WO2022116714A1 - Compound and medical use thereof for novel coronavirus pneumonia - Google Patents

Compound and medical use thereof for novel coronavirus pneumonia Download PDF

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WO2022116714A1
WO2022116714A1 PCT/CN2021/124330 CN2021124330W WO2022116714A1 WO 2022116714 A1 WO2022116714 A1 WO 2022116714A1 CN 2021124330 W CN2021124330 W CN 2021124330W WO 2022116714 A1 WO2022116714 A1 WO 2022116714A1
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compound
amino
halogen
hydrogen
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余文颖
来茂德
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中国药科大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D275/00—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings
    • C07D275/04—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D275/06—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings condensed with carbocyclic rings or ring systems with hetero atoms directly attached to the ring sulfur atom
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention relates to the fields of medicinal chemistry and medicinal therapy, in particular to a class of compounds and their medicinal uses for novel coronavirus pneumonia, and belongs to the technical field of pharmacy.
  • 3CLpro is highly conserved among all coronaviruses and plays an important role in mediating viral replication and transcription, so it is considered an ideal protein target for the development of broad-spectrum antiviral drugs. Cys145 and His41 are key residues in the catalytic site of 3CLpro. In addition, there are several pockets in the active site of 3CLpro, such as a glutamine-specific S1 site, a hydrophobic S2 site, and a small S4 site, which together provide ample opportunities for drug design. PLpro of SARS-CoV-2 can inhibit viral replication and reactivate innate immune responses.
  • the active site of PLpro of SARS-CoV-2 contains the classical Cys-His-Asp ternary catalytic molecule, the side chain of Trp106 is located in the oxyanion pore, and it is suggested that the indole ring nitrogen is involved in the whole process. Stabilization of negatively charged tetrahedral intermediates generated during catalysis.
  • LY1 is a novel and potent small molecule inhibitor and a new antiviral drug candidate.
  • the chemical name is 3-((2-(piperazin-1-yl)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide, research shows LY1 can effectively inhibit the viral activity of COVID-19 new coronary pneumonia, and at the same time, it does not cause obvious toxicity at multiple effective doses.
  • the structure is as follows:
  • LY1 may become a therapeutic drug for COVID-19 by inhibiting the activities of 3CLpro and PLpro.
  • the present invention synthesizes a series of compounds with similar structures and effective against new coronary pneumonia.
  • the invention provides a preparation method of a series of compounds, the preparation method is efficient, practical and economical, has a short production cycle, a high yield and is easy for industrial production.
  • the invention verifies the anti-new crown pneumonia activity of a series of compounds, and systematically verifies the compound LY1, thereby providing potential drug molecules for anti-new crown drugs.
  • a class of compounds which is a compound represented by formula I or formula II, or a pharmaceutically acceptable salt or ester thereof:
  • the A ring can be a five-, six-, seven-membered saturated or unsaturated ring, and there are one to two heteroatoms in the ring, and the heteroatoms refer to N, O, and S;
  • R1 is one or more substituents, each independently selected from hydrogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl;
  • R2 is one or two substituents selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, and the halogen refers to fluorine, chlorine, bromine or iodine.
  • R3 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine.
  • R4 is one or more substituents selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, aromatic, heteroaromatic , ring or heterocycle, and the halogen refers to fluorine, chlorine, bromine or iodine,
  • the R1 is one or more substituents, each independently selected from hydrogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, preferably as hydrogen.
  • the R2 is one or two substituents selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine, It is preferably methyl.
  • the R3 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, Bromine or iodine, preferably hydrogen, methyl, hydroxyl, and halogen.
  • the R4 is one or more substituents selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, aromatic, hetero Aromatic, cyclic or heterocyclic,
  • the halogen refers to fluorine, chlorine, bromine or iodine, preferably as
  • the compound is 3-((2-(piperazin-1-yl)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1- Dioxide, referred to as compound LY1, has the following structural formula
  • One of the objects of the present invention is to provide the preparation method of the compound shown in formula I/formula II general formula, and the synthetic route is as follows:
  • Another object of the present invention is to provide the preparation method of compound LY1, and the synthetic route is as follows:
  • the preparation method of compound LY1 is characterized in that, the synthetic route is as follows:
  • step (1) specifically refers to: adding compound 1 into a reactor filled with a solvent, stirring, and dropping ammonia water at 0° C. in an ice bath, after the dropwise addition, the thin-layer tracking detection reaction is completed, and the reaction is completed. After that, it was distilled under reduced pressure to precipitate a solid, which was filtered and dried in vacuo to obtain compound 2.
  • the solvent is one or more of aprotic solvents.
  • Step (2) specifically refers to: adding (compound 2) 1-naphthalenesulfonamide into a reactor filled with an organic solvent, controlling the temperature to 65°C-70°C and stirring; adding ten times the equivalent of cerium sulfate with 2mol/L Dissolve dilute sulfuric acid, control the temperature to 65°C-70°C and stir, slowly add dropwise to the system, start timing from the dropwise addition, stop the reaction after 25-35 minutes, stand to cool and filter with suction, then add dichloromethane to the filtrate to extract , take the dichloromethane layer and dry in vacuo to obtain 5,8-dioxo-dihydronaphthalene. More preferably, the temperature is 65°C, and the reaction time is 27 minutes.
  • the organic solvent is one or more of glacial acetic acid and trifluoroacetic acid.
  • Step (3) specifically refers to: dissolving compound 3, compound 4 and the catalyst with glacial acetic acid, and stirring the reaction at room temperature for 25-30 hours; distilling off the solvent under reduced pressure to obtain a crude product, and the crude product is purified to obtain compound 5;
  • the catalyst is one or more of copper acetate monohydrate, cerium trichloride and triethylamine; more preferably copper acetate monohydrate.
  • Step (4) specifically refers to: compound 5 is deprotected from the amino group to obtain compound LY1; it specifically includes: dissolving compound 5 with organic solvent dichloromethane, passing hydrochloric acid gas, stirring at room temperature, and thin-layer tracking detection reaction; after the reaction, filtering , the compound LY1 is obtained.
  • the organic solvent is one or more of dichloromethane, chloroform and dioxane.
  • step (3) the crude product is purified to obtain compound 5, including:
  • the organic solvent is one or more of dichloromethane and ethyl acetate, preferably dichloromethane;
  • the alcoholic organic solvent is one or more of methanol, ethanol, and isopropanol. Several, preferably isopropanol.
  • the preparation and purification method of the compound LY1 series compounds provided by the invention has the advantages of simple preparation method, low cost and high yield.
  • the compound LY1 with good antitumor activity can be conveniently prepared at low cost by using naphthalenesulfonyl chloride as a raw material.
  • the application of the compound in the preparation of a medicine for preventing and/or treating diseases related to novel coronavirus pneumonia is provided.
  • the compound inhibits the activities of 3CLpro and PLpro with high specificity, and is used as a drug for the treatment of novel coronavirus pneumonia.
  • It can be used as a drug for the treatment of diseases that are resistant to STAT3 inhibitors or gefitinib, including colorectal cancer, lung cancer, and novel coronavirus pneumonia.
  • compound LY1 is particularly effective.
  • Figure 1 The efficacy of LY1 on the reproduction of the new coronavirus was evaluated by quantitative real-time RT-PCR, and the compound LY1 showed good antiviral activity;
  • Figure 2 shows: MTT method, the cytotoxicity of compound LY1 was assessed by MTT method, and the CC50 value of compound LY1 was higher than 15 ⁇ M. as shown in picture 2.
  • the experimental results show that LY1 has excellent antiviral activity and less toxicity in vitro;
  • FIG. 3 Fluorescence resonance energy transfer (FRET) protease assay results show that LY1 can effectively inhibit the activities of SARS-CoV-2 3CLpro and PLpro;
  • FRET Fluorescence resonance energy transfer
  • FIG. 4 Surface plasmon resonance (SPR) analysis results show that LY1 mainly targets 3CLpro protein.
  • N-methyl-5,8-dihydronaphthalene-1-sulfonamide (56.5 g, 238.17 mmol), tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (60 g, 217.90 mmol) and copper acetate monohydrate (5.6g, 28.05mmol) were added to the 2L round-bottomed flask filled with 1200ml of glacial acetic acid, reacted at 25°C, and stirred for 26 hours at this temperature; the solvent was distilled off under reduced pressure to obtain the crude product.
  • the open reading frame of 3CLpro was cloned into the pET28a vector between the multiple cloning sites of Nde I and Xho I. This construct ensures that the expressed protein can be automatically cleaved by 3CLpro itself and can be treated with PreScission protease to remove the 6 x His tag.
  • codon-optimized SARS-CoV-2 nsp3 was inserted into Nde I and EcoR I of pET28a in the aa746-1062 portion, which was fused to the N-terminal SAVLQ protein sequence.
  • the first amino acid (Glu) was mutated to Ser to facilitate digestion of PLP by 3CLpro to remove the N-terminal His tag. All mutants were generated using KOD-plus-neo (TOYOBO, Osaka, Japan).
  • the 3CLpro and PLP constructs were respectively transformed into E. coli BL21(DE3) (Novagen) for expression.
  • individual clones were pre-cultured overnight at 37°C in LB medium containing kanamycin (50 ⁇ g/mL) to generate seed cultures. Then, the seed cultures were inoculated at a ratio of 0.5% isopropyl-D-thiogalactoside (IPTG) at a ratio of 1% to generate sufficient culture to express the protein. After 12 hours of induction of expression, cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4°C.
  • IPTG isopropyl-D-thiogalactoside
  • the pellet was then resuspended in buffer A (20 mM HEPES, 500 mM NaCl, pH 7.5) prior to lysis by sonication. After sonication, lysates were clarified by ultracentrifugation at 18000 rpm for 1.5 h at 4°C and supernatants were used for protein purification using HisTrap FF columns (GE Healthcare). The obtained protein was cleaved overnight at 4°C with PreScission protease against 3CLpro at a molar ratio of 20:1, and then loaded on a Superdex 200 column ( GE Healthcare) was further purified by gel filtration chromatography.
  • buffer A (20 mM HEPES, 500 mM NaCl, pH 7.5
  • lysates were clarified by ultracentrifugation at 18000 rpm for 1.5 h at 4°C and supernatants were used for protein purification using HisTrap FF columns (GE Healthcare).
  • the obtained protein was cleaved overnight at
  • the eluted fractions were concentrated to 15 mg/ml using Amicon (10 kDa cutoff, Millipore). The concentrated protein was then stored at -80°C for crystallization and activity assays.
  • Amicon 10 kDa cutoff, Millipore
  • 3CLpro was used at a molar ratio of 20:1 to remove the N-terminal His tag.
  • Fluorescence resonance energy transfer (FRET) protease assays were performed using Dabcyl-KLSAVLQSGFRKM-Edans-NH2 and CBZ-RLRGG-AMC as fluorogenic substrates, respectively.
  • FRET Fluorescence resonance energy transfer
  • Fluorescence signals for excitation at 320 nm and emission at 405 nm were then immediately measured every 3 seconds using a BioTek Synergy4 plate reader.
  • PLP inhibition assay a similar procedure was performed while monitoring the fluorescence signal at 340 nm (excitation) and 450 nm (emission).
