WO2023169119A1 - Solid form of compound, preparation method therefor, and use thereof - Google Patents

Abstract

The present application relates to a solid form of ((3-carbamoyl-5-fluoropyrazine-2-yl) oxy) methyl isobutyrate, a method for preparing the solid form, a pharmaceutical composition comprising the solid form, and a use of the solid form in the prevention or treatment of a disease caused by a virus.

Description

化合物的固体形式及其制备方法和用途Solid forms of compounds and their preparation and uses

本申请是以CN申请号为202210243856.6，申请日为2022年3月10日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。This application is based on the application with CN application number 202210243856.6 and the filing date is March 10, 2022, and claims its priority. The disclosure content of the CN application is hereby incorporated into this application as a whole.

技术领域Technical field

本申请涉及((3-氨甲酰-5-氟吡嗪-2-基)氧基)甲基异丁酸酯(在下文中称作“化合物I”)的固体形式，制备所述固体形式的方法，包含所述固体形式的药物组合物，以及所述固体形式用于治疗布尼亚病毒目的病毒，例如克里米亚-刚果出血热病毒(CCHFV)，引起的疾病或感染的用途。
The present application relates to the solid form of ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methylisobutyrate (hereinafter referred to as "Compound I"), the preparation of which Methods, pharmaceutical compositions comprising said solid form, and use of said solid form for the treatment of diseases or infections caused by viruses of the order Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus (CCHFV).

背景技术Background technique

克里米亚-刚果出血热(CCHF)是一种广泛流行于非洲、亚洲和欧洲等地区的急性传染病，以发热、胃肠道出血、呕血、休克为主要特征，病死率可达50％，CCHF致病病原体为克里米亚-刚果出血热病毒(CCHFV)。克里米亚-刚果出血热病毒属于布尼亚病毒目(Bunyavirales)，内罗病毒科(Nairoviridae)，正内罗病毒属(Orthonairovirus)蜱传病毒。CCHF患者临床症状广泛，典型的病程经历四个阶段：潜伏期、出血前期、出血期和恢复期。潜伏期时间长短与接触途径、病毒载量等因素相关；潜伏期之后，出血前阶段表现为发热疾病，患者伴有严重头痛、恶心、腹泻、肌肉酸痛、畏光等症状当患者进入出血阶段，机体会依次出现瘀点、大面积瘀斑及大量出血，9％～50％的病例在该阶段死亡。除了这些表征，研究还发现CCHF在发病的同时伴随着严重的肝、神经***，呼吸***以及心脏的损伤等病变。目前没有上市的抗克里米亚-刚果出血热的药物和疫苗，严重威胁着人类健康。Crimean-Congo hemorrhagic fever (CCHF) is an acute infectious disease widely prevalent in Africa, Asia, Europe and other regions. It is characterized by fever, gastrointestinal bleeding, hematemesis, and shock. The mortality rate can reach 50%. , The causative pathogen of CCHF is Crimean-Congo hemorrhagic fever virus (CCHFV). Crimean-Congo hemorrhagic fever virus belongs to the order Bunyavirales, family Nairoviridae, and genus Orthonairovirus, a tick-borne virus. Patients with CCHF have a wide range of clinical symptoms, and the typical disease course goes through four stages: latent period, pre-hemorrhagic period, hemorrhagic period and recovery period. The length of the incubation period is related to the route of exposure, viral load and other factors; after the incubation period, the pre-hemorrhagic stage manifests as a febrile illness, with the patient accompanied by severe headache, nausea, diarrhea, muscle aches, photophobia and other symptoms. When the patient enters the hemorrhage stage, the body will Petechiae, large-area ecchymosis, and massive bleeding appear sequentially, and 9% to 50% of cases die at this stage. In addition to these symptoms, studies have also found that the onset of CCHF is accompanied by severe liver, nervous system, respiratory system and heart damage and other diseases. There are currently no drugs or vaccines against Crimean-Congo hemorrhagic fever on the market, posing a serious threat to human health.

发明内容 Contents of the invention

在一个方面中，本申请提供如下所示的化合物I((3-氨甲酰-5-氟吡嗪-2-基)氧基)甲基异丁酸酯的晶型I：
In one aspect, the application provides crystalline Form I of Compound I ((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methylisobutyrate as shown below:

本申请所述的化合物I的晶型I不仅在预防或治疗克里米亚-刚果出血热病毒引起的疾病中具有优异的效果，还具有其它优点。例如，本申请的优选晶型具有优良的物理性质(包括溶解度、溶出率、耐光照性、低吸湿性、耐高温性、耐高湿性、流动性等)，并且在诸如生物利用度、物理和/或化学稳定性及易于制备性等性质上，本申请所述的化合物I的晶型I可具有更优异的性质。本申请所述的化合物I的晶型I具有良好的粉体学性质，更适合和便于大量制造和用于形成制剂，可减少刺激性并提高吸收，解决了代谢速度方面的问题，显著降低了药物蓄积带来的毒性，提高了安全性，有效保证了药物产品的质量和效能。Crystal Form I of Compound I described in the present application not only has excellent effects in preventing or treating diseases caused by Crimean-Congo hemorrhagic fever virus, but also has other advantages. For example, the preferred crystalline form of the present application has excellent physical properties (including solubility, dissolution rate, light resistance, low hygroscopicity, high temperature resistance, high humidity resistance, fluidity, etc.), and has excellent properties such as bioavailability, physical and / Or in terms of properties such as chemical stability and ease of preparation, the crystal form I of compound I described in the present application may have more excellent properties. The crystal form I of Compound I described in the present application has good powder properties, is more suitable and convenient for mass manufacturing and preparation, can reduce irritation and improve absorption, solves the problem of metabolic speed, and significantly reduces The toxicity caused by drug accumulation improves safety and effectively ensures the quality and efficacy of drug products.

在另一方面中，本申请提供制备本申请所述的化合物I的晶型I的方法。In another aspect, the present application provides methods of preparing Form I of Compound I described herein.

在另一方面中，本申请提供药物组合物，其包含本申请所述的化合物I的晶型I，以及一种或多种药学上可接受的载体。In another aspect, the present application provides a pharmaceutical composition comprising Form I of Compound I described herein, and one or more pharmaceutically acceptable carriers.

在另一方面中，本申请提供本申请所述的化合物I的晶型I在制备用于治疗或预防布尼亚病毒目病毒引起的疾病或感染的药物中的用途。In another aspect, the present application provides the use of Form I of Compound I described herein in the preparation of a medicament for the treatment or prevention of diseases or infections caused by viruses of the order Bunyavirales.

发明详细描述Detailed description of the invention

晶型及其制备方法Crystal form and preparation method thereof

在一个实施方案中，本申请提供化合物I的晶型I：
In one embodiment, the application provides Form I of Compound I:

所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱包括在约7.6±0.2°、22.7±0.2°和30.5±0.2°的衍射角(2θ)处的特征峰。The XRPD pattern in 2θ obtained using Cu-Kα radiation for Form I includes characteristic peaks at diffraction angles (2θ) of approximately 7.6±0.2°, 22.7±0.2° and 30.5±0.2°.

在某些实施方案中，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱还包括在约13.7±0.2°、17.6±0.2°、18.7±0.2°、19.0±0.2°、19.9±0.2°和22.4±0.2°的衍射角(2θ)处的特征峰。In certain embodiments, the XRPD pattern of Form I obtained using Cu-Kα radiation in 2θ angles also includes at about 13.7±0.2°, 17.6±0.2°, 18.7±0.2°, 19.0±0.2° , the characteristic peaks at the diffraction angle (2θ) of 19.9±0.2° and 22.4±0.2°.

在某些实施方案中，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱还包括在约8.7±0.2°、10.3±0.2°、15.1±0.2°、20.6±0.2°、23.9±0.2°和24.0±0.2°的衍射角(2θ)处的特征峰。In certain embodiments, the XRPD pattern of Form I obtained using Cu-Kα radiation in 2θ angles also includes at about 8.7±0.2°, 10.3±0.2°, 15.1±0.2°, 20.6±0.2° , the characteristic peaks at the diffraction angle (2θ) of 23.9±0.2° and 24.0±0.2°.

在某些实施方案中，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱包括在以下衍射角(2θ)处的峰：
In certain embodiments, the XRPD pattern in 2θ obtained using Cu-Kα radiation of Form I includes peaks at the following diffraction angles (2θ):

在某些实施方案中，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱包括与图1所示基本上相同的衍射角(2θ)处的峰。In certain embodiments, the XRPD pattern in 2θ obtained using Cu-Kα radiation of Form I includes peaks at substantially the same diffraction angle (2θ) as shown in FIG. 1 .

在某些实施方案中，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD峰位与图1所示基本上相同。In certain embodiments, the XRPD peak positions at 2θ obtained using Cu-Kα radiation of the Form I are substantially the same as shown in Figure 1 .

在某些实施方案中，所述晶型I为无水合物。In certain embodiments, the Form I is an anhydrate.

在某些实施方案中，所述晶型I的DSC图谱包括在约88.39±5℃(例如约88.39℃)处的吸热/放热峰。In certain embodiments, the DSC pattern of Form I includes an endothermic/exothermic peak at about 88.39 ± 5°C (eg, about 88.39°C).

在某些实施方案中，在热重分析中，所述晶型I在加热至约265.35±5℃(例如约265.35℃)时有约97.69％的失重。In certain embodiments, the Form I has a weight loss of about 97.69% when heated to about 265.35±5°C (eg, about 265.35°C) in thermogravimetric analysis.

在某些实施方案中，所述晶型I的DSC图谱包括与图2所示基本上相同的特征峰。在某些实施方案中，所述晶型I的DSC图谱与图2所示基本上相同。In certain embodiments, the DSC spectrum of Form I includes substantially the same characteristic peaks as shown in Figure 2. In certain embodiments, the DSC pattern of Form I is substantially the same as shown in Figure 2.

在某些实施方案中，所述晶型I的TGA图谱与图3所示基本上相同。 In certain embodiments, the TGA pattern of Form I is substantially the same as shown in Figure 3.

在某些实施方案中，本申请提供制备所述晶型I的方法，包括：In certain embodiments, the application provides methods of preparing the Form I, comprising:

1)将化合物I在良溶剂中溶解，形成溶液；1) Dissolve compound I in a good solvent to form a solution;

2)向所述溶液中添加反溶剂，在搅拌下析出固体。2) Add an anti-solvent to the solution and precipitate a solid under stirring.

