WO2022262820A1 - 一组具有抗冠状病毒活性的imb-c5系列化合物及其应用 - Google Patents

一组具有抗冠状病毒活性的imb-c5系列化合物及其应用 Download PDF

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WO2022262820A1
WO2022262820A1 PCT/CN2022/099223 CN2022099223W WO2022262820A1 WO 2022262820 A1 WO2022262820 A1 WO 2022262820A1 CN 2022099223 W CN2022099223 W CN 2022099223W WO 2022262820 A1 WO2022262820 A1 WO 2022262820A1
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imb
coronavirus
alkyl
benzyl
substituted
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French (fr)
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洪斌
陈晓芳
杜郁
李玉环
丁晓添
乔梦倩
李怡华
王琨
卞聪
颜海燕
吴硕
王辉强
高荣梅
王丽
王丽非
孙红敏
李星星
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中国医学科学院医药生物技术研究所
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Publication of WO2022262820A1 publication Critical patent/WO2022262820A1/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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/04—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms
    • C07D473/06—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3
    • C07D473/08—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two oxygen atoms with radicals containing only hydrogen and carbon atoms, attached in position 1 or 3 with methyl radicals in positions 1 and 3, e.g. theophylline

Definitions

  • the invention belongs to the field of medical biotechnology, and specifically relates to a group of IMB-C5 series compounds with anti-coronavirus activity and applications thereof.
  • Human coronavirus is one of the common pathogens causing acute respiratory infection, which can cause zoonosis and may cause a global pandemic. Since the 21st century, the world has experienced three outbreaks caused by highly pathogenic coronaviruses, namely severe acute respiratory syndrome (SARS) in 2002 and Middle East respiratory syndrome (Middle East respiratory syndrome) in 2012. Respiratory syndrome, MERS) and COVID-19 caused by a new type of coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) in late 2019.
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • COVID-19 caused by a new type of coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) in late 2019.
  • COVID-19 has become the fifth recorded global pandemic since the influenza pandemic in 1918, and it is also the first coronavirus pandemic in human history, which has caused unprecedented damage to global health and economic development [1 ] .
  • the "two-pronged approach" of vaccine prevention and drug treatment will become the main means of fighting the virus in the next stage.
  • Small molecule drugs have become the focus of antiviral drug research and development because of their advantages such as clear targets, easy scale production and distribution, and convenient oral administration.
  • Coronaviruses are a class of enveloped single-stranded positive-sense RNA viruses.
  • coronaviruses that can infect humans, namely HCoV-229E and HCoV-NL63 of the genus Alphacoronavirus, and HCoV-NL63 of the genus Betacoronavirus.
  • coronavirus The genome of coronavirus is about 26-32Kb, encoding four kinds of structural proteins, including spike (spike, S) protein, envelope (envelope, E) protein, membrane (membrane, M) protein and nucleocapsid (nucleocapsid, N ) proteins and non-structural proteins such as RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRp), 3-chymotrypsin-like protease (3C-like protease, 3CLpro, also known as the main protease Mpro), papain-like protease (papain-like protease, PLpro) and so on.
  • RNA-dependent RNA polymerase RNA-dependent RNA polymerase, RdRp
  • 3-chymotrypsin-like protease 3C-like protease, 3CLpro, also known as the main protease Mpro
  • papain-like protease papa
  • Viruses need to invade host cells to survive and proliferate. Therefore, antiviral drug discovery strategies targeting host targets have attracted increasing attention from researchers. It is possible to discover new antiviral drugs that have broad-spectrum antiviral effects and are less prone to drug resistance. Viruses replicate themselves by hijacking and utilizing specific host proteins, and their energy supply and material metabolism also depend on the host. Lipids are basic cellular components that play important biological roles in forming cell structures, acting as signaling molecules and energy storage, and are crucial in the virus life cycle [2] . SARS-CoV-2 is a virus encapsulated by a lipid bilayer.
  • lipids participate in in the virus life cycle include the fusion of the virus membrane and the host cell, virus replication, and endocytosis and exocytosis of the virus.
  • Targeting host cell lipid metabolism can interfere with coronavirus replication, which is a new way to discover new anti-coronavirus drugs.
  • Clinical data studies have found that certain lipid levels in the plasma of patients with COVID-19 correlate with the severity of their infection [3] .
  • Some experts have suggested that lipid-lowering therapy such as statins can be used as one of the treatment strategies for complications in patients with severe new coronary pneumonia [4] .
  • IMB-C5 is a new small molecule inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9) obtained by our laboratory using the PCSK9 transcription inhibitor screening model in the early stage. It has been verified that it has good Lowering blood lipids and inhibiting the formation of atherosclerotic plaques [5] .
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • a series of anti-coronavirus pharmacodynamic studies have found that IMB-C5 has a better effect of inhibiting the replication of common coronaviruses (HCoV-229E and HCoV-OC43), and also has an inhibitory effect on SARS-CoV-2.
  • the viral mechanism may be related to the regulation of lipid metabolism.
  • IMB-C5 compound Based on the structure of the IMB-C5 compound, we have carried out a series of chemical structure modifications and transformations, and obtained some active compounds with better antiviral activity and higher therapeutic index, which are expected to be developed into new anti-coronavirus drugs.
  • this type of compound can also be combined with inhibitors targeting viral targets such as 3CLpro and RdRp to form an antiviral "cocktail" to further improve the treatment level of COVID-19.
  • the present invention first relates to the compound represented by formula (1).
  • R1 is alkyl, substituted alkyl, substituents containing alkenyl, alkynyl, epoxyalkyl, benzyl, pyridine, etc.; preferably, R1 is C1 -C5 alkyl, C1-C5 monoalkenyl , C1-C5 monoalkynyl, pyridine ring-containing alkyl, benzene ring-containing alkyl, substituted benzene ring-containing alkyl, Boc-NH-linked C1-C3 alkyl;
  • R2 is alkyl, substituted alkyl, substituents containing alkenyl, alkynyl, benzyl, etc.; preferably, R2 is methyl or benzyl;
  • R3 is alkyl, substituted alkyl, epoxyalkyl, benzyl, substituted benzyl, thienyl methyl and other substituents, substituted phenethyl etc.; preferably, R3 is epoxyhexanemethyl, methyl Ester benzyl, methoxy or fluoromethoxy or fluoromethyl modified benzyl, monohalogen substituted or polyhalogen substituted benzyl, C1-C5 alkyl modified benzyl, nitro substituted benzyl group, thienyl group, substituted phenethyl group;
  • R 4 is -NH-R 5 , benzyl or substituted benzyl, imidazolyl, substituted piperazinyl;
  • R 5 is alkyl, substituted alkyl, alkyl alcohols, amide-containing alkyl, ester-containing alkyl, alkoxy, etc.; preferably, R 5 is C1 ⁇ C5 straight-chain or branched-chain alkyl alcohols, Alkoxyalkyl, halogen atom and/or methyl or fluoromethyl substituted benzene ring and/or benzyl, amide, ester containing alkyl.
  • the compound is IMB-ZH-2, IMB-ZH-11, IMB-ZH-12, IMB-ZHC-2, IMB-ZHC-15, IMB-2-26, IMB-2-31 , IMB-2-32, IMB-3-19, IMB-4-6, IMB-4-12, IMB-4-13, IMB-ZHB-4x, IMB-2-3, IMB-2-8, IMB -68, IMB-82, IMB-83, IMB-84, IMB-85, IMB-92, IMB-93, IMB-3-6, IMB-3-13, IMB-3-15, IMB-3-16 , IMB-3-30, IMB-3-45, IMB-3-46, IMB-3-47, IMB-3-57, IMB-3-58, IMB-3-71, IMB-3-72, IMB -3-81.
  • the present invention also relates to the application of the compound represented by formula (1) in the preparation of medicines, and the medicines are medicines for inhibiting coronavirus.
  • the coronavirus is a human coronavirus; more preferably, the coronavirus is a human alpha coronavirus and a human beta coronavirus; most preferably, the coronavirus is HCoV-229E, HCoV-OC43 , SARS-CoV-2.
  • the present invention also relates to the application of the compound represented by formula (1) in the preparation of a combination drug, the drug is a drug for inhibiting coronavirus, and the combination drug also includes drugs targeting other viral targets, Preferably, the other viral targets are 3CLpro and/or RdRp, etc.
  • the coronavirus is a human coronavirus; preferably, the coronavirus is a human ⁇ -coronavirus, a human ⁇ -coronavirus; most preferably, the coronavirus is HCoV-229E, HCoV-OC43, SARS-CoV-2.
  • the present invention also relates to a pharmaceutical composition and/or pharmaceutical preparation for treating coronavirus, which contains a therapeutically effective amount of the compound represented by formula (1), and necessary pharmaceutical adjuvants/diluents.
  • the present invention also relates to a method for treating diseases caused by coronavirus infection, the method comprising: administering a therapeutically effective amount of a compound represented by formula (1) to a patient, or a compound containing a compound represented by formula (1) Pharmaceutical compositions and/or pharmaceutical preparations.
  • the present invention also relates to a method for synthesizing the compound shown in formula (1), specifically, comprising the following steps:
  • reaction conditions for Reaction 1 are:
  • reaction conditions for Reaction 2 are:
  • reaction conditions for reaction 3 are:
  • Fig. 1 Effect of IMB-C5 on coronavirus HCoV-229E N protein mRNA level in Huh7 and Huh7.5 cells.
  • Con is Control, referring to the virus control without drug; RBV, Ribavirin.
  • Con is Control, referring to the virus control without drug; RBV, Ribavirin.
  • Fig. 4 Effect of IMB-C5 on coronavirus HCoV-OC43 N protein level in C3A cells (WB experiment).
  • Fig. 5 Effect of IMB-C5 series compounds on human coronavirus HCoV-229E N protein level in Huh7 cells.
  • Fig. 6 Toxicity assay results of IMB-C5 and IMB-85 on different cells.
  • Fig. 7 Effect of IMB-C5 and IMB-85 on human coronavirus HCoV-229E N protein mRNA level in Huh7 cells.
  • Con is Control, referring to the virus control without drug; RBV, Ribavirin.
  • IMB-C5 and IMB-85 are to the influence (WB experiment) of human coronavirus HCoV-229E N protein level in Huh7 cell.
  • FIG. 10 Effects of IMB-C5 and IMB-85 on the level of coronavirus HCoV-229E N protein in Huh7 cells (A) and Huh7.5 cells (B) (WB experiment).
  • Control which refers to the untreated virus control
  • Mock which is the blank control of untreated cells.
  • FIG. 13 Time-course experiment of IMB-85 acting on coronavirus (WB experiment to detect virus N protein level).
  • Fig. 14 Time course experiment of IMB-85 acting on coronavirus (immunofluorescence experiment to detect the dsRNA level of the virus).
  • the positive control drug ribavirin (ribavirin, RBV) injection was purchased from Tianjin Jinyao Group Hubei Tianyao Pharmaceutical Co., Ltd., the batch number is 31712252, and the specification is 100 mg/ml; the positive control drug Remdesivir (RDV ) was purchased from MedChemExpress Company, product number HY-104077; the positive control drug Molnupiravir (MNP) was purchased from Shanghai Taoshu Biotechnology Co., Ltd., product number T8309.
  • RBV positive control drug Remdesivir
  • MNP positive control drug Molnupiravir
  • Coronavirus HCoV-OC43N protein (Nucleocapsid protein, NP) antibody (mouse monoclonal antibody, MAB9013, Millipore), HCoV-229E NP antibody (rabbit polyclonal antibody, 40640-T62, Yiqiao Shenzhou),
  • glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase, GAPDH
  • GAPDH human glyceraldehyde-3-phosphate dehydrogenase
  • Double strand RNA (double strand RNA, dsRNA) antibody mouse monoclonal antibody J2, SCICONS
  • Fluorescein Isothiocyanate labeled goat anti-mouse IgG (H+L) (HS211, full gold).
