毒的半数抑制浓度(公式如下)及选择指数(SI＝CC 50/IC 50)。
Figure PCTCN2022116003-appb-000026
其中：A＝累积抑制率<50％的药物浓度，B＝累积抑制率>50％的抑制率，C＝ 累积抑制率<50％的抑制率，D＝log稀释倍数
(四)实验结果计算
1.化合物Ⅲ在H460细胞中毒性及对HCoV-OC43毒株的抑制作用
在H460细胞中，CPE法测定化合物Ⅲ的CC 50为>100μM，其对HCoV-OC43的IC 50为0.09μM；选择指数SI为>1111；阳性对照药RBV半数有毒浓度CC 50为>0.36±0.06μM，对HCoV-OC43的IC 50为0.015±0.01μM，选择指数SI为>24。阳性药RBV抗HCoV-OC43活性与文献的结果相当，说明实验系统成立。
结果表明，化合物Ⅲ对HCoV-OC43具有明显的抑制活性。
实施例3.化合物Ⅲ体外抗新冠病毒2019-nCoV(COVID-19)活性和细胞毒性的测试
(一)实验目的
评价化合物Ⅲ在细胞水平对2019-nCoV(COVID-19)的抑制效果。
(二)实验材料与试剂
1.细胞系：Huh7细胞，本实验室保存；
2.病毒株：2019-nCoV(COVID-19)；
3.测试药物：化合物Ⅲ
4.阳性对照药物：瑞德西韦；
5.试剂：DMEM培养基(Gibco)，胎牛血清(Gibco)，双抗，胰酶，MTT(Amresco)等；
6.试剂盒：QIAamp viral RNA mini kit(52906，Qiagen)，One Step TB Green PrimeScript PLUS RT-PCR Kit(Perfect Real Time)(RR096A TaKaRa)
7.耗材：细胞培养板，96孔酶标板等；
8.仪器：多功能酶标仪，StepOnePlus荧光定量PCR仪，二氧化碳培养箱等。
(三)实验步骤
1.化合物Ⅲ对细胞毒性测定
利用MTT法检测化合物Ⅲ对Huh7细胞的毒性。MTT全称为3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide，是一种黄颜色的染料。MTT比色法，是一种检测细胞存活和生长的方法。其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒(Formazan)并沉积在细胞中，而死细胞无此功能。10％SDS(溶于0.01mol/L HCl溶液中)能溶解细胞中的甲瓒，用多功能酶标仪在570nm波长处测定其光吸收 值，可间接反映活细胞数量。在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。通过检测不同干扰素浓度下570nm波长处光吸收值，即可计算化合物Ⅲ浓度下细胞的存活率，从而计算化合物Ⅲ的半数中毒浓度(CC 50)。
提前一天接种Huh7细胞于96孔板中，每孔1×10 4个(注意96孔板最边缘的孔勿用作实验孔，加PBS防止其他孔培养基挥发)；观察细胞状态，达到约50％时，将化合物Ⅲ用含2％FBS的DMEM培养基进行2倍比稀释后，以100μL/孔加至细胞板中，每种浓度设6个重复；同时设置对照组(不含药物组)和空白组(不含细胞组)，置于37℃，5％CO 2的培养箱中培养；于加药后48h，每孔加入25μL MTT溶液(5mg/mL)，继续培养4h后，每孔加入125μL 10％SDS(溶于0.01mol/L HCl溶液中)，轻轻吹打，放置2h，使结晶物充分溶解，以空白组调零，测定OD570，按以下公式计算：存活率(％)＝加药组OD570/对照组OD570×100％。同时计算药物的半数中毒浓度(CC 50)。
2.化合物Ⅲ对2019-nCoV(COVID-19)病毒的抑制效果评价
在Huh7细胞模型上进行抗病毒活性测定，每次试验均设3复孔，共重复3次。
1)于24孔细胞培养板每孔中接种5×10 4个Huh7细胞，在37℃，5％CO 2培养条件下，待汇合度达到60％时，按照感染复数MOI为0.005，分别向每孔中加入200μL用含2％FBS的DMEM培养基稀释后的2019-nCoV(COVID-19)病毒液，37℃、5％CO 2培养箱中吸附1h后，弃病毒液，将阳性对照药物和化合物用含2％FBS的DMEM培养基从最大无毒浓度开始进行2倍比稀释，以500μL/孔加至细胞板中，同时设置对照组(不含药物组)，于感染后48h收集上清病毒液。
2)利用real-time RT-PCR(qRT-PCR)对收集的病毒进行RNA定量：
收集的上清病毒液分别取140μL，按照QIAamp viral RNA mini kit试剂盒说明书进行RNA提取。用One Step TB Green PrimeScript PLUS RT-PCR Kit(Perfect Real Time)试剂盒进行qRT-PCR检测，引物为RBD-qF1:5’-CAATGGTTTAACAGGCACAGG-3’；RBD-qR1:5’-CTCAAGTGTCTGTGGATCACG-3’。反应体系总体积为20μL：10μL 2X One Step TB Green RT-PCR Buffer 4，1.2μL TaKaRa Ex Taq HS Mix，0.4μL PrimeScript PLUS RTase Mix，RBD-qF1和RBD-qR1各0.8μL，0.4μL ROX Reference Dye(50X)，2μL病毒RNA，4.4μL RNase Free dH2O。反应参数为： 反转录42℃5min，预变性95℃10s，PCR 40个循环包括变性95℃10s，退火和延伸60℃30s。
3)计算每种浓度下的药物抑制率。抑制率(％)＝1-实验组病毒RNA拷贝数/不含药物组病毒RNA拷贝数×100％。同时计算药物的半数有效浓度(EC 50)和治疗指数(TI)＝半数中毒浓度(CC 50)/半数有效浓度(EC 50)。
(四)实验结果计算
表1.化合物Ⅲ抑制新冠病毒(COVID-19)活性
受试药物 EC 50 CC 50 TI
化合物Ⅲ 0.08μM >100μM >1250
瑞德西韦 1.50μM >45.0μM >30.0
根据表1结果，化合物Ⅲ抑制50％新冠病毒所需要的浓度是0.08μM，而瑞德西韦需要1.5μM，化合物Ⅲ抑制新冠病毒的活性是瑞德西韦的18倍，而且细胞毒性更小。
因此，化合物Ⅲ及其类似物具有预防或治疗冠状病毒感染性疾病的效果。