在某些实施方案中，本申请提供制备所述晶型I的方法，其包括将化合物I在良溶剂中溶解(可在室温或者加热条件(例如加热至30-60℃，优选50℃)下进行)，形成溶液(视需要可将混合物进行过滤以得到溶液)，然后向所述溶液中添加反溶剂，在搅拌(所述添加反溶剂和搅拌可在室温或者冷却条件(例如冷却至0-10℃，优选5℃)下进行)下析出固体，将其过滤得到晶型I。In certain embodiments, the application provides a method for preparing the crystalline form I, which includes dissolving compound I in a good solvent (can be at room temperature or under heating conditions (eg, heated to 30-60°C, preferably 50°C) Carry out), form a solution (if necessary, the mixture can be filtered to obtain a solution), then add an anti-solvent to the solution, and stir (the adding of anti-solvent and stirring can be at room temperature or under cooling conditions (for example, cooled to 0- The solid precipitates at 10°C, preferably 5°C), and is filtered to obtain crystal form I.

在某些实施方案中，本申请提供制备所述晶型I的方法，其包括以下步骤：In certain embodiments, the application provides a method of preparing the Form I, comprising the steps of:

1)将化合物I加入良溶剂(例如乙酸乙酯)中，搅拌使化合物I溶解得到溶液；1) Add compound I to a good solvent (such as ethyl acetate) and stir to dissolve compound I to obtain a solution;

2)向步骤1)中所得溶液中加入反溶剂(例如石油醚)，通过过滤收集析出的固体，任选地将其干燥，得到晶型I。2) Add an antisolvent (such as petroleum ether) to the solution obtained in step 1), collect the precipitated solid by filtration, and optionally dry it to obtain crystalline form I.

在某些实施方案中，本申请提供制备所述晶型I的方法，其包括以下步骤：In certain embodiments, the application provides a method of preparing the Form I, comprising the steps of:

1)将化合物I加入良溶剂(例如乙醇)中，搅拌使化合物I溶解得到溶液；1) Add compound I to a good solvent (such as ethanol) and stir to dissolve compound I to obtain a solution;

2)向步骤1)中所得溶液中加入反溶剂(例如水)，通过过滤收集析出的固体，任选地将其干燥，得到晶型I。2) Add an antisolvent (such as water) to the solution obtained in step 1), collect the precipitated solid by filtration, and optionally dry it to obtain crystalline form I.

在某些实施方案中，所述良溶剂为具有3-10个碳原子的酯，优选为乙酸乙酯；所述反溶剂为具有5-10个碳原子的烃(其包括烷烃类、卤代烷烃类、烯烃类、炔烃类和芳烃类，具体包括但不限于二氯甲烷、三氯甲烷(氯仿)、正己烷、正庚烷和甲苯)或者具有2-6个碳原子的醚(优选为链状醚，例如石油醚、***、二异丙基醚或甲基叔丁基醚)，优选为石油醚。In certain embodiments, the good solvent is an ester with 3-10 carbon atoms, preferably ethyl acetate; the anti-solvent is a hydrocarbon with 5-10 carbon atoms (including alkanes, halogenated alkanes olefins, alkynes and aromatic hydrocarbons, specifically including but not limited to methylene chloride, chloroform (chloroform), n-hexane, n-heptane and toluene) or ethers with 2-6 carbon atoms (preferably Chain ethers, such as petroleum ether, diethyl ether, diisopropyl ether or methyl tert-butyl ether), preferably petroleum ether.

在某些实施方案中，所述良溶剂为具有2-10个碳原子的醇，优选为乙醇；所述反溶剂为水。In certain embodiments, the good solvent is an alcohol having 2 to 10 carbon atoms, preferably ethanol; the antisolvent is water.

在某些实施方案中，化合物I和良溶剂的重量体积比(g/mL)为约1:(8-120)，例如约1:10、约1:15、约1:20、约1:30、约1:40、约1:50、约1:60、约1:70、约1:80、约1:90或约1:100。In certain embodiments, the weight-to-volume ratio (g/mL) of Compound 1 and good solvent is about 1:(8-120), such as about 1:10, about 1:15, about 1:20, about 1:30 , about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90 or about 1:100.

在某些实施方案中，所述良溶剂和反溶剂的体积比为约1:0.5至1:5(例如约1:1、约1:2、约1:3、约1:4)。In certain embodiments, the volume ratio of the good solvent and antisolvent is about 1:0.5 to 1:5 (eg, about 1:1, about 1:2, about 1:3, about 1:4).

药物组合物、治疗方法和用途Pharmaceutical compositions, methods of treatment and uses

在某些实施方案中，本申请提供药物组合物，其包含本申请所述的晶型I，以及一种 或多种药学上可接受的载体。In certain embodiments, the application provides pharmaceutical compositions comprising Form I as described herein, and a or a variety of pharmaceutically acceptable carriers.

在某些实施方案中，所述的晶型I在药物组合物中以治疗布尼亚病毒目病毒引起的疾病或感染的有效量存在。In certain embodiments, the Form I is present in the pharmaceutical composition in an amount effective to treat a disease or infection caused by a virus of the order Bunyavirales.

本申请所述的药物组合物可以***地作用和/或局部地作用。为此目的，它们可以适合的途径给药，例如通过注射、静脉内、动脉内、皮下、腹膜内、肌内或经皮给药；或通过口服、含服、经鼻、透粘膜、局部、以眼用制剂的形式或通过吸入给药。The pharmaceutical compositions described herein may act systemically and/or locally. For this purpose, they may be administered by suitable routes, for example by injection, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or transdermally; or by oral, buccal, nasal, transmucosal, topical, Administered as an ophthalmic preparation or by inhalation.

对于这些给药途径，可以适合的剂型给药本申请所述的药物组合物。For these routes of administration, the pharmaceutical compositions described herein may be administered in suitable dosage forms.

所述剂型可为固体制剂、半固体制剂、液体制剂或气态制剂，具体包括但不限于片剂、胶囊剂、散剂、颗粒剂、锭剂、硬糖剂、散剂、喷雾剂、乳膏剂、软膏剂、栓剂、凝胶剂、糊剂、洗剂、软膏剂、水性混悬剂、可注射溶液剂、混悬剂、酏剂、糖浆剂。The dosage form may be a solid preparation, a semi-solid preparation, a liquid preparation or a gaseous preparation, specifically including but not limited to tablets, capsules, powders, granules, lozenges, hard candies, powders, sprays, creams, and ointments. Agents, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, suspensions, elixirs, syrups.

本申请所述的药物组合物可以通过本领域熟知的任何方法来制备，例如通过混合、溶解、制粒、糖包衣、碾磨、乳化、冻干等处理来制备。The pharmaceutical compositions described in the present application can be prepared by any method well known in the art, such as by mixing, dissolving, granulating, sugar coating, grinding, emulsifying, lyophilizing and other processes.

在某些实施方案中，本申请提供所述的晶型I在制备用于预防或治疗布尼亚病毒目病毒引起的疾病或感染的药物中的用途。In certain embodiments, the present application provides the use of the crystalline Form I in the preparation of a medicament for preventing or treating diseases or infections caused by viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述的晶型I在制备用于预防或治疗克里米亚-刚果出血热中的药物中用途。In certain embodiments, the application provides the use of the crystalline form I in the preparation of a medicament for preventing or treating Crimean-Congo hemorrhagic fever.

在某些实施方案中，本申请提供所述的晶型I在制备作为布尼亚病毒目病毒抑制剂的药物中的用途。In certain embodiments, the present application provides the use of Form I in the preparation of a medicament as an inhibitor of viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述的晶型I在制备用于抑制布尼亚病毒目病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途。In certain embodiments, the application provides the use of the crystalline form I in the preparation of a medicament for inhibiting the replication or propagation of Bunyavirales viruses in cells (eg, mammalian cells).

在某些实施方案中，本申请提供所述药物组合物在制备用于治疗布尼亚病毒目病毒引起的疾病或感染的药物中的用途。In certain embodiments, the application provides the use of the pharmaceutical composition in the manufacture of a medicament for the treatment of diseases or infections caused by viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述药物组合物在制备作为布尼亚病毒目病毒抑制剂的药物中的用途。In certain embodiments, the application provides the use of the pharmaceutical composition in the manufacture of a medicament that is an inhibitor of viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述药物组合物在制备用于抑制布尼亚病毒目病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途。In certain embodiments, the application provides the use of the pharmaceutical composition in the manufacture of a medicament for inhibiting the replication or propagation of viruses of the order Bunyavirales in cells, such as mammalian cells.

在某些实施方案中，本申请提供预防或治疗布尼亚病毒目病毒引起的疾病或感染的方法，其包括向需要其的个体(优选哺乳动物)施用预防或治疗有效量的本申请所述的晶型I。In certain embodiments, the present application provides methods for preventing or treating diseases or infections caused by viruses of the order Bunyavirales, which comprise administering to an individual (preferably a mammal) in need thereof a prophylactically or therapeutically effective amount of a method described herein. of crystal form I.

在某些实施方案中，本申请提供在有需要的个体中抑制布尼亚病毒目病毒复制或繁殖的方法，其包括向需要其的个体(优选哺乳动物)施用预防或治疗有效量的本申请所述的 晶型I。In certain embodiments, the present application provides a method for inhibiting replication or propagation of Bunyavirales viruses in an individual in need thereof, comprising administering to an individual (preferably a mammal) in need thereof a prophylactically or therapeutically effective amount of the present application. described Form I.

在某些实施方案中，本申请提供所述的晶型I，其用于治疗布尼亚病毒目病毒引起的疾病或感染。In certain embodiments, the application provides said Form I for use in the treatment of diseases or infections caused by viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述的晶型I，其用作布尼亚病毒目病毒抑制剂。In certain embodiments, the application provides Form I as described for use as a Bunyavirales virus inhibitor.

在某些实施方案中，本申请提供所述的晶型I，其用于抑制布尼亚病毒目病毒在细胞(例如哺乳动物细胞)中复制或繁殖。In certain embodiments, the present application provides Form I as described for use in inhibiting replication or propagation of Bunyavirales viruses in cells, such as mammalian cells.

在某些实施方案中，本申请提供所述的药物组合物，其用于治疗布尼亚病毒目病毒引起的疾病或感染。In certain embodiments, the application provides pharmaceutical compositions for use in treating diseases or infections caused by viruses of the order Bunyavirales.