  • IC50 curves were calculated using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). IC50 values from three independent experiments are expressed as mean ⁇ SD.
  • African green monkey kidney Vero E6 cell line was obtained from the American Type Culture Collection (ATCC, No. 1586) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS). ; Gibco Invitrogen) and 1% penicillin-streptomycin (Gibco Invitrogen).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Gibco Invitrogen Gibco Invitrogen
  • penicillin-streptomycin Gabco Invitrogen
  • a clinical isolate of SARS-CoV-2 was propagated in Vero E6 cells and viral titers were determined as previously described (11). All infection experiments were performed under Biosafety Level 3 (BLS-3).
  • LY1 efficacy of LY1 on viral yield was assessed by quantitative real-time RT-PCR.
  • Vero E6 cells were seeded in 96-well plates and infected with SARS-CoV-2 at an MOI of 0.05. Two hours after infection, virus-containing medium was removed, and cells were treated with 0.1% DMSO as a control or 2.5-15 ⁇ M LL1. After 48 hours of treatment, the copy number of viral RNA in the cell supernatant was detected using real-time PCR, and the response was indicated by the expression of RdRP, N and E.
  • Example 2 LY1 inhibits the activity of 3CLpro or PLpro
  • Recombinant SARS-CoV-2 3CLpro and PLpro were purified from E. coli fermentation broth.
  • a fluorescence resonance energy transfer (FRET) protease assay was performed using Dabcyl-KLSAVLQSGFRKM-Edans-NH2 and CBZ-RLRGG-AMC as fluorogenic substrates, respectively.
  • the results are shown in Figure 3, when the concentration was 10 ⁇ M, the inhibitory effects on 3CLpro and PLpro reached 94.4% and 88.6%, respectively.
  • the results showed that compound LY1 could effectively inhibit the activities of SARS-CoV-2 3CLpro and PLpro.
  • FRET fluorescence resonance energy transfer
  • Example 3 LY1 mainly targets 3CLpro protein

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Abstract

Provided in the present application are a compound and the medical use thereof for novel coronavirus pneumonia, which belongs to the technical field of pharmaceuticals. The compound is a compound represented by formula I or formula II, or a pharmaceutically acceptable salt or ester thereof. Further provided is the use of the compound in the preparation of a drug for preventing and/or treating a disease associated with novel coronavirus pneumonia.

Description

一类化合物及其用于新型冠状病毒肺炎的医药用途A class of compounds and their medicinal uses for novel coronavirus pneumonia 技术领域technical field
本发明涉及药物化学和药物治疗学领域，具体涉及一类化合物及其用于新型冠状病毒肺炎的医药用途，属于药学技术领域。The invention relates to the fields of medicinal chemistry and medicinal therapy, in particular to a class of compounds and their medicinal uses for novel coronavirus pneumonia, and belongs to the technical field of pharmacy.
背景技术Background technique
自从SARS-CoV-2引起的COVID-19爆发以来，它已夺走了全球数百万人的生命。此外，尽管先前有人畜共患病暴发，但仍未批准针对紧密相关的冠状病毒，SARS-CoV-1或MERS-CoV的抗病毒药物或疫苗。对于COVID-19的抗病毒药物的设计和开发，了解复制周期和关键基因组要素仍然至关重要。冠状病毒是有包膜的，正义的，单链RNA病毒。两种病毒半胱氨酸蛋白酶3CLpro和PLpro扮演关键的角色。3CLpro在所有冠状病毒中高度保守，并且在介导病毒复制和转录中起着重要作用，因此被认为是理想的蛋白目标是开发广谱抗病毒药物。Cys145和His41是3CLpro催化位点的关键残基。此外，3CLpro活性位点上有几个口袋，例如谷氨酰胺特异性S1位点，疏水性S2位点和小的S4位点，这些共同为药物设计提供了充足的机会。SARS-CoV-2的PLpro可以抑制病毒复制并重新激活固有的免疫应答。与SARS-CoV PLpro相似，SARS-CoV-2的PLpro的活性位点包含经典的Cys-His-Asp三元催化分子，Trp106的侧链位于氧阴离子孔内，并建议吲哚环氮参与在整个催化过程中产生的带负电荷的四面体中间体的稳定化。Since the outbreak of COVID-19, caused by SARS-CoV-2, it has claimed millions of lives around the world. Furthermore, despite previous zoonotic outbreaks, no antiviral drugs or vaccines have been approved against the closely related coronaviruses, SARS-CoV-1 or MERS-CoV. Understanding the replication cycle and key genomic elements remains critical for the design and development of antiviral drugs for COVID-19. Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses. Two viral cysteine proteases, 3CLpro and PLpro, play key roles. 3CLpro is highly conserved among all coronaviruses and plays an important role in mediating viral replication and transcription, so it is considered an ideal protein target for the development of broad-spectrum antiviral drugs. Cys145 and His41 are key residues in the catalytic site of 3CLpro. In addition, there are several pockets in the active site of 3CLpro, such as a glutamine-specific S1 site, a hydrophobic S2 site, and a small S4 site, which together provide ample opportunities for drug design. PLpro of SARS-CoV-2 can inhibit viral replication and reactivate innate immune responses. Similar to SARS-CoV PLpro, the active site of PLpro of SARS-CoV-2 contains the classical Cys-His-Asp ternary catalytic molecule, the side chain of Trp106 is located in the oxyanion pore, and it is suggested that the indole ring nitrogen is involved in the whole process. Stabilization of negatively charged tetrahedral intermediates generated during catalysis.
发明内容SUMMARY OF THE INVENTION
LY1是一种新型有效的小分子抑制剂，是抗病毒候选新药。化学名为3-((2-(哌嗪-1-基)苯基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物，研究表明LY1可以有效抑制COVID-19新冠肺炎的病毒活性，同时在多次有效剂量下，它不会引起明显的毒性。结构式如下：LY1 is a novel and potent small molecule inhibitor and a new antiviral drug candidate. The chemical name is 3-((2-(piperazin-1-yl)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide, research shows LY1 can effectively inhibit the viral activity of COVID-19 new coronary pneumonia, and at the same time, it does not cause obvious toxicity at multiple effective doses. The structure is as follows:
Figure PCTCN2021124330-appb-000001
Figure PCTCN2021124330-appb-000001
因此LY1可以通过抑制3CLpro和PLpro的活性，可能成为COVID-19新冠肺炎的治疗药物。Therefore, LY1 may become a therapeutic drug for COVID-19 by inhibiting the activities of 3CLpro and PLpro.
本发明合成一系列结构类似，对新冠肺炎有效的化合物。The present invention synthesizes a series of compounds with similar structures and effective against new coronary pneumonia.
本发明提供一系列化合物的制备方法，该制备方法高效实用经济、生产周期短，收率较高，易于工业化生产。The invention provides a preparation method of a series of compounds, the preparation method is efficient, practical and economical, has a short production cycle, a high yield and is easy for industrial production.
本发明验证了系列化合物抗新冠肺炎活性，对化合物LY1进行了系统的验证，为抗新冠药物提供潜在药物分子。The invention verifies the anti-new crown pneumonia activity of a series of compounds, and systematically verifies the compound LY1, thereby providing potential drug molecules for anti-new crown drugs.
技术方案：为解决上述技术问题，本发明采用的技术方案为：Technical scheme: in order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is:
一种化合物，所述化合物的结构式如下A compound whose structural formula is as follows
Figure PCTCN2021124330-appb-000002
Figure PCTCN2021124330-appb-000002
本发明采用的技术方案为：The technical scheme adopted in the present invention is:
第一方面，提供一类化合物，为式I或式II所示的化合物，或其药学上可接受的盐或酯：In the first aspect, a class of compounds is provided, which is a compound represented by formula I or formula II, or a pharmaceutically acceptable salt or ester thereof:
Figure PCTCN2021124330-appb-000003
Figure PCTCN2021124330-appb-000003
其中，A环可以是五、六、七元饱和或者不饱和环，环内存在一到两个杂原子，杂原子指N、O、S；Among them, the A ring can be a five-, six-, seven-membered saturated or unsaturated ring, and there are one to two heteroatoms in the ring, and the heteroatoms refer to N, O, and S;
R1为一个或多个取代基，各自独立地选自氢、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基；R1 is one or more substituents, each independently selected from hydrogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl;
R2为一个或者两个取代基，选自氢、卤素、C1～C3烷基、C1～C3烷氧基、所述卤素指氟、氯、溴或碘。R2 is one or two substituents selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, and the halogen refers to fluorine, chlorine, bromine or iodine.
R3为取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，所述卤素指氟、氯、溴或碘。R3 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine.
R4为一个或多个取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，芳香族、杂芳族、环或杂环，所述卤素指氟、氯、溴或碘、
Figure PCTCN2021124330-appb-000004
Figure PCTCN2021124330-appb-000005
R4 is one or more substituents selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, aromatic, heteroaromatic , ring or heterocycle, and the halogen refers to fluorine, chlorine, bromine or iodine,
Figure PCTCN2021124330-appb-000004
Figure PCTCN2021124330-appb-000005
在一些实施例中，A环吡咯基、咪唑基、吡唑基、呋喃基、四氢呋喃基、噻吩基、四氢呋喃基、噻唑基、苯环、吡嗪基、嘧啶基、哒嗪基、
Figure PCTCN2021124330-appb-000006
作为优选为苯环。所述R1为一个或多个取代基，各自独立地选自氢、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，作为优选为氢。 In some embodiments, A-ring pyrrolyl, imidazolyl, pyrazolyl, furanyl, tetrahydrofuranyl, thienyl, tetrahydrofuranyl, thiazolyl, benzene ring, pyrazinyl, pyrimidinyl, pyridazinyl,
Figure PCTCN2021124330-appb-000006
Preferably, it is a benzene ring. The R1 is one or more substituents, each independently selected from hydrogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, preferably as hydrogen.
所述R2为一个或者两个取代基，选自氢、卤素、C1～C3烷基、C1～C3烷氧基、或C1～C3烷氧酰基，所述卤素指氟、氯、溴或碘，作为优选为甲基。The R2 is one or two substituents selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine, It is preferably methyl.
所述R3为取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，所述卤素指氟、氯、溴或碘，作为优选为氢、甲基、羟基、卤素。The R3 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, Bromine or iodine, preferably hydrogen, methyl, hydroxyl, and halogen.
所述R4为一个或多个取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，芳香族、杂芳族、环或杂环，
Figure PCTCN2021124330-appb-000007
Figure PCTCN2021124330-appb-000008
所述卤素指氟、氯、溴或碘，作为优选为
Figure PCTCN2021124330-appb-000009
The R4 is one or more substituents selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, aromatic, hetero Aromatic, cyclic or heterocyclic,
Figure PCTCN2021124330-appb-000007
Figure PCTCN2021124330-appb-000008
The halogen refers to fluorine, chlorine, bromine or iodine, preferably as
Figure PCTCN2021124330-appb-000009
在上述优选的基础上，化合物为3-((2-(哌嗪-1-基)苯基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物，简称化合物LY1，结构式如下On the basis of the above preference, the compound is 3-((2-(piperazin-1-yl)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1- Dioxide, referred to as compound LY1, has the following structural formula
Figure PCTCN2021124330-appb-000010
Figure PCTCN2021124330-appb-000010
药理实验证明，该化合物对新冠病毒有明显的抑制作用。Pharmacological experiments have shown that the compound has a significant inhibitory effect on the new coronavirus.
