WO2023159808A1 - 化合物阿瑞吡坦在制备预防或治疗非洲猪瘟药物中的应用 - Google Patents

化合物阿瑞吡坦在制备预防或治疗非洲猪瘟药物中的应用 Download PDF

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WO2023159808A1
WO2023159808A1 PCT/CN2022/098311 CN2022098311W WO2023159808A1 WO 2023159808 A1 WO2023159808 A1 WO 2023159808A1 CN 2022098311 W CN2022098311 W CN 2022098311W WO 2023159808 A1 WO2023159808 A1 WO 2023159808A1
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aprepitant
compound
swine fever
african swine
asfv
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PCT/CN2022/098311
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French (fr)
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张克山
郑海学
崔卉梅
杨博
�田宏
朱紫祥
冯涛
杨帆
曹伟军
马旭升
茹毅
郭建宏
刘湘涛
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中国农业科学院兰州兽医研究所
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Priority to US17/895,390 priority Critical patent/US11883411B2/en
Publication of WO2023159808A1 publication Critical patent/WO2023159808A1/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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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
    • A61P31/20—Antivirals for DNA viruses

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  • the invention belongs to the technical field of treating African swine fever, and in particular relates to the application of compound aprepitant in the preparation of drugs for preventing or treating African swine fever.
  • African swine fever is an acute, highly contagious disease caused by African swine fever virus (ASFV) infection, with a mortality rate of up to 100%. Since it was introduced into China in 2018, it has caused significant economic losses to the domestic pig industry. There is currently no commercial vaccine, so the development of effective antiviral drugs at a reasonable cost is of great significance for the treatment of ASF. Although various types of active anti-ASFV drugs have been reported, the in vivo efficacy of these compounds has not been evaluated.
  • ASFV is a double-stranded DNA virus that replicates mainly in monocytes and macrophages and encodes more than 50 structural proteins and more than 100 nonstructural proteins.
  • Aprepitant (Aprepitant or Emend), also known as Aprepitant, has a molecular formula of C 23 H 21 F 7 N 4 O 3 and a CAS number of 170729-80-3.
  • the chemical name is 5-[2(R)-[1(R)-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3(S)-(4-fluorophenyl) Lin-4-ylmethyl]-3,4-dihydro-2H-1,2,4-triazol-3-one.
  • the structural formula is
  • Aprepitant was approved by the FDA in 2003 for the treatment of chemotherapy-induced vomiting. It is the first marketed NK-1 receptor antagonist, and achieves specific anti-tumor effects through NK-1 receptors. It can be used as a broad-spectrum antitumor drug. tumor drugs. In addition, aprepitant also exhibits anti-HIV-1 activity. Miguel and Manak Mark M and other researchers in academic articles (see Miguel, Rosso Marisa, The NK-1 receptor antagonist as a broad spectrum antitumor drug [J]. Invest New Drugs, 2010, 28:187-93.; Manak Mark M, Moshkoff Dmitry A, Nguyen Lequan T et al.
  • the present invention shows that the compound aprepitant can significantly inhibit the replication of ASFV.
  • Aprepitant not only inhibits the transcription and protein expression of D1133L, but also reduces the transcription and protein expression levels of p30 and p72, preventing The virus invades the host cell and can be used to inhibit the early and late infection of ASFV. Therefore, the compound aprepitant can be used to prevent or treat African swine fever.
  • the molecular formula of the compound aprepitant is C 23 H 21 F 7 N 4 O 3 .
  • the CAS number is 170729-80-3.
  • the chemical name is 5-[2(R)-[1(R)-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3(S)-(4-fluorophenyl) Lin-4-ylmethyl]-3,4-dihydro-2H-1,2,4-triazol-3-one, the structural formula is shown in the following formula (I):
  • one of the objectives of the present invention is to provide the application of the compound aprepitant in the preparation of a drug for treating African swine fever.
  • Another object of the present invention is to provide the application of the compound aprepitant in the preparation of drugs for preventing African swine fever.
  • Another object of the present invention is to provide the application of the compound aprepitant in the preparation of a drug for inhibiting African swine fever virus gene transcription.
  • the genes are African swine fever virus D1133L gene, p30 gene and p72 gene.
  • Another object of the present invention is to provide the application of the compound aprepitant in the preparation of a drug for inhibiting the expression of African swine fever virus protein.
  • the proteins are African swine fever virus D1133L protein, p30 protein and p72 protein.
  • the drug is the compound aprepitant added with pharmaceutically acceptable carriers and/or excipients to make any one of tablets, sprays, granules, capsules, oral liquids, injections, and suspensions dosage form.