  • CCK-8 Cell Counting Kit (referred to as CCK-8 kit) was purchased from Nanjing Nuoweizan Biotechnology Co., Ltd., item number A311.
  • Embodiment 1 the synthetic method of target compound and NMR data
  • step 1 the product obtained in step 1 (compound 9 in the formula, 1.0 equiv) was added to a round bottom flask, dissolved with dry DMF (3.0 mL), DBU (1.2 equiv) was added, and 2-( Trimethylsilyl) ethoxymethyl chloride (1.0 equiv), capped and reacted at 0°C for about 24 hours, monitored by TLC, until the reaction was complete and the raw material point disappeared.
  • the reaction solution was extracted with EA, washed with water and saturated NaCl solution, dried over anhydrous Na 2 SO 4 , and concentrated to obtain a crude product (compound 10 in the reaction formula).
  • step 2 put the crude product obtained in step 2 (compound 10 in the reaction formula, 1.0 equiv) into a round bottom flask, dissolve it with dry DMF (3.0 mL), add anhydrous K 2 CO 3 or NaH (1.2 equiv) , add methyl iodide (1.0 equiv) to the solution, cap it and react at 60°C for about 2h, monitor by TLC, until the reaction is complete and the raw material point disappears.
  • the reaction solution was extracted with EA, washed with water and saturated NaCl solution, dried over anhydrous Na 2 SO 4 , and concentrated to obtain a crude product (compound 11 in the reaction formula).
  • step 3 compound 11 in the reaction formula, 1.0 equiv
  • step 3 compound 11 in the reaction formula, 1.0 equiv
  • DCM dry DCM
  • TFA 1.0 mL
  • Reaction formula 1 the modification route to N7 and C8 positions
  • Embodiment 2 application causes cytopathic effect (cytopathic effect, CPE) method to measure IMB-C5 series compound anti-coronavirus HCoV-229E activity
  • IMB-C5 series compounds were diluted three-fold with DMEM culture solution containing 2% FBS and 1% double antibody to obtain 8 doses of samples.
  • the cell death ratio is marked as 4+ (cell death ratio 75%-100%), 3+ (cell death ratio 50%-75%), 2+ (cell death ratio 25%-50%), 1+ (0-25% cell death ratio), 0+ (all cells survived).
  • IMB-C5 series compounds have better activity of inhibiting HCoV-229E on Huh7 and Huh7.5 cells, and the activity of most of the compounds is better than that of the positive control drug ribavirin ( RBV), which is equivalent to or better than the activity of the marketed RdRp inhibitor Molnupiravir (MNP).
  • RBV positive control drug ribavirin
  • MNP Molnupiravir
  • compound IMB-85 has the best activity, and its EC 50 for inhibiting HCoV-229E on Huh7 and Huh7.5 cells is 0.09 ⁇ M and 4.05 ⁇ M, respectively, which is superior to the positive control drug and other homologous compounds of IMB-C5.
  • Embodiment 3 IMB-C5 anti-alpha coronavirus HCoV-229E activity assay
  • IMB-C5 can dose-dependently reduce the mRNA level of HCoV-229E N protein in both Huh7 and Huh7.5 cells, and its antiviral activity on Huh7 cells is better than that of Huh7.5.
  • BSA bovine serum albumin
  • IMB-C5 has an inhibitory effect on the production of dsRNA during the replication of HCoV-229E in Huh7 cells.
  • Embodiment 4 IMB-C5 anti-beta coronavirus HCoV-OC43 activity assay
  • C3A cells Using C3A cells as the virus host, the effect of IMB-C5 on the mRNA level of coronavirus HCoV-OC43N protein was detected (Figure 3).
  • IMB-C5 The effect of IMB-C5 on the protein level of coronavirus HCoV-OC43N was further detected (Fig. 4).
  • IMB-C5 has a good inhibitory effect on HCoV-OC43 and can inhibit the expression of viral N protein in a dose-dependent manner.
  • Embodiment 5 IMB-C5 series compound inhibits coronavirus HCoV-229E N protein level
  • IMB-C5 series compounds In the in vitro drug efficacy test, the effect of IMB-C5 series compounds on the level of coronavirus HCoV-229E N protein was detected.
  • Select 8 compounds with SI>80 from the CPE results including IMB-2-5, IMB-2-8, IMB-2-14, IMB-68, IMB-92, IMB-93, IMB-3-19 and IMB-85, IMB-C5 served as homolog controls.
  • 5 ⁇ M of IMB-C5 series compounds and 15 ⁇ M of MNP (Molnupiravir, a marketed oral RdRp inhibitor for the new crown) were administered as a positive control.
  • Embodiment 7 IMB-C5 and IMB-85 inhibit coronavirus HCoV-229E activity
  • IMB-C5 and IMB-85 The effects of IMB-C5 and IMB-85 on the mRNA level and protein level of coronavirus HCoV-229E N protein were detected.
  • Infect Huh7 cells with a viral load of MOI 17, and at the same time administer 3 ⁇ M IMB-C5, 3 ⁇ M, 0.6 ⁇ M and 0.12 ⁇ M three concentrations of IMB-85 and 200 ⁇ M positive drug RBV, and extract RNA and protein respectively after 24 hours.
  • RT-qPCR and Western blot detection As shown in Figure 7 and Figure 8, IMB-85 can dose-dependently reduce the mRNA and protein levels of HCoV-229E N protein in Huh7 cells, and its activity is significantly better than that of IMB-C5 at the same concentration.
  • Embodiment 8 on Huh7 and Huh7.5 cells, the impact of IMB-C5 and IMB-85 on coronavirus HCoV-229E N protein level
  • Embodiment 9 IMB-C5 and IMB-85 suppress coronavirus HCoV-OC43N protein level
  • Embodiment 10 IMB-C5 anti-SARS-CoV-2 activity assay
  • Embodiment 11 IMB-C5 and IMB-85 act on the early stages of coronavirus infection
  • the results showed that adding the medicine at the same time of infection had the most obvious inhibitory effect on the virus, and administration after 1h to 4h of infection had better antiviral effect, while the antiviral effect produced by administration after infection for 6h and later was greatly weakened . This suggests that IMB-C5 may act in the early stages of coronavirus infection.
  • the results showed that when the drug was added at the same time as the virus infection or administered within 1-5 hours after the virus infection, the inhibitory effect on the virus was more obvious, while the antiviral effect produced by administration after 6 hours of infection was weakened. This suggests that IMB-85 may mainly act on the early stages of coronavirus infection.

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Abstract

本发明涉及一组具有抗冠状病毒活性的IMB-C5系列化合物及其应用，所述IMB-C5系列化合物结构如式(1)所示。所述应用为其在制备药物中的应用，所述的药物为抑制冠状病毒的药物，优选的，所述的药物为治疗人冠状病毒感染的药物，所述的冠状病毒优选为人α属冠状病毒、人β属冠状病毒尤其是新型冠状病毒(SARS-CoV-2)。

Description

一组具有抗冠状病毒活性的IMB-C5系列化合物及其应用 技术领域
本发明属于医药生物技术领域，具体的，涉及一组具有抗冠状病毒活性的IMB-C5系列化合物及其应用。
背景技术
急性传染病对公共卫生构成重大威胁，其中急性呼吸道感染是世界范围内传染病发病和死亡的重要原因。人冠状病毒是引起急性呼吸道感染的常见病原体之一，可引发人畜共患病并可能造成全球性大流行。自21世纪以来，全球已经历三次高致病性冠状病毒引起的疫情，分别是2002年爆发的严重急性呼吸综合征(severe acute respiratory syndrome，SARS)，2012年爆发的中东呼吸综合征(Middle East respiratory syndrome，MERS)和2019年末开始由新型冠状病毒(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)引发的COVID-19。其中COVID-19已成为1918年流感大流行以来有记载的第五次全球大流行病，这也是人类历史上第一个冠状病毒大流行，对全球卫生和经济的发展造成了前所未有的破坏 [1]。疫苗预防与药物治疗“双管齐下”将成为下一阶段对抗该病毒的主要手段。小分子药物因具有靶点明确、易于规模化生产分发和口服给药便利等优势成为抗病毒药物研发的重点。
冠状病毒是一类有包膜的单股正链RNA病毒，目前已知有7种可以感染人类的冠状病毒，分别是α冠状病毒属的HCoV-229E和HCoV-NL63，以及β冠状病毒属的HCoV-OC43、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2，其中SARS-CoV、MERS-CoV以及SARS-CoV-2具有高致病性。冠状病毒的基因组约为26-32Kb，编码4种结构蛋白，包括棘突(spike，S)蛋白、包膜(envelope，E)蛋白、膜(membrane，M)蛋白和核衣壳(nucleocapsid，N)蛋白以及非结构蛋白如RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase，RdRp)、3-胰凝乳蛋白酶样蛋白酶(3C-like protease，3CLpro，亦称主蛋白酶Mpro)、木瓜蛋白酶样蛋白酶(papain-like protease，PLpro)等。近期已有两款分别针对3CLpro和RdRp的小分子口服药物Paxlovid和莫努匹韦(Molnupiravir，MNP)获准上市，两者能够一定程度上降低住院风险高的轻症/非重症患者的住院率和死亡率。这些直接靶向病毒靶点的药物特异性高，但也面临病毒变异迅速、易产生耐药的难题。
病毒需要入侵宿主细胞才能生存和增殖，因此，靶向宿主靶标的抗病毒药物发现策略日益受到研究者的重视，有可能发现具有广谱抗病毒作用且不易产生耐药的新型抗病毒药物。病毒通过劫持和利用特定的宿主蛋白来进行自身的复制，其能量供应和物质代谢等也都依赖于宿主。脂质是基本的细胞成分，在组成细胞结构、充当信号分子及能量存储等方面发挥着重要的生物学作用，在病毒生命周期中至关重要 [2]。SARS-CoV-2是一种被脂质双分子层包裹的病毒，脂质在病毒生命周期中参与的过程包括病毒膜与宿主细胞的融合、病毒复制和病毒的胞吞、胞吐作用等，靶向宿主细胞脂质代谢可以干扰冠状病毒复制，是发现新型抗冠状病毒药物的一种新思路。临床数据研究发现，新冠患者血浆中的某些脂质水平与其感染的严重性相关 [3]。有专家提出他汀类药物等降脂疗法可作为重症新冠肺炎患者并发症的治疗策略之一 [4]。
IMB-C5为本实验室前期利用PCSK9转录抑制剂筛选模型得到的一个全新的前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)小分子抑制剂，经验证其具有良好的降血脂、抑制动脉粥样硬化斑块形成的效果 [5]。经一系列抗冠状病毒的药效学研究发现，IMB-C5具有较好的抑制普通冠状病毒(HCoV-229E和HCoV-OC43)复制的效果，对SARS-CoV-2也有抑制作用，推测其抗病毒机制可能与调控脂质代谢有关。在IMB-C5化合物结构的基础上，我们进行了一系列的化学结构修饰和改造，获得了一些抗病毒活性更好、治疗指数更高的活性化合物，有望发展为新型抗冠状病毒药物。该类化合物 作为宿主靶向药物，也将可以与靶向病毒靶标如3CLpro和RdRp等的抑制剂组合成抗病毒“鸡尾酒”，进一步提升COVID-19的治疗水平。
[参考文献]
[1]Liu YC,Kuo RL,Shih SR.COVID-19:The first documented coronavirus pandemic in history.Biomed J,2020,43(4):328-333.