在某些实施方案中，本申请提供所述的药物组合物，其用作布尼亚病毒目病毒抑制剂。In certain embodiments, the application provides pharmaceutical compositions for use as inhibitors of Bunyavirales viruses.

在某些实施方案中，本申请提供所述的药物组合物，其用于抑制布尼亚病毒目病毒在细胞(例如哺乳动物细胞)中复制或繁殖。In certain embodiments, the application provides pharmaceutical compositions for inhibiting replication or propagation of Bunyavirales viruses in cells, such as mammalian cells.

在某些实施方案中，本申请所述的布尼亚病毒目病毒为内罗病毒科病毒。In certain embodiments, the Bunyavirales viruses described herein are Nairoviridae viruses.

在某些实施方案中，本申请所述的布尼亚病毒目病毒为正内罗病毒属病毒。In certain embodiments, a Bunyavirales virus described herein is a virus of the genus Orthonarovirus.

在某些实施方案中，本申请所述的布尼亚病毒目病毒为克里米亚-刚果出血热病毒(CCHFV)。In certain embodiments, a Bunyavirales virus described herein is Crimean-Congo hemorrhagic fever virus (CCHFV).

在某些实施方案中，本申请所述的布尼亚病毒目病毒引起的疾病为布尼亚病毒目病毒引起的出血热。In certain embodiments, the disease caused by a Bunyavirales virus described herein is a hemorrhagic fever caused by a Bunyavirales virus.

在某些实施方案中，本申请所述的布尼亚病毒目病毒引起的疾病为内罗病毒科病毒引起的出血热。In certain embodiments, the disease caused by a Bunyavirales virus described herein is a hemorrhagic fever caused by a virus in the family Nairoviridae.

在某些实施方案中，本申请所述的布尼亚病毒目病毒引起的疾病为正内罗病毒属病毒引起的出血热。In certain embodiments, the disease caused by a virus of the order Bunyavirales described herein is a hemorrhagic fever caused by a virus of the genus Orthonarovirus.

在某些实施方案中，本申请所述的布尼亚病毒目病毒引起的疾病为克里米亚-刚果出血热病毒引起的出血热。In certain embodiments, the disease caused by a Bunyavirales virus described herein is a hemorrhagic fever caused by Crimean-Congo hemorrhagic fever virus.

在某些实施方案中，本申请所述的布尼亚病毒目病毒引起的疾病为布尼亚病毒目病毒引起的单纯性感染、发热、头痛、肌肉疼痛、呕吐、胃肠道出血、鼻出血、呕血或休克。In certain embodiments, the diseases caused by Bunyavirales viruses described in the present application are simple infections, fever, headache, myalgia, vomiting, gastrointestinal bleeding, and epistaxis caused by Bunyavirales viruses. , vomiting blood or shock.

在某些实施方案中，本申请所述布尼亚病毒目病毒引起的疾病为克里米亚-刚果出血热。In certain embodiments, the disease caused by a virus of the order Bunyavirales described herein is Crimean-Congo hemorrhagic fever.

在某些实施方案中，本申请所述布尼亚病毒目病毒引起的疾病为克里米亚-刚果出 血热病毒引起的疾病。In certain embodiments, the disease caused by a Bunyavirales virus described herein is a disease originating from Crimea-Congo. Disease caused by blood fever virus.

在某些实施方案中，本申请所述哺乳动物包括牛科动物、马科动物、羊亚科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，例如是人、猫、狗或猪。In certain embodiments, mammals described herein include bovine, equid, ovine, porcine, canine, feline, rodent, primate, for example, Human, cat, dog or pig.

定义definition

除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本申请。Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technology as used herein are intended to mean technology as commonly understood in the art, including those variations or equivalent technology that would be apparent to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain this application.

如本文中所使用的术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。As used herein, the terms "includes," "includes," "has," "contains," or "involves" and their other variations herein are inclusive or open-ended and do not Exclude other elements or method steps not listed.

如本文中所使用的词语“约”是指本领域的普通技术人员认为在所述值的可接受的标准误差内，例如±0.05、±0.1、±0.2、±0.3、±1、±2或±3等。除非另外根据上下文显而易见，否则本文提供的所有数值都由术语“约”修饰。The word "about" as used herein means that what one of ordinary skill in the art considers to be within the acceptable standard error of the stated value, such as ±0.05, ±0.1, ±0.2, ±0.3, ±1, ±2, or ±3 etc. All numerical values provided herein are modified by the term "about" unless otherwise apparent from context.

如本文中所使用的术语“固体形式”包括化合物I的所有固态形式，例如晶体形式或无定形形式。The term "solid form" as used herein includes all solid forms of Compound I, such as crystalline or amorphous forms.

如本文中所使用的术语“无定形”是指三维上无排序的任意固体物质。在一些情况中，无定形固体可通过已知技术表征，所述技术包括XRPD晶体学、固态核磁共振(ssNMR)波谱学、DSC或这些技术的一些组合。如以下所说明，无定形固体产生弥散的XRPD图谱，其通常包括一个或两个宽峰(即具有约5°2θ或更大的基宽的峰)。The term "amorphous" as used herein refers to any solid substance that is not ordered in three dimensions. In some cases, amorphous solids can be characterized by known techniques including XRPD crystallography, solid-state nuclear magnetic resonance (ssNMR) spectroscopy, DSC, or some combination of these techniques. As explained below, amorphous solids produce diffuse XRPD patterns that typically include one or two broad peaks (ie, peaks with a base width of about 5° 2θ or greater).

如本文中所使用的术语“晶型”或“晶体”是指呈现三维排序的任意固体物质，与无定形固体物质相反，其产生具有边界清楚的峰的特征性XRPD图谱。The term "crystalline form" or "crystal" as used herein refers to any solid material exhibiting a three-dimensional order that, in contrast to amorphous solid materials, produces a characteristic XRPD pattern with well-defined peaks.

如本文中所使用的术语“X射线粉末衍射图谱(XRPD图谱)”是指实验观察的衍射图或源于其的参数。XRPD图谱通常由峰位(横坐标)和/或峰强度(纵坐标)表征。The term "X-ray powder diffraction pattern (XRPD pattern)" as used herein refers to an experimentally observed diffraction pattern or parameters derived therefrom. XRPD patterns are usually characterized by peak position (abscissa) and/or peak intensity (ordinate).

如本文中所使用的术语“2θ”是指基于X射线衍射实验的实验设置的以度数表示的峰位，并且通常是在衍射图谱中的横坐标单位。如果当入射束与某晶格面形成θ角时反射被衍射，则实验设置需要以2θ角记录反射束。应当理解，在本文中提到的特定晶体形式的特定2θ值意图表示使用本文所述的X射线衍射实验条件所测量的2θ值(以度数表示)。例如，如本文所述，使用Cu-Kα(Kα11.540598和Kα21.544426)作为辐射源。The term "2θ" as used herein refers to the peak position expressed in degrees based on an experimental setup of an X-ray diffraction experiment, and is typically the abscissa unit in a diffraction pattern. If the reflection is diffracted when the incident beam forms an angle θ with a lattice plane, the experimental setup requires recording the reflected beam at an angle 2θ. It should be understood that reference herein to a specific 2Θ value for a particular crystalline form is intended to mean the 2Θ value (expressed in degrees) measured using the X-ray diffraction experimental conditions described herein. For example, using Cu-Kα(Kα1 1.540598 and Kα2 1.544426) as a radiation source.

如本文中所使用，“I％”表示峰强度百分比。As used herein, "I%" means peak intensity percentage.

如本文中所使用的术语“差示扫描量热(DSC)图谱”是指由差示扫描量热仪记录到的曲 线。除非另外说明，在描述DSC图谱中特征峰时所提及的温度是指峰的起始温度。As used herein, the term "differential scanning calorimetry (DSC) spectrum" refers to the curve recorded by a differential scanning calorimeter. Wire. Unless otherwise stated, the temperatures mentioned when describing characteristic peaks in DSC spectra refer to the peak onset temperature.

如本文中所使用的术语“热重分析(TGA)图谱”是指由热重分析仪记录到的曲线。The term "thermogravimetric analysis (TGA) profile" as used herein refers to the curve recorded by a thermogravimetric analyzer.

如本文中所使用的，对于X射线衍射峰位的术语“基本上相同”意指将代表性峰位和强度变化考虑在内。例如，本领域技术人员会理解峰位(2θ)会显示一些变化，通常多达0.1-0.3度，并且用于测量衍射的仪器也会显示一些变化。另外，本领域技术人员会理解相对峰强度会显示仪器间的变化以及由于结晶性程度、择优取向、制备的样品表面以及本领域技术人员已知的其它因素的变化。相似地，如本文中所使用，对于DSC图谱的“基本上相同”也意图涵盖本领域技术人员已知的与这些分析技术有关的变化。例如，对于边界清楚的峰，在差示扫描量热图谱通常会具有多达±0.2℃的变化，对于宽峰甚至更大(例如多达±1℃)。As used herein, the term "substantially the same" with respect to X-ray diffraction peak positions means taking representative peak positions and intensity variations into account. For example, those skilled in the art will understand that the peak position (2θ) will show some variation, typically as much as 0.1-0.3 degrees, and that the instruments used to measure diffraction will also show some variation. Additionally, those skilled in the art will understand that relative peak intensities will exhibit inter-instrument variation as well as variations due to degree of crystallinity, preferred orientation, prepared sample surface, and other factors known to those skilled in the art. Similarly, as used herein, "substantially the same" with respect to a DSC profile is also intended to encompass variations known to those of skill in the art associated with these analytical techniques. For example, there will typically be a variation in a differential scanning calorimetry spectrum of up to ±0.2°C for well-defined peaks, and even larger (e.g., up to ±1°C) for broad peaks.

本申请中的液态核磁谱图优选在Bruker 400M核磁共振仪上采集，除非另外说明，以DMSO-d6作为溶剂。The liquid NMR spectra in this application are preferably collected on a Bruker 400M NMR instrument, using DMSO-d6 as the solvent unless otherwise stated.

本申请中的偏光显微数据优选通过Polarizing Microscope ECLIPSE LV100POL(Nikon,JPN)进行采集。The polarizing microscopy data in this application are preferably collected by Polarizing Microscope ECLIPSE LV100POL (Nikon, JPN).