  • the present invention found that the compound aprepitant can significantly inhibit the replication of ASFV, and found that aprepitant can not only inhibit the transcription and protein expression of D1133L, but also reduce the transcription and protein expression levels of p30 and p72, and prevent the virus from invading host cells. To inhibit the early and late infection of ASFV. Therefore, the compound aprepitant can be used to prevent or treat African swine fever.
  • Figure 1 is the fluorescence diagram of the compound aprepitant inhibiting African swine fever virus, wherein Figure 1A is the effect of DMSO on the infection and replication of the virus after the cells are treated, and Figure 2B is the infection and replication of the virus after the cells are treated with aprepitant
  • the GFP and TRANS rows indicate the observation under green fluorescence and white light, respectively, and the ASFV column, DMSO column, 18 ⁇ M, 22 ⁇ M, and 24 ⁇ M indicate that the cells were dissolved with ASFV fluorescent virus, ASFV fluorescent virus + DMSO, and ASFV fluorescent virus mixed with DMSO Treatment with different concentrations of aprepitant (18 ⁇ M, 22 ⁇ M, 24 ⁇ M);
  • Figure 2 is a graph showing the results of compound aprepitant inhibiting the genome copy number of African swine fever virus, wherein Figure 2A is the inhibitory effect of DMSO on virus replication, and Figure 2B is a comparison chart of the inhibitory effect of different concentrations of compound aprepitant on ASFV replication;
  • Fig. 3 is the result graph that compound aprepitant inhibits African swine fever virus TCID 50 ;
  • Figure 4 is the result of compound aprepitant inhibiting African swine fever virus D1133L transcription
  • Figure 5 shows the results of compound aprepitant inhibiting African swine fever virus p30 and p72 transcription
  • Figure 5A shows the inhibition of compound aprepitant on African swine fever virus p30 transcription
  • Figure 5B shows the effect of compound aprepitant on African swine fever virus Inhibition of viral p72 transcription
  • Figure 6 shows that the compound aprepitant inhibits the expression of African swine fever virus D1133L protein
  • Figure 7 shows that the compound aprepitant inhibits the expression of African swine fever virus p30 and p72 proteins, wherein Figure 7A is the result of p30 protein expression level, and Figure 7B is the result of p72 protein expression level;
  • Figure 8 is a graph showing the cytotoxicity test results of the compound aprepitant.
  • 0 represents the negative control
  • 20, 40, and 80 represent different concentrations of aprepitant (20 ⁇ M, 40 ⁇ M, 80 ⁇ M) dissolved in DMSO.
  • depository unit China Center for Type Culture Collection
  • the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has passed the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Experimental Animal Ethics Committee, and the Biosafety Committee of the Chinese Academy of Agricultural Sciences in accordance with the biosafety level 3 laboratory (BSL-3) and the relevant biosafety requirements of African swine fever.
  • BSL-3 biosafety level 3 laboratory
  • the Experimental Animal Ethics Committee of Lanzhou Veterinary Research Institute, and the Biosafety Committee of Lanzhou Veterinary Research Institute reported step by step, and obtained the permission from the Ministry of Agriculture to carry out research on highly pathogenic ASFV pathogens and animals, and has been filed with the Ministry of Agriculture and rural Affairs, which meets the national biosafety level requirements.
  • PAM Primary porcine alveolar macrophages
  • BMDM primary bone marrow macrophages
  • ASFV is the virus strain CN/GS 2018, and the isolate of ASFV CN/GS 2018 comes from the National African Swine Fever Regional Laboratory (Lanzhou), belongs to genotype II, and the virus titer is 5 ⁇ 10 7 TCID 50 /mL, which is the propagation of PAMs
  • the fourth-generation virus was deposited in the China Center for Type Culture Collection on December 21, 2020, with the preservation number CCTCC NO:V202096.
  • the ASFV fluorescent virus is an ASFV fluorescent virus containing an eGFP screening expression box gene fragment and missing the MGF-360-9L gene. For its preparation method, refer to the Chinese invention patent application with publication number CN 111593028 A.
  • the compound aprepitant was purchased from Shanghai Taoshu Biotechnology Co., Ltd.
  • DMSO DMSO
  • Figure 1A shows that there is no significant difference between the infection control group and the infection blank group under fluorescent light and white light, indicating that DMSO has no effect on virus infection and replication.
  • Figure 1B shows that the fluorescence in the infection control group (DMSO) was brighter, but the fluorescence gradually became darker after treatment with different concentrations of aprepitant, and the fluorescence disappeared completely after treatment with 24 ⁇ M aprepitant.