[2]Abu-Farha M,Thanaraj TA,Qaddoumi MG,Hashem A,Abubaker J,Al-Mulla F.The role of lipid metabolism in COVID-19 virus infection and as a drug target.Int J Mol Sci,2020,21(10):3544.
[3]Hu X,Chen D,Wu L,He G,Ye W.Declined serum high density lipoprotein cholesterol is associated with the severity of COVID-19 infection.Clin Chim Acta,2020,510:105-110.
[4]Morawietz H,Julius U,Bornstein SR.Cardiovascular diseases,lipid-lowering therapies and European registries in the COVID-19 pandemic.Cardiovasc Res,2020,116(10):e122-e125.
[5]Wang X,Chen X,Zhang X,et al.A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α.EBioMedicine,2020,52:102650.
发明内容
本发明首先涉及式(1)所示化合物。
Figure PCTCN2022099223-appb-000001
式中，
R 1为烷基、取代烷基、含烯基、炔基、环氧烷基、苄基、吡啶等的取代基；优选的，R1为C1-C5的烷基、C1-C5的单烯基、C1-C5的单炔基、含吡啶环的烷基、含苯环的烷基、含取代苯环的烷基、Boc-NH-连接的C1-C3的烷基；
R 2为烷基、取代烷基、含烯基、炔基、苄基等的取代基；优选的，R2为甲基或苄基；
R 3为烷基、取代烷基、环氧烷基、苄基、取代苄基、噻吩甲基等的取代基、取代苯乙基等；优选的，R 3为环氧己烷甲基、甲酸酯苄基、甲氧基或氟代甲氧基或氟代甲基修饰的苄基、单卤素取代或多卤素取代的苄基、C1-C5的烷基修饰的苄基、硝基取代苄基、噻吩甲基、取代苯乙基；
R 4为-NH-R 5、苄基或取代的苄基、咪唑基、取代哌嗪基；
R 5为烷基、取代烷基、烷基醇类、含酰胺烷基、含酯烷基、烷氧基等；优选的，R 5为C1～C5的直链或支链烷基醇类、烷氧烷基、卤素原子和/或甲基或氟甲基取代的苯环和/或苄基、含酰胺、酯的烷基。
更优选的，所述的化合物为IMB-ZH-2、IMB-ZH-11、IMB-ZH-12、IMB-ZHC-2、IMB-ZHC-15、IMB-2-26、IMB-2-31、IMB-2-32、IMB-3-19、IMB-4-6、IMB-4-12、IMB-4-13、IMB-ZHB-4x、IMB-2-3、IMB-2-8、IMB-68、IMB-82、IMB-83、IMB-84、IMB-85、IMB-92、IMB-93、IMB-3-6、IMB-3-13、IMB-3-15、IMB-3-16、IMB-3-30、IMB-3-45、IMB-3-46、IMB-3-47、IMB-3-57、IMB-3-58、IMB-3-71、IMB-3-72、IMB-3-81。
本发明还涉及式(1)所示化合物在制备药物中的应用，所述的药物为抑制冠状病毒的药物。
Figure PCTCN2022099223-appb-000002
优选的，所述的冠状病毒为人冠状病毒；更优选的，所述的冠状病毒为人α属冠状病毒、人β属冠状病毒；最优选的，所述的冠状病毒为HCoV-229E、HCoV-OC43、SARS-CoV-2。
本发明还涉及式(1)所示化合物在制备联用型药物中的应用，所述的药物为抑制冠状病毒的药物，所述的联用型药物中还包括靶向其他病毒靶标的药物，优选的，所述的其他病毒靶标为3CLpro和/或RdRp等。
Figure PCTCN2022099223-appb-000003
优选的，所述的冠状病毒为人冠状病毒；优选的，所述的冠状病毒为人α属冠状病毒、人β属冠状病毒；最优选的，所述的冠状病毒为HCoV-229E、HCoV-OC43、SARS-CoV-2。
本发明还涉及一种治疗冠状病毒的药物组合物和/或药物制剂，其含有治疗有效量的式(1)所示的化合物，以及必要的药用辅料/稀释剂。
本发明还涉及一种治疗因冠状病毒感染导致的疾病的方法，所述的方法包括，向患者施用治疗有效量的式(1)所示的化合物，或含有式(1)所示的化合物的药物组合物和/或药物制剂。
本发明还涉及合成式(1)所示化合物的方法，具体的，包括如下步骤：
Figure PCTCN2022099223-appb-000004
如反应式1所示合成路线，
反应1的反应条件为：
a.R 3-X,DMF,K 2CO 3,0-100℃；
b.R 4H,DMSO,60-150℃；
或如反应式2所示合成路线，
Figure PCTCN2022099223-appb-000005
反应2的反应条件为：
a.R 3-Br,DIPEA,DMF,0-100℃；
b.R 1-X,K 2CO 3,DMF,0-100℃；或R 1-OH,PPh 3,DEAD,THF,rt；
c.R 4H,DMSO,60-150℃.
或如反应式3所示合成路线，
Figure PCTCN2022099223-appb-000006
反应3的反应条件为：
a.3-Methoxybenzyl chloride,DIPEA,DMF,rt,24h；
b.SEMCl,DBU,DMF,rt-60℃；
c.CH 3I,DMF,K 2CO 3,60℃,1h；
d.HCl；
e.R 2-X,DMF,K 2CO 3,60℃,2h；
f.Ethanolamine,DMSO,130℃。
附图说明
图1、IMB-C5对Huh7及Huh7.5细胞中冠状病毒HCoV-229E N蛋白mRNA水平的影响。
Con为Control，指未加药病毒对照；RBV，Ribavirin。
图2、IMB-C5对HCoV-229E在Huh7细胞复制过程中dsRNA产生的影响(免疫荧光实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；RBV，Ribavirin；RDV，Remdesivir。
图3、IMB-C5对C3A细胞中冠状病毒HCoV-OC43 N蛋白mRNA水平的影响。
Con为Control，指未加药病毒对照；RBV，Ribavirin。
图4、IMB-C5对C3A细胞中冠状病毒HCoV-OC43 N蛋白水平的影响(WB实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；RBV，Ribavirin。
图5、IMB-C5系列化合物对Huh7细胞中人冠状病毒HCoV-229E N蛋白水平的影响。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；MNP，Molnupiravir。图中化合物IMB-2-5标注为2-5，余同。
图6、IMB-C5和IMB-85在不同细胞上的毒性测定结果。
图7、IMB-C5和IMB-85对Huh7细胞中人冠状病毒HCoV-229E N蛋白mRNA水平的影响。
Con为Control，指未加药病毒对照；RBV，Ribavirin。
图8、IMB-C5和IMB-85对Huh7细胞中人冠状病毒HCoV-229E N蛋白水平的影响(WB实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；RBV，Ribavirin。
图9、IMB-C5和IMB-85对HCoV-229E在Huh7细胞复制过程中dsRNA产生的影响(免疫荧光实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；RBV，Ribavirin。
图10、IMB-C5和IMB-85对Huh7细胞(A)、Huh7.5细胞(B)中冠状病毒HCoV-229E N蛋白水平的影响(WB实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；MNP，Molnupiravir。
图11、IMB-C5和IMB-85对C3A细胞中冠状病毒HCoV-OC43 N蛋白水平的影响(WB实验)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照；MNP，Molnupiravir。
图12、IMB-C5作用于冠状病毒的时间进程实验(免疫荧光实验检测病毒的dsRNA水平)。
Con为Control，指未加药病毒对照；Mock，未处理细胞空白对照。
图13、IMB-85作用于冠状病毒的时间进程实验(WB实验检测病毒的N蛋白水平)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照。
图14、IMB-85作用于冠状病毒的时间进程实验(免疫荧光实验检测病毒的dsRNA水平)。
Mock，未处理细胞空白对照；Con为Control，指未加药病毒对照。
具体实施方式
实验材料
1、细胞株和病毒
人肝癌细胞C3A(ATCC，CRL-10741)，本实验室保存。
人肝癌细胞Huh7，本实验室保存。
人肝癌细胞Huh7.5，本实验室保存。
非洲绿猴肾细胞Vero E6(ATCC，CRL-1586)，本实验室保存。
人冠状病毒HCoV-229E(ATCC，VR-740)，本实验室保存。
人冠状病毒HCoV-OC43(ATCC，VR-1558)，本实验室保存。
人冠状病毒SARS-CoV-2Beta变异株，广东省疾病控制中心保存。
2、化合物
阳性对照药利巴韦林(ribavirin，RBV)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；阳性对照药瑞德西韦(Remdesivir，RDV)购自MedChemExpress公司，货号HY-104077；阳性对照药物莫努匹韦(Molnupiravir，MNP)购自上海陶术生物科技有限公司，货号T8309。
3、反转录-定量PCR(reverse transcription quantitative PCR，RT-qPCR)引物
F＝Forward primer，R＝Reverse primer，P＝Probe，NP＝Nucleocapsid protein
Figure PCTCN2022099223-appb-000007
4、抗体
冠状病毒HCoV-OC43N蛋白(Nucleocapsid protein,NP)抗体(小鼠单克隆抗体，MAB9013，Millipore)，HCoV-229E NP抗体(兔多克隆抗体，40640-T62，义翘神州)，
人甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)抗体(小鼠单克隆抗体，ZB2305，中杉金桥)，
双链RNA(double strand RNA，dsRNA)抗体(小鼠单克隆抗体J2，SCICONS)，
异硫氰酸荧光素(Fluorescein Isothiocyanate，FITC)标记山羊抗小鼠IgG(H+L)(HS211，全式金)。
5、试剂
CCK-8Cell Counting Kit(简称CCK-8试剂盒)购自南京诺唯赞生物科技有限公司，货号A311。
实施例1、目标化合物的合成方法与核磁数据
1.1、N3位改造反应路线及中间体12的合成
Figure PCTCN2022099223-appb-000008
反应通式如上式所示，反应条件如下：
a.3-Methoxybenzyl chloride,DIPEA,DMF,rt,24h；
b.SEMCl,DBU,DMF,rt-60℃；
c.CH 3I,DMF,K 2CO 3,60℃,1h；
d.HCl；
e.R 2-X,DMF,K 2CO 3,60℃,2h；
f.Ethanolamine,DMSO,130℃。
具体步骤描述如下：
步骤一：
氮气保护下，将8-溴黄嘌呤(反应式中化合物8，1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入间甲氧基苄氯(1.0equiv)，封盖后于0℃下反应约24h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离纯化得到产物(反应式中化合物9)(PE/EA＝3/1～1/1)。
步骤二：
氮气保护下，将步骤一得到的产物(式中化合物9，1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入DBU(1.2equiv)，向该溶液中加入2-(三甲硅烷基)乙氧甲基氯(1.0equiv)，封盖后于0℃下反应约24h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩得到粗产物(反应式中化合物10)。
步骤三：
氮气保护下，将步骤二得到的粗产物(反应式中化合物10，1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入碘甲烷(1.0equiv)，封盖后于60℃下反应约2h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，得到粗产物(反应式中化合物11)。
步骤四：
氮气保护下，将步骤三得到的粗产物(反应式中化合物11，1.0equiv)加入圆底烧瓶中，用干燥的DCM(3.0mL)溶解，加入TFA(1.0mL)，在室温下搅拌约1h，TLC监测，至反应完全原料点消失。反应液用0.5N NaOH调PH至中性，用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离纯化得到产物(反应式中化合物12)(PE/EA＝3/1～1/1)。
1.2、合成通法：
合成通法一:
具体的，氮气保护下，将8-溴茶碱(化合物1,1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入卤代物(1.2equiv)，封盖后于60℃下反应约2h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离纯化得到产物(化合物2)(PE/EA＝3/1－1/1)。
合成通法二(改造N1位)：
具体的，氮气保护下，将8-溴-3-甲基-3,7-二氢-嘌呤-2,6-二酮(化合物4,1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入间甲氧基苄溴(1.0equiv)，封盖后于60℃下反应约2h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离纯化得到产物(化合物5)(PE/EA＝3/1－1/1)。
合成通法三：
氮气保护下，将由合成通法二获得的化合物5或中间体12(1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入不同的卤代物(3.0equiv)，封盖后于70℃下反应约7h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离得到产物(化合物6，(化合物13)(PE/EA＝3/1－1/1)。
合成通法四：
氮气保护下，将由合成通法一或三获得的化合物2，6，13(1.0equiv)加入圆底烧瓶中，用干燥的DMF(3.0mL)溶解，加入无水K 2CO 3或NaH(1.2equiv)，向该溶液中加入胺类化合物(3.0equiv)，封盖后于130℃下反应约13h，TLC监测，至反应完全原料点消失。反应液用EA萃取、水洗、饱和NaCl溶液洗，无水Na 2SO 4干燥，浓缩，进行柱层析分离得到终产物3，7，14(DCM/MeOH＝30/1－20/1)。
1.3、合成路线
反应式1：对N7和C8位的改造路线
Figure PCTCN2022099223-appb-000009
反应条件：
a.R 3-X,DMF,K 2CO 3,0-100℃；
b.R 4H,DMSO,60-150℃；
反应式2：对N1位的改造路线
Figure PCTCN2022099223-appb-000010
反应条件：
a.R 3-Br,DIPEA,DMF,0-100℃；
b.R 1-X,K 2CO 3,DMF,0-100℃；或R 1-OH,PPh 3,DEAD,THF,rt；
c.R 4H,DMSO,60-150℃.