如本文中所使用的数值范围(如“1-10个”、“1-6个”、“2-10个”、“2-6个”、“3-10个”、“5-10个”、“3-6个”)等涵盖所述数值范围中的任意个(例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)。As used herein, numerical ranges (such as "1-10", "1-6", "2-10", "2-6", "3-10", "5-10 ", "3-6"), etc. cover any number in the stated numerical range (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ).

可将制备的晶体形式通过包括倾析、离心、蒸发、重力过滤、抽滤或者在加压下或在减压下的任何其它用于固体回收的技术在内的方法进行回收。可将回收的固体任选地进行干燥。本申请中的“干燥”是在减压(优选真空)下进行直到残留溶剂的含量降低至International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use(“ICH”)指南所给出的限度的范围内。残留溶剂含量取决于溶剂的类型，但不超过约5000ppm、或优选约4000ppm、或更优选约3000ppm。所述干燥可以在盘式干燥器、真空烘箱、空气烘箱、锥形真空干燥器(cone vacuum dryer)、旋转式真空干燥器、流化床干燥器、旋转闪蒸干燥器、快速干燥器等中进行。所述干燥可以在低于约100℃、低于约80℃、低于约60℃、低于约50℃、低于约30℃的温度或任何其它合适的温度下，在大气压或减压(优选真空)下在能够实现期望的结果的任何期望的时间内(如约1、2、3、5、10、15、20、24小时或者过夜)进行，只要盐的品质不劣化。所述干燥可以进行任何期望的次数，直到实现所需的产物品质。干燥的产物可以任选地经历粉碎操作，以产生期望的粒度。可在产物的干燥前或干燥完成后进行研磨或微粉化。可用于减 小粒度的技术包括但不限于球磨、辊磨和锤磨，以及喷射研磨(jet milling)。The prepared crystalline form may be recovered by methods including decantation, centrifugation, evaporation, gravity filtration, suction filtration, or any other technique for solids recovery under pressure or under reduced pressure. The recovered solids can optionally be dried. "Drying" in this application is carried out under reduced pressure (preferably vacuum) until the content of residual solvent is reduced to the limit given by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use ("ICH") within the range. Residual solvent content depends on the type of solvent but does not exceed about 5000 ppm, or preferably about 4000 ppm, or more preferably about 3000 ppm. The drying can be in a tray dryer, vacuum oven, air oven, cone vacuum dryer, rotary vacuum dryer, fluidized bed dryer, spin flash dryer, rapid dryer, etc. conduct. The drying may be at a temperature below about 100°C, below about 80°C, below about 60°C, below about 50°C, below about 30°C, or any other suitable temperature, at atmospheric pressure or reduced pressure ( Preferably under vacuum) for any desired period of time capable of achieving the desired results (such as about 1, 2, 3, 5, 10, 15, 20, 24 hours or overnight) as long as the quality of the salt is not degraded. The drying can be carried out any desired number of times until the desired product quality is achieved. The dried product may optionally undergo comminution operations to produce the desired particle size. Grinding or micronization can be carried out before drying of the product or after drying is complete. Can be used to reduce Small particle size technologies include, but are not limited to, ball milling, roller milling, and hammer milling, as well as jet milling.

如本文中所使用的术语“无水合物”优选意指其中不含有水分子作为结构要素的晶型。The term "anhydrate" as used herein preferably means a crystalline form which does not contain water molecules as a structural element.

如本文中所使用的术语“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险比相应的其它问题或并发症。The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered and which, within the scope of sound medical judgment, is suitable for contact human and/or other animal tissue without undue toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit/risk ratio.

在本申请所述的药物组合物中可使用的药学上可接受的载体包括但不限于无菌液体，例如水和油，包括那些石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、芝麻油等。当所述药物组合物通过静脉内给药时，水是示例性载体。还可以使用生理盐水和葡萄糖及甘油水溶液作为液体载体，特别是用于注射液。适合的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽糖、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等。所述组合物还可以视需要包含少量的湿润剂、乳化剂或pH缓冲剂。口服制剂可以包含标准载体，如药物级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(1990)中所述。Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions described herein include, but are not limited to, sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil , mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous glucose and glycerol solutions may also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol etc. The compositions may also, if desired, contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

如本文中所使用的术语“治疗有效量”指被给药后会在一定程度上缓解所治疗病症的一或多种症状的化合物的量。The term "therapeutically effective amount" as used herein refers to an amount of a compound that, when administered, alleviates to a certain extent one or more symptoms of the condition being treated.

可调整给药方案以提供最佳所需响应。例如，可给药单次推注，可随时间给药数个分剂量，或可如治疗情况的急需所表明而按比例减少或增加剂量。要注意，剂量值可随要减轻的病况的类型及严重性而变化，且可包括单次或多次剂量。要进一步理解，对于任何特定个体，具体的给药方案应根据个体需要及给药组合物或监督组合物的给药的人员的专业判断来随时间调整。Dosage regimens can be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased as the exigencies of the therapeutic situation indicate. It is noted that dosage values may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that, for any particular individual, specific dosage regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions.

所给药的本申请所述的化合物I的晶型I的量会取决于所治疗的个体、病症或病况的严重性、给药的速率、化合物的处置及处方医师的判断。一般而言，有效剂量在每日每kg体重约0.0001至约50mg，例如约0.01至约10mg/kg/日(单次或分次给药)。对70kg的人而言，这会合计为约0.007mg/日至约3500mg/日，例如约0.7mg/日至约700mg/日。在一些情况下，不高于前述范围的下限的剂量水平可以是足够的，而在其它情况下，仍可在不引起任何有害副作用的情况下采用较大剂量，条件是首先将所述较大剂量分成数个较小剂量以在一整天中给药。The amount of Form I of Compound I described herein that is administered will depend on the individual being treated, the severity of the disorder or condition, the rate of administration, disposition of the compound, and the judgment of the prescribing physician. Generally speaking, the effective dose is about 0.0001 to about 50 mg per kg of body weight per day, for example, about 0.01 to about 10 mg/kg/day (single or divided administration). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, for example about 0.7 mg/day to about 700 mg/day. In some cases, dosage levels no higher than the lower end of the foregoing ranges may be sufficient, while in other cases, larger dosages may still be employed without causing any deleterious side effects, provided that the larger dosage is first The dose is divided into several smaller doses to be administered throughout the day.

本申请所述的化合物I的晶型I在药物组合物中的含量或用量可以是约0.01mg至约1000mg，适合地是0.1-500mg，优选0.5-300mg，更优选1-150mg。 The content or amount of the crystalline Form I of Compound I described in the present application in the pharmaceutical composition may be about 0.01 mg to about 1000 mg, suitably 0.1-500 mg, preferably 0.5-300 mg, more preferably 1-150 mg.

除非另外说明，否则如本文中所使用，术语“治疗(treating)”意指逆转、减轻、抑制这样的术语所应用的病症或病况或者这样的病症或病况的一或多种症状的进展，或预防这样的病症或病况或者这样的病症或病况的一或多种症状。Unless otherwise stated, the term "treating" as used herein means reversing, alleviating, inhibiting the disorder or condition to which such term applies or the progression of one or more symptoms of such disorder or condition, or Preventing such a disease or condition or one or more symptoms of such a disease or condition.

如本文所使用的“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本申请中“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。"Individual" as used herein includes humans or non-human animals. Exemplary human subjects include human subjects (referred to as patients) suffering from a disease, such as those described herein, or normal subjects. "Non-human animals" as used herein includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (e.g., sheep, dogs, etc.) , cats, cows, pigs, etc.).

附图说明Description of the drawings

图1示出了化合物I的晶型I的XRPD图谱；Figure 1 shows the XRPD pattern of Form I of Compound I;

图2示出了化合物I的晶型I的差示扫描量热(DSC)图谱；Figure 2 shows a differential scanning calorimetry (DSC) spectrum of Form I of Compound I;

图3示出了化合物I的晶型I的的热重分析(TGA)图谱；Figure 3 shows a thermogravimetric analysis (TGA) spectrum of Form I of Compound I;

图4示出了化合物I的¹H-NMR谱图；Figure 4 shows the ¹ H-NMR spectrum of compound I;

图5示出了化合物I的¹³C-NMR谱图；Figure 5 shows the ¹³ C-NMR spectrum of compound I;

图6示出了化合物I的HRMS谱图；Figure 6 shows the HRMS spectrum of compound I;

图7示出了化合物I体外抗CCHFV的效果，其中A显示了化合物I抗CCHFV初筛结果；B示出了化合物I的EC₅₀及CC₅₀；Figure 7 shows the anti-CCHFV effect of Compound I in vitro, where A shows the preliminary screening results of Compound I against CCHFV; B shows the EC ₅₀ and CC ₅₀ of Compound I;

图8示出了化合物I能有效抑制CCHFV的体内感染，其中A示出了小鼠体重变化曲线及存活率；B示出了小鼠组织中病毒拷贝数检测结果；C示出了EIDD-2801无法抑制CCHFV的体内感染；Figure 8 shows that compound I can effectively inhibit CCHFV infection in vivo, where A shows the mouse body weight change curve and survival rate; B shows the detection results of virus copy number in mouse tissue; C shows EIDD-2801 Unable to suppress CCHFV infection in vivo;

图9示出了低剂量化合物I能有效抑制CCHFV的体内感染，其中A示出了小鼠体重变化曲线及存活率；B示出了小鼠组织中病毒拷贝数检测结果；Figure 9 shows that low-dose Compound I can effectively inhibit CCHFV infection in vivo, where A shows the mouse body weight change curve and survival rate; B shows the detection results of virus copy number in mouse tissues;

图10示出了延迟给药化合物I能有效抑制CCHFV的体内感染，其中A示出了小鼠体重变化曲线及存活率；B示出了小鼠组织中病毒拷贝数检测结果；C示出了小鼠肝脏及脾脏病理变化。Figure 10 shows that delayed administration of Compound I can effectively inhibit CCHFV infection in vivo, where A shows the mouse body weight change curve and survival rate; B shows the detection results of virus copy number in mouse tissue; C shows Pathological changes in mouse liver and spleen.

实施例Example

以下将结合实施例更详细地解释本申请，本申请的实施例仅用于说明本申请的技术方案，并非用于限定本申请的范围，本领域技术人员可进行一些非本质的改进和调整，仍属于本申请的保护范围。The present application will be explained in more detail below with reference to the examples. The examples of the present application are only used to illustrate the technical solutions of the present application and are not used to limit the scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments. still fall within the protection scope of this application.