本发明的目的之一在于提供式I/式II通式所示的化合物的制备方法，合成路线如下：One of the objects of the present invention is to provide the preparation method of the compound shown in formula I/formula II general formula, and the synthetic route is as follows:
Figure PCTCN2021124330-appb-000011
Figure PCTCN2021124330-appb-000011
本发明的另一目的在于提供化合物LY1的制备方法，合成路线如下：化合物LY1的制备方法，其特征在于，合成路线如下：Another object of the present invention is to provide the preparation method of compound LY1, and the synthetic route is as follows: The preparation method of compound LY1 is characterized in that, the synthetic route is as follows:
Figure PCTCN2021124330-appb-000012
Figure PCTCN2021124330-appb-000012
包括：include:
(1)(化合物1)1-萘磺酰氯经取代反应制得(化合物2)1-萘磺酰胺；(1) (Compound 1) 1-naphthalenesulfonyl chloride is subjected to substitution reaction to obtain (compound 2) 1-naphthalenesulfonamide;
(2)(化合物2)1-萘磺酰胺经氧化反应制得(化合物3)5，8-二氧代-二氢萘；(2) (Compound 2) 1-naphthalenesulfonamide is oxidized to obtain (compound 3) 5,8-dioxo-dihydronaphthalene;
(3)(化合物3)5，8-二氧代-二氢萘再与(化合物4)叔丁基4-(2-氨基苯基)哌嗪-1-甲酸酯发生取代反应得到(化合物5)(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)；(3) (compound 3) 5,8-dioxo-dihydronaphthalene is then substituted with (compound 4) tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate to obtain (compound 4) 5) (4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylic acid tert-butyl ester);
(4)(化合物5)(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)氨基脱保护得到目标化合物(化合物LY1)4-((2-(哌嗪-1-基)苯基)氨)-5H-萘并[1，8-cd]异噻唑-5-酮1，1-二氧化物。(4) (Compound 5) (4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1 - Deprotection of amino acid tert-butyl ester) to obtain the target compound (compound LY1) 4-((2-(piperazin-1-yl)phenyl)amino)-5H-naphtho[1,8-cd]isothiazole -5-keto 1,1-dioxide.
进一步的，步骤(1)具体是指：将化合物1加入到盛有溶剂的反应器中，搅拌，在冰浴中滴加0℃的氨水，滴加完毕后，薄层跟踪检测反应，反应完成后，减压蒸馏，析出固体，过滤，真空干燥，得化合物2。Further, step (1) specifically refers to: adding compound 1 into a reactor filled with a solvent, stirring, and dropping ammonia water at 0° C. in an ice bath, after the dropwise addition, the thin-layer tracking detection reaction is completed, and the reaction is completed. After that, it was distilled under reduced pressure to precipitate a solid, which was filtered and dried in vacuo to obtain compound 2.
优选的，所述的溶剂为非质子性溶剂中的一种或几种。Preferably, the solvent is one or more of aprotic solvents.
步骤(2)具体是指：将(化合物2)1-萘磺酰胺加入盛有有机溶剂的反应器中，控温65℃-70℃并搅拌；将十倍当量硫酸高铈用2mol/L的稀硫酸溶解，控温65℃-70℃并搅拌，缓慢滴加入至体系中，从滴加开始计时，25-35分钟后停止反应，放置冷却后抽滤，随后在滤液中加入二氯甲烷萃取，取二氯甲烷层，真空干燥，得5，8-二氧代-二氢萘。作为更优选，温度为65℃，反应时间为27分钟。Step (2) specifically refers to: adding (compound 2) 1-naphthalenesulfonamide into a reactor filled with an organic solvent, controlling the temperature to 65°C-70°C and stirring; adding ten times the equivalent of cerium sulfate with 2mol/L Dissolve dilute sulfuric acid, control the temperature to 65℃-70℃ and stir, slowly add dropwise to the system, start timing from the dropwise addition, stop the reaction after 25-35 minutes, stand to cool and filter with suction, then add dichloromethane to the filtrate to extract , take the dichloromethane layer and dry in vacuo to obtain 5,8-dioxo-dihydronaphthalene. More preferably, the temperature is 65°C, and the reaction time is 27 minutes.
进一步的，步骤(2)中，所述有机溶剂为冰乙酸、三氟醋酸中的一种或几种。Further, in step (2), the organic solvent is one or more of glacial acetic acid and trifluoroacetic acid.
步骤(3)具体是指：将化合物3、化合物4与催化剂用冰乙酸溶解，常温下搅拌反应25-30小时；减压蒸馏除去溶剂，得粗品，粗品经纯化得化合物5；Step (3) specifically refers to: dissolving compound 3, compound 4 and the catalyst with glacial acetic acid, and stirring the reaction at room temperature for 25-30 hours; distilling off the solvent under reduced pressure to obtain a crude product, and the crude product is purified to obtain compound 5;
所述催化剂为一水合乙酸铜、三氯化铈、三乙胺中的一种或几种；更优选为一水合乙酸铜。The catalyst is one or more of copper acetate monohydrate, cerium trichloride and triethylamine; more preferably copper acetate monohydrate.
步骤(4)具体是指：化合物5脱去氨基保护得到化合物LY1；具体包括：用有机溶剂二氯甲烷溶解化合物5，通入盐酸气体，室温搅拌，薄层跟踪检测反应；反应结束后，过滤，即得化合物LY1。Step (4) specifically refers to: compound 5 is deprotected from the amino group to obtain compound LY1; it specifically includes: dissolving compound 5 with organic solvent dichloromethane, passing hydrochloric acid gas, stirring at room temperature, and thin-layer tracking detection reaction; after the reaction, filtering , the compound LY1 is obtained.
步骤(4)中，所述有机溶剂为二氯甲烷、氯仿，二氧六环中的一种或几种。In step (4), the organic solvent is one or more of dichloromethane, chloroform and dioxane.
优选的，步骤(3)中，粗品经纯化得化合物5，包括：Preferably, in step (3), the crude product is purified to obtain compound 5, including:
在粗品中加入无水乙醇，110℃加热沸腾，液相监测反应产物全部转化后，停止加热，自然冷却搅拌6h，过滤得滤饼，柱层析后，加入10倍当量的有机溶剂完全溶解后，滴加入25倍当量的醇类有机溶剂，旋蒸后除去有机溶剂，待晶体全部析出，过滤得化合物5。Add absolute ethanol to the crude product, heat and boil at 110 °C, and monitor the liquid phase after the reaction product is completely converted, stop heating, cool and stir naturally for 6 hours, filter to obtain a filter cake, after column chromatography, add 10 times the equivalent of organic solvent to dissolve completely , 25 times the equivalent of an alcohol organic solvent was added dropwise, and the organic solvent was removed after rotary evaporation. After all the crystals were precipitated, compound 5 was obtained by filtration.
进一步的，纯化过程中，所述有机溶剂为二氯甲烷、乙酸乙酯中的一种或几种，优选为二氯甲烷；醇类有机溶剂为甲醇、乙醇、异丙醇中的一种或几种，优选为异丙醇。Further, in the purification process, the organic solvent is one or more of dichloromethane and ethyl acetate, preferably dichloromethane; the alcoholic organic solvent is one or more of methanol, ethanol, and isopropanol. Several, preferably isopropanol.
本发明提供的化合物LY1系列化合物的制备纯化方法，制备方法简单，成本低，收率高，以萘磺酰氯为原料，以较低的成本方便地制备了有良好抗肿瘤活性的化合物LY1。The preparation and purification method of the compound LY1 series compounds provided by the invention has the advantages of simple preparation method, low cost and high yield. The compound LY1 with good antitumor activity can be conveniently prepared at low cost by using naphthalenesulfonyl chloride as a raw material.
第三方面，提供所述的化合物在制备预防和/或治疗与新型冠状病毒肺炎有关疾病的药物中的应用。In a third aspect, the application of the compound in the preparation of a medicine for preventing and/or treating diseases related to novel coronavirus pneumonia is provided.
该化合物通过高特异性来抑制3CLpro和PLpro的活性，用作治疗新型冠状病毒肺炎的药物。The compound inhibits the activities of 3CLpro and PLpro with high specificity, and is used as a drug for the treatment of novel coronavirus pneumonia.
能够作为治疗包括结直肠癌，肺癌、新型冠状病毒肺炎在内的对STAT3抑制剂类药物或者吉非 替尼产生耐药性的疾病的药物。It can be used as a drug for the treatment of diseases that are resistant to STAT3 inhibitors or gefitinib, including colorectal cancer, lung cancer, and novel coronavirus pneumonia.
其中化合物LY1效果尤佳。Among them, compound LY1 is particularly effective.
附图说明Description of drawings
图1为：通过定量实时RT-PCR评估LY1对新冠病毒繁殖的功效，化合物LY1表现出良好的抗病毒活性；Figure 1: The efficacy of LY1 on the reproduction of the new coronavirus was evaluated by quantitative real-time RT-PCR, and the compound LY1 showed good antiviral activity;
图2为：MTT法，化合物LY1的细胞毒性通过MTT法评估，化合物LY1的CC50值高于15μM。如图2所示。实验结果表明，LY1在体外具有优秀的抗病毒活性和较小的毒性；Figure 2 shows: MTT method, the cytotoxicity of compound LY1 was assessed by MTT method, and the CC50 value of compound LY1 was higher than 15 μM. as shown in picture 2. The experimental results show that LY1 has excellent antiviral activity and less toxicity in vitro;
图3为：荧光共振能量转移(FRET)蛋白酶测定结果表明LY1能有效抑制SARS-CoV-2 3CLpro和PLpro的活性；Figure 3: Fluorescence resonance energy transfer (FRET) protease assay results show that LY1 can effectively inhibit the activities of SARS-CoV-2 3CLpro and PLpro;
图4为：表面等离振子共振(SPR)分析结果表明LY1主要靶向3CLpro蛋白。Figure 4: Surface plasmon resonance (SPR) analysis results show that LY1 mainly targets 3CLpro protein.
具体实施方式Detailed ways
为了进一步阐明本发明，下面给出一系列实施例，这些实施例完全是例证性的，它们仅用来对本发明具体描述，不应当理解为对本发明的限制。In order to further illustrate the present invention, a series of examples are given below, which are purely illustrative, and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.