反应式3：对N3位的改造路线
Figure PCTCN2022099223-appb-000011
反应条件：
a.3-Methoxybenzyl chloride,DIPEA,DMF,rt,24h；
b.SEMCl,DBU,DMF,rt-60℃；
c.CH 3I,DMF,K 2CO 3,60℃,1h；
d.HCl；
e.R 2-X,DMF,K 2CO 3,60℃,2h；
f.Ethanolamine,DMSO,130℃.
1.4、合成的化合物结构和核磁数据
以下为C8位改造的化合物：
IMB-ZH-2
(S)-8-((1-hydroxypropan-2-yl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione性状：白色固体 产率：75％.
1H NMR(500MHz,DMSO-d 6)δ＝7.22(t,J＝7.8,1H),6.88(d,J＝7.8,1H),6.82(s,2H),6.77(d,J＝7.3,1H),5.28(q,J＝16.0,2H),4.74(s,1H),3.98–3.92(m,1H),3.71(s,3H),3.47(d,J＝4.9,1H),3.35(s,3H),3.15(s,3H),1.14(d,J＝6.3,3H). 13C NMR(126MHz,DMSO-d 6)δ＝159.38,153.67,152.81,151.10,149.00,138.85,129.74,119.23,113.04,112.68,101.15,64.49,55.07,50.64,45.26,29.45,27.32,17.64.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1828；Found 374.1822.
IMB-ZH-6
8-((3-hydroxypropyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：86％.
1H NMR(500MHz,DMSO-d 6)δ＝7.23(t,J＝8.1,1H),7.14(s,1H),6.82(s,2H),6.76(d,J＝7.3,1H),5.26(s,2H),4.48(s,1H),3.71(s,3H),3.44(d,J＝5.1,2H),3.39(d,J＝5.8,2H),3.36(s,3H),3.34(s,1H),3.16(s,3H),1.77–1.61(m,2H). 13C NMR(126MHz,DMSO-d 6)δ＝159.41,153.97,152.87,151.08,148.93,138.70,129.78,119.16,113.15,112.58,101.29,58.31,55.09,45.23,40.51,32.52,29.44,27.33.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1828；Found 374.1824.
IMB-ZH-10
7-(3-methoxybenzyl)-8-((2-methoxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：73％.
1H NMR(500MHz,DMSO-d 6)δ＝7.28(d,J＝5.0,1H),7.22(t,J＝7.8,1H),6.82(d,J＝7.4,2H),6.77(d,J＝7.5,1H),5.26(s,2H),3.71(s,3H),3.48(dd,J＝9.8,4.5,4H),3.35(s,3H),3.24(s,3H),3.16(s,3H). 13C NMR(126MHz,DMSO-d 6)δ＝159.29,153.85,152.76,150.99,148.87,138.59,129.67,119.02,113.17,112.38,101.19,62.02,55.52,54.97,45.10,42.05,29.33,27.22.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1828；Found 374.2.
IMB-ZH-11
8-((4-fluoro-3-(trifluoromethyl)benzyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：73％.
1H NMR(500MHz,DMSO-d 6)δ＝7.83(t,J＝5.5,1H),7.70(d,J＝6.7,1H),7.65(s,1H),7.43(t,J＝9.6,1H),7.21(t,J＝7.8,1H),6.83(d,J＝8.2,1H),6.79(s,1H),6.74(d,J＝7.4,1H),5.29(s,2H),4.58(d,J＝5.4,2H),3.68(s,3H),3.33(d,J＝7.8,4H),3.16(s,3H). 13C NMR(151MHz,DMSO-d 6)δ＝159.33,153.30,152.93,150.91,148.45,138.29,136.69,134.22,129.61,126.12,123.52,118.90,117.05,116.91,116.21,113.06,112.42,101.59,54.89,45.27,44.61,29.23,27.19.HRMS(ESI)Calcd for C 23H 22N 5O 3F 4[M+H] +270.1658；Found 492.1645.
IMB-ZH-12
(R)-8-((1-hydroxy-3-methylbutan-2-yl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：82％.
1H NMR(500MHz,DMSO-d 6)δ＝7.21(t,J＝7.8,1H),6.85(s,1H),6.80(t,J＝8.6,2H),6.70(d,J＝8.6,1H),5.41(d,J＝16.0,1H),5.28(d,J＝15.9,1H),4.59(s,1H),3.74(s,1H),3.70(s,3H),3.51(d,J＝5.9,2H),3.33(d,J＝13.5,5H),3.16(s,3H),1.91(dd,J＝13.3,6.7,1H),0.80(dd,J＝14.1,6.7,6H). 13C NMR(151MHz,DMSO-d 6)δ＝159.28,154.28,152.67,150.99,148.78,138.71,129.52,119.25,112.59,112.59,101.02,61.04,60.05,54.94,45.08,29.26,28.49,27.15,19.43,18.28.HRMS(ESI)Calcd for C 20H 28N 5O 4[M+H] +402.2141；Found 402.2137.
IMB-ZHC-2
methyl(7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)glycinate
性状：棕色固体 产率：76％.
1H NMR(500MHz,CDCl 3)δ＝6.97–6.88(m,2H),6.84(d,J＝8.1,1H),5.52(d,J＝5.2,2H),3.79(s,3H),3.60–3.53(m,3H),3.39(t,J＝8.1,3H),1.54(s,2H). 13C NMR(151MHz,CDCl 3)δ＝160.07,154.41,151.41,148.42,147.47,136.53,130.13,127.98,120.08,113.91,113.70,109.07,55.40,50.26,49.26,49.26,30.01,28.24.HRMS(ESI)Calcd for C 18H 22N 5O 5[M+H] +388.1615；Found 388.1632.
IMB-ZHC-6
(R)-8-((2-hydroxypropyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：76％.
1H NMR(600MHz,CDCl 3)δ＝6.82(dd,J＝11.3,5.0,2H),6.78(s,1H),5.32(s,2H),4.71(s,1H),3.98–3.88(m,1H),3.77(s,3H),3.60–3.48(m,4H),3.37(s,3H),3.30–3.20(m,1H),1.14(d,J＝6.3,3H). 13C NMR(151MHz,CDCl 3)δ＝160.42,154.31,153.55,151.77,147.99,136.78,130.47,119.33,113.63,113.18,103.15,67.63,55.45,50.64,46.88,29.99,27.87,20.83.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1823；Found 374.1832.
IMB-ZHC-7
(S)-8-((2-hydroxypropyl)amino)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：68％.
1H NMR(600MHz,CDCl 3)δ＝7.29–7.27(m,1H),6.84(dd,J＝11.3,5.3,2H),6.80(d,J＝1.7,1H),5.33(s,2H),4.74(t,J＝5.4,1H),3.97–3.89(m,1H),3.78(s,3H),3.59–3.52(m,4H),3.39(s,3H),3.27(ddd,J＝13.8,7.6,4.9,1H),1.16(d,J＝6.3,3H). 13C NMR(151MHz,CDCl 3)δ＝160.40,154.30,153.66,151.79,148.19,136.84,130.44,119.30,113.57,113.16,103.16,67.62,55.43,50.62,46.84,29.91,27.85,20.83.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1823；Found 374.1829.
IMB-ZHC-15
((7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)amino)acetamide
性状：棕黄色粉末产率：85％.
1H NMR(500MHz,CDCl 3)δ＝7.84(s,1H),7.34(d,J＝7.8,1H),6.89(dd,J＝16.4,8.9,4H),5.35(s,2H),3.82(d,J＝4.9,5H),3.61–3.50(m,6H). 13C NMR(151MHz,DMSO-d 6)δ＝171.26,159.30,154.29,153.13,150.95,150.59,138.54,129.64,119.41,113.52,112.45,101.05,55.01,45.44,44.98,28.41,28.32.
IMB-2-26
8-(1H-imidazol-1-yl)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：66％.
1H NMR(500MHz,CDCl 3)δ＝7.86(s,1H),7.24–7.18(m,2H),6.82(d,J＝8.2,1H),6.58(d,J＝7.6,1H),6.54(s,1H),5.53(s,2H),3.74(s,3H),3.59(s,3H),3.43(s,3H). 13C NMR(151MHz,CDCl 3)δ＝160.33(s,1H),155.15(s,1H),151.53(s,1H),146.93(s,1H),141.80(s,1H),137.36(s,2H),136.62(s,1H),131.10(s,2H),130.58(s,2H),119.24(s,2H),118.79(s,2H),113.86(s,2H),112.62(s,2H),106.94(s,1H),55.39(s,2H),48.84(s,2H),30.07(s,2H),28.30(s,2H).HRMS(ESI)Calcd for C 18H 19N 6O 3[M+H] +367.1519；Found 367.1527.
IMB-2-31
8-(4-(4-fluorophenyl)piperazin-1-yl)-7-(3-methoxybenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione性状：浅棕色固体 产率：82％.
1H NMR(500MHz,CDCl 3)δ＝7.24(d,J＝7.9,1H),6.98(t,J＝8.6,2H),6.92–6.89(m,2H),6.82–6.77(m,3H),5.38(s,2H),3.77(s,3H),3.56(s,3H),3.37(s,4H),3.36(d,J＝4.7,4H),3.18(d,J＝4.4,4H). 13C NMR(151MHz,CDCl 3)δ＝160.11,158.45,156.86,156.34,154.86,151.91,147.81,138.39,130.07,119.03,115.88,115.88,115.73,115.73,112.97,112.90,105.27,55.36,50.53,50.08,48.68,29.90,27.99.HRMS(ESI)Calcd for C 25H 28N 6O 3F[M+H] +479.2207；Found 479.2203.
IMB-2-32
Methyl-1-(7-(3-methoxybenzyl)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1H-indole-5-carboxylate
性状：浅棕色固体 产率：65％.