除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。 Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

以下实施例中使用的检测仪器及条件如下：The detection instruments and conditions used in the following examples are as follows:

(1)X-射线粉末衍射(XRPD)(1)X-ray powder diffraction (XRPD)

仪器型号：Bruker D8advance，配备LynxEye检测器Instrument model: Bruker D8advance, equipped with LynxEye detector

测试条件：阳极靶材料为铜，光管设定为(40KV 40mA)，样品的2θ扫描角度从3°到40°，扫描步长为0.02°。Test conditions: The anode target material is copper, the light tube is set to (40KV 40mA), the 2θ scanning angle of the sample is from 3° to 40°, and the scanning step is 0.02°.

(2)差示扫描量热分析(DSC)(2) Differential scanning calorimetry (DSC)

仪器型号：TA Discovery DSC 250(TA Instruments,US)Instrument model: TA Discovery DSC 250 (TA Instruments, US)

测试条件：升温速率为10℃/min，干燥氮气用作吹扫气体。Test conditions: The heating rate is 10°C/min, and dry nitrogen is used as the purge gas.

(3)热重分析(TGA)(3) Thermogravimetric analysis (TGA)

仪器型号：Discovery TGA 55(TA Instruments,US)Instrument model: Discovery TGA 55 (TA Instruments, US)

测试条件：加热炉内自动称量，升温速率为10℃/min，干燥氮气用作吹扫气体。Test conditions: Automatic weighing in the heating furnace, the heating rate is 10°C/min, and dry nitrogen is used as the purge gas.

实施例1：化合物I的晶型I的制备一Example 1: Preparation of Crystal Form I of Compound I -

在反应罐中加入化合物I粗品3-4公斤、35升无水乙醇，加热至溶液澄清，再加入0.5Kg 767#针用活性炭，在70～80℃温度下回流0.5小时，过滤，用5L无水乙醇洗涤滤饼。滤液保持温度在55～65℃、真空度≤-0.08Mpa下进行减压浓缩至有大量固体析出时停止，向浓缩后的反应液中加入15L蒸馏水，冷却至0～10℃，搅拌结晶3h，过滤，用5L饮用水洗涤滤饼，滤饼再放入真空干燥箱，并控制干燥箱内温在40～50℃、真空度≤-0.08MPa下干燥8～16小时，得固体晶型I，其为无水合物。Add 3-4 kg of compound I crude product and 35 liters of absolute ethanol into the reaction tank, heat until the solution is clear, then add 0.5Kg 767# needle activated carbon, reflux at 70-80°C for 0.5 hours, filter, and use Wash the filter cake with water and ethanol. The filtrate is concentrated under reduced pressure at a temperature of 55-65°C and a vacuum degree of ≤-0.08Mpa until a large amount of solid is precipitated. Add 15L of distilled water to the concentrated reaction solution, cool to 0-10°C, and stir for 3 hours to crystallize. Filter, wash the filter cake with 5L of drinking water, put the filter cake into a vacuum drying oven, and control the temperature inside the drying oven to dry at 40-50°C and a vacuum degree of ≤-0.08MPa for 8-16 hours to obtain solid crystal form I. It is anhydrous.

经X-射线粉末衍射检测，所得固体晶型I的XRPD图谱如图1所示。对所得固体晶型I进行差示扫描量热分析(DSC)和热重分析(TGA)，所得DSC图谱和TGA图谱分别如图2和图3所示。After X-ray powder diffraction detection, the XRPD pattern of the obtained solid crystal form I is shown in Figure 1. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were performed on the obtained solid crystal form I, and the obtained DSC spectra and TGA spectra are shown in Figure 2 and Figure 3 respectively.

实施例2：化合物I的晶型I的制备二Example 2: Preparation II of Crystalline Form I of Compound I

将1.3g化合物I加入到100mL乙酸乙酯中，室温下搅拌至完全溶解，将所得溶液过滤并收集滤液。向滤液中缓慢滴加300ml石油醚，室温下搅拌过夜，析出固体，过滤悬浮液，得到固体晶型I，其为无水合物。经X-射线粉末衍射检测，所得固体晶型I的XRPD图谱基本与图1相同。对所得固体晶型I进行差示扫描量热分析(DSC)和热重分析(TGA)，所得DSC图谱基本与图2相同，所得TGA图谱基本与图3相同。Add 1.3 g of compound I to 100 mL of ethyl acetate, stir at room temperature until completely dissolved, filter the resulting solution and collect the filtrate. Slowly add 300 ml of petroleum ether dropwise to the filtrate, stir at room temperature overnight, solids precipitate, and the suspension is filtered to obtain solid crystal form I, which is an anhydrate. After X-ray powder diffraction detection, the XRPD pattern of the obtained solid crystal form I was basically the same as Figure 1. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were performed on the obtained solid crystal form I. The obtained DSC spectrum was basically the same as Figure 2, and the obtained TGA spectrum was basically the same as Figure 3.

以上实施例1和2用到的化合物I按照实施例3记载的方法制备获得。Compound I used in Examples 1 and 2 above was prepared according to the method described in Example 3.

实施例3：化合物I(((3-氨甲酰-5-氟吡嗪-2-基)氧基)甲基异丁酸酯)的制备
Example 3: Preparation of compound I (((3-carbamoyl-5-fluoropyrazin-2-yl)oxy)methylisobutyrate)

在氮气保护下，将T-705(1.57g，10mmol)溶于无水乙腈(10mL)中，溶液在室温搅拌15min后，滴加入三乙胺(3.03g，30mmol)，并将反应液降温至-20℃，随后滴入溴甲基异丁酸酯(3.41g，25mmol)。反应液继续在-20℃下反应24h后，将反应液倾倒入冷水(100mL)中，混合物采用二氯甲烷萃取三次，合并的有机相分别采用1N盐酸、饱和碳酸氢钠溶液、饱和氯化钠溶液洗涤两次后，采用硫酸钠干燥过夜。将干燥后的有机相浓缩至干，经硅胶拌样后采用Flash柱层析进行分离纯化(二氯甲烷/甲醇洗脱)得到目标产物化合物I。¹H-NMR(400MHz,CDCl₃):δ(ppm)8.19(d,J＝8.0Hz,1H,pyrazine H),7.37(brs,1H,CONH₂),6.21(s,2H,CH₂),6.18–6.05(brs,1H,CONH₂),2.58(dt,J＝14.0,7.0Hz,1H,CH),1.16(d,J＝4.0Hz,6H,CH3).¹³C-NMR(100MHz,CDCl₃)δ176.09(O＝C-O),163.08(O＝C-NH₂),154.90(C-F,¹J_C-F＝247Hz),154.50(C-F,⁴J_C-F＝2Hz),132.19(C-C-F,J_C-C-F＝41Hz),129.20(C-F,³J_C-F＝7Hz),82.83(CH₂),33.88(CH),8.72(CH₃).HRMS(ESI⁺)m/z[M+H]⁺calculated for C₁₀H₁₂FN₃O₄:258.0885；found:258.0893.HRMS(ESI⁺)m/z[M+Na]⁺calculated for C₁₀H₁₂FN₃NaO₄:280.0710；found:280.0713.Under nitrogen protection, T-705 (1.57g, 10mmol) was dissolved in anhydrous acetonitrile (10mL). After the solution was stirred at room temperature for 15min, triethylamine (3.03g, 30mmol) was added dropwise, and the reaction solution was cooled to -20°C, followed by dropwise addition of bromomethyl isobutyrate (3.41 g, 25 mmol). After the reaction solution continued to react at -20°C for 24 hours, the reaction solution was poured into cold water (100 mL). The mixture was extracted three times with dichloromethane. The combined organic phases were treated with 1N hydrochloric acid, saturated sodium bicarbonate solution, and saturated sodium chloride. After the solution was washed twice, it was dried over sodium sulfate overnight. The dried organic phase was concentrated to dryness, mixed with silica gel and then separated and purified by Flash column chromatography (elution with dichloromethane/methanol) to obtain the target product compound I. ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 8.19 (d, J = 8.0Hz, 1H, pyrazine H), 7.37 (brs, 1H, CONH ₂ ), 6.21 (s, 2H, CH ₂ ), 6.18–6.05 (brs, 1H, CONH ₂ ), 2.58 (dt, J＝14.0, 7.0Hz, 1H, CH), 1.16 (d, J＝4.0Hz, 6H, CH3). ¹³ C-NMR (100MHz, CDCl ₃ ) δ176.09 (O＝CO), 163.08 (O＝C-NH ₂ ), 154.90 (CF, ¹ J _CF ＝247Hz), 154.50 (CF, ⁴ J _CF ＝2Hz), 132.19 (CCF, J _CCF ＝ 41Hz),129.20(CF, ³ J _CF =7Hz),82.83(CH ₂ ),33.88(CH),8.72(CH ₃ ).HRMS(ESI ⁺ )m/z[M+H] ⁺ calculated for C ₁₀ H ₁₂ FN ₃ O ₄ :258.0885; found: 258.0893. HRMS(ESI ⁺ )m/z[M+Na] ⁺ calculated for C ₁₀ H ₁₂ FN ₃ NaO ₄ :280.0710; found: 280.0713.

化合物I的¹H-NMR、¹³C-NMR、HRMS谱图如图4-6所示。 ^{The 1} H-NMR, ¹³ C-NMR and HRMS spectra of compound I are shown in Figure 4-6.

实施例4：化合物I降低克里米亚-刚果出血热病毒核酸载量实验Example 4: Experiment on compound I reducing nucleic acid load of Crimean-Congo hemorrhagic fever virus

所有CCHFV感染实验均在BLS-3实验室进行。All CCHFV infection experiments were performed in the BLS-3 laboratory.

1.材料和方法1. Materials and methods

1.1受试化合物：化合物I用DMSO配成100mM母液。实验时用细胞培养液(含2％FBS的MEM培养基)稀释成实验所需的浓度。1.1 Test compound: Compound I was prepared into a 100mM stock solution with DMSO. During the experiment, cell culture medium (MEM medium containing 2% FBS) was used to dilute it to the concentration required for the experiment.

1.2细胞：Vero E6细胞(ATCC编号：1586)为非洲绿猴肾细胞，由中国科学院武汉病毒研究所传代保存。1.2 Cells: Vero E6 cells (ATCC number: 1586) are African green monkey kidney cells, which are passaged and preserved by the Wuhan Institute of Virology, Chinese Academy of Sciences.