  • DMSO infection control group
  • the cell plate was cultured at 37°C and 5% CO 2 for 48h, and then the cell plate was frozen and thawed three times at -80°C and then inactivated by RT-qPCR to detect the copy number of the viral genome.
  • the total volume of the RT-qPCR reaction system is 20 ⁇ L, including 10 ⁇ L of Premix Ex Taq (2 ⁇ ), 0.2 ⁇ L of ROX Reference Dye II (50 ⁇ ), 0.6 ⁇ L of primers, 0.1 ⁇ L of ASFV probe primers, 2 ⁇ L of templates, supplemented without Bacterial deionized water to 20 ⁇ L.
  • the reaction conditions are: 50°C, 2min; 95°C, 2min; 95°C, 15s; 58°C, 1min; a total of 45 cycles.
  • ASFV-p72 upstream primer 5'-GATACCACAAGATCAGCCGT-3' (SEQ ID NO: 1); downstream primer: 5'-CTGCTCATGGTATCAATCTTATCGA-3' (SEQ ID NO: 2); ASFV probe primer: 5'-CCACGGGAGGAATACCAACCCAGTG -3' (SEQ ID NO: 3).
  • Figure 2A is a comparison chart of virus replication between the infection control group and the infection blank group, which shows that the inhibitory effect of DMSO on virus replication is not significantly different from that of the blank group.
  • Figure 2B is a comparison chart of the inhibitory effect of different concentrations of compound aprepitant on ASFV replication, which shows that the ASFV genome copy number decreased after adding the compound aprepitant, and when the dose of the compound aprepitant was 22 ⁇ M, the effect on The suppression rate of ASFV genome copy number was higher than 50%.
  • the cell plate was cultured at 37°C and 5% CO 2 for 48 hours, and then the cell plate was frozen and thawed three times at -80°C as samples, which were serially diluted 10 times with serum-free RPMI 1640 to make 6 dilutions, each 8 wells were repeated for each dilution, inoculated into BMDMs for culture, and the cell plate was cultured at 37°C and 5% CO 2 for 5 days. Fluorescence changes in each cell culture well were observed every day, and TCID 50 was calculated.
  • the total volume of the RT-qPCR reaction system was 10 ⁇ L, including 0.4 ⁇ L of upstream and downstream primers, 2 ⁇ L of cDNA, 5 ⁇ L of TB Green TM Premix Ex Taq (TaKaRa), and supplemented with sterile deionized water to 10 ⁇ L.
  • the reaction conditions are: 95°C, 2min; 95°C, 10s, 60°C, 34s, 40 cycles.
  • the primer sequence for amplifying D1133L is: upstream primer: 5'-CTTCTGGAAAACGGGGTACA-3' (SEQ ID NO: 4); downstream primer: 5'-CAAGATAAGAACCCCCGACA-3' (SEQ ID NO: 5).
  • the results are shown in Figure 4.
  • the compound aprepitant can inhibit the RNA expression level of D1133L in the African swine fever virus gene.
  • the inhibition rate of the D1133L RNA expression level is higher than 50%.
  • the total volume of the RT-qPCR reaction system was 10 ⁇ L, including 0.4 ⁇ L of upstream and downstream primers, 2 ⁇ L of cDNA, 5 ⁇ L of TB Green TM Premix Ex Taq (TaKaRa), and supplemented with sterile deionized water to 10 ⁇ L.
  • the reaction conditions are: 95°C, 2min; 95°C, 10s, 60°C, 34s, 40 cycles.
  • the primer sequence for amplifying p30 is: upstream primer: 5'-CTCCGATGAGGGCTCTTGCT-3' (SEQ ID NO: 6); downstream primer: 5'-AGACGGAATCCTCAGCATCTTC-3' (SEQ ID NO: 7);
  • the primer sequence for amplifying p72 is: upstream primer: 5'-TGCGATGATGATTACCTT-3' (SEQ ID NO: 8); downstream primer: 5'-ATTCTCTTGCTCTGGATAC-3' (SEQ ID NO: 9).
  • the compound aprepitant can inhibit the RNA expression levels of p30 and p72 in the African swine fever virus gene, and when the compound aprepitant dose is 22 ⁇ M, the inhibition rate of the p30 RNA expression level is higher than 50%. %, when the dose of compound aprepitant was 44 ⁇ M, the inhibition rate of p30 RNA expression level was higher than 95%; when the dose of compound aprepitant was 22 ⁇ M, the inhibition rate of p72 RNA expression level was higher than 50%, the compound When the dose of prepitant was 44 ⁇ M, the inhibition rate of p72 RNA expression level was higher than 95%.