1H NMR(500MHz,CDCl 3)δ＝8.05(s,1H),7.94(d,J＝8.3,1H),7.71(d,J＝8.3,1H),7.26(s,1H),7.10(t,J＝7.9,1H),6.77(d,J＝2.9,1H),6.72(d,J＝8.2,1H),6.50(d,J＝7.5,1H),6.36(s,1H),5.45(s,2H),3.92(s,3H),3.62(s,3H),3.59(s,3H),3.47(s,3H). 13C NMR(151MHz,CDCl 3)δ＝167.45,159.97,155.31,151.65,147.37,143.29,136.81,136.12,132.65,130.16,129.97,126.02,123.33,121.34,119.57,114.05,113.26,112.89,106.87,106.70,55.20,52.29,49.14,30.14,28.32.HRMS(ESI)Calcd for C 25H 24N 5O 5[M+H] +474.1774；Found 474.1770.
以下为N7位改造的化合物IMB-ZHB-4X
7-(3-chloro-2-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：72％.
1H NMR(500MHz,DMSO-d 6)δ＝7.49(s,1H),7.25(s,1H),7.14(s,1H),6.71(s,1H),5.39(s,2H),4.72(s,1H),3.53(s,2H),3.38(d,J＝1.6,5H),3.32(s,3H),3.11(s,3H). 13C NMR(151MHz,DMSO-d 6)δ＝153.42,152.41,151.05,148.40,135.14,130.74,126.91,126.46,125.54,123.96,101.88,57.90,53.40,44.47,29.57,27.33.HRMS(ESI)Calcd for C 16H 18N 5O 3FCl[M+H] +382.1082；Found 382.1073.
IMB-ZHB-13X
7-benzyl-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：53％.
1H NMR(500MHz,DMSO-d 6)δ＝7.18(s,2H),5.26(s,2H),4.70(s,1H),3.51(s,2H),3.36(s,2H),3.31(d,J＝6.1,3H),3.11(d,J＝6.1,3H). 13C NMR(126MHz,DMSO-d 6)δ＝153.90,151.00,148.78,137.19,128.51,128.51,127.33,127.02,127.02,101.28,59.74,45.29,45.11,29.33,27.22.HRMS(ESI)Calcd for C 16H 20N 5O 3[M+H] +330.1566；Found 330.1572.
IMB-68
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(2-methylbenzyl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：86％.
1H NMR(500MHz,DMSO-d 6)δ＝7.21(d,J＝7.3,1H),7.14(t,J＝7.4,1H),7.08(dd,J＝13.7,6.3,2H),6.37(d,J＝7.2,1H),5.27(s,2H),4.70(t,J＝5.3,1H),3.52(dd,J＝11.2,5.4,2H),3.44–3.37(m,5H),3.10(s,3H),2.33(s,3H). 13C NMR(151MHz,DMSO-d 6)δ＝153.87,152.71,150.97,148.71,137.52,137.04,128.38,127.91,127.53,123.90,101.27,59.68,45.21,45.03,29.29,27.16,21.02.
IMB-79
7-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-nitrobenzyl)-3,7-dihydro-1H-purine-2,6-dione
8-性状：黄色固体 产率：41％.
1H NMR(500MHz,DMSO-d 6)δ＝8.22–8.09(m,2H),7.63(d,J＝5.1,2H),7.37(t,J＝5.5,1H),5.43(s,2H),4.73(t,J＝5.4,1H),3.53(dd,J＝11.5,5.8,2H),3.43–3.39(m,2H),3.37(s,3H),3.16(s,3H). 13C NMR(126MHz,DMSO-d 6)δ153.87,152.83,150.99,148.98,147.80,139.38,133.78,130.25,122.47,122.05,101.11,59.63,45.08,44.77,29.41,27.27.HRMS(ESI)Calcd for C 16H 19N 6O 5[M+H] +375.1411；Found 375.1416.
IMB-80
7-(3-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：灰色固体 产率：81％.
1H NMR(500MHz,DMSO-d 6)δ＝7.37(s,1H),7.25(s,1H),7.06(d,J＝10.3,3H),5.30(s,2H),4.73(d,J＝3.9,1H),3.53(s,2H),3.38(dd,J＝16.4,4.4,5H),3.16(d,J＝3.4,3H). 13C NMR(126MHz,DMSO-d 6)δ＝163.15,161.21,153.86,152.79,151.01,148.88,140.01,130.61,123.10,114.11,101.17,59.67,45.10,44.85,29.38,27.25.Calcd for C 16H 19N 5O 3F[M+H] +348.1466；Found 348.1475.
IMB-81
7-(3-bromobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：灰色固体 产率：78％.
1H NMR(500MHz,DMSO-d 6)δ＝7.40–7.34(m,1H),7.25(d,J＝3.4,1H),7.06(d,J＝10.3,3H),5.30(s,2H),4.73(d,J＝3.9,1H),3.57–3.51(m,2H),3.42–3.38(m,2H),3.36(d,J＝3.5,2H),3.32(d,J＝3.7,1H),3.16(d,J＝3.4,3H). 13C NMR(126MHz,DMSO-d 6)δ＝153.84,152.78,150.99,148.88,139.91,130.82,130.29,129.90,126.02,121.72,101.15,59.66,45.08,44.75,29.39,27.26.HRMS(ESI)Calcd for C 16H 19N 6O 5[M+H] +375.1411；Found 375.1422.
IMB-82
7-(2,3-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：89％.
1H NMR(500MHz,DMSO-d 6)δ＝7.34(dd,J＝17.4,8.4,1H),7.24(t,J＝5.4,1H),7.12(dd,J＝12.8,7.6,1H),6.58(t,J＝6.8,1H),5.41(s,2H),4.71(t,J＝5.4,1H),3.53(dd,J＝11.5,5.7,2H),3.43–3.34(m,5H),3.12(s,3H). 13C NMR(126MHz,DMSO-d 6)δ＝154.20,152.66,151.02,148.89,127.12,127.12,125.01,122.34,116.29,116.29,116.22,101.22,59.65,45.12,29.40,27.17.HRMS(ESI)Calcd for C 16H 18N 5O 3F 2[M+H] +366.1372；Found 366.1374.
IMB-83
7-(2,4-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：灰色固体 产率：84％.
1H NMR(500MHz,DMSO-d 6)δ＝7.28(t,J＝10.0,1H),7.20(d,J＝5.3,1H),7.00(t,J＝8.3,1H),6.85(dd,J＝14.9,8.3,1H),5.33(s,2H),4.71(t,J＝5.4,1H),3.39(d,J＝11.4,2H),3.31(s,3H),3.12(s,2H),2.50(s,3H). 13C NMR(101MHz,DMSO-d 6)δ＝160.53,158.26,154.18,152.66,151.03,148.91,128.70,120.67,111.53,103.93,101.21,59.68,45.11,29.38,27.17.HRMS(ESI)Calcd for C 16H 18N 5O 3F 2[M+H] +366.1372；Found 366.1378.
IMB-84
7-(4-(tert-butyl)benzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：棕色固体 产率：58％.
1H NMR(500MHz,DMSO-d 6)δ＝7.33(d,J＝8.1,2H),7.21(d,J＝10.7,1H),7.15(d,J＝8.0,2H),5.25(s,2H),4.73(t,J＝5.4,1H),3.55(dd,J＝11.6,5.8,2H),3.42–3.38(m,2H),3.35(s,2H),3.32(s,1H),3.15(s,3H),1.24(s,9H). 13C NMR(101MHz,DMSO-d 6)δ＝154.34,153.20,151.44,150.16,149.23,134.66,127.18,125.72,101.70,60.16,45.54,45.38,34.65,31.54,29.77,27.66.HRMS(ESI)Calcd for C 20H 28N 5O 3[M+H] +386.2187；Found 386.2193.
IMB-85
7-(4-butylbenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：浅黄色固体 产率：85％.
1H NMR(500MHz,DMSO-d 6)δ＝7.15(d,J＝29.4,5H),5.25(s,2H),4.72(s,1H),3.54(s,2H),3.37(d,J＝19.8,5H),3.31(s,2H),3.15(s,3H),1.50(s,2H),1.28(s,2H),0.87(s,3H). 13C NMR(101MHz,DMSO-d 6)δ ＝153.88,152.77,151.01,148.79,141.47,134.39,128.40,128.40,127.03,127.03,101.28,59.72,45.08,34.47,33.15,29.34,27.23,21.78,13.77.HRMS(ESI)Calcd for C 20H 28N 5O 3[M+H] +386.2187；Found 386.2195.
IMB-88
7-(3,5-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：91％.
1H NMR(500MHz,DMSO-d 6)δ＝7.22(d,J＝8.5,3H),6.87(s,2H),5.21(s,2H),4.73(d,J＝3.8,1H),3.70(d,J＝3.5,3H),3.54(s,2H),3.38(d,J＝14.4,2H),3.36(s,4H),3.17(d,J＝3.3,2H). 13C NMR(101MHz,DMSO-d 6)δ＝158.62,153.79,152.80,151.01,148.82,129.11,128.74,128.74,113.88,113.88,101.19,59.75,55.07,45.09,44.74,29.33,27.25.HRMS(ESI)Calcd for C 17H 22N 5O 4[M+H] +360.1666；Found 360.1575.
IMB-92
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-(trifluoromethoxy)benzyl)-3,7-dihydro-1H-purine-2,6-dione性状：白色固体 产率：91％.
1H NMR(500MHz,DMSO-d 6)δ＝7.45(t,J＝7.9,1H),7.34–7.24(m,3H),7.21(d,J＝7.5,1H),5.34(s,2H),4.73(t,J＝5.3,1H),3.55–3.49(m,2H),3.39(d,J＝5.7,2H),3.36(s,3H),3.15(s,3H). 13C NMR(101MHz,CDCl 3)δ＝159.09,158.02,156.21,154.14,153.65,145.03,135.83,135.83,131.21,125.05,124.90,106.29,64.85,50.25,50.01,34.56,32.43.HRMS(ESI)Calcd for C 17H 19N 5O 4F 3[M+H] +414.1384；Found 414.1380.
IMB-93
7-(2,6-difluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：灰色固体 产率：73％.
1H NMR(500MHz,DMSO-d 6)δ＝7.43–7.30(m,1H),7.12(t,J＝5.4,1H),7.04(t,J＝8.1,2H),5.35(s,2H),4.74(t,J＝5.4,1H),3.55(dd,J＝11.6,5.8,2H),3.46–3.39(m,2H),3.35(s,1H),3.31(s,3H),3.08(s,2H). 13C NMR(101MHz,DMSO-d 6)δ＝161.93(d,J＝8.1,1H),159.46(d,J＝8.0,1H),154.48,152.53,151.02,148.76,129.96,112.77,111.94,111.51,101.51,59.85,45.20,36.54,29.34,27.18.HRMS(ESI)Calcd for C 16H 18N 5O 3F 2[M+H] +366.1372；Found 366.1365.
IMB-2-2
7-(4-fluorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：90％.
1H NMR(500MHz,DMSO-d 6)δ＝7.29(d,J＝25.5,2H),7.17(s,2H),5.30(s,2H),4.75(t,J＝5.4,1H),3.55(dd,J＝10.3,5.2,2H),3.41(dd,J＝9.4,3.7,2H),3.38(s,3H),3.18(s,3H). 13C NMR(101MHz,CDCl 3)δ＝165.49,159.02,158.01,156.21,154.09,138.52,134.53,120.51,106.35,64.88,50.27,49.81,34.56,32.45.HRMS(ESI)Calcd for C 16H 19N 5O 3F[M+H]+348.1466；Found 348.1459.
IMB-2-3
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(4-(trifluoromethoxy)benzyl)-3,7-dihydro-1H-purine-2,6-dione性状：白色固体 产率：84％.