一、化合物及其制备1. Compounds and their preparation
前体准备Precursor preparation
1)、制备1-萘磺酰胺1), preparation of 1-naphthalenesulfonamide
Figure PCTCN2021124330-appb-000013
Figure PCTCN2021124330-appb-000013
将1-萘磺酰氯(5g，21.9mmol)加入到盛有200ml丙酮的1L圆底烧瓶中，搅拌，在冰浴中滴加240ml 0℃的氨水，滴加完毕后，薄层跟踪检测反应，反应完成后，减压蒸馏出有机溶剂和氨水分解出的氨气，析出固体，过滤，真空干燥，得1-萘磺酰胺(4.35g，收率96.7％)。该化合物无需进一步纯化，直接用于下一步反应。实验数据如下：1-Naphthalenesulfonyl chloride (5g, 21.9mmol) was added to a 1L round-bottomed flask containing 200ml of acetone, stirred, and 240ml of ammonia water at 0°C was added dropwise in an ice bath. After the reaction was completed, the organic solvent and ammonia gas decomposed by ammonia water were distilled off under reduced pressure, and a solid was precipitated, filtered, and dried in vacuo to obtain 1-naphthalenesulfonamide (4.35 g, yield 96.7%). This compound was used directly in the next reaction without further purification. The experimental data is as follows:
Mp 147～149℃. 1H NMR(500MHz,DMSO-d6)δ:8.65(d,J＝8.5Hz,1H),8.19(d,J＝8.2Hz,1H),8.14(d,J＝6.8Hz,1H),8.08(d,J＝7.4Hz,1H),7.76–7.59(m,5H).HR-MS(ESI)calcd for C 10H 9NO 2S[M+Na] +230.0246,found 230.0244 Mp 147～149℃. 1 H NMR(500MHz, DMSO-d6)δ:8.65(d,J=8.5Hz,1H),8.19(d,J=8.2Hz,1H),8.14(d,J=6.8Hz ,1H),8.08(d,J=7.4Hz,1H),7.76–7.59(m,5H).HR-MS(ESI)calcd for C 10 H 9 NO 2 S[M+Na] + 230.0246,found 230.0244
2)、制备5，8-二氧代-二氢萘2), preparation 5,8-dioxo-dihydronaphthalene
Figure PCTCN2021124330-appb-000014
Figure PCTCN2021124330-appb-000014
将无水硫酸高铈(160g，677.45mmol)用750ml 2mol/L稀硫酸溶解，控温65℃。将1-萘磺酰胺(10g，48.49mmol)加入盛有200ml冰乙酸的500ml圆底烧瓶中，控温65℃，该温度下搅拌，缓慢滴加入至硫酸高铈水相体系中，从滴加开始计时，薄层色谱监测，27分钟后停止反应，待反应液冷却后，过滤，滤液用1200ml二氯甲烷萃取，干燥除水后低压旋蒸，得到淡黄色固体，真空干燥，得5，8-二氧代-二氢萘(6.72g，收率58.43％)。直接用于下一步反应无需纯化。实验数据如下：Anhydrous cerium sulfate (160g, 677.45mmol) was dissolved in 750ml of 2mol/L dilute sulfuric acid, and the temperature was controlled to 65°C. 1-Naphthalenesulfonamide (10g, 48.49mmol) was added to a 500ml round-bottomed flask containing 200ml of glacial acetic acid, and the temperature was controlled to 65°C. Start timing, monitor by thin-layer chromatography, stop the reaction after 27 minutes, after the reaction solution is cooled, filter, extract the filtrate with 1200 ml of dichloromethane, dry and remove water, and then rotate at low pressure to obtain a pale yellow solid, which is dried in vacuo to obtain 5,8 -Dioxo-dihydronaphthalene (6.72 g, 58.43% yield). It was used directly in the next reaction without purification. The experimental data is as follows:
Mp 186～188℃.HR-MS(ESI)calcd for C 10H 7NO 4S[M+Na] +259.9988,found 259.9989 Mp 186～188℃.HR-MS(ESI)calcd for C 10 H 7 NO 4 S[M+Na] + 259.9988, found 259.9989
实施例1Example 1
Figure PCTCN2021124330-appb-000015
Figure PCTCN2021124330-appb-000015
1.1制备(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)1.1 Preparation of (4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalene-2-yl)amino)phenyl)piperazine-1-carboxylic acid tert-butyl ester)
Figure PCTCN2021124330-appb-000016
Figure PCTCN2021124330-appb-000016
将5，8-二氧代-二氢萘(56.5g，238.17mmol)、叔丁基4-(2-氨基苯基)哌嗪-1-甲酸酯(60g，217.90mmol)与一水合乙酸铜(5.6g，28.05mmol)加入到盛有1200ml冰乙酸的2L圆底烧瓶中，25℃反应，该温度下搅拌反应26小时；减压蒸馏除去溶剂，得粗品。Combine 5,8-dioxo-dihydronaphthalene (56.5 g, 238.17 mmol), tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (60 g, 217.90 mmol) with acetic acid monohydrate Copper (5.6g, 28.05mmol) was added to a 2L round-bottomed flask containing 1200ml of glacial acetic acid, and the reaction was carried out at 25°C. The reaction was stirred at this temperature for 26 hours; the solvent was distilled off under reduced pressure to obtain a crude product.
在粗品中加入1500ml的无水乙醇，110℃加热沸腾，液相监测反应产物全部转化后，停止加热，自然冷却搅拌6h，过滤得滤饼，柱层析后除去一般杂质，后加入10倍当量的二氯甲烷完全溶解后，缓慢滴加25倍当量的异丙醇，33℃低压旋蒸后除去相同量的二氯甲烷，待晶体全部析出，过滤，得紫红色晶体，低压干燥得4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯，纯度为97％.(67.53g，收率83％)。1500ml of absolute ethanol was added to the crude product, heated and boiled at 110°C, and the liquid phase monitoring reaction product was completely converted, then stopped heating, cooled and stirred for 6 hours, filtered to obtain a filter cake, and after column chromatography, general impurities were removed, and 10 times the equivalent was added. After the dichloromethane was completely dissolved, 25 times the equivalent of isopropanol was slowly added dropwise, and the same amount of dichloromethane was removed after low-pressure rotary evaporation at 33°C. (2-((1,4-Dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl ester, purity 97 %. (67.53 g, 83% yield).
实验数据如下：The experimental data is as follows:
Mp 189～190℃. 1H NMR(500MHz,DMSO-d 6)δ8.85(s,1H),8.39(dd,J＝8.0,1.3Hz,1H),8.29(dd,J＝7.8,1.3Hz,1H),8.05(t,J＝7.8Hz,1H),7.46–7.40(m,3H),7.23(d,J＝4.2Hz,2H),6.12(s,1H),3.45(t,J＝4.8Hz,4H),2.82(t,J＝5.0Hz,4H),1.40(s,9H).HR-MS(ESI)calcd for C 25H 28N 4O 6S,[M+Na] +535.1622,found 535.1619 Mp 189～190℃. 1 H NMR(500MHz, DMSO-d 6 )δ8.85(s,1H),8.39(dd,J=8.0,1.3Hz,1H),8.29(dd,J=7.8,1.3Hz ,1H),8.05(t,J＝7.8Hz,1H),7.46-7.40(m,3H),7.23(d,J＝4.2Hz,2H),6.12(s,1H),3.45(t,J＝ 4.8Hz, 4H), 2.82(t, J=5.0Hz, 4H), 1.40(s, 9H).HR-MS(ESI) calcd for C 25 H 28 N 4 O 6 S, [M+Na] + 535.1622 ,found 535.1619
1.2制备4-((2-(哌嗪-1-基)苯基)胺)-5H-萘并[1，8-cd]异噻唑-5-酮1，1-二氧化物1.2 Preparation of 4-((2-(piperazin-1-yl)phenyl)amine)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000017
Figure PCTCN2021124330-appb-000017
将(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)(2g，3.78mmol)加入到盛有2ml二氯甲烷的50ml圆底烧瓶中，随后通入盐酸气，薄层跟踪检测反应；反应结束后，过滤得滤饼，减压干燥分离出4-((2-(哌嗪-1-基)苯基)胺)-5H-萘并[1，8-cd]异噻唑-5-酮1，1-二氧化物(1.95g，收率97％)。实验数据如下：(4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl ester ) (2g, 3.78mmol) was joined in the 50ml round-bottomed flask containing 2ml of dichloromethane, then passed into hydrochloric acid gas, and the thin layer tracked and detected the reaction; ((2-(Piperazin-1-yl)phenyl)amine)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide (1.95 g, 97% yield ). The experimental data is as follows:
Mp 200～201℃. 1H NMR(500MHz，DMSO-d6)δ：9.11(s，1H)，8.74(s，2H)，8.42(d，J＝7.4Hz，1H)，8.08(m，2H)，7.44(d，J＝7.8Hz，1H)，7.32(t，J＝7.7Hz，1H)，7.28–7.19(m，2H)，5.86(s，1H)，3.18–3.10(m，8H).HR-MS(ESI)calcd for C20H18N4O3S,[M+H]+395.1172,found 395.1171 Mp 200～201℃. 1 H NMR (500MHz, DMSO-d6)δ: 9.11(s, 1H), 8.74(s, 2H), 8.42(d, J=7.4Hz, 1H), 8.08(m, 2H) , 7.44 (d, J=7.8Hz, 1H), 7.32 (t, J=7.7Hz, 1H), 7.28–7.19 (m, 2H), 5.86 (s, 1H), 3.18–3.10 (m, 8H). HR-MS(ESI)calcd for C20H18N4O3S,[M+H]+395.1172,found 395.1171
实施例2Example 2
Figure PCTCN2021124330-appb-000018
Figure PCTCN2021124330-appb-000018
2.1制备(1,4-二氧代5-氨磺酰基-1,4-二氢萘-2-基)氨基)-烟酸2.1 Preparation of (1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)-nicotinic acid
Figure PCTCN2021124330-appb-000019
Figure PCTCN2021124330-appb-000019
将5，8-二氧代-二氢萘1(237mg,1mmol)、5-氨基烟酸(172mg,1.2mmol).与一水合乙酸铜(20mg,0.1mmol)加入到盛有5ml冰乙酸的25ml圆底烧瓶中，加热回流，该温度下搅拌反应3小时；减压蒸馏除去溶剂，液相色谱分离出3-(萘-2-基氨基)-5H-萘[1,8-cd]异噻唑-5-酮1,1-二氧化物(221mg，收率71％)。5,8-dioxo-dihydronaphthalene 1 (237mg, 1mmol), 5-aminonicotinic acid (172mg, 1.2mmol). and copper acetate monohydrate (20mg, 0.1mmol) were added to a solution containing 5ml of glacial acetic acid. In a 25ml round-bottomed flask, heated to reflux, and the reaction was stirred for 3 hours at this temperature; the solvent was distilled off under reduced pressure, and 3-(naphthalene-2-ylamino)-5H-naphthalene[1,8-cd]iso was separated by liquid chromatography. Thiazol-5-one 1,1-dioxide (221 mg, 71% yield).
实验数据如下The experimental data is as follows
1H NMR(500MHz,DMSO):δ8.31(s,1H),8.04(d,J＝7.6Hz,1H),7.98(d,J＝2.5Hz,1H),7.94(d,J＝7.5Hz,1H),7.78(t,J＝7.5Hz,1H),7.44(s,1H),7.40(s,2H),5.62(s,1H),5.34(s,2H).HRMS(ESI)for C 16H 11N 3O 6SNa[M+Na]+:calcd,396.0266；found,396.0270. 1 H NMR (500MHz, DMSO): δ8.31 (s, 1H), 8.04 (d, J=7.6Hz, 1H), 7.98 (d, J=2.5Hz, 1H), 7.94 (d, J=7.5Hz) ,1H),7.78(t,J=7.5Hz,1H),7.44(s,1H),7.40(s,2H),5.62(s,1H),5.34(s,2H).HRMS(ESI)for C 16 H 11 N 3 O 6 SNa[M+Na]+: calcd, 396.0266; found, 396.0270.