  • the cell plate was cultured at 37° C. and 5% CO 2 for 48 hours, and then the cell culture was collected, centrifuged, and the supernatant was discarded. The total protein was extracted, and the expression difference of p30 and p72 protein was detected by western blotting method.
  • the compound aprepitant can significantly inhibit the RNA transcription and protein expression levels of ASFV, prevent the virus from invading host cells, and can be used to inhibit early and late infection of ASFV.
  • Cytotoxicity assay of the small molecule compound aprepitant by MTT method on PAMs Porcine alveolar macrophages (PAM, 2 ⁇ 10 5 /well) were cultured in 96-well plate with RPMI 1640+10% FBS medium, and different concentrations of aprepitant (20 ⁇ M, 40 ⁇ M, 80 ⁇ M) treated cells as the experimental group, while setting a blank control group (medium+DMSO) and a negative control group (cells+medium+DMSO).
  • the compound aprepitant has less toxicity to cells, and even when the dose reaches 80 ⁇ M, the cell survival rate can still reach more than 50%, with low cytotoxicity and good safety.
  • the compound aprepitant of the present invention has good inhibitory effect on African swine fever virus, has low cytotoxicity and good safety, and can be used to prevent or treat African swine fever.

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Abstract

本发明属于非洲猪瘟治疗技术领域，具体涉及化合物阿瑞吡坦在预防或治疗非洲猪瘟中的应用。本发明发现化合物阿瑞吡坦能够显著抑制ASFV复制，并且发现阿瑞吡坦不仅抑制D1133L的转录和蛋白表达，而且可以降低p30和p72的转录和蛋白表达水平，阻止病毒入侵宿主细胞，可用于抑制ASFV的早、晚期感染。因此，化合物阿瑞吡坦可用于预防或治疗非洲猪瘟。

Description

化合物阿瑞吡坦在制备预防或治疗非洲猪瘟药物中的应用
本申请要求于2022年02月25日提交中国专利局、申请号为202210181584.1、发明名称为“化合物阿瑞吡坦在制备预防或治疗非洲猪瘟药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于非洲猪瘟治疗技术领域，具体涉及化合物阿瑞吡坦在制备预防或治疗非洲猪瘟药物中的应用。
背景技术
非洲猪瘟(African swine fever，ASF)是由非洲猪瘟病毒(African swine fevervirus，ASFV)感染引起的的一种急性、高度接触性传染性疾病，死亡率高达100％。自2018年传入中国，对国内养猪业造成了重大的经济损失。目前还无商品化疫苗，所以以合理的成本开发有效的抗病毒药物对治疗非洲猪瘟具有重要意义。尽管目前已报道了各种类型的活性抗ASFV药物，但尚未对这些化合物的体内药效进行评估。
ASFV是一种双链DNA病毒，主要在单核细胞和巨噬细胞中复制，编码50多种结构蛋白和100多种非结构蛋白。
阿瑞吡坦(Aprepitant或Emend)别名阿瑞匹坦，分子式为C 23H 21F 7N 4O 3，CAS号为170729-80-3。化学名为5-[2(R)-[1(R)-[3,5-二(三氟甲基)苯基]乙氧基]-3(S)-(4-氟苯基)吗啉-4-基甲基]-3,4-二氢-2H-1,2,4-三唑-3-酮。结构式为
Figure PCTCN2022098311-appb-000001