Claims (8)

  1. 式(I)所示的化合物或其药学上可接受的盐在制备预防或治疗冠状病毒感染性疾病的药物中的用途；
    Figure PCTCN2022116003-appb-100001
    其中，
    X和Y独立地选自：-H、烷基或取代烷基、卤素、-CN、-N 3、-OH、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3或SR 3；
    R'和R各自独立地选自：-H、-COR 3、-COOR 3、-CONHR 3或
    Figure PCTCN2022116003-appb-100002
    Ar选自：芳基或取代芳基、杂芳基或取代杂芳基；
    R 1和R 2独立地选自：-H、烷基或取代烷基、炔基或取代炔基、烯基或取代烯基、或1-3卤代烷基；
    或R 1和R 2形成3-6元环饱和脂肪环；
    各个R 3各自独立地选自-H、烷基或取代烷基、炔基或取代炔基、烯基或取代烯基、1-3卤代烷基或取代烷基、芳基或取代芳基、或者杂芳基或取代杂芳基；
    所述“取代烷基”、“取代芳基”、“取代杂芳基”、“取代炔基”和“取代烯基”中的取代基各自独立的选自：烷基、卤素、-CN、-N 3、-OH、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3、-SR 3；
    所述“卤素”和“卤代烷基”中的卤原子选自F、Cl、Br或I；
    所述“烷基”和“卤代烷基”中的烷基为C 1-20直链或支链烷基，可选为C 1-10直链或支链烷基，可选为C 1-6直链或支链烷基；可选为：甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、或异戊基；可选地，所述卤代烷基为2-氯乙基、2-氟乙基或三氟乙基；
    所述“炔基”为C 2-C 20炔基；可选为C 2-C 10炔基；可选为乙炔；
    所述“烯基”为C 2-C 20烯基；可选为C 2-C 10烯基；可选为乙烯；
    所述“芳基”为6-10元芳基；可选为苯基或萘基；
    所述“杂芳基”为环上含有选自N、O或S中至少一种杂原子的5-10元杂芳基。
  2. 根据权利要求1所述的用途，其特征在于，式(I)中，
    X选自-H、-NH 2、-NHR 3、-NR 3(R 3)、-OR 3或-SR 3；
    Y为-NH 2；
    R'为-H；
    R为-H或
    Figure PCTCN2022116003-appb-100003
    其中，
    R 1和R 2独立地选自：-H或C 1-6直链或支链烷基；
    各个R 3各自独立地为C 1-6直链或支链烷基；
    Ar选自苯基或取代苯基，萘基或取代萘基；
    可选地：所述C 1-6直链或支链烷基选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、或异戊基。
  3. 根据权利要求1或2所述的用途，其特征在于，所述式(I)所示的化合物或其药学上可接受的盐为式(II)化合物或其药学上可接受的盐，
    Figure PCTCN2022116003-appb-100004
    其中，X、Y、R'和R如权利要求1或2所定义。
  4. 根据权利要求1-3任一项所述的用途，其特征在于，所述式(I)所示的化合物或其药学上可接受的盐选自化合物(I-a)、(I-b)、及其药学上可接受的盐，
    Figure PCTCN2022116003-appb-100005
    Figure PCTCN2022116003-appb-100006
    式(I-a)和(I-b)中，X为-H、-NH 2、-NHMe、-N(Me) 2、-N(Me)(Et)、-N(Et) 2、-OMe、-OEt、-O(iPr)、-SMe、-SEt或-S(iPr)；
    式(I-b)中，Ar为苯基或萘基。
  5. 根据权利要求1-4任一项所述的用途，其中，所述药学上可接受的盐包括式(I)化合物与无机酸或者有机酸形成的盐；
    可选地，所述无机酸包括盐酸、氢溴酸、氢碘酸、硫酸、半硫酸、硝酸、磷酸、或碳酸；