1.3病毒：CCHFV YL16070分离株(GenBank登录号：KY354082)由中国科学院武 汉病毒研究所分离传代培养。1.3 Virus: CCHFV YL16070 isolate (GenBank accession number: KY354082) was developed by Wuhan University of Science and Technology, Chinese Academy of Sciences Isolated and subcultured at the Institute of Chinese Virology.

1.4试剂、实验用品及仪器：1.4 Reagents, experimental supplies and instruments:

1.4.1试剂：minimum Eagle’s medium(MEM)培养基，美国GIBCO公司产品；胎牛血清，美国GIBCO公司产品；碳酸氢钠，国药集团产品；青霉素、链霉素和卡那霉素：均为华北制药厂产品。1.4.1 Reagents: minimum Eagle's medium (MEM) culture medium, product of GIBCO Company of the United States; fetal bovine serum, product of GIBCO Company of the United States; sodium bicarbonate, product of Sinopharm Group; penicillin, streptomycin and kanamycin: all from North China Pharmaceutical factory products.

1.4.2实验用品及仪器：培养瓶，美国Corning公司产品；培养板96孔板，美国Corning公司产品；二氧化碳孵箱，美国Thermo公司产品；1.4.2 Experimental supplies and instruments: culture flask, product of Corning Company of the United States; 96-well culture plate, product of Corning Company of the United States; carbon dioxide incubator, product of Thermo Company of the United States;

1.4.3细胞培养液及试剂配制：MEM培养液100ml：含胎牛血清(fetal bovine serum,FBS)10％，青霉素，链霉素和卡那霉素各100U/ml，NaHCO₃ 5％。细胞消化液：0.25％胰酶，用Hanks液配制，0.02％EDTA。1.4.3 Preparation of cell culture medium and reagents: 100 ml of MEM culture medium: containing 10% fetal bovine serum (FBS), 100 U/ml each of penicillin, streptomycin and kanamycin, and 5% NaHCO ₃ . Cell digestion solution: 0.25% trypsin, prepared with Hanks solution, 0.02% EDTA.

1.5.实验方法：1.5. Experimental methods:

1.5.1Vero E6细胞培养：在长满细胞的培养瓶内加0.25％胰酶0.1ml，0.02％EDTA5ml，37℃消化5分钟，弃消化液，加培养液轻轻吹打，1:3传代，3天长满，供化合物I毒性试验及抑制实验用。1.5.1 Vero E6 cell culture: Add 0.1ml of 0.25% trypsin and 5ml of 0.02% EDTA into a culture flask full of cells. Digest at 37°C for 5 minutes. Discard the digestive fluid, add culture medium and pipet gently. Passage 1:3, 3 Tianchangman is used for toxicity testing and inhibition experiments of compound I.

1.5.2细胞毒性实验1.5.2 Cytotoxicity experiment

化合物I对细胞毒性的检测利用CCK-8试剂盒(Beyotime)测定。具体步骤如下：The cytotoxicity of Compound I was measured using CCK-8 kit (Beyotime). Specific steps are as follows:

①96孔板中接种约2×10⁴个Vero-E6(ATCC)细胞，37℃培养8小时。① Inoculate approximately 2×10 ⁴ Vero-E6 (ATCC) cells in a 96-well plate and culture at 37°C for 8 hours.

②将化合物I的母液用细胞培养液(含2％FBS的MEM培养基)稀释到药物使用浓度，弃96孔板中原培养基，取100μL含化合物I的细胞培养液加入到细胞中，每个浓度做三个复孔。注意设置阴性对照(细胞孔中加含对应浓度DMSO而不含化合物I的细胞培养液)和空白对照(不含细胞，仅加DMSO和细胞培养液)。加药完毕，细胞37℃培养24小时。② Dilute the stock solution of compound I to the drug concentration with cell culture medium (MEM medium containing 2% FBS), discard the original medium in the 96-well plate, and add 100 μL of cell culture medium containing compound I to the cells. Make three replicate wells according to the concentration. Pay attention to setting negative controls (cell culture medium containing corresponding concentration of DMSO without compound I is added to the cell wells) and blank controls (cells are not included, only DMSO and cell culture medium are added). After adding the drug, the cells were cultured at 37°C for 24 hours.

③向待测孔中加入20μL CCK-8溶液(Beyotime)，轻轻混匀，不要产生气泡，37℃继续培养2小时。在酶标仪上读取OD450，计算细胞活性：③Add 20μL CCK-8 solution (Beyotime) into the wells to be tested, mix gently without generating bubbles, and continue to incubate at 37°C for 2 hours. Read OD450 on a microplate reader and calculate cell viability:

细胞活性(％)＝(A(化合物I处理组)-A(空白对照))/(A(阴性对照)-A(空白对照))×100％Cell activity (%)=(A(Compound I treated group)-A(blank control))/(A(negative control)-A(blank control))×100%

1.5.3抗病毒实验：1.5.3 Anti-virus experiment:

①将Vero E6细胞接种到48孔板中，每孔约6×10⁴个细胞，待第二天实验。先将100μL含相应浓度化合物I的细胞培养液加入细胞板中，预处理细胞1小时，然后加入20μL稀释的病毒(MOI＝0.01)，置于培养箱孵育1小时。然后弃病毒培养液，用PBS洗去未感 染的残留病毒，再用加入含相应浓度化合物I的细胞培养液，然后放37℃、5％CO₂孵化箱继续培养72h，细胞对照组加入终浓度0.5％DMSO的细胞培养液。① Inoculate Vero E6 cells into a 48-well plate, approximately 6×10 ⁴ cells per well, and wait for the experiment the next day. First, add 100 μL of cell culture medium containing compound I at the corresponding concentration to the cell plate, pretreat the cells for 1 hour, then add 20 μL of diluted virus (MOI=0.01), and incubate in an incubator for 1 hour. Then discard the virus culture medium and wash away the uninfected virus with PBS. After staining the residual virus, add cell culture medium containing corresponding concentration of Compound I, and then place it in a 37°C, 5% CO ₂ incubator to continue culturing for 72 hours. In the cell control group, add cell culture medium with a final concentration of 0.5% DMSO.

②RNA提取，采用TaKaRa公司生产的试剂盒(TaKaRa MiniBEST Viral RNA/DNA Extraction Kit，货号9766)，步骤如下：② RNA extraction, using the kit produced by TaKaRa Company (TaKaRa MiniBEST Viral RNA/DNA Extraction Kit, Cat. No. 9766), the steps are as follows:

1)取受试培养板的上清液100μL，加入无核酸酶EP管中，然后每孔加入321μL裂解液(100μL PBS,200μL buffer VGB,20μL proteinase K,1μL carrier RNA)，混匀后置于56℃消化30min；1) Take 100μL of the supernatant of the test culture plate, add it to a nuclease-free EP tube, then add 321μL lysis solution (100μL PBS, 200μL buffer VGB, 20μL proteinase K, 1μL carrier RNA) to each well, mix well and place Digest at 56°C for 30 minutes;

2)所得混合液加200μL无水乙醇，混匀；2) Add 200 μL of absolute ethanol to the resulting mixture and mix evenly;

3)将上述混合液转入无RNA酶的离心柱中，12000rpm离心15s，弃废液；3) Transfer the above mixture into an RNase-free spin column, centrifuge at 12,000 rpm for 15 seconds, and discard the waste liquid;

4)加入500μL Buffer RW1，12000rpm离心15s，弃废液；4) Add 500μL Buffer RW1, centrifuge at 12000rpm for 15s, and discard the waste liquid;

5)加入650μL Buffer RW2，12000rpm离心15s，弃废液；5) Add 650μL Buffer RW2, centrifuge at 12000rpm for 15s, and discard the waste liquid;

6)加入650μL Buffer RW2，12000rpm离心2min，弃废液；6) Add 650μL Buffer RW2, centrifuge at 12000rpm for 2 minutes, and discard the waste liquid;

7)换新的无RNA酶的2ml收集管，12000rpm离心1min，使离心柱干燥；7) Replace with a new RNase-free 2ml collection tube and centrifuge at 12000rpm for 1 minute to dry the spin column;

8)换上新的1.5ml收集管，每管加入30μl不含RNA酶的水，12000rpm离心2min，洗脱液即含有相应的RNA。8) Replace with a new 1.5ml collection tube, add 30μl RNase-free water to each tube, and centrifuge at 12000rpm for 2 minutes. The eluate will contain the corresponding RNA.

③RNA反转录，步骤如下：③RNA reverse transcription, the steps are as follows:

实验采用TaKaRa公司生产的反转录试剂盒(PrimeScript^TM RT reagent Kit with gDNA Eraser，货号RR047Q)进行RNA反转录。首先进行gDNA去除：收集各实验组RNA样品，分别取3μL RNA进行反转录。向各实验组RNA中加入2μl 5×gDNA Eraser Buffer，用RNase Free水补足反应体系至10μl，充分混匀，42℃水浴2min去除样品中可能存在的gDNA。然后进行逆转录：向所得样品中加入适量的酶和引物Mix及反应缓冲液，用RNase Free水补足体积至20μl，37℃水浴反应15min，之后投入85℃水中5s，既可转录得到cDNA。The experiment used the reverse transcription kit (PrimeScript ^TM RT reagent Kit with gDNA Eraser, Cat. No. RR047Q) produced by TaKaRa Company for RNA reverse transcription. First, gDNA removal was performed: RNA samples from each experimental group were collected, and 3 μL RNA was taken for reverse transcription. Add 2 μl of 5×gDNA Eraser Buffer to the RNA of each experimental group, make up the reaction system to 10 μl with RNase Free water, mix thoroughly, and take a 42°C water bath for 2 minutes to remove possible gDNA in the sample. Then perform reverse transcription: add an appropriate amount of enzyme, primer mix and reaction buffer to the obtained sample, make up the volume to 20 μl with RNase Free water, react in a 37°C water bath for 15 minutes, and then put it into 85°C water for 5 seconds, and the cDNA can be transcribed.