阿瑞吡坦于2003年获FDA批准用于治疗化疗引起的呕吐，是首个上市的NK-1受体拮抗剂，且通过NK-1受体实现特异性抗肿瘤作用，可作为广谱抗肿瘤药物。除此之外，阿瑞吡坦还表现出抗HIV-1活性。Miguel和Manak Mark M等研究学者在学术文章(具体参见Miguel,Rosso Marisa,The NK-1 receptor antagonistaprepitant as a broad spectrum antitumor drug[J].Invest New Drugs,2010,28:187-93.；Manak Mark M,Moshkoff Dmitry A,Nguyen Lequan T et al.Anti-HIV-1activity of the neurokinin-1receptor antagonist aprepitant and synergisticinteractions with other antiretrovirals[J].AIDS,2010,24:2789-96.)中报道过该化合物，但目前并未有研究表明阿瑞吡坦具有抗ASFV的作用。
发明内容
为了克服现有技术的上述缺点，本发明表明化合物阿瑞吡坦能够显著抑制ASFV复制，阿瑞吡坦不仅抑制D1133L的转录和蛋白表达，而且可以降低p30和p72的转录和蛋白表达水平，阻止病毒入侵宿主细胞，可用于抑制ASFV的早、晚期感染。因此，化合物阿瑞吡坦可用于预防或治疗非洲猪瘟。
所述化合物阿瑞吡坦的分子式为C 23H 21F 7N 4O 3。CAS号为170729-80-3。化学名为5-[2(R)-[1(R)-[3,5-二(三氟甲基)苯基]乙氧基]-3(S)-(4-氟苯基)吗啉-4-基甲基]-3,4-二氢-2H-1,2,4-三唑-3-酮，结构式如下式(Ⅰ)所示：
Figure PCTCN2022098311-appb-000002
首先，本发明的目的之一在于提供化合物阿瑞吡坦在制备治疗非洲猪瘟药物中的应用。
其次，本发明的另一目的在于提供化合物阿瑞吡坦在制备预防非洲猪瘟药物中的应用。
本发明的又一目的在于提供化合物阿瑞吡坦在制备抑制非洲猪瘟病毒基因转录药物中的应用。所述基因为非洲猪瘟病毒D1133L基因、p30基因、p72基因。
本发明的再一目的在于提供化合物阿瑞吡坦在制备抑制非洲猪瘟病毒蛋白表达药物中的应用。所述蛋白为非洲猪瘟病毒D1133L蛋白、p30蛋白、p72蛋白。
优选地，所述药物为化合物阿瑞吡坦加入药学上可接受的载体和/或辅料，制成片剂、喷雾剂、颗粒剂、胶囊剂、口服液、针剂、混悬剂的任一种剂型。
本发明的有益效果是：
本发明发现化合物阿瑞吡坦能够显著抑制ASFV复制，并且发现阿瑞吡坦不仅能抑制D1133L的转录和蛋白表达，而且可以降低p30和p72的转录和蛋白表达水平，阻止病毒入侵宿主细胞，可用于抑制ASFV的早、晚期感染。因此，化合物阿瑞吡坦可用于预防或治疗非洲猪瘟。
附图说明
图1为化合物阿瑞吡坦抑制非洲猪瘟病毒的荧光图，其中图1A为 DMSO处理细胞后对病毒的感染和复制的影响，图2B为阿瑞吡坦处理细胞后对病毒的感染和复制的影响，GFP和TRANS行分别表示在绿色荧光和白光下观察，ASFV列、DMSO列、18μM、22μM、24μM表示细胞分别用ASFV荧光毒、ASFV荧光毒+DMSO、ASFV荧光毒混合用DMSO溶解的不同浓度的阿瑞吡坦(18μM、22μM、24μM)处理；
图2为化合物阿瑞吡坦抑制非洲猪瘟病毒基因组拷贝数结果图，其中图2A为DMSO对病毒复制的抑制效果，图2B为不同浓度化合物阿瑞吡坦对ASFV复制的抑制效果对比图；
图3为化合物阿瑞吡坦抑制非洲猪瘟病毒TCID 50结果图；
图4为化合物阿瑞吡坦抑制非洲猪瘟病毒D1133L转录结果；
图5为化合物阿瑞吡坦抑制非洲猪瘟病毒p30和p72转录结果，图5A为化合物阿瑞吡坦对非洲猪瘟病毒p30转录的抑制情况，图5B为化合物阿瑞吡坦对非洲猪瘟病毒p72转录的抑制情况；
图6为化合物阿瑞吡坦抑制非洲猪瘟病毒D1133L蛋白表达；
图7为化合物阿瑞吡坦抑制非洲猪瘟病毒p30和p72蛋白表达，其中图7A为p30蛋白表达水平结果，图7B为p72蛋白表达水平结果；
图8为化合物阿瑞吡坦细胞毒性检测结果图。图中0表示阴性对照，20、40、80表示DMSO溶解的不同浓度的阿瑞吡坦(20μM、40μM、80μM)。
生物保藏说明：
保藏时间：2020年12月21日；
保藏单位名称：中国典型培养物保藏中心；
保藏编号：CCTCC NO:V202096；
保藏单位地址：中国武汉大学；
分类命名：II型非洲猪瘟病毒株ASFV CN/GS 2018。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
以下实施例中所述的实验获得生物安全许可和非洲猪瘟实验室活动许可：
中国农业科学院兰州兽医研究所根据生物安全3级实验室(BSL-3)和非洲猪瘟相关生物安全的相关要求，经兰州兽医研究所生物安全委员会、 实验动物伦理委员会、中国农业科学院生物安全委员会、兰州兽医研究所实验动物伦理委员会、兰州兽医研究所生物安全委员会逐级上报，获得农业部关于开展高致病性ASFV病原及动物研究许可，并已在农业农村部备案，符合国家生物安全等级的要求。