实施例3Example 3
Figure PCTCN2021124330-appb-000020
Figure PCTCN2021124330-appb-000020
3.1制备(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)3.1 Preparation of (4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylic acid tert-butyl ester)
Figure PCTCN2021124330-appb-000021
Figure PCTCN2021124330-appb-000021
将5，8-二氧代-二氢萘(0.5g，2.11mmol)、叔丁基4-(2-氨基苯基)哌嗪-1-甲酸酯(0.787g，2.53mmol)与一水合乙酸铜(42mg，0.21mmol)加入到盛有12ml冰乙酸的25ml圆底烧瓶中，118℃加热回流，该温度下搅拌反应3小时；减压蒸馏除去溶剂，液相色谱分离出化合物(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)(0.568g，收率51％)。实验数据如下：5,8-Dioxo-dihydronaphthalene (0.5 g, 2.11 mmol), tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (0.787 g, 2.53 mmol) were combined with monohydrate Copper acetate (42mg, 0.21mmol) was added to the 25ml round-bottomed flask containing 12ml of glacial acetic acid, heated to reflux at 118°C, and stirred for 3 hours at this temperature; the solvent was distilled off under reduced pressure, and the compound (4- (2-((1,4-Dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl ester) (0.568g , the yield is 51%). The experimental data is as follows:
Mp 189～190℃. 1H NMR(500MHz,DMSO-d 6)δ8.85(s,1H),8.39(dd,J＝8.0,1.3Hz,1H),8.29(dd,J＝7.8,1.3Hz,1H),8.05(t,J＝7.8Hz,1H),7.46–7.40(m,3H),7.23(d,J＝4.2Hz,2H),6.12(s,1H),3.45(t,J＝4.8Hz,4H),2.82(t,J＝5.0Hz,4H),1.40(s,9H).HR-MS(ESI)calcd for C 25H 28N 4O 6S,[M+Na] +535.1622,found 535.1619 Mp 189～190℃. 1 H NMR (500MHz, DMSO-d 6 )δ8.85(s,1H),8.39(dd,J=8.0,1.3Hz,1H),8.29(dd,J=7.8,1.3Hz ,1H),8.05(t,J＝7.8Hz,1H),7.46–7.40(m,3H),7.23(d,J＝4.2Hz,2H),6.12(s,1H),3.45(t,J＝ 4.8Hz, 4H), 2.82(t, J=5.0Hz, 4H), 1.40(s, 9H).HR-MS(ESI) calcd for C 25 H 28 N 4 O 6 S, [M+Na] + 535.1622 ,found 535.1619
3.2制备4-((2-(哌嗪-1-基)苯基)胺)-5H-萘并[1，8-cd]异噻唑-5-酮1，1-二氧化物3.2 Preparation of 4-((2-(piperazin-1-yl)phenyl)amine)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000022
Figure PCTCN2021124330-appb-000022
将(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)(200mg，0.378mmol)加入到盛有2ml二氯甲烷的10ml圆底烧瓶中，随后加入2ml三氟醋酸，室温搅拌，薄层跟踪检测反应；反应结束后，减压蒸馏除去溶剂，液相色谱分离出化合物制备4-((2-(哌嗪-1-基)苯基)胺)-5H-萘并[1，8-cd]异噻唑-5-酮1，1-二氧化物(149mg，收率95％)。实验数据如下：(4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl ester ) (200mg, 0.378mmol) was added to the 10ml round-bottomed flask containing 2ml of dichloromethane, followed by adding 2ml of trifluoroacetic acid, stirring at room temperature, and the thin-layer tracking detection reaction; after the reaction, the solvent was distilled off under reduced pressure, and the liquid phase The compound was isolated by chromatography to prepare 4-((2-(piperazin-1-yl)phenyl)amine)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide ( 149 mg, 95% yield). The experimental data is as follows:
Mp 200～201℃. 1H NMR(500MHz，DMSO-d 6)δ：9.11(s，1H)，8.74(s，2H)，8.42(d，J＝7.4Hz，1H)，8.08(m，2H)，7.44(d，J＝7.8Hz，1H)，7.32(t，J＝7.7Hz，1H)，7.28–7.19(m，2H)，5.86(s，1H)，3.18–3.10(m，8H).HR-MS(ESI)calcd for C 20H 18N 4O 3S,[M+H] +395.1172,found 395.1171。 Mp 200～201°C. 1 H NMR (500 MHz, DMSO-d 6 ) δ: 9.11 (s, 1H), 8.74 (s, 2H), 8.42 (d, J=7.4 Hz, 1H), 8.08 (m, 2H) ), 7.44 (d, J=7.8Hz, 1H), 7.32 (t, J=7.7Hz, 1H), 7.28–7.19 (m, 2H), 5.86 (s, 1H), 3.18–3.10 (m, 8H) .HR-MS (ESI) calcd for C20H18N4O3S , [ M + H] + 395.1172 , found 395.1171.
实施例4Example 4
Figure PCTCN2021124330-appb-000023
Figure PCTCN2021124330-appb-000023
制备7-((4-羟苯基)氨基)-5,8-二氧-5,8-二氢萘-1-磺酰胺Preparation of 7-((4-hydroxyphenyl)amino)-5,8-dioxo-5,8-dihydronaphthalene-1-sulfonamide
Figure PCTCN2021124330-appb-000024
Figure PCTCN2021124330-appb-000024
将5，8-二氧代-二氢萘(100mg，0.421mmol)、4-氨基苯酚(55.2mg，0.506mmol)与一水合乙酸铜(16.83mg，0.084mmol)加入25mL圆底烧瓶中，加入5ml冰乙酸，使之溶解，在室温下反应3小时，反应完全后，减压蒸馏除去溶剂，液相色谱分离得到7-((4-羟苯基)氨基)-5,8-二氧-5,8-二氢萘-1-磺酰胺。5,8-dioxo-dihydronaphthalene (100 mg, 0.421 mmol), 4-aminophenol (55.2 mg, 0.506 mmol) and copper acetate monohydrate (16.83 mg, 0.084 mmol) were added to a 25 mL round-bottomed flask. 5ml of glacial acetic acid was dissolved and reacted at room temperature for 3 hours. After the reaction was complete, the solvent was distilled off under reduced pressure, and 7-((4-hydroxyphenyl)amino)-5,8-dioxo- 5,8-Dihydronaphthalene-1-sulfonamide.
实验数据如下.The experimental data are as follows.
1H NMR(400MHz,DMSO)δ9.67(s,1H),)δ9.63(s,1H)8.39(dd,J＝8.0,1.3Hz,1H),8.07(dd,J＝7.8,1.3Hz,1H),8.05(t,J＝7.8Hz,1H)7.23(d,J＝4.2Hz,2H))6.93(s,1H)7.23(d,J＝4.2Hz,2H),5.88(S,H)HR-MS(ESI)calcd forC 16H 12N 2O 5S,[M+H]345.0467,found 345.0460 1 H NMR (400MHz, DMSO)δ9.67(s,1H),)δ9.63(s,1H)8.39(dd,J=8.0,1.3Hz,1H),8.07(dd,J=7.8,1.3Hz ,1H),8.05(t,J＝7.8Hz,1H)7.23(d,J＝4.2Hz,2H))6.93(s,1H)7.23(d,J＝4.2Hz,2H),5.88(S,H )HR-MS(ESI)calcd forC 16 H 12 N 2 O 5 S,[M+H]345.0467,found 345.0460
实施例5Example 5
Figure PCTCN2021124330-appb-000025
Figure PCTCN2021124330-appb-000025
5.1制备3-((4-(二乙氨基)苯基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物5.1 Preparation of 3-((4-(diethylamino)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000026
Figure PCTCN2021124330-appb-000026
将5,8-二氧代-二氢萘3(300mg，1.26mmol)、N,N-二乙基对苯二胺4(249.26mg，1.52mmol)与一水合乙酸铜(20mg，0.1mmol)加入到盛有15ml冰乙酸的100ml圆底烧瓶中，室温下搅拌24小时； 减压蒸馏除去溶剂，加入无水乙醇加热，冷凝回流，将大部分溶剂蒸去后过滤，柱层析分离滤饼出3-((4-(二乙氨基)苯基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物(37mg，收率7.67％)。Combine 5,8-dioxo-dihydronaphthalene 3 (300 mg, 1.26 mmol), N,N-diethyl-p-phenylenediamine 4 (249.26 mg, 1.52 mmol) with copper acetate monohydrate (20 mg, 0.1 mmol) Add it to a 100ml round-bottomed flask containing 15ml of glacial acetic acid, stir at room temperature for 24 hours; remove the solvent by distillation under reduced pressure, add absolute ethanol for heating, condense and reflux, evaporate most of the solvent and filter, and separate the filter cake by column chromatography 3-((4-(diethylamino)phenyl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide (37 mg, 7.67% yield) .