④Real-time PCR。荧光定量PCR采用TB Green Premix(Takara，Cat#RR820A)混好反应体系，在StepOne Plus Real-time PCR仪(品牌：ABI)进行扩增反应和读数。计算原病毒液每毫升所含拷贝数。步骤如下：④Real-time PCR. Fluorescence quantitative PCR uses TB Green Premix (Takara, Cat#RR820A) to mix the reaction system, and perform amplification reaction and reading on StepOne Plus Real-time PCR instrument (Brand: ABI). Calculate the number of copies per milliliter of the original virus solution. Proceed as follows:

1)首先建立标准品：将质粒pMT-S稀释成5×10⁸,5×10⁷,5×10⁶,5×10⁵,5×10⁴,5×10³和5×10²copies/μL。取2μL标准品或cDNA模板用于qPCR反应；1) First establish the standard: dilute plasmid pMT-S into 5×10 ⁸ , 5×10 ⁷ , 5×10 ⁶ , 5×10 ⁵ , 5×10 ⁴ , 5×10 ³ and 5×10 ² copies/ μL. Take 2 μL of standard or cDNA template for qPCR reaction;

2)实验过程中所用引物序列如下(均为5’-3’方向表示)：2) The primer sequences used during the experiment are as follows (all expressed in the 5’-3’ direction):

S-qF:TCAAGTGGAGGAAGGACATAGG S-qF:TCAAGTGGAGGAAGGACATAGG

S-qR:TCCACATGTTCACGGCTCACTGGGS-qR:TCCACATGTTCACGGCTCACTGGG

3)反应程序如下：3) The reaction procedure is as follows:

预变性：95℃5分钟；Pre-denaturation: 95°C for 5 minutes;

循环参数：95℃15秒，54℃15秒，72℃30秒。共40个循环。Cycle parameters: 95℃ for 15 seconds, 54℃ for 15 seconds, 72℃ for 30 seconds. 40 cycles in total.

2.结果2. Results

2.1化合物I对Vero E6细胞的毒性2.1 Toxicity of compound I to Vero E6 cells

细胞毒性结果显示，化合物I的CC₅₀为242.50μM(表1)。Cytotoxicity results showed that the CC ₅₀ of compound I was 242.50 μM (Table 1).

表1.化合物I的细胞毒性实验结果
Table 1. Cytotoxicity experimental results of compound I

2.2化合物I的抗病毒活性2.2 Antiviral activity of compound I

抗病毒实验的结果显示，化合物I在有效浓度下能够显著抑制CCHFV YL16070株病毒感染上清中病毒基因组的复制(表2)。The results of the antiviral experiment showed that compound I can significantly inhibit the replication of the viral genome in the CCHFV YL16070 strain virus infection supernatant at effective concentrations (Table 2).

表2.化合物I的抗CCHFV YL16070株的效果评价结果
Table 2. Evaluation results of the effect of Compound I against CCHFV YL16070 strain

2.3计算其EC₅₀和CC₅₀ 2.3 Calculate its EC ₅₀ and CC ₅₀

公式：细胞活性(％)＝(A(药物处理组)-A(空白对照))/(A(阴性对照)-A(空白对照))×100％Formula: Cell activity (%) = (A (drug treatment group) - A (blank control)) / (A (negative control) - A (blank control)) × 100%

在病毒感染复数MOI＝0.01的条件下：Under the condition of multiplicity of virus infection MOI=0.01:

化合物I对CCHFV YL16070株的抗病毒效果为：The antiviral effect of compound I against CCHFV YL16070 strain is:

EC₅₀＝8.08μM，CC₅₀＝101.4μM，SI＝12.55。EC ₅₀ =8.08 μM, CC ₅₀ =101.4 μM, SI =12.55.

在初步药物筛选中，用IFA对感染细胞进行染色。结果显示，化合物I在体外20μM时显著抑制了CCHFV的感染(图7中的A)。经计算，化合物I的EC₅₀值为8.08±2.47μM，CC₅₀值为101.4μM,SI为12.55(图7中的B)。In a preliminary drug screen, infected cells were stained with IFA. The results showed that compound I significantly inhibited CCHFV infection at 20 μM in vitro (A in Figure 7). After calculation, the EC ₅₀ value of compound I was 8.08±2.47 μM, the CC ₅₀ value was 101.4 μM, and the SI was 12.55 (B in Figure 7).

实施例5：化合物Ⅰ保护克里米亚-刚果出血热病毒感染小鼠免于死亡的实验Example 5: Experiment of compound I protecting mice infected with Crimean-Congo hemorrhagic fever virus from death

2.1体内实验设计 2.1 In vivo experimental design

所有动物实验均使用C57BL/6J背景的I型IFN受体敲除(IFNAR^-/-)小鼠。12-18周龄的雄性或雌性小鼠通过腹腔注射(IP)感染CCHFV(3000或5000TCID₅₀)。小鼠以IP给药方式给予不同剂量的化合物Ⅰ或EIDD-2801(购自翰香生物科技公司，货号BCP32744)，每日1次，对照组给予类似的溶剂注射。每天监测小鼠的体重和临床症状。当老鼠体重减轻超过20％，对触摸刺激无反应，爬行困难时，用异氟烷麻醉剂对其实施安乐死。对照组死亡后，处死药物治疗组小鼠，并采集小鼠器官组织(肝、脾)进行进一步的病毒载量测定和病理分析。All animal experiments used type I IFN receptor knockout (IFNAR ^−/− ) mice on a C57BL/6J background. Male or female mice aged 12-18 weeks were infected with CCHFV (3000 or 5000 TCID ₅₀ ) by intraperitoneal injection (IP). Mice were given different doses of Compound I or EIDD-2801 (purchased from Hanxiang Biotechnology Company, Cat. No. BCP32744) via IP administration once a day, and the control group was given similar solvent injections. Mice were monitored daily for body weight and clinical signs. When the mice lost more than 20% of their body weight, were unresponsive to touch stimulation, and had difficulty crawling, they were euthanized with isoflurane anesthesia. After the death of the control group, the mice in the drug treatment group were killed, and the mouse organs and tissues (liver, spleen) were collected for further viral load determination and pathological analysis.

2.2实验结果2.2 Experimental results

IFNAR^-/-每只小鼠通过腹腔注射3000TCID₅₀CCHFV。小鼠腹腔注射化合物Ⅰ的剂量为300mg/kg，对照组小鼠腹腔注射生理盐水。从攻毒当天连续给药8天，每天一次。结果显示，对照组出现严重的体重减轻，而化合物Ⅰ治疗组仅出现轻微的体重减轻或体重波动(图8中的A)。对照组在第7天死亡率为80％(6/8)，而化合物Ⅰ治疗组显示出100％的保护(图8中的A)。300mg/kg化合物Ⅰ治疗后肝脏和脾脏的病毒载量减少了约5log10copies/g RNA(P<0.001)(图8中的B)。剂量为150mg/kg的化合物Ⅰ具有类似的效果。EIDD-2801无法抑制CCHFV的体内感染(图8中的C)。此次实验CCHFV感染小鼠组织中病毒拷贝数的结果见表3。IFNAR ^−/− mice were injected intraperitoneally with 3000 TCID ₅₀ CCHFV. Mice were intraperitoneally injected with compound I at a dose of 300 mg/kg, and mice in the control group were intraperitoneally injected with physiological saline. Administration was given continuously for 8 days from the day of challenge, once a day. The results showed that the control group experienced severe weight loss, while the compound I treatment group only experienced slight weight loss or weight fluctuation (A in Figure 8). The control group had a mortality rate of 80% (6/8) on day 7, while the Compound I treated group showed 100% protection (A in Figure 8). The viral load in liver and spleen was reduced by approximately 5 log10 copies/g RNA after treatment with 300 mg/kg compound I (P<0.001) (B in Figure 8). Compound I had a similar effect at a dose of 150 mg/kg. EIDD-2801 was unable to inhibit CCHFV infection in vivo (Fig. 8, C). The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 3.

表3.CCHFV感染小鼠组织中病毒拷贝数(Copies/g tissue)
Table 3. Virus copy number (Copies/g tissue) in CCHFV-infected mouse tissues

为了更详细地表征化合物Ⅰ的保护作用，将病毒感染剂量增加到5000TCID₅₀，同时减少药物剂量。与3000TCID₅₀病毒感染时相似，对照组的存活率为14％(1/7)(图9中 的A)。化合物Ⅰ在150mg/kg或更低剂量75mg/kg时有效地保护小鼠的体重减轻，小鼠存活率为100％。病毒载量检测显示，化合物Ⅰ的两种剂量治疗均显著降低了CCHFV在肝脏和脾脏中的复制(图9中的B)。因此，低剂量(75mg/kg)的化合物Ⅰ有效地保护IFNAR^-/-小鼠免受致命的CCHFV攻击。此次实验CCHFV感染小鼠组织中病毒拷贝数的结果见表4。In order to characterize the protective effect of compound I in more detail, the viral infection dose was increased to 5000 TCID ₅₀ while the drug dose was reduced. Similar to 3000TCID ₅₀ virus infection, the survival rate of the control group was 14% (1/7) (Figure 9 A). Compound I effectively protected mice from weight loss at 150 mg/kg or a lower dose of 75 mg/kg, and the mouse survival rate was 100%. Viral load detection showed that treatment with Compound I at both doses significantly reduced CCHFV replication in the liver and spleen (B in Figure 9). Therefore, a low dose (75 mg/kg) of Compound I effectively protected IFNAR ^−/− mice from lethal CCHFV challenge. The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 4.

表4.CCHFV感染小鼠组织中病毒拷贝数(Copies/g tissue)
Table 4. Virus copy number (Copies/g tissue) in CCHFV-infected mouse tissues

为了评估化合物Ⅰ的延迟治疗是否也提供了有效的保护，CCHFV攻毒小鼠24或48小时后，给药化合物Ⅰ(150mg/kg)，每天一次，直到实验结束。对于24小时的治疗组，化合物Ⅰ完全保护小鼠免受致命的CCHFV感染(图10中的A)。化合物Ⅰ处理的小鼠肝脏和脾脏病毒载量的平均值也显著降低(图10中的B)。然而，当给药时间为攻毒后48小时，化合物Ⅰ处理组的生存率为71％(5/7)(图10中的A)。化合物Ⅰ处理组的小鼠组织在48小时的病毒拷贝数均显著下降(图10中的B)。这些数据表明，150mg/kg剂量下，延迟24小时给药化合物Ⅰ对感染CCHFV的小鼠具有完全的保护作用。此次实验CCHFV感染小鼠组织中病毒拷贝数的结果见表5。To evaluate whether delayed treatment with Compound I also provided effective protection, mice were dosed with Compound I (150 mg/kg) 24 or 48 hours after CCHFV challenge, once a day until the end of the experiment. For the 24-hour treatment group, Compound I completely protected mice from lethal CCHFV infection (Figure 10, A). The average viral load in the liver and spleen of mice treated with Compound I was also significantly reduced (Fig. 10, B). However, when the administration time was 48 hours after challenge, the survival rate of the compound I-treated group was 71% (5/7) (A in Figure 10). The number of virus copies in the mouse tissues of the compound I-treated group decreased significantly at 48 hours (B in Figure 10). These data indicate that compound I at a dose of 150 mg/kg with a 24-hour delay in administration has a complete protective effect on mice infected with CCHFV. The results of the virus copy number in CCHFV-infected mouse tissues in this experiment are shown in Table 5.