    可选地，所述有机酸包括甲酸、抗坏血酸、乙酸、丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、柠檬酸、枸橼酸、酒石酸、葡萄糖酸、酒石氢酸、葡萄糖醒酸、碳酸、苦味酸、甲磺酸、乙磺酸、对甲苯磺酸、苯甲酸、苯磺酸、对溴苯磺酸、谷氨酸、水杨酸、或双羟萘酸；
    可选地，所述药学上可接受的盐为式(I)化合物的盐酸盐；
    可选地，所述药学上可接受的盐为式(I)化合物的半硫酸盐。
  6. 根据权利要求1-5任一项所述的用途，其中，所述冠状病毒感染性疾病包括感染人或其他动物所引起的疾病；
    可选地，所述冠状病毒包括HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV或COVID-19。
  7. 根据权利要求1-6中任一项所述的用途，其中，所述药物的剂型为速释剂型、缓释剂型、或控释剂型；
    可选地，所述药物的剂型为片剂、胶囊剂、水性或油性混悬剂、颗粒剂、乳剂、糖浆剂、酏剂、注射液、或粉针剂。
  8. 式(I)化合物或其药学上可接受的盐的制备方法，包括以下步骤：包括将化合物5经过步骤e的双羟化反应，然后经步骤f的环磺酯化反应，然后经步骤g采用三乙胺三氢氟酸盐开环，然后经步骤h的水解后环化反应以及苯甲酰化，得到中间体化合物9的步骤；
    Figure PCTCN2022116003-appb-100007
    可选地，步骤e中，采用以下试剂和条件：KMnO 4，NaHCO 3，丙酮；
    可选地，步骤f中，采用以下试剂和条件：SOCl 2，EtN 3，NaClO；
    可选地，步骤g中，采用以下试剂和条件：Et 3N·3HF，Et 3N，HCl；
    可选地，步骤h中，采用以下试剂和条件：BaCl，BzCl，DMAP，Et 3N，CH 3CN；
    可选地，该方法还包括：采用中间体化合物9依次经步骤i的还原反应及步骤j的溴化反应得到化合物11，
    Figure PCTCN2022116003-appb-100008
    然后化合物11与
    Figure PCTCN2022116003-appb-100009
    反应得到
    Figure PCTCN2022116003-appb-100010
    嘌呤核苷，然后经过酯化的步骤得到
    Figure PCTCN2022116003-appb-100011
    可选地，步骤i中，采用以下试剂和条件：LiAlH[OC(CH 3) 3] 3，THF；
    可选地，步骤j中，采用以下试剂和条件：PPh 3，CBr 4，DCM；
    可选地，式(IV)反应得到式(VI)的反应步骤的试剂和条件：t-BuOK,t-BuOH/CH 3CN；
    可选地，该方法还包括：将化合物1与丙烯酸乙酯经步骤a反应得到化合物2，再经步骤b的溴化，步骤c的水解反应，步骤d的氟化反应得到化合物5的反应步骤：
    Figure PCTCN2022116003-appb-100012
    Figure PCTCN2022116003-appb-100013
    可选地，步骤a中，采用以下试剂和条件：三乙烯二胺(DABCO)；
    可选地，步骤b中，采用以下试剂和条件：NBS，DMS，DCM；
    可选地，步骤c中，采用以下试剂和条件：乙酸钠，回流条件；
    可选地，步骤d中，采用以下试剂和条件：全氟丁基磺酰氟，Et 3N·3HF。
PCT/CN2022/116003 2021-09-01 2022-08-31 嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途 Ceased WO2023030347A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111018773.9A CN115721661A (zh) 2021-09-01 2021-09-01 嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途
CN202111018773.9 2021-09-01