1H NMR(400MHz,DMSO-d 6)δ9.66(s,1H),8.39(dd,J＝8.0,1.3Hz,1H),8.07(dd,J＝7.8,1.3Hz,1H),8.05(t,J＝7.8Hz,1H)7.20(d,J＝8.9Hz 2H),6.78–6.70(d,J＝8.9Hz,2H),5.99(s,1H),3.37(d,J＝7.0Hz,4H),,1.11(t,J＝7.0Hz,6H).HR-MS(ESI)calcd forC 20H 19N 3O 3S,[M+H]382.1147,found 382.1147 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.66 (s, 1H), 8.39 (dd, J=8.0, 1.3 Hz, 1H), 8.07 (dd, J=7.8, 1.3 Hz, 1H), 8.05 ( t, J=7.8Hz, 1H) 7.20 (d, J=8.9Hz 2H), 6.78–6.70 (d, J=8.9Hz, 2H), 5.99 (s, 1H), 3.37 (d, J=7.0Hz, 4H),,1.11(t,J=7.0Hz,6H).HR-MS(ESI)calcd forC 20 H 19 N 3 O 3 S,[M+H]382.1147,found 382.1147
实施例6Example 6
Figure PCTCN2021124330-appb-000027
Figure PCTCN2021124330-appb-000027
6.1制备3-((2-氨基苯基)氨基)-5H-萘[1,8-cd]异噻唑-5-酮1,1-二氧化物6.1 Preparation of 3-((2-aminophenyl)amino)-5H-naphthalene[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000028
Figure PCTCN2021124330-appb-000028
将5,8-二氧代-二氢萘1(237mg,1mmol)、1,2-二氨基苯胺(130mg,1.2mmol)。与一水合乙酸铜(20mg,0.1mmol)加入到盛有10ml冰乙酸的50ml茄形瓶中，室温搅拌反应16小时；减压蒸馏除去溶剂，液相色谱测得3-((2-氨基苯基)氨基)-5H-萘[1,8-cd]异噻唑-5-酮1,1-二氧化物(234mg，收率72％)5,8-Dioxo-dihydronaphthalene 1 (237 mg, 1 mmol), 1,2-diaminoaniline (130 mg, 1.2 mmol). Join with copper acetate monohydrate (20mg, 0.1mmol) in the 50ml eggplant-shaped flask filled with 10ml glacial acetic acid, and the reaction was stirred at room temperature for 16 hours; the solvent was distilled off under reduced pressure, and liquid chromatography recorded 3-((2-aminobenzene (yl)amino)-5H-naphthalene[1,8-cd]isothiazol-5-one 1,1-dioxide (234 mg, 72% yield)
实验数据如下；黄色粉末状固体mp(262℃-267℃). 1H NMR(300MHz,DMSO)δ8.70(dd,J＝1.0Hz,2H),8.24-7.90(m,7H),7.23(s,1H),6.43-6.58(m,1H) The experimental data are as follows; yellow powdery solid mp (262°C-267°C). 1 H NMR (300MHz, DMSO) δ8.70 (dd, J=1.0Hz, 2H), 8.24-7.90 (m, 7H), 7.23 ( s,1H),6.43-6.58(m,1H)
HRMS(ESI)of C 16H 12N 3O 3S[M +H] +326.0 HRMS(ESI) of C 16 H 12 N 3 O 3 S[M + H] + 326.0
实施例7Example 7
Figure PCTCN2021124330-appb-000029
Figure PCTCN2021124330-appb-000029
7.1制备3-(4-氟-2-(三氟甲基)苄基)-5H-萘[1,8-cd]异噻唑-5-酮1,1-二氧化物7.1 Preparation of 3-(4-fluoro-2-(trifluoromethyl)benzyl)-5H-naphthalene[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000030
Figure PCTCN2021124330-appb-000030
将5，8-二氧代-二氢萘1(100mg)、4-氟-2-(三氟甲基)苯胺(90.6mg).与一水合乙酸铜(28.4mg)加入到盛有5ml冰乙酸的25ml圆底烧瓶中，加热回流，该温度下搅拌反应34小时；反应结束后，得到产物粗产品47.6mg(收率47.6％)。随后加入DCM溶解后加入5g100目硅胶制砂，制砂结束后进行柱层析分离纯化。分离结束后得到初步纯化产物，进LC-MS中分析得到里面有397分子量产物，可以进一步纯化。进一步柱层析纯化得到纯化后产物3-(4-氟-2-(三氟甲基)苄基)-5H-萘[1,8-cd]异噻唑-5-酮1,1-二氧化物26.2mg(收率26.2％)。5,8-dioxo-dihydronaphthalene 1 (100mg), 4-fluoro-2-(trifluoromethyl)aniline (90.6mg). and copper acetate monohydrate (28.4mg) were added to a solution containing 5ml of ice. In a 25 ml round-bottomed flask of acetic acid, heated to reflux, and the reaction was stirred at this temperature for 34 hours; after the reaction was completed, 47.6 mg of crude product was obtained (yield 47.6%). Subsequently, DCM was added to dissolve, and then 5 g of 100 mesh silica gel sand was added. After the sand production was completed, column chromatography was performed for separation and purification. After the separation, a preliminary purified product was obtained, which was analyzed by LC-MS to obtain a product with a molecular weight of 397, which could be further purified. Further purification by column chromatography gave the purified product 3-(4-fluoro-2-(trifluoromethyl)benzyl)-5H-naphthalene[1,8-cd]isothiazol-5-one 1,1-dioxide Compound 26.2 mg (yield 26.2%).
1H NMR(400MHz,DMSO-d 6)δ10.20(s,1H),8.37(d,J＝7.6Hz,1H),8.26(d,J＝7.6Hz,1H),8.02(t,J＝7.6Hz,1H),7.64(dd,J＝8.8,5.1Hz,3H),5.76(s,1H),HRMS(ESI)of C 17H 8F 4N 2O 3S[M +H]397.3152 1 H NMR (400MHz, DMSO-d 6 )δ10.20(s, 1H), 8.37(d, J=7.6Hz, 1H), 8.26(d, J=7.6Hz, 1H), 8.02(t, J= 7.6Hz, 1H), 7.64(dd, J=8.8, 5.1Hz, 3H), 5.76(s, 1H), HRMS(ESI) of C 17 H 8 F 4 N 2 O 3 S[M + H] 397.3152
实施例8Example 8
Figure PCTCN2021124330-appb-000031
Figure PCTCN2021124330-appb-000031
8.1制备3-((6-氟吡啶-3-基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物8.1 Preparation of 3-((6-fluoropyridin-3-yl)amino)-5H-naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide
Figure PCTCN2021124330-appb-000032
Figure PCTCN2021124330-appb-000032
将5，8-二氧代-二氢萘(100mg，0.421mmol)、2-氟-5氨基吡啶(56.71mg，0.506mmol)与一水合乙酸铜(16.83mg，0.084mmol)加入25mL圆底烧瓶中，加入5ml冰乙酸，使之溶解，在室温下反应3小时，反应完全后，减压蒸馏除去溶剂，液相色谱分离得到3-((6-氟吡啶-3-基)氨基)-5H-萘并[1,8-cd]异噻唑-5-酮1,1-二氧化物。5,8-Dioxo-dihydronaphthalene (100 mg, 0.421 mmol), 2-fluoro-5aminopyridine (56.71 mg, 0.506 mmol) and copper acetate monohydrate (16.83 mg, 0.084 mmol) were added to a 25 mL round bottom flask 5ml of glacial acetic acid was added to dissolve it, and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, the solvent was distilled off under reduced pressure, and 3-((6-fluoropyridin-3-yl)amino)-5H was obtained by liquid chromatography. - Naphtho[1,8-cd]isothiazol-5-one 1,1-dioxide.
实验数据如下The experimental data is as follows
1H NMR(400MHz,DMSO-d 6)δ9.87(s,1H),8.64(dd,J＝7.9,1.1Hz,1H)8.37(d,J＝7.6Hz,1H),8.26(d,J＝7.6Hz,1H),8.02(t,J＝7.6Hz,1H)7.67(dd,J＝7.4,2.7Hz,1H)7.40(d,J＝7.4Hz,1H)6.06(s,1H)HR-MS(ESI)calcd for C 15H 8FN 3O 3S,[M+H]330.02,found330.02 1 H NMR (400MHz, DMSO-d 6 ) δ 9.87 (s, 1H), 8.64 (dd, J=7.9, 1.1 Hz, 1H) 8.37 (d, J=7.6 Hz, 1H), 8.26 (d, J =7.6Hz,1H),8.02(t,J=7.6Hz,1H)7.67(dd,J=7.4,2.7Hz,1H)7.40(d,J=7.4Hz,1H)6.06(s,1H)HR- MS(ESI)calcd for C 15 H 8 FN 3 O 3 S,[M+H]330.02,found330.02
实施例9Example 9
Figure PCTCN2021124330-appb-000033
Figure PCTCN2021124330-appb-000033
9.1制备N-甲基萘-1-磺酰胺9.1 Preparation of N-methylnaphthalene-1-sulfonamide
Figure PCTCN2021124330-appb-000034
Figure PCTCN2021124330-appb-000034
将1-萘磺酰氯(5g，21.9mmol)加入到盛有200ml丙酮的1L圆底烧瓶中，搅拌，在冰浴中滴加240ml 0℃的甲胺，滴加完毕后，薄层跟踪检测反应，反应完成后，减压蒸馏出有机溶剂和氨水分解出的氨气，析出固体，过滤，真空干燥，得N-甲基萘-1-磺酰胺(4.82g，收率95.66％)。该化合物无需进一步纯化，直接用于下一步反应。1-Naphthalenesulfonyl chloride (5g, 21.9mmol) was added to a 1L round-bottomed flask containing 200ml of acetone, stirred, and 240ml of methylamine at 0°C was added dropwise in an ice bath. , after the completion of the reaction, the organic solvent and ammonia gas decomposed by ammonia water were distilled off under reduced pressure, the solid was precipitated, filtered and dried in vacuo to obtain N-methylnaphthalene-1-sulfonamide (4.82g, yield 95.66%). This compound was used directly in the next reaction without further purification.
9.2制备N-甲基-5,8-二氢萘-1-磺酰胺9.2 Preparation of N-methyl-5,8-dihydronaphthalene-1-sulfonamide
Figure PCTCN2021124330-appb-000035
Figure PCTCN2021124330-appb-000035
将无水硫酸高铈(160g，677.45mmol)用750ml 2mol/L稀硫酸溶解，控温65℃。将N-甲基萘-1-磺酰胺(10g，48.49mmol)加入盛有200ml冰乙酸的500ml圆底烧瓶中，控温65℃，该温度下搅拌，缓慢滴加入至硫酸高铈水相体系中，从滴加开始计时，薄层色谱监测，27分钟后停止反应，待反应液 冷却后，过滤，滤液用1200ml二氯甲烷萃取，干燥除水后低压旋蒸，得到淡黄色固体，真空干燥，得N-甲基-5,8-二氢萘-1-磺酰胺(6g，收率54.22％)。直接用于下一步反应无需纯化Anhydrous cerium sulfate (160g, 677.45mmol) was dissolved in 750ml of 2mol/L dilute sulfuric acid, and the temperature was controlled to 65°C. N-methylnaphthalene-1-sulfonamide (10g, 48.49mmol) was added to a 500ml round-bottomed flask containing 200ml of glacial acetic acid, the temperature was controlled to 65°C, stirred at this temperature, and slowly added dropwise to the cerium sulfate aqueous phase system , the time was started from the dropwise addition, monitored by thin layer chromatography, the reaction was stopped after 27 minutes, the reaction solution was cooled, filtered, the filtrate was extracted with 1200 ml of dichloromethane, dried to remove water, and evaporated at low pressure to obtain a pale yellow solid, which was dried in vacuo , to obtain N-methyl-5,8-dihydronaphthalene-1-sulfonamide (6g, yield 54.22%). Directly used in the next reaction without purification
9.3 4-(2-(((8-(N-甲基氨磺酰基)-1,4-二氧代-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯9.3 4-(2-(((8-(N-methylsulfamoyl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1 - tert-butyl carboxylate
Figure PCTCN2021124330-appb-000036
Figure PCTCN2021124330-appb-000036
将N-甲基-5,8-二氢萘-1-磺酰胺(56.5g，238.17mmol)、叔丁基4-(2-氨基苯基)哌嗪-1-甲酸酯(60g，217.90mmol)与一水合乙酸铜(5.6g，28.05mmol)加入到盛有1200ml冰乙酸的2L圆底烧瓶中，25℃反应，该温度下搅拌反应26小时；减压蒸馏除去溶剂，得粗品。N-methyl-5,8-dihydronaphthalene-1-sulfonamide (56.5 g, 238.17 mmol), tert-butyl 4-(2-aminophenyl)piperazine-1-carboxylate (60 g, 217.90 mmol) and copper acetate monohydrate (5.6g, 28.05mmol) were added to the 2L round-bottomed flask filled with 1200ml of glacial acetic acid, reacted at 25°C, and stirred for 26 hours at this temperature; the solvent was distilled off under reduced pressure to obtain the crude product.