以下实施例中所述的实验细胞、病毒来源：
原代猪肺泡巨噬细胞(PAM)及原代骨髓巨噬细胞(BMDM)取自5周龄左右的仔猪，无菌采集细胞后，用红细胞裂解液(购自Biosharp公司)去除红细胞，低速离心后，弃上清，将细胞沉淀重悬于含有10％FBS(购自Gibco公司)的RPMI 1640完全培养基(购自Gibco公司)中，置于37℃、5％CO 2培养箱中培养。
ASFV为病毒株CN/GS 2018，ASFV CN/GS 2018分离株来自国家非洲猪瘟区域实验室(兰州)，属于基因II型，病毒效价为5×10 7TCID 50/mL，为PAMs扩繁后的第4代毒，于2020年12月21日保藏于中国典型培养物保藏中心，保藏编号CCTCC NO:V202096。ASFV荧光毒为含有eGFP筛选表达盒基因片段、缺失了MGF-360-9L基因的ASFV荧光病毒，其制备方法参考公开号为CN 111593028 A的中国发明专利申请。
化合物阿瑞吡坦购自上海陶术生物科技有限公司。
实施例中其他试剂如果没有特别说明均为常见的市售试剂；实施例中的操作如果没有特别说明均是本领域知晓的操作方法。
实施例1
化合物阿瑞吡坦对非洲猪瘟病毒复制和基因的转录表达的影响
1.荧光下观察化合物阿瑞吡坦对ASFV感染和复制的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)，实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(18μM、22μM、24μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。另外，将只用ASFV荧光毒(MOI＝0.1)处理细胞的作为感染空白组。然后将细胞板置于37℃，5％CO 2条件培养48h后显微镜下观察荧光变化。
结果如图1所示，图1A表明，在荧光下和白光下，感染对照组和感染空白组均没有显著差异，表明DMSO对病毒的感染和复制没有影响。
图1B显示，感染对照组(DMSO)中荧光较亮，而用不同浓度的阿瑞吡坦处理后荧光逐渐变暗，其中当用24μM的阿瑞吡坦处理后，荧光已完全消失。上述结果表明，化合物阿瑞吡坦能够显著抑制ASFV的感染和复制，且随着化合物浓度的增加，抑制效果愈加明显。
2.化合物阿瑞吡坦对ASFV感染和复制过程中的病毒基因组拷贝数的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)，实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(18μM、20μM、22μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。另外，将只用ASFV荧光毒(MOI＝0.1)处理细胞的孔作为感染空白组。然后将细胞板置于37℃，5％CO 2条件培养48h，然后将细胞板置于-80℃冻融三次灭活后用RT-qPCR检测病毒基因组拷贝数。RT-qPCR反应体系总体积20μL，包括10μL的Premix Ex Taq(2×)，0.2μL的ROX Reference DyeⅡ(50×)，0.6μL的引物，0.1μL的ASFV探针引物，2μL的模板，补充无菌去离子水到20μL。反应条件为：50℃，2min；95℃，2min；95℃，15s；58℃，1min；共45个循环。
其中，ASFV-p72上游引物：5’-GATACCACAAGATCAGCCGT-3’(SEQ ID NO:1)；下游引物：5’-CTGCTCATGGTATCAATCTTATCGA-3’(SEQ ID NO:2)；ASFV探针引物：5’-CCACGGGAGGAATACCAACCCAGTG-3’(SEQ ID NO:3)。
ASFV基因组拷贝数检测结果如图2所示，图2A是感染对照组与感染空白组病毒复制的比较图，该图表明DMSO对病毒复制的抑制效果与空白组相比差异不显著。
图2B是不同浓度化合物阿瑞吡坦对ASFV复制的抑制效果对比图，该图表明，加入化合物阿瑞吡坦后ASFV基因组拷贝数有所降低，且化合物阿瑞吡坦剂量为22μM时，对ASFV基因组拷贝数的抑制率高于50％。
3.化合物阿瑞吡坦对ASFV的TCID 50的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪骨髓巨噬细胞 (BMDM，1×10 6/孔)，实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(22μM、44μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。然后将细胞板置于37℃，5％CO 2条件培养48h后将细胞板置于-80℃冻融三次后作为样品，分别用无血清RPMI 1640连续10倍稀释，做6个稀释度，每个稀释度重复8个孔，接种到BMDMs进行培养，将细胞板置于37℃，5％CO 2条件培养5天，每天观察各细胞培养孔中荧光变化，计算TCID 50。