1H NMR(500MHz,DMSO-d 6)δ＝7.33(s,4H),7.28(t,J＝4.6,1H),5.33(s,2H),4.73(t,J＝5.1,1H),3.53(dd,J＝11.1,5.1,2H),3.42–3.38(m,2H),3.36(s,3H),3.15(s,3H). 13C NMR(101MHz,CDCl 3)δ＝159.08,158.01,156.22,154.10,152.75,152.75,141.83,134.15,134.15,126.40,126.40,106.37,64.85,50.28,49.85,34.57,32.44.HRMS(ESI)Calcd for C 17H 19N 5O 4F 3[M+H] +414.1384；Found 414.1375.
IMB-2-5
7-(3-chlorobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：棕色固体 产率：88％.
1H NMR(500MHz,DMSO-d 6)δ＝7.38–7.31(m,3H),7.26(s,1H),7.15(d,J＝6.9,1H),5.30(s,2H),4.73(t,J＝5.4,1H),3.53(t,J＝5.6,2H),3.53(t,J＝5.6,2H),3.43–3.37(m,2H),3.31(s,4H),3.16(s,2H). 13C NMR(151MHz,DMSO-d 6)δ＝153.92,152.85,151.07,148.93,139.65,133.15,130.54(,127.44,127.01,125.71,101.24,59.72,45.13,44.87,29.41,27.29.HRMS(ESI)Calcd for C 16H 19N 5O 3Cl[M+H] +364.1171；Found364.1163.
IMB-2-6
7-(2-bromobenzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：乳白色固体 产率：92％.
1H NMR(500MHz,DMSO-d 6)δ＝7.67(d,J＝7.8,1H),7.32–7.26(m,2H),7.22(t,J＝7.6,1H),6.44(d,J＝7.3,1H),5.32(s,2H),4.69(t,J＝5.4,1H),3.52(d,J＝5.7,2H),3.41(s,5H),3.10(s,3H). 13C NMR(101MHz,DMSO-d 6)δ＝154.33,152.66,151.08,148.92,136.06,132.60,128.98,128.12,125.81,121.23,101.14,59.68,46.33,45.11,29.45,27.16.HRMS(ESI)Calcd for C 16H 19N 5O 3Br[M+H] +408.0666；Found 408.0657.
IMB-2-8
7-(4-(difluoromethoxy)benzyl)-8-((2-hydroxyethyl)amino)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione性状：白色固体 产率：94％.
1H NMR(500MHz,DMSO-d 6)δ＝7.29(s,3H),7.12(d,J＝8.8,2H),5.28(s,2H),3.54(s,2H),3.39(s,2H),3.35(d,J＝7.3,3H),3.16(s,3H). 13C NMR(151MHz,DMSO-d 6)δ＝153.80,152.75,150.96,150.15,148.83,134.11,128.85,128.85,118.85,118.85,116.30,101.11,59.65,45.06,44.61,29.28,27.18.HRMS(ESI)Calcd for C 17H 20N 5O 4F 2[M+H] +396.1478；Found 396.1469.
IMB-2-14
8-bromo-7-(4-chlorobenzyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：96％.
1H NMR(500MHz,DMSO-d 6)δ＝7.39(s,2H),7.25(s,3H),5.29(s,2H),4.74–4.71(m,1H),3.53(s,2H),3.35(d,J＝4.4,5H),3.15(s,3H).HRMS(ESI)Calcd for C 16H 19N 5O 3Cl[M+H] +364.1176；Found 364.1172.
IMB-3-19
((2-hydroxyethyl)amino)-1,3-dimethyl-7-((tetrahydro-2H-pyran-4-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：76％. 1H NMR(500MHz,DMSO-d 6)δ＝7.00(s,1H),5.75(s,1H),4.73(s,1H),3.90(d,J＝5.0,2H),3.81(d,J＝9.8,2H),3.54(s,2H),3.39(s,2H),3.34(s,4H),3.26–3.08(m,5H),1.99(s,1H),1.32(dd,J＝38.0,11.2,4H). 13C NMR(126MHz,DMSO-d 6)δ＝153.82,152.57,150.98,148.47,101.73,66.58,66.58,59.77,47.29,45.06,34.92,29.38,29.38,29.28,27.22.HRMS(ESI)Calcd for C 15H 24N 5O 4[M+H] +338.1828；Found 338.1824.
IMB-4-3
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(3-methylbenzyl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：83％.
1H NMR(500MHz,CDCl 3)δ＝7.24(d,J＝8.0,1H),7.12(d,J＝7.6,1H),7.04(d,J＝12.0,2H),5.33(s,2H),4.50(s,1H),3.76–3.69(m,2H),3.53(d,J＝6.7,5H),3.40(s,3H),2.33(s,3H). 13C NMR(151MHz,CDCl 3)δ＝154.29,153.73,151.81,148.28,139.21,135.22,129.20,129.17,127.74,124.09,103.29,62.69,46.85,46.00,29.84,27.85,21.52.HRMS(ESI)Calcd for C 17H 22N 5O 3[M+H] +344.1723；Found 344.1701.
IMB-4-6
ethyl3-((8-((2-hydroxyethyl)amino)-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)methyl)benzoate
性状：白色固体 产率：88％.
1H NMR(500MHz,CDCl 3)δ＝8.01–7.91(m,2H),7.51(d,J＝7.2,1H),7.45(d,J＝7.6,1H),5.40(s,2H),4.66(s,1H),4.38(q,J＝7.2,2H),3.78(t,J＝3.8,2H),3.59(dd,J＝9.9,5.7,2H),3.52(s,3H),3.38(s,3H),1.39(t,J＝7.1,3H). 13C NMR(151MHz,CDCl 3)δ＝166.58,154.25,153.63,151.80,148.65,136.08,131.92,131.19,129.48,129.38,128.15,102.95,62.27,61.54,46.35,45.80,29.85,27.83,14.37.HRMS(ESI)Calcd for C 19H 24N 5O 5[M+H] +402.1777；Found 402.1775.
IMB-4-12
8-((2-hydroxyethyl)amino)-1,3-dimethyl-7-(thiophen-3-ylmethyl)-3,7-dihydro-1H-purine-2,6-dione
性状：浅棕色固体 产率：72％.
1H NMR(500MHz,CDCl 3)δ＝7.41–7.36(m,1H),7.27(s,1H),7.08(d,J＝4.9,1H),5.40(s,2H),4.65(s,1H),3.84–3.76(m,2H),3.61(dd,J＝9.5,5.3,2H),3.55(s,3H),3.43(s,3H). 13C NMR(151MHz,DMSO-d 6)δ＝153.56,152.77,150.97,148.75,137.56,127.41,126.59,123.21,101.02,59.70,45.03,40.85,29.27,27.19.HRMS(ESI)Calcd for C 14H 18N 5O 3S[M+H] +336.1130；Found 336.1124.
IMB-4-13
8-((2-hydroxyethyl)amino)-7-(3-methoxyphenethyl)-1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：55％.
1H NMR(500MHz,CDCl 3)δ＝7.27(d,J＝8.0,1H),6.85(d,J＝6.4,1H),6.75(d,J＝7.4,1H),6.66(s,1H),4.27(t,J＝6.2,2H),3.80(s,3H),3.75(s,1H),3.61–3.56(m,2H),3.53(s,3H),3.45(s,3H),3.27(dd,J＝9.5,5.2,2H),3.10(t,J＝6.1,2H). 13C NMR(151MHz,DMSO-d 6)δ＝159.22,153.46,152.66,151.00,148.61,139.48,129.24,121.14,114.50,111.84,101.20,59.75,54.89,44.95,43.59,35.11,29.24,27.21.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1828；Found 374.1821.
以下为N1位改造的化合物：
IMB-3-6
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-((tetrahydro-2H-pyran-4-yl)methyl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：77％.
1H NMR(500MHz,CDCl 3)δ＝7.28(d,J＝7.9,1H),6.87–6.79(m,3H),5.35(s,2H),4.79(s,1H),3.93(t,J＝11.6,4H),3.78(s,3H),3.77–3.73(m,2H),3.60(s,2H),3.54(s,3H),3.34(t,J＝11.6,2H),2.61(s,1H),2.07(ddd,J＝11.4,7.4,4.0,1H),1.56(d,J＝12.4,2H),1.51–1.42(m,2H). 13C NMR(151MHz,CDCl 3)δ＝160.40,154.40,153.78,151.83,148.43,136.83,130.44,119.30,113.58,113.20,103.19,67.82,67.82,62.74,55.40,46.86,46.26,46.01,34.43,30.91,30.91,29.84.HRMS(ESI)Calcd for C 22H 30N 5O 5[M+H] +444.2247；Found 444.2241.
IMB-3-13
1-(4-fluorobenzyl)-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：18％.
1H NMR(600MHz,CDCl 3)δ＝7.48–7.44(m,2H),7.28(d,J＝7.9,1H),6.87–6.77(m,5H),5.33(s,2H),5.30(s,1H),5.12(s,2H),4.59(s,1H),3.76(s,3H),3.74–3.71(m,2H),3.53(dd,J＝9.7,5.0,2H),3.51–3.49(m,3H). 13C NMR(151MHz,CDCl 3)δ＝160.41,159.01,154.16,153.72,151.69,148.39,136.80,130.47,130.47,130.42,130.25,119.28,113.80,113.80,113.77,113.03,103.33,62.76,55.38,46.88,46.02,43.74,29.84.
IMB-3-15
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-1-(4-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：20％.
1H NMR(500MHz,CDCl 3)δ＝7.16(dt,J＝20.1,8.0,2H),6.98–6.92(m,2H),6.80–6.68(m,4H),5.46(s,1H),5.25(s,2H),5.09(s,2H),3.71(s,3H),3.68(d,J＝9.1,5H),3.49(d,J＝3.8,2H),3.44(s,3H).
IMB-3-16
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-(pyridin-4-ylmethyl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：20％.
1H NMR(500MHz,CDCl 3)δ＝7.40(d,J＝7.5,2H),7.25–7.15(m,4H),6.78(dd,J＝13.4,8.1,3H),5.45(s,1H),5.26(s,2H),5.12(s,2H),3.68(d,J＝9.4,5H),3.50(s,2H),3.45(s,3H). 13C NMR(151MHz,CDCl 3)δ＝160.40,154.11,153.76,151.69,148.47,137.94,136.80,130.40,128.76,128.76,128.46,128.46,127.42,119.28,113.78,113.03,103.27,62.72,55.39,46.87,46.00,44.31,29.88.HRMS(ESI)Calcd for C 22H 25N 5O 4[M+H] +437.1937；Found437.1931.
IMB-3-30
8-bromo-1-ethyl-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione性状：白色固体 产率：45％.
1H NMR(500MHz,CDCl 3)δ＝6.94(d,J＝12.0,2H),6.85(d,J＝8.2,1H),5.53(s,2H),4.07(q,J＝7.0,2H),3.78(s,2H),3.55(s,3H),1.24(t,J＝7.0,3H). 13C NMR(151MHz,CDCl 3)δ＝160.07,154.15,151.03,148.52,136.64,130.11,127.88,120.19,113.81,109.17,55.38,50.25,36.87,29.90,13.36.HRMS(ESI)Calcd for C 16H 18N 4O 3Br[M+H] +393.0562；Found 393.0570.
IMB-3-45
1-(but-3-yn-1-yl)-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：黄色固体 产率：28％.