在粗品中加入1500ml的无水乙醇，110℃加热沸腾，液相监测反应产物全部转化后，停止加热，自然冷却搅拌6h，过滤得滤饼，柱层析后除去一般杂质，后加入10倍当量的二氯甲烷完全溶解后，缓慢滴加25倍当量的异丙醇，33℃低压旋蒸后除去相同量的二氯甲烷，待晶体全部析出，过滤，得紫红色晶体，低压干燥得4-(2-(((8-(N-甲基氨磺酰基)-1,4-二氧代-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯.(54.33g，收率78.32％)。1500ml of absolute ethanol was added to the crude product, heated and boiled at 110°C, and the liquid phase monitoring reaction product was completely converted, then stopped heating, cooled and stirred for 6 hours, filtered to obtain a filter cake, and after column chromatography, general impurities were removed, and 10 times the equivalent was added. After the dichloromethane was completely dissolved, 25 times the equivalent of isopropanol was slowly added dropwise, and the same amount of dichloromethane was removed after low-pressure rotary evaporation at 33°C. (2-(((8-(N-Methylsulfamoyl)-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylic acid tert-Butyl ester. (54.33 g, 78.32% yield).
9.4制备N-甲基-5,8-二氧杂-7-((2-(哌嗪-1-基)苯基)氨基)-5,8-二氢萘-1-磺酰胺9.4 Preparation of N-methyl-5,8-dioxa-7-((2-(piperazin-1-yl)phenyl)amino)-5,8-dihydronaphthalene-1-sulfonamide
Figure PCTCN2021124330-appb-000037
Figure PCTCN2021124330-appb-000037
将(4-(2-((1,4-二氧代-5-氨磺酰基-1,4-二氢萘-2-基)氨基)苯基)哌嗪-1-羧酸叔丁酯)(2g，3.78mmol)加入到盛有20ml二氯甲烷的50ml圆底烧瓶中，随后通入盐酸气，薄层跟踪检测反应；反应结束后，过滤得滤饼，减压干燥分离出N-甲基-5,8-二氧杂-7-((2-(哌嗪-1-基)苯基)氨基)-5,8-二氢萘-1-磺酰胺(1.82g，收率98％)。实验数据如下：(4-(2-((1,4-dioxo-5-sulfamoyl-1,4-dihydronaphthalen-2-yl)amino)phenyl)piperazine-1-carboxylate tert-butyl ester ) (2g, 3.78mmol) was joined in the 50ml round-bottomed flask containing 20ml of dichloromethane, then passed into hydrochloric acid, and the thin layer tracked and detected the reaction; Methyl-5,8-dioxa-7-((2-(piperazin-1-yl)phenyl)amino)-5,8-dihydronaphthalene-1-sulfonamide (1.82g, yield 98 %). The experimental data is as follows:
1H NMR(400MHz,Chloroform-d)δ8.58(s,1H),8.47(dq,J＝7.9,1.4Hz,2H),7.93(t,J＝7.8Hz,1H),7.46(tq,J＝4.6,2.7Hz,1H),7.21(t,J＝3.4Hz,3H),6.65(s,1H),6.37(s,1H),3.17(t,J＝4.6Hz,4H),2.93(dd,J＝5.9,3.4Hz,4H),2.80(d,J＝3.0Hz,3H),2.04(s,1H)HR-MS(ESI)calcd forC 21H 22N 4O 4S,[M+H]+427.1362,found 427.1361 1 H NMR (400MHz, Chloroform-d) δ 8.58 (s, 1H), 8.47 (dq, J=7.9, 1.4 Hz, 2H), 7.93 (t, J=7.8 Hz, 1H), 7.46 (tq, J =4.6,2.7Hz,1H),7.21(t,J=3.4Hz,3H),6.65(s,1H),6.37(s,1H),3.17(t,J=4.6Hz,4H),2.93(dd , J=5.9, 3.4Hz, 4H), 2.80(d, J=3.0Hz, 3H), 2.04(s, 1H) HR-MS(ESI) calcd for C 21 H 22 N 4 O 4 S, [M+H ]+427.1362,found 427.1361
二、药理部分(新型冠状病毒肺炎)2. Pharmacological part (new coronavirus pneumonia)
1.质粒1. Plasmids
将3CLpro的开放阅读框克隆到Nde I和Xho I多个克隆位点之间的pET28a载体中。该构建体确保了表达的蛋白可以被3CLpro自身自动切割，并可以用PreScission蛋白酶处理以去除6 ⅹ His标签。对于PLP构建，将密码子优化的SARS-CoV-2nsp3在aa746-1062部分中插入到pET28a的Nde I和EcoR I中，该肽与N端的SAVLQ蛋白序列融合。将第一个氨基酸(Glu)突变为Ser，以促进3CLpro对PLP的消化，以去除N端His标签。所有突变体均使用KOD-plus-neo(TOYOBO，日本大阪)产生。The open reading frame of 3CLpro was cloned into the pET28a vector between the multiple cloning sites of Nde I and Xho I. This construct ensures that the expressed protein can be automatically cleaved by 3CLpro itself and can be treated with PreScission protease to remove the 6 ⅹ His tag. For PLP construction, codon-optimized SARS-CoV-2 nsp3 was inserted into Nde I and EcoR I of pET28a in the aa746-1062 portion, which was fused to the N-terminal SAVLQ protein sequence. The first amino acid (Glu) was mutated to Ser to facilitate digestion of PLP by 3CLpro to remove the N-terminal His tag. All mutants were generated using KOD-plus-neo (TOYOBO, Osaka, Japan).
2.重组蛋白的生产与纯化2. Production and purification of recombinant proteins
将3CLpro和PLP的构建体分别转化到大肠杆菌BL21(DE3)(Novagen)中进行表达。有关详细信息，将单个克隆在含有卡那霉素(50μg/mL)的LB培养基中于37℃预培养过夜，以产生种子培养物。然后，以0.5％的异丙基-D-硫代半乳糖苷(IPTG)的比例，以1％的比例接种种子培养物以产生足够的培养物以表达蛋白质。诱导表达12小时后，通过在4℃以5000rpm离心10分钟收获细胞。然后，在通过超声处理裂解之前，将沉淀重悬于缓冲液A(20mM HEPES，500mM NaCl，pH 7.5)中。超声处理后，通过在4℃下以18000rpm超离心1.5h来澄清裂解物，并使用HisTrap FF柱(GE Healthcare)将上清液用于蛋白质纯化。将获得的蛋白质在4℃下用PreScission蛋白酶以20：1的摩尔比对3CLpro裂解一整夜，然后在用缓冲液B(20mM HEPES，100mM NaCl，1mM DTT，pH 7.5)平衡的Superdex 200柱(GE Healthcare)上通过凝胶过滤色谱法进一步纯化。之后，使用Amicon(10kDa临界值，Millipore)将洗脱的级分浓缩至15mg/ml。然后将浓缩的蛋白质储存在-80℃，用于结晶和活性测定。对于PLP纯化，以20：1的摩尔比使用3CLpro来去除N端His标签。The 3CLpro and PLP constructs were respectively transformed into E. coli BL21(DE3) (Novagen) for expression. For details, individual clones were pre-cultured overnight at 37°C in LB medium containing kanamycin (50 μg/mL) to generate seed cultures. Then, the seed cultures were inoculated at a ratio of 0.5% isopropyl-D-thiogalactoside (IPTG) at a ratio of 1% to generate sufficient culture to express the protein. After 12 hours of induction of expression, cells were harvested by centrifugation at 5000 rpm for 10 minutes at 4°C. The pellet was then resuspended in buffer A (20 mM HEPES, 500 mM NaCl, pH 7.5) prior to lysis by sonication. After sonication, lysates were clarified by ultracentrifugation at 18000 rpm for 1.5 h at 4°C and supernatants were used for protein purification using HisTrap FF columns (GE Healthcare). The obtained protein was cleaved overnight at 4°C with PreScission protease against 3CLpro at a molar ratio of 20:1, and then loaded on a Superdex 200 column ( GE Healthcare) was further purified by gel filtration chromatography. Afterwards, the eluted fractions were concentrated to 15 mg/ml using Amicon (10 kDa cutoff, Millipore). The concentrated protein was then stored at -80°C for crystallization and activity assays. For PLP purification, 3CLpro was used at a molar ratio of 20:1 to remove the N-terminal His tag.
3.SARS-CoV-2 3CLpro和PLP的抑制分析3. Inhibition analysis of SARS-CoV-2 3CLpro and PLP
分别使用Dabcyl-KLSAVLQSGFRKM-Edans-NH2和CBZ-RLRGG-AMC作为荧光底物，进行了荧光共振能量转移(FRET)蛋白酶测定。为了进行3CLpro抑制分析，将100nM 3CLpro重组蛋白(化合物的系列稀释液)在反应缓冲液1(50mM Tris-HCl，1mM EDTA，pH 7.3)中混合，共孵育50h，混合后的总体积为50μL，加入20μM底物以引发反应。然后立即用BioTek Synergy4读板器每3秒钟测量320nm激发和405nm发射的荧光信号。对于PLP抑制测定，进行了类似的步骤，同时在340nm (激发)和450nm(发射)监测荧光信号。使用GraphPad Prism 5.0软件(GraphPad Software，Inc.，San Diego,CA,USA)来计算IC50曲线。来自三个独立实验的IC 50值表示为平均值±SD。Fluorescence resonance energy transfer (FRET) protease assays were performed using Dabcyl-KLSAVLQSGFRKM-Edans-NH2 and CBZ-RLRGG-AMC as fluorogenic substrates, respectively. For the 3CLpro inhibition assay, 100 nM 3CLpro recombinant protein (serial dilutions of compounds) were mixed in reaction buffer 1 (50 mM Tris-HCl, 1 mM EDTA, pH 7.3) for 50 h of incubation, and the total volume after mixing was 50 μL, 20 μM substrate was added to initiate the reaction. Fluorescence signals for excitation at 320 nm and emission at 405 nm were then immediately measured every 3 seconds using a BioTek Synergy4 plate reader. For the PLP inhibition assay, a similar procedure was performed while monitoring the fluorescence signal at 340 nm (excitation) and 450 nm (emission). IC50 curves were calculated using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). IC50 values from three independent experiments are expressed as mean ± SD.
4.LY1的抗病毒测定4. Antiviral assay of LY1
非洲绿猴肾Vero E6细胞系获自美国典型培养物保藏中心(ATCC，编号1586)，并保存在Dulbecco改良的Eagle培养基(DMEM；Gibco Invitrogen)中，并补充了10％胎牛血清(FBS；Gibco Invitrogen)和1％青霉素-链霉素(Gibco Invitrogen)。将SARS-CoV-2的临床分离株(nCoV-2019BetaCoV/WIV04/2019)在Vero E6细胞中繁殖，并按照先前的描述确定病毒滴度(11)。所有感染实验均在生物安全等级3(BLS-3)下进行。African green monkey kidney Vero E6 cell line was obtained from the American Type Culture Collection (ATCC, No. 1586) and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS). ; Gibco Invitrogen) and 1% penicillin-streptomycin (Gibco Invitrogen). A clinical isolate of SARS-CoV-2 (nCoV-2019BetaCoV/WIV04/2019) was propagated in Vero E6 cells and viral titers were determined as previously described (11). All infection experiments were performed under Biosafety Level 3 (BLS-3).