结果如图3所示，22μM、44μM化合物阿瑞吡坦均可降低ASFV荧光毒TCID 50，当化合物阿瑞吡坦浓度为44μM时，ASFV荧光毒TCID 50最低。
4.化合物阿瑞吡坦对ASFV的D1133L RNA转录水平的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)；实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(22μM、44μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。然后将细胞板置于37℃，5％CO 2条件培养48h后收集细胞培养物，离心，弃上清。用TrizoL法提取总RNA，使用RT Primer Mix试剂盒合成cDNA后，用RT-qPCR方法检测D1133L RNA的表达差异。
RT-qPCR反应体系总体积10μL，包括0.4μL的上下游引物，2μL的cDNA，5μL的TB Green TM Premix Ex Taq(TaKaRa)，补充无菌去离子水到10μL。反应条件为：95℃，2min；95℃，10s，60℃，34s，40个循环。
其中，扩增D1133L的引物序列为：上游引物：5’-CTTCTGGAAAACGGGGTACA-3’(SEQ ID NO:4)；下游引物：5’-CAAGATAAGAACCCCCGACA-3’(SEQ ID NO:5)。
结果如图4所示，化合物阿瑞吡坦能够抑制非洲猪瘟病毒基因中D1133L的RNA表达水平，当化合物阿瑞吡坦剂量为22μM时，对D1133L RNA表达水平的抑制率高于50％。
5.化合物阿瑞吡坦对ASFV的p30和p72的RNA转录水平的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)；实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％) 溶解的不同浓度的阿瑞吡坦(22μM和44μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。然后将细胞板置于37℃，5％CO 2条件培养48h后收集细胞培养物，离心，弃上清。用TrizoL法提取总RNA，使用RT Primer Mix试剂盒合成cDNA后，用RT-qPCR方法检测p30和p72 RNA的表达差异。
RT-qPCR反应体系总体积10μL，包括0.4μL的上下游引物，2μL的cDNA，5μL的TB Green TMPremix Ex Taq(TaKaRa)，补充无菌去离子水到10μL。反应条件为：95℃，2min；95℃，10s，60℃，34s，40个循环。
其中，扩增p30的引物序列为：上游引物：5’-CTCCGATGAGGGCTCTTGCT-3’(SEQ ID NO:6)；下游引物：5’-AGACGGAATCCTCAGCATCTTC-3’(SEQ ID NO:7)；
扩增p72的引物序列为：上游引物：5’-TGCGATGATGATTACCTT-3’(SEQ ID NO:8)；下游引物：5’-ATTCTCTTGCTCTGGATAC-3’(SEQ ID NO:9)。
结果如图5所示，化合物阿瑞吡坦能够抑制非洲猪瘟病毒基因中p30和p72的RNA表达水平，且化合物阿瑞吡坦剂量为22μM时，对p30 RNA表达水平的抑制率高于50％，化合物阿瑞吡坦剂量为44μM时，对p30 RNA表达水平的抑制率高于95％；化合物阿瑞吡坦剂量为22μM时，对p72 RNA表达水平的抑制率高于50％，化合物阿瑞吡坦剂量为44μM时，对p72 RNA表达水平的抑制率高于95％。
6.化合物阿瑞吡坦对ASFV的D1133L的蛋白水平的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)，实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(20μM、22μM)处理细胞，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。另外，将只用ASFV荧光毒(MOI＝0.1)处理细胞的作为感染空白组。然后将细胞板置于37℃，5％CO 2条件培养48h后收集细胞培养物，离心，弃上清。提取总蛋白，用western blotting方法检测D1133L蛋白的表达差异。
结果如图6所示，与未感染对照组(Mock组)相比，感染空白组(ASFV)、感染对照组(DMSO)和实验组(阿瑞吡坦+ASFV)的D1133L蛋白 表达水平增加；但是，相对于感染对照组(DMSO)，实验组(阿瑞吡坦+ASFV)中D1133L蛋白表达水平显著降低。结果表明，化合物阿瑞吡坦能够显著抑制非洲猪瘟病毒基因中D1133L蛋白表达水平。
7.化合物阿瑞吡坦对ASFV的p30和p72的蛋白表达水平的影响