Publications (1)

Publication Number Publication Date
WO2023030347A1 true WO2023030347A1 (zh) 2023-03-09

Family

ID=85291956

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/116003 Ceased WO2023030347A1 (zh) 2021-09-01 2022-08-31 嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途

Country Status (2)

Country Link
CN (1) CN115721661A (zh)
WO (1) WO2023030347A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11963967B2 (en) 2020-10-16 2024-04-23 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
US12030904B2 (en) 2020-08-24 2024-07-09 Gilead Sciences, Inc. Phospholipid compounds and uses thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040259934A1 (en) * 2003-05-01 2004-12-23 Olsen David B. Inhibiting Coronaviridae viral replication and treating Coronaviridae viral infection with nucleoside compounds
CN103980332A (zh) * 2013-12-09 2014-08-13 南京迈勒克生物技术研究中心 2′-氟-2′-(氟甲基)-2′-脱氧核苷类化合物及其磷酸酯潜药
CN106573011A (zh) * 2014-06-24 2017-04-19 艾丽奥斯生物制药有限公司 取代的核苷、核苷酸和其类似物
US20190085013A1 (en) * 2016-03-07 2019-03-21 Emory University Nucleotide and nucleoside therapeutic compositions and uses related thereto
US10874687B1 (en) * 2020-02-27 2020-12-29 Atea Pharmaceuticals, Inc. Highly active compounds against COVID-19

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5716500B2 (ja) * 2010-06-03 2015-05-13 セントラル硝子株式会社 （２Ｒ）−２−フルオロ−２−Ｃ−メチル−Ｄ−リボノ−γ−ラクトン類前駆体の製造方法
CN103987712B (zh) * 2011-07-19 2016-05-04 河南美泰宝生物制药有限公司 2’，3’-二脱氧-2’-α-氟-2’-β-C-甲基核苷和其前药
JP2017137267A (ja) * 2016-02-05 2017-08-10 セントラル硝子株式会社 環状硫酸エステルの製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040259934A1 (en) * 2003-05-01 2004-12-23 Olsen David B. Inhibiting Coronaviridae viral replication and treating Coronaviridae viral infection with nucleoside compounds
CN103980332A (zh) * 2013-12-09 2014-08-13 南京迈勒克生物技术研究中心 2′-氟-2′-(氟甲基)-2′-脱氧核苷类化合物及其磷酸酯潜药
CN106573011A (zh) * 2014-06-24 2017-04-19 艾丽奥斯生物制药有限公司 取代的核苷、核苷酸和其类似物
US20190085013A1 (en) * 2016-03-07 2019-03-21 Emory University Nucleotide and nucleoside therapeutic compositions and uses related thereto
US10874687B1 (en) * 2020-02-27 2020-12-29 Atea Pharmaceuticals, Inc. Highly active compounds against COVID-19
CN113784721A (zh) * 2020-02-27 2021-12-10 阿堤亚制药公司 抗covid-19的高活性化合物