5.RT-PCR5. RT-PCR
通过定量实时RT-PCR评估LY1对病毒产量的功效。将Vero E6细胞接种在96孔板中，并以0.05的MOI感染SARS-CoV-2。感染2小时后，去除含有病毒的培养基，然后用0.1％DMSO作为对照或2.5-15μMLL1处理细胞。处理48小时后，使用实时荧光定量PCR检测细胞上清液中病毒RNA的拷贝数，该反应由RdRP，N和E的表达指示。The efficacy of LY1 on viral yield was assessed by quantitative real-time RT-PCR. Vero E6 cells were seeded in 96-well plates and infected with SARS-CoV-2 at an MOI of 0.05. Two hours after infection, virus-containing medium was removed, and cells were treated with 0.1% DMSO as a control or 2.5-15 μM LL1. After 48 hours of treatment, the copy number of viral RNA in the cell supernatant was detected using real-time PCR, and the response was indicated by the expression of RdRP, N and E.
表面等离子体共振分析Surface Plasmon Resonance Analysis
6.SPR分析6. SPR analysis
SPR分析是在BiaCore T200系统(GE Healthcare，瑞典)上进行的。将目标蛋白稀释在10mM乙酸钠中，并通过胺偶联方法固定在CM5传感器芯片上。然后将样品在运行缓冲液(PBS)中稀释。然后，将经过分析物的药物以30μL/min的流速通过参比通道和活性通道注入。关联和解离时间均设置为120s。在Biacore T200评估软件上，通过稳态亲和模型通过全局拟合进行亲和拟合，以获得平衡解离常数K D。 SPR analysis was performed on a BiaCore T200 system (GE Healthcare, Sweden). The target protein was diluted in 10 mM sodium acetate and immobilized on the CM5 sensor chip by amine coupling method. The samples were then diluted in running buffer (PBS). The analyte-passing drug was then injected through the reference and active channels at a flow rate of 30 μL/min. The association and dissociation times were both set to 120 s. Affinity fitting was performed by global fitting via a steady-state affinity model on Biacore T200 evaluation software to obtain the equilibrium dissociation constant KD .
实施例1.LY1体外抗病毒活性Example 1. In vitro antiviral activity of LY1
并行使用免疫荧光染色测定法和通过定量RT-PCR评估的抗病毒作用，以证明LY1的体外抗病毒能力。我们发现在组装好的化合物库中，化合物LY1表现出强大的抗病毒活性，在30μM时抑制率达到100％。使用免疫荧光染色测定法，用抗核蛋白(NP)抗体和DAPI染色，在显微镜下观察到结果LY1使得NP明显减少，如图1所示。通过定量实时RT-PCR，化合物LY1表现出良好的抗病毒活性，EC50值为3.93μM，见图2。化合物LY1的细胞毒性通过MTT法评估，化合物LY1的CC50值高于15μM。如图2所示。实验结果表明，LY1在体外具有优秀的抗病毒活性和较小的毒性。An immunofluorescence staining assay and antiviral effects assessed by quantitative RT-PCR were used in parallel to demonstrate the in vitro antiviral capacity of LY1. We found that in the assembled compound library, compound LY1 exhibited potent antiviral activity with 100% inhibition at 30 μM. Using an immunofluorescence staining assay, stained with an anti-nucleoprotein (NP) antibody and DAPI, it was observed under the microscope that LY1 caused a significant reduction in NP, as shown in Figure 1. By quantitative real-time RT-PCR, compound LY1 exhibited good antiviral activity with an EC50 value of 3.93 μM, see Figure 2. The cytotoxicity of compound LY1 was assessed by MTT assay, and the CC50 value of compound LY1 was higher than 15 μM. as shown in picture 2. The experimental results show that LY1 has excellent antiviral activity and less toxicity in vitro.
实施例2.LY1抑制3CLpro或PLpro的活性Example 2. LY1 inhibits the activity of 3CLpro or PLpro
从大肠杆菌(E.coli)发酵液纯化得到重组SARS-CoV-2 3CLpro和PLpro。为了测量化合物LY1对3CLpro或PLpro的抑制能力，分别使用Dabcyl-KLSAVLQSGFRKM-Edans-NH2和 CBZ-RLRGG-AMC作为荧光底物，进行了荧光共振能量转移(FRET)蛋白酶测定。结果如图3所示，当浓度为10μM时，对3CLpro和PLpro的抑制效果分别达到94.4％和88.6％。结果表明化合物LY1能有效抑制SARS-CoV-2 3CLpro和PLpro的活性。我们使用了基于荧光共振能量转移(FRET)的裂解测定法来确定IC 50值，IC 50值分别为0.51μM与2.97μM。 Recombinant SARS-CoV-2 3CLpro and PLpro were purified from E. coli fermentation broth. To measure the inhibitory ability of compound LY1 against 3CLpro or PLpro, a fluorescence resonance energy transfer (FRET) protease assay was performed using Dabcyl-KLSAVLQSGFRKM-Edans-NH2 and CBZ-RLRGG-AMC as fluorogenic substrates, respectively. The results are shown in Figure 3, when the concentration was 10 μM, the inhibitory effects on 3CLpro and PLpro reached 94.4% and 88.6%, respectively. The results showed that compound LY1 could effectively inhibit the activities of SARS-CoV-2 3CLpro and PLpro. We used a fluorescence resonance energy transfer (FRET) -based cleavage assay to determine IC50 values, which were 0.51 μM and 2.97 μM, respectively.
实施例3.LY1主要靶向3CLpro蛋白Example 3. LY1 mainly targets 3CLpro protein
为了进一步确定3CLpro和PLpro作为潜在的靶标，我们然后通过表面等离振子共振(SPR)分析来表征3CLpro和PLpro与化合物LY1之间的亲和力。K D值为0.51μM，这个结果表明3CLpro和前导化合物LY1之间具有很强的结合亲和力(图4。但是，化合物LY1与PLpro结合的K D值为2.97μM。结果表明，与3CLpro相比，LY1与PLpro结合亲和力较小(图4)。因此，由于3CLpro与先导化合物Y8之间的高亲和力，我们推断3CLpro是主要的靶标。 To further identify 3CLpro and PLpro as potential targets, we then characterized the affinity between 3CLpro and PLpro and the compound LY1 by surface plasmon resonance (SPR) analysis. The KD value was 0.51 μM, which indicated a strong binding affinity between 3CLpro and the lead compound LY1 (Fig. 4). However, compound LY1 bound to PLpro with a KD value of 2.97 μM. The results showed that, compared with 3CLpro, the LY1 binds PLpro with less affinity (Figure 4). Therefore, due to the high affinity between 3CLpro and the lead compound Y8, we infer that 3CLpro is the primary target.

Claims (9)

  1. 一类化合物，为式I或式II所示的化合物，或其药学上可接受的盐或酯：A class of compounds is a compound represented by formula I or formula II, or a pharmaceutically acceptable salt or ester thereof:
    Figure PCTCN2021124330-appb-100001
    Figure PCTCN2021124330-appb-100001
    其中，A环为五、六、七元饱和、不饱和碳环或杂环，杂环环内存在一到两个杂原子，杂原子各自独立地选自N、O、S；Wherein, the A ring is a five-, six-, seven-membered saturated, unsaturated carbocyclic ring or a heterocyclic ring, and there are one to two heteroatoms in the heterocyclic ring, and the heteroatoms are independently selected from N, O, and S;
    R1为一个或多个取代基，各自独立地选自氢、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基；R1 is one or more substituents, each independently selected from hydrogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl;
    R2为取代基，选自氢、卤素、C1～C3烷基、C1～C3烷氧基、或C1～C3烷氧酰基，所述卤素指氟、氯、溴或碘；R2 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, C1-C3 alkoxy, or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine;
    R3为取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，所述卤素指氟、氯、溴或碘；R3 is a substituent selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, and the halogen refers to fluorine, chlorine, bromine or iodine;
    R4为一个或多个取代基，选自氢、卤素、C1～C3烷基、羟基、C1～C3烷氧基、硝基、氨基、羧基或C1～C3烷氧酰基，芳香族、杂芳族的碳环或杂环，杂环环内存在一到两个杂原子，杂原子各自独立地选自N、O、S；所述卤素指氟、氯、溴或碘；R4 is one or more substituents selected from hydrogen, halogen, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro, amino, carboxyl or C1-C3 alkoxyacyl, aromatic, heteroaromatic The carbocyclic or heterocyclic ring, there are one to two heteroatoms in the heterocyclic ring, and the heteroatoms are independently selected from N, O, S; the halogen refers to fluorine, chlorine, bromine or iodine;
    或R4选自
    Figure PCTCN2021124330-appb-100002
    or R4 is selected from
    Figure PCTCN2021124330-appb-100002
  2. 根据权利要求1所述的化合物，其特征在于，所述A环选自苯环、吡咯基、咪唑基、吡唑基、呋喃基、四氢呋喃基、噻吩基、噻唑基、吡嗪基、嘧啶基、哒嗪基、
    Figure PCTCN2021124330-appb-100003
    优选为苯环。     The compound according to claim 1, wherein the A ring is selected from benzene ring, pyrrolyl, imidazolyl, pyrazolyl, furanyl, tetrahydrofuranyl, thienyl, thiazolyl, pyrazinyl, pyrimidinyl , pyridazinyl,
    Figure PCTCN2021124330-appb-100003
    Preferably it is a benzene ring.
  3. 根据权利要求1所述的化合物，其特征在于，所述R1为氢。The compound of claim 1, wherein the R1 is hydrogen.
  4. 根据权利要求1所述的化合物，其特征在于，所述R2为甲基。The compound of claim 1, wherein the R2 is methyl.
  5. 根据权利要求1所述的化合物，其特征在于，所述R3为氢、甲基、羟基、卤素。The compound according to claim 1, wherein the R3 is hydrogen, methyl, hydroxyl or halogen.
  6. 根据权利要求1所述的化合物，其特征在于，所述R4为一个取代基，优选为
    Figure PCTCN2021124330-appb-100004
    The compound according to claim 1, wherein the R4 is a substituent, preferably
    Figure PCTCN2021124330-appb-100004
  7. 根据权利要求1所述的化合物，其特征在于，式I所示的化合物选自：compound according to claim 1, is characterized in that, the compound shown in formula I is selected from:
    Figure PCTCN2021124330-appb-100005
    Figure PCTCN2021124330-appb-100005
  8. 根据权利要求1所述的化合物，其特征在于，式II所示的化合物选自：compound according to claim 1, is characterized in that, the compound shown in formula II is selected from:
    Figure PCTCN2021124330-appb-100006
    Figure PCTCN2021124330-appb-100006
  9. 如权利要求1-8任一项所述的化合物在制备预防和/或治疗与新型冠状病毒肺炎有关疾病的药物中的应用。Application of the compound according to any one of claims 1-8 in the preparation of a medicine for preventing and/or treating diseases related to novel coronavirus pneumonia.
PCT/CN2021/124330 2021-02-20 2021-10-18 Compound and medical use thereof for novel coronavirus pneumonia WO2022116714A1 (en)

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