在12孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，1×10 6/孔)，实验组用ASFV荧光毒(MOI＝0.1)混合用DMSO(＜1％)溶解的不同浓度的阿瑞吡坦(p30：4.5μM、9μM、12μM、18μM；p72：20μM、22μM)处理细胞后，感染对照组用ASFV荧光毒(MOI＝0.1)混合DMSO(＜1％)处理细胞。另外，将只用ASFV荧光毒(MOI＝0.1)处理的细胞作为感染空白组。然后将细胞板置于37℃，5％CO 2条件培养48h后收集细胞培养物，离心，弃上清。提取总蛋白，用western blotting方法检测p30和p72蛋白的表达差异。
结果如图7所示，与未感染对照组(Mock组)相比，感染空白组(ASFV)、感染对照组(DMSO)和实验组(阿瑞吡坦+ASFV)的p30和p72蛋白表达水平增加；但是，相对于感染对照组(DMSO)，实验组(阿瑞吡坦+ASFV)中p30和p72蛋白表达水平显著降低。结果表明，化合物阿瑞吡坦能够显著抑制非洲猪瘟病毒基因中p30和p72蛋白表达水平。
上述结果表明，化合物阿瑞吡坦能够显著抑制ASFV的RNA转录和蛋白表达水平，阻止病毒入侵宿主细胞，可用于抑制ASFV的早、晚期感染。
实施例2化合物阿瑞吡坦的细胞毒性
在PAMs上用MTT法对小分子化合物阿瑞吡坦进行细胞毒性检测。在96孔板中用RPMI 1640+10％FBS培养基培养猪肺泡巨噬细胞(PAM，2×10 5/孔)，向孔中加入DMSO溶解的不同浓度的阿瑞吡坦(20μM、40μM、80μM)处理细胞作为实验组，同时设置空白对照组(培养基+DMSO)和阴性对照组(细胞+培养基+DMSO)。将培养板在培养箱中孵育48h后，向板的每个孔中加入10μL的MTT溶液，将培养板在培养箱中继续孵育4h后，向板的每个孔中加入100μL的Formazan溶解液，适当混匀，在细胞培养箱内再继续孵育。直至在普通光学显微镜下观察发现Formazan全部溶解。然后读取酶标仪测量570nm处的吸光度，根据公式1计算每种浓度的细 胞存活率。
细胞存活率＝((OD 实验组-OD 空白对照组)/(OD 阴性对照组-OD 空白对照组))×100％公式1
结果如图8所示，化合物阿瑞吡坦对细胞的毒性较小，甚至使用剂量达到80μM时，细胞存活率活依然可达到50％以上，细胞毒性小，安全性较好。
综上所述，本发明所述化合物阿瑞吡坦对非洲猪瘟病毒具有较好的抑制作用，并且细胞毒性小，安全性较好，可用于预防或治疗非洲猪瘟。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (14)

  1. 阿瑞吡坦在制备治疗非洲猪瘟药物中的应用，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100001
  2. 化合物阿瑞吡坦在制备预防非洲猪瘟药物中的应用，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100002
  3. 化合物阿瑞吡坦在制备抑制非洲猪瘟病毒基因转录药物中的应用，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100003
  4. 根据权利要求3所述的应用，其特征在于，所述非洲猪瘟病毒基因为以下一种或几种基因：非洲猪瘟病毒D1133L基因、p30基因和p72基因。
  5. 化合物阿瑞吡坦在制备抑制非洲猪瘟病毒蛋白表达药物中的应用，其特征在于，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100004
  6. 根据权利要求5所述的应用，其特征在于，所述非洲猪瘟病毒蛋白为以下一种或几种蛋白：非洲猪瘟病毒D1133L蛋白、p30蛋白和p72蛋白。
  7. 根据权利要求1～6任一项所述的应用，所述药物为化合物阿瑞吡坦加入药学上可接受的载体和/或辅料，制成片剂、喷雾剂、颗粒剂、胶囊剂、口服液、针剂或混悬剂。
  8. 化合物阿瑞吡坦在预防和/或治疗非洲猪瘟中的应用，所述化合物 阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100005
  9. 化合物阿瑞吡坦在抑制非洲猪瘟病毒基因转录中的应用，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100006
  10. 化合物阿瑞吡坦在抑制非洲猪瘟病毒蛋白表达中的应用，其特征在于，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100007
  11. 根据权利要求10所述的应用，其特征在于，所述蛋白为以下一种或几种蛋白：非洲猪瘟病毒D1133L蛋白、p30蛋白和p72蛋白。
  12. 化合物阿瑞吡坦在抑制非洲猪瘟的感染和复制中的应用，所述化合物阿瑞吡坦的结构式如下式(Ⅰ)所示：
    Figure PCTCN2022098311-appb-100008
  13. 根据权利要求8～12任意一项所述应用，其特征在于，所述化合物阿瑞吡坦的处理浓度不低于20μM。
  14. 根据权利要求13所述应用，其特征在于，所述化合物阿瑞吡坦的处理浓度为22～80μM。
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