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12030904B2 (en) 2020-08-24 2024-07-09 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
US12473314B2 (en) 2020-08-24 2025-11-18 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
US11963967B2 (en) 2020-10-16 2024-04-23 Gilead Sciences, Inc. Phospholipid compounds and uses thereof
US12208110B2 (en) 2020-10-16 2025-01-28 Gilead Sciences, Inc. Phospholipid compounds and uses thereof

Also Published As

Publication number Publication date
CN115721661A (zh) 2023-03-03

Similar Documents

Publication Publication Date Title
EP2940031B1 (en) Nucleoside phosphoramidate compounds for use in the treatment of hcv
JP7624093B2 (ja) ウイルス増殖阻害活性を有するウラシル誘導体およびそれらを含有する医薬組成物
WO2023030347A1 (zh) 嘌呤核苷类药物预防或治疗冠状病毒感染性疾病的用途
CN110105348A (zh) 新型迈克尔受体类肠病毒71型抑制剂的制备及用途
CN101058535B (zh) 二(7-羟基-2,3-二氢-1-1h-茚基)醚类及其类似物、合成方法及应用
CN102526087B (zh) 核苷类化合物在制备治疗肠病毒71(ev71)感染疾病药物的应用
CA3124852A1 (en) Indanone derivatives, pharmaceutically acceptable salts or optical isomers thereof, preparation method for same, and pharmaceutical compositions containing same as active ingredient for preventing or treating viral diseases
CN112771048B (zh) 流感病毒复制抑制剂及其中间体和用途
ES2744982T3 (es) Derivado de 1,3-di-oxo-indeno, sal farmacéuticamente aceptable o isómero óptico del mismo, método de preparación del mismo, y composición farmacéutica que contiene al mismo, principio activo, antiviral
CN107459511B (zh) 抗肠病毒71(ev71)4-亚氨基恶唑烷-2-酮类化合物及其制备方法和用途
CN104876860B (zh) 一种二芳基吡啶衍生物及其制备方法与应用
CN103130730B (zh) 喹唑啉类衍生物及其制备方法、抗hiv活性和抗tmv活性
CN118076589A (zh) 一种酰胺类化合物、其制备方法和制药用途
CN108350007B (zh) 一种取代的腺嘌呤化合物及其药物组合物
CN111675661B (zh) 一种含有反式双键的二芳基嘧啶类hiv-1逆转录酶抑制剂及其制备方法和应用
CN109384727B (zh) 酞嗪酮类化合物、其制备方法、药物组合物及用途
CN107129517A (zh) 一种具有α,β‑不饱和酮结构片段的孕烯醇酮衍生物及其用途
CN115916763B (zh) 抗病毒素1,3-二氧代茚化合物
CN117924242A (zh) 一种吡啶-2（1h）-酮类hiv-1非核苷类逆转录酶抑制剂及其制备方法与应用
TWI404709B (zh) 4- (3-benzamidophenyl) -6,7-dimethoxy-2-methylamine quinazoline derivatives
CN114989131B (zh) N’-(2-(苯并[1,3]二氧戊烷-5-基)酰基)芳酰肼类化合物及其合成和用途
CN115197164B (zh) 新型噻唑类化合物及其制备方法和用途
CN107074876B (zh) 一类抑制丙肝病毒的大环状杂环化合物及其制备和用途
CN115109083B (zh) Pyridostatin类化合物及其制备方法、应用、药物组合物
CN103130722B (zh) 哒嗪类衍生物及其制备方法、抗hiv活性和抗tmv活性

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22863482

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22863482

Country of ref document: EP

Kind code of ref document: A1