表5.CCHFV感染小鼠组织中病毒拷贝数(Copies/g tissue)

Table 5. Virus copy number in CCHFV-infected mouse tissues (Copies/g tissue)

对对照组和24h药物处理组小鼠的肝脏和脾脏组织进行病理学观察。组织病理学分析显示，对照组肝、脾损伤明显(图10中的C)。感染CCHFV的小鼠肝脏出现广泛的凝固性坏死，周围有成纤维细胞增生，肝细胞多灶性点状坏死和水肿(图10中的C)。此外，肝窦单核细胞/淋巴细胞浸润明显增加。感染小鼠脾脏白髓和红髓界限模糊，脾窦单核细胞明显增多(图10中的C)。化合物Ⅰ治疗组仅有少量肝细胞坏死，炎症反应轻微，脾脏白髓和红髓结构较为正常和清晰。同样，在对照组中，通过IFA检测到肝脏和脾脏中丰富的CCHFV NP蛋白，而在化合物Ⅰ治疗组中，病毒蛋白几乎未检测到(图10中的C)。因此，化合物Ⅰ能有效保护小鼠免受病毒感染和组织损伤。Pathological observations were made on the liver and spleen tissues of mice in the control group and 24h drug treatment group. Histopathological analysis showed that the liver and spleen in the control group were significantly damaged (C in Figure 10). The livers of mice infected with CCHFV showed extensive coagulative necrosis, surrounded by fibroblast proliferation, multifocal punctate necrosis and edema of hepatocytes (C in Figure 10). In addition, hepatic sinusoidal mononuclear cell/lymphocyte infiltration was significantly increased. The boundaries between the white pulp and red pulp of the spleen of infected mice were blurred, and mononuclear cells in the splenic sinus were significantly increased (C in Figure 10). In the Compound Ⅰ treatment group, there was only a small amount of liver cell necrosis, mild inflammatory reaction, and the structure of the white pulp and red pulp of the spleen was relatively normal and clear. Similarly, in the control group, abundant CCHFV NP protein was detected in the liver and spleen by IFA, while in the compound I treatment group, the viral protein was almost not detected (C in Figure 10). Therefore, compound I can effectively protect mice from viral infection and tissue damage.

综上，化合物Ⅰ能够有效抑制克里米亚-刚果出血热病毒CCHFV，并能够高效的治疗克里米亚-刚果出血热病毒CCHFV感染引起的疾病、显著提高感染后的存活率。 In summary, compound I can effectively inhibit Crimean-Congo hemorrhagic fever virus CCHFV, and can effectively treat diseases caused by Crimean-Congo hemorrhagic fever virus CCHFV infection and significantly improve the survival rate after infection.

Claims (10)

1. 化合物I的晶型I：
   Form I of Compound I:
   

   所述晶型I的所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱包括在约7.6±0.2°、22.7±0.2°和30.5±0.2°的衍射角(2θ)处的特征峰，The XRPD pattern in 2θ obtained using Cu-Kα radiation of the Form I includes diffraction angles (2θ) of about 7.6±0.2°, 22.7±0.2° and 30.5±0.2°. The characteristic peaks of

   优选地，包括在约7.6±0.2°、13.7±0.2°、17.6±0.2°、18.7±0.2°、19.0±0.2°、19.9±0.2°、22.4±0.2°、22.7±0.2°和30.5±0.2°的衍射角(2θ)处的特征峰，Preferably, included at about 7.6±0.2°, 13.7±0.2°, 17.6±0.2°, 18.7±0.2°, 19.0±0.2°, 19.9±0.2°, 22.4±0.2°, 22.7±0.2° and 30.5±0.2° The characteristic peak at the diffraction angle (2θ),

   优选地，包括在约7.6±0.2°、8.7±0.2°、10.3±0.2°、13.7±0.2°、15.1±0.2°、17.6±0.2°、18.7±0.2°、19.0±0.2°、19.9±0.2°、20.6±0.2°、22.4±0.2°、22.7±0.2°、23.9±0.2°、24.0±0.2°和30.5±0.2°的衍射角(2θ)处的特征峰，Preferably, it is included at about 7.6±0.2°, 8.7±0.2°, 10.3±0.2°, 13.7±0.2°, 15.1±0.2°, 17.6±0.2°, 18.7±0.2°, 19.0±0.2°, 19.9±0.2° , the characteristic peaks at the diffraction angle (2θ) of 20.6±0.2°, 22.4±0.2°, 22.7±0.2°, 23.9±0.2°, 24.0±0.2° and 30.5±0.2°,

   优选地，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD图谱包括与图1所示基本上相同的衍射角(2θ)处的峰，Preferably, the XRPD pattern in 2θ obtained using Cu-Kα radiation of the Form I includes peaks at substantially the same diffraction angle (2θ) as shown in Figure 1,

   优选地，所述晶型I的使用Cu-Kα辐射获得的以2θ角度表示的XRPD峰位与图1所示基本上相同，Preferably, the XRPD peak position expressed in 2θ angle obtained using Cu-Kα radiation of the crystalline form I is substantially the same as shown in Figure 1,

   优选地，所述晶型I为无水合物。Preferably, the crystalline Form I is an anhydrate.
2. 权利要求1所述的晶型I，其DSC图谱包括在约88.39±5℃(例如约88.39℃)处的吸热/放热峰，The crystalline Form I of claim 1, the DSC pattern of which includes an endothermic/exothermic peak at about 88.39±5°C (for example, about 88.39°C),

   优选地，其DSC图谱包括与图2所示基本上相同的特征峰，Preferably, its DSC spectrum includes substantially the same characteristic peaks as shown in Figure 2,

   优选地，其DSC图谱与图2所示基本上相同。Preferably, its DSC pattern is substantially the same as shown in Figure 2.
3. 权利要求1所述的晶型I，其在热重分析中，加热至约265.35±5℃(例如约265.35℃)时有约97.69％的失重，The crystal form I of claim 1 has a weight loss of about 97.69% when heated to about 265.35±5°C (for example, about 265.35°C) in thermogravimetric analysis,

   优选地，TGA图谱与图3所示基本上相同。Preferably, the TGA spectrum is substantially the same as shown in Figure 3.
4. 制备权利要求1所述的晶型I的方法，包括：A method for preparing the crystal form I of claim 1, comprising:

   1)将化合物I在良溶剂中溶解，形成溶液； 1) Dissolve compound I in a good solvent to form a solution;

   2)向所述溶液中添加反溶剂，在搅拌下析出固体。2) Add an anti-solvent to the solution and precipitate a solid under stirring.
5. 药物组合物，其包含权利要求1所述的晶型I，以及一种或多种药学上可接受的载体。A pharmaceutical composition comprising the crystal form I of claim 1 and one or more pharmaceutically acceptable carriers.
6. 权利要求1所述的晶型I在制备用于预防或治疗布尼亚病毒目病毒引起的疾病或感染的药物中的用途，或在制备用于抑制布尼亚病毒目病毒在细胞(例如哺乳动物细胞)中复制或繁殖的药物中的用途，或在制备作为布尼亚病毒目病毒抑制剂的药物中的用途。The use of the crystal form I according to claim 1 in the preparation of medicaments for preventing or treating diseases or infections caused by bunyavirales, or in the preparation of drugs for inhibiting the growth of bunyavirales in cells (such as lactation) Use in drugs that replicate or reproduce in animal cells) or in the preparation of drugs that are inhibitors of viruses of the order Bunyavirales.
7. 权利要求6所述的用途，其中所述的布尼亚病毒目病毒为内罗病毒科病毒，The use of claim 6, wherein the Bunyavirales virus is a Nairoviridae virus,

   优选地，所述的布尼亚病毒目病毒为正内罗病毒属病毒，Preferably, the Bunyavirales virus is a virus of the genus Orthonarovirus,

   优选地，所述的布尼亚病毒目病毒为克里米亚-刚果出血热病毒(CCHFV)。Preferably, the virus of the order Bunyavirales is Crimean-Congo hemorrhagic fever virus (CCHFV).
8. 权利要求6所述的用途，其中所述的布尼亚病毒目病毒引起的疾病为布尼亚病毒目病毒引起的出血热，The use of claim 6, wherein the disease caused by the Bunyavirales virus is hemorrhagic fever caused by the Bunyavirales virus,

   优选地，所述的布尼亚病毒目病毒引起的疾病为布尼亚病毒目病毒引起的单纯性感染、发热、头痛、肌肉疼痛、呕吐、胃肠道出血、鼻出血、呕血或休克；Preferably, the diseases caused by Bunyavirales viruses are simple infections, fever, headache, muscle pain, vomiting, gastrointestinal bleeding, epistaxis, hematemesis or shock caused by Bunyavirales viruses;

   优选地，所述的布尼亚病毒目病毒引起的疾病为内罗病毒科病毒引起的出血热；Preferably, the disease caused by a virus of the order Bunyavirales is a hemorrhagic fever caused by a virus of the family Nairoviridae;

   优选地，所述的布尼亚病毒目病毒引起的疾病为正内罗病毒属病毒引起的出血热；Preferably, the disease caused by a virus of the order Bunyavirales is a hemorrhagic fever caused by a virus of the genus Orthonarovirus;

   优选地，所述的布尼亚病毒目病毒引起的疾病为克里米亚-刚果出血热病毒引起的出血热。Preferably, the disease caused by a Bunyavirales virus is a hemorrhagic fever caused by Crimean-Congo hemorrhagic fever virus.
9. 权利要求6所述的用途，其中所述布尼亚病毒目病毒引起的疾病为克里米亚-刚果出血热。The use of claim 6, wherein the disease caused by the Bunyavirales virus is Crimean-Congo hemorrhagic fever.
10. 权利要求6的用途，其中所述哺乳动物包括牛科动物、马科动物、羊亚科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物，例如是人、猫、狗或猪。 The use of claim 6, wherein the mammal includes bovine, equid, ovine, porcine, canine, feline, rodent, primate, for example, human, Cat, dog or pig.