1H NMR(500MHz,CDCl 3)δ＝7.33–7.27(m,1H),6.83(dd,J＝15.7,7.6,3H),5.32(s,2H),4.57(s,1H),4.21(t,J＝6.2,2H),3.78(d,J＝1.9,3H),3.75(s,2H),3.55(s,2H),3.51(d,J＝1.9,3H),2.886(s,J＝2.886,1H),1.96(d,J＝2.2,1H),1.26(s,1H). 13C NMR(151MHz,CDCl 3)δ＝160.38,153.84,153.76,151.42,148.51,136.84,130.41,119.31,113.49,113.28,103.16,81.26,69.78,62.71,55.42,46.84,46.00,39.43,29.80,17.91.HRMS(ESI)Calcd for C 20H 24N 5O 4[M+H] +398.1828；Found 398.1826.
IMB-3-46
ethyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：54％.
1H NMR(500MHz,CDCl 3)δ＝7.28(d,J＝7.9,1H),6.83(dd,J＝15.4,7.7,3H),5.33(s,2H),4.55(s,1H),4.07(q,J＝6.8,2H),3.78(s,3H),3.74(s,2H),3.53(dd,J＝9.6,5.3,2H),3.50(s,3H),3.00(s,1H),1.24(t,J＝6.9,3H). 13C NMR(151MHz,CDCl 3)δ＝160.39,154.05,153.66,151.42,148.24,136.89,130.43,119.28,113.57,113.13,103.35,62.80,55.41,46.83,46.05,36.35,29.75,13.56.HRMS(ESI)Calcd for C 18H 24N 5O 4[M+H] +374.1828；Found 374.1807.
IMB-3-47
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-propyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：36％.
1H NMR(500MHz,CDCl 3)δ＝7.28(d,J＝7.8,1H),6.83(dd,J＝14.7,7.0,3H),5.33(s,2H),4.58(s,1H),4.00–3.92(m,2H),3.78(s,3H),3.74(s,2H),3.54(dd,J＝9.6,5.2,2H),3.50(s,3H),3.04(s,1H),1.73–1.64(m,2H),0.95(t,J＝7.4,3H). 13C NMR(151MHz,CDCl 3)δ＝160.37,154.24,153.67,151.57,148.25,136.91,130.40,119.27,113.58,113.09,103.29,62.78,55.40,46.80,46.04,42.78,29.77,21.55,11.47.HRMS(ESI)Calcd for C 19H 26N 5O 4[M+H] +388.1985；Found 388.1979.
IMB-3-57
tert-butyl(2-(8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)ethyl)carbamate
性状：白色晶体 产率：12％.
1H NMR(500MHz,CDCl 3)δ＝7.36–7.30(m,1H),6.87(t,J＝7.4,2H),6.83(s,1H),5.34(d,J＝3.9,2H),4.21(s,2H),3.82(s,3H),3.80–3.74(m,2H),3.58(dd,J＝9.6,5.2,2H),3.54(s,3H),3.46(s,2H),1.64(s,1H),1.42(s,9H). 13C NMR(151MHz,CDCl 3)δ＝160.27,156.17,154.23,153.79,151.64,148.51,136.78,130.32,119.19,113.49,113.04,103.04,78.94,62.48,55.30,46.71,45.86,38.23,37.26,29.75,28.49,28.49,28.49.
IMB-3-58
tert-butyl(3-(8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)propyl)carbamate
性状：白色晶体 产率：40％.
1H NMR(500MHz,CDCl 3)δ＝7.28(d,J＝6.8,1H),6.86–6.81(m,2H),6.79(s,1H),5.31(s,2H),4.68(t,J＝5.4,1H),4.07(t,J＝6.1,2H),3.78(s,3H),3.76–3.71(m,2H),3.54(dd,J＝9.7,5.2,2H),3.50(s,3H),3.09(d,J＝5.3,2H),1.87–1.79(m,2H),1.43(s,9H). 13C NMR(151MHz,CDCl 3)δ＝160.22,156.15,154.18,153.80,151.61,148.53,136.78,130.27,119.15,113.43,113.00,102.99,78.93,62.40,55.26,46.65,45.82,38.20,37.25,29.71,28.46,28.46,28.46,28.36.HRMS(ESI)Calcd for C 24H 35N 6O 6[M+H] +503.2618；Found 503.2603.
IMB-3-71
allyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：黄色固体 产率：52％.
1H NMR(500MHz,CDCl 3)δ＝7.24(d,J＝7.9,1H),6.80(dd,J＝15.9,7.5,3H),5.91(dq,J＝10.7,5.6,1H),5.29(d,J＝3.8,2H),5.22(d,J＝17.2,1H),5.14(d,J＝10.2,1H),4.70(t,J＝5.3,1H),4.59(d,J＝5.5,2H),3.75(s,3H),3.72(dd,J＝13.6,8.9,2H),3.52(dd,J＝9.8,5.2,3H). 13C NMR(151MHz,CDCl 3)δ＝160.38,153.84,153.78,151.41,148.51,136.83,132.92,130.41,119.27,117.11,113.62,113.10,103.22,62.72,55.41,46.84,46.00,43.18,29.80.HRMS(ESI)Calcd for C 19H 24N 5O 4[M+H] +386.1828；Found 386.1816.
IMB-3-72
8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-3-methyl-1-(prop-2-yn-1-yl)-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：16％.
1H NMR(500MHz,CDCl 3)δ＝7.25(d,J＝4.3,1H),6.81(dd,J＝15.1,7.3,3H),5.30(s,2H),5.29(s,1H),4.76(d,J＝2.2,2H),4.66(t,J＝5.3,1H),3.77(s,3H),3.75–3.68(m,2H),3.54(dd,J＝9.5,4.9,2H),3.48(s,3H),2.15(t,J＝2.1,1H). 13C NMR(151MHz,CDCl 3)δ＝160.44,153.87,153.11,151.04,148.73,136.67,130.49,119.34,113.75,113.17,103.16,79.32,70.29,62.67,55.46,46.99,45.98,30.34,29.91.HRMS(ESI)Calcd for C 19H 22N 5O 4[M+H] +384.1672；Found 384.1662.
对N3位的改造：
IMB-3-81
benzyl-8-((2-hydroxyethyl)amino)-7-(3-methoxybenzyl)-1-methyl-3,7-dihydro-1H-purine-2,6-dione
性状：白色固体 产率：98％.
1H NMR(500MHz,CDCl 3)δ＝7.51(d,J＝7.1,2H),7.31(d,J＝8.0,3H),6.87–6.82(m,2H),6.81(s,1H),5.32(s,2H),5.21(s,2H),3.78(s,3H),3.76(dd,J＝7.1,2.8,2H),3.55(dd,J＝9.5,4.5,2H),3.39(s,3H). 13C NMR(151MHz,CDCl 3)δ＝160.43,154.38,153.61,151.59,147.92,136.83,136.81,130.51,128.97,128.97,128.63,128.63,127.92,119.40,113.64,113.25,103.39,62.70,55.45,46.95,46.75,46.05,27.91.HRMS(ESI)Calcd for C 23H 26N 5O 4[M+H] +436.1985；Found 436.1987.
实施例2、应用致细胞病变效应(cytopathic effect，CPE)法测定IMB-C5系列化合物抗冠状病毒HCoV-229E活性
(1)实验在Huh7和Huh7.5细胞中进行，细胞接种96孔培养板，置5％CO 2，37℃培养24h左右；
(2)用含2％FBS及1％双抗的DMEM培养液三倍比稀释IMB-C5系列化合物，得到8个剂量的样品。
(3)以100TCID 50的HCoV-229E病毒液感染细胞，同时加入含有不同稀释度的样品，同时设置细胞对照孔和病毒对照孔，于5％CO 2，35℃培养约48h。待病毒对照组病变程度(CPE)达4+时观察各组细胞病变程度，用Reed-Muench法分别计算样品对细胞的半数有毒浓度(TC 50)和对病毒的半数抑制浓度(EC 50)，同时计算选择指数(SI＝TC 50/EC 50)。
CPE评价标准：以细胞死亡比例分别标记为4+(细胞死亡比例75％～100％)、3+(细胞死亡比例50％～75％)、2+(细胞死亡比例25％～50％)、1+(细胞死亡比例0～25％)、0+(细胞全部存活)。
结果如表1和表2所示，IMB-C5系列化合物在Huh7及Huh7.5细胞上有较好的抑制HCoV-229E的活性，且大部分化合物的活性优于阳性对照药利巴韦林(RBV)，与已上市的RdRp抑制剂莫努匹韦(Molnupiravir，MNP)活性相当或更优。其中，化合物IMB-85活性最好，在Huh7、Huh7.5细胞上抑制HCoV-229E的EC 50分别为0.09μM和4.05μM，优于阳性对照药和IMB-C5其他同系化合物。
表中化合物IMB-ZH-2、IMB-ZH-11、IMB-ZH-12、IMB-ZHC-2、IMB-ZHC-15、IMB-2-26、IMB-2-31、IMB-2-32、IMB-3-19、IMB-4-6、IMB-4-12、IMB-4-13、IMB-ZHB-4x、IMB-2-3、IMB-2-8、IMB-68、IMB-82、IMB-83、IMB-84、IMB-85、IMB-92、IMB-93、IMB-3-6、IMB-3-13、IMB-3-15、IMB-3-16、IMB-3-30、IMB-3-45、IMB-3-46、IMB-3-47、IMB-3-57、IMB-3-58、IMB-3-71、IMB-3-72、IMB-3-81为新结构化合物。
表1 CPE法测定IMB-C5系列化合物在Huh7细胞中抗HCoV-229E活性
Figure PCTCN2022099223-appb-000012
Figure PCTCN2022099223-appb-000013
Figure PCTCN2022099223-appb-000014
Figure PCTCN2022099223-appb-000015
Figure PCTCN2022099223-appb-000016
Figure PCTCN2022099223-appb-000017
表2 CPE法测定IMB-C5系列化合物在Huh7.5细胞中抗HCoV-229E活性
Figure PCTCN2022099223-appb-000018
Figure PCTCN2022099223-appb-000019
实施例3、IMB-C5抗α属冠状病毒HCoV-229E活性测定
在体外药效检测中，首先检测IMB-C5对冠状病毒HCoV-229E N蛋白的mRNA水平的影响(图1)。
以MOI＝0.035的病毒载量感染Huh7及Huh7.5细胞，同时以三个浓度的IMB-C5或200μM阳性药RBV给药，24h后提取RNA并进行RT-qPCR检测。如图1A、B所示，IMB-C5在Huh7和Huh7.5细胞中均能剂量依赖性地降低HCoV-229E N蛋白的mRNA的水平，在Huh7细胞上的抗病毒活性优于Huh7.5。
随后通过免疫荧光实验检测IMB-C5对HCoV-229E双链RNA(double strand RNA，dsRNA)的影响(图2)。
以MOI＝0.035的病毒载量感染Huh7细胞，感染同时加入梯度浓度IMB-C5或阳性药，35℃培养24h后，将处理好的细胞用PBS洗3次，随后加入4％多聚甲醛室温孵育15min进行固定，清洗后用含0.5％Triton X-100的PBS缓冲液室温孵育20min透化细胞，清洗后加入含1％牛血清白蛋白(bovine serum albumin，BSA)的TBST缓冲液室温封闭1h。加入抗dsRNA抗体于4℃孵育过夜。PBST缓冲液清洗细胞后加入FITC荧光标记二抗，室温避光孵育1h。最后加入染核液(Hoechst 33342)室温孵育10min，用荧光显微镜观察病毒dsRNA的水平。如图2所示，IMB-C5对HCoV-229E在Huh7细胞复制过程中dsRNA的产生具有抑制作用。
实施例4、IMB-C5抗β属冠状病毒HCoV-OC43活性测定
以C3A细胞为病毒宿主，检测了IMB-C5对冠状病毒HCoV-OC43N蛋白mRNA水平的影响(图3)。以MOI＝0.023的病毒载量感染C3A细胞，同时以三个浓度的IMB-C5或200μM阳性药RBV给药，24h后提取RNA并进行RT-qPCR检测。结果表明IMB-C5在C3A细胞中可剂量依赖性地降低HCoV-OC43N蛋白mRNA的水平。
进一步检测了IMB-C5对冠状病毒HCoV-OC43N蛋白水平的影响(图4)。以MOI＝0.023的病毒载量感染C3A细胞，同时以三个浓度的IMB-C5或200μM阳性药RBV给药，24h后检测病毒N蛋白的水平。如图所示，IMB-C5对HCoV-OC43具有很好的抑制作用，可剂量依赖性地抑制病毒N蛋白的表达。
实施例5、IMB-C5系列化合物抑制冠状病毒HCoV-229E N蛋白水平
在体外药效检测中，检测了IMB-C5系列化合物对冠状病毒HCoV-229E N蛋白水平的影响。从CPE结果中挑选SI>80的化合物8个，包括IMB-2-5、IMB-2-8、IMB-2-14、IMB-68、IMB-92、IMB-93、IMB-3-19和IMB-85，IMB-C5作为同系物对照。以MOI＝1的病毒载量感染Huh7细胞，同时以5μM的IMB-C5系列化合物以及阳性对照15μM MNP(Molnupiravir，已上市口服新冠RdRp抑制剂)给药，24h后提取蛋白进行Western blot检测。如图5所示(图中化合物IMB-2-5标注为2-5，余同)，5μM浓度下，这8个化合物均可在Huh7细胞中不同程度抑制HCoV-229E；同样浓度下，IMB-85降低HCoV-229E N蛋白水平活性最好，明显优于IMB-C5和其他化合物，与阳性对照15μM MNP作用相当。
实施例6、化合物IMB-C5和IMB-85的细胞毒性测定
为明确IMB-C5和IMB-85可能存在的细胞毒性，首先利用CCK-8法检测了两种化合物在不同浓度下给药48h对不同细胞存活率的影响。如图5所示，IMB-C5和IMB-85浓度在200μM及以下时，给药48h后两种肝癌细胞(Huh7和Huh7.5)的存活率均在对照组的95％以上(TC 50>200μM)，显示化合物对上述细胞的毒性较低(图6)。
实施例7、IMB-C5和IMB-85抑制冠状病毒HCoV-229E活性
检测IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白mRNA水平和蛋白水平的影响。以MOI＝17的病毒载量感染Huh7细胞，同时以3μM的IMB-C5，3μM、0.6μM和0.12μM三个浓度的IMB-85以及200μM阳性药RBV给药，24h后分别提取RNA和蛋白进行RT-qPCR和Western blot检测。如图7和图8所示，IMB-85在Huh7细胞中能剂量依赖性地降低HCoV-229E N蛋白的mRNA和蛋白水平，且活性明显优于同浓度的IMB-C5。
在相同的实验条件下，通过免疫荧光实验检测IMB-C5和IMB-85对HCoV-229E双链RNA(double strand RNA，dsRNA)的影响(图9)，结果表明，IMB-C5和IMB-85均可以抑制HCoV-229E在Huh7细胞复制过程中产生的dsRNA，且在同样浓度下IMB-85的活性优于IMB-C5。
实施例8、在Huh7和Huh7.5细胞上，IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白水平的影响
(1)在Huh7细胞上，IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白水平的影响
以Huh7细胞为病毒宿主，检测了IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白水平的影响(图10A)。以MOI＝1的病毒载量感染Huh7细胞，同时以不同浓度的IMB-85和IMB-C5给药，MNP作为阳性对照。24h后提取细胞总蛋白进行Western blot检测。结果表明IMB-85在Huh7细胞中可剂量依赖性地降低HCoV-229E N蛋白的水平，且在同样浓度下(10μM)IMB-85比IMB-C5对冠状病毒的抑制效果更佳，与阳性对照15μM MNP效果相当。
(2)在Huh7.5细胞上，IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白水平的影响
以Huh7.5细胞为病毒宿主，检测了IMB-C5和IMB-85对冠状病毒HCoV-229E N蛋白水平的影响(图10B)。以MOI＝1的病毒载量感染Huh7.5细胞，同时以不同浓度的IMB-85和IMB-C5给药，MNP作为阳性对照。24h后提取细胞总蛋白进行Western blot检测。结果表明IMB-85在Huh7.5细胞中同样可剂量依赖性地降低HCoV-229E N蛋白的水平，且IMB-85比IMB-C5对冠状病毒的抑制效果更佳，与阳性对照15μM MNP效果相当。
实施例9、IMB-C5和IMB-85抑制冠状病毒HCoV-OC43N蛋白水平
检测了IMB-C5和IMB-85对冠状病毒HCoV-OC43N蛋白水平的影响。以MOI＝0.037的病毒载量感染C3A细胞，同时以10μM的IMB-C5，10μM、2μM、0.4μM和0.08μM四个浓度的IMB-85以及阳性对照15μM MNP给药，24h后提取蛋白进行Western blot检测。如图11所示，IMB-85在C3A细胞中能剂量依赖性地降低HCoV-OC43N蛋白水平。
实施例10、IMB-C5抗SARS-CoV-2活性测定
通过CPE法测定IMB-C5抗SARS-CoV-2的活性。Vero E6细胞接种到96孔培养板，37℃培养过夜后弃培养基，以SARS-CoV-2Beta变异株(MOI＝0.05)感染细胞，感染同时用不含FBS的DMEM培养基稀释药物并加入96孔培养板，感染1h后弃去培养液，将以2％FBS的DMEM培养基按浓度梯度稀释的药物加入96孔培养板继续培养，待病毒对照组CPE达4+时观察各组细胞病变程度。结果表明IMB-C5对SARS-CoV-2Beta变异株具有抑制活性。
表3 CPE法测定IMB-C5在Vero E6细胞中抗SARS-CoV-2活性
Figure PCTCN2022099223-appb-000020
实施例11、IMB-C5及IMB-85作用于冠状病毒感染的早期阶段
在HCoV-OC43(MOI＝0.28)感染C3A细胞后的不同时间点加入50μM IMB-C5，通过免疫荧光检测病毒的N蛋白水平(图12)。结果显示在感染病毒的同时加药对病毒的抑制效果最为明显，在感染1h至4h后给药均具有较好的抗病毒作用，而在感染6h及以后给药所产生的抗病毒效果大大减弱。这提示IMB-C5可能作用于冠状病毒感染的早期阶段。
在HCoV-229E(MOI＝10)感染Huh7细胞2h，并在感染时和感染后不同时间点加入5μM IMB-85，通过Western blot和免疫荧光分别检测病毒的N蛋白水平(图13)及dsRNA水平(图14)。结果显示在感染病毒 的同时加药或感染病毒后1-5h之内给药，对病毒的抑制效果较为明显，而在感染6h及以后给药所产生的抗病毒效果有所减弱。这提示IMB-85可能主要作用于冠状病毒感染的早期阶段。
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。

Claims (6)

  1. 式(1)所示的具有抑制冠状病毒活性的化合物。
    Figure PCTCN2022099223-appb-100001
    R1为烷基、取代烷基、含烯基、炔基、环氧烷基、苄基、吡啶等的取代基；优选的，R1为C1-C5的烷基、C1-C5的单烯基、C1-C5的单炔基、含吡啶环的烷基、含苯环的烷基、含取代苯环的烷基、Boc-NH-连接的C1-C3的烷基；
    R2为烷基、取代烷基、含烯基、炔基、苄基等的取代基；优选的，R2为甲基或苄基；
    R3为烷基、取代烷基、环氧烷基、苄基、取代苄基、噻吩甲基等的取代基、取代苯乙基等；优选的，R3为环氧己烷甲基、甲酸酯苄基、甲氧基或氟代甲氧基或氟代甲基修饰的苄基、单卤素取代或多卤素取代的苄基、C1-C5的烷基修饰的苄基、硝基取代苄基、噻吩甲基、取代苯乙基；
    R4为-NH-R5、苄基或取代的苄基、咪唑基、取代哌嗪基；
    R5为烷基、取代烷基、烷基醇类、含酰胺烷基、含酯烷基、烷氧基等；优选的，R5为C1～C5的直链或支链烷基醇类、烷氧烷基、卤素原子和/或甲基或氟甲基取代的苯环和/或苄基、含酰胺、酯的烷基。
    更优选的，所述的化合物为IMB-ZH-2、IMB-ZH-11、IMB-ZH-12、IMB-ZHC-2、IMB-ZHC-15、IMB-2-26、IMB-2-31、IMB-2-32、IMB-3-19、IMB-4-6、IMB-4-12、IMB-4-13、IMB-ZHB-4x、IMB-2-3、IMB-2-8、IMB-68、IMB-82、IMB-83、IMB-84、IMB-85、IMB-92、IMB-93、IMB-3-6、IMB-3-13、IMB-3-15、IMB-3-16、IMB-3-30、IMB-3-45、IMB-3-46、IMB-3-47、IMB-3-57、IMB-3-58、IMB-3-71、IMB-3-72、IMB-3-81。
  2. 权利要求1所述的化合物在制备药物中的应用，所述的药物为抑制冠状病毒的药物，优选的，所述的冠状病毒为人冠状病毒；更优选的，所述的冠状病毒为人α属冠状病毒、人β属冠状病毒；最优选的，所述的冠状病毒为HCoV-229E、HCoV-OC43、SARS-CoV-2。
  3. 权利要求1所述的化合物在制备联用型药物中的应用，所述的药物为抑制冠状病毒的药物，所述的联用型药物中还包括靶向其他病毒靶标的药物，
    优选的，所述的其他病毒靶标为3CLpro和/或RdRp等；
    优选的，所述的冠状病毒为人冠状病毒；优选的，所述的冠状病毒为人α属冠状病毒、人β属冠状病毒；最优选的，所述的冠状病毒为HCoV-229E、HCoV-OC43、SARS-CoV-2。
  4. 一种治疗冠状病毒的药物组合物和/或药物制剂，其含有治疗有效量的权利要求1所述的化合物，以及必要的药用辅料/稀释剂。
  5. 一种治疗因冠状病毒感染导致的疾病的方法，所述的方法包括，向患者施用治疗有效量的权利要求1所述的化合物，或权利要求4所述的药物组合物和/或药物制剂。
  6. 合成权利要求1所述化合物的方法，其特征在于，包括如下三个合成路线：
    Figure PCTCN2022099223-appb-100002
    (1)如反应式1所示合成路线1，
    反应1的反应条件为：
    a.R 3-X,DMF,K 2CO 3,0-100℃；
    b.R 4H,DMSO,60-150℃；
    或(2)如反应式2所示合成路线2，
    Figure PCTCN2022099223-appb-100003
    反应2的反应条件为：
    a.R 3-Br,DIPEA,DMF,0-100℃；
    b.R 1-X,K 2CO 3,DMF,0-100℃；或R 1-OH,PPh 3,DEAD,THF,rt；
    c.R 4H,DMSO,60-150℃.
    或(3)如反应式3所示合成路线3，
    Figure PCTCN2022099223-appb-100004
    反应3的反应条件为：
    a.3-Methoxybenzyl chloride,DIPEA,DMF,rt,24h；
    b.SEMCl,DBU,DMF,rt-60℃；
    c.CH 3I,DMF,K 2CO 3,60℃,1h；
    d.HCl；
    e.R 2-X,DMF,K 2CO 3,60℃,2h；
    f.Ethanolamine,DMSO,130℃。
PCT/CN2022/099223 2021-06-16 2022-06-16 一组具有抗冠状病毒活性的imb-c5系列化合物及其应用 WO2022262820A1 (zh)

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