WO2019154192A1 - 一种3,4-二氢噻吩并[3,2-d]嘧啶类化合物的晶型及其制备方法 - Google Patents

一种3,4-二氢噻吩并[3,2-d]嘧啶类化合物的晶型及其制备方法 Download PDF

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WO2019154192A1
WO2019154192A1 PCT/CN2019/073700 CN2019073700W WO2019154192A1 WO 2019154192 A1 WO2019154192 A1 WO 2019154192A1 CN 2019073700 W CN2019073700 W CN 2019073700W WO 2019154192 A1 WO2019154192 A1 WO 2019154192A1
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compound
crystalline form
preparation
water
crystal form
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French (fr)
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熊剑
王晶晶
胡伯羽
谭海忠
陈新海
黎健
陈曙辉
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南京明德新药研发股份有限公司
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Priority to JP2020542935A priority Critical patent/JP7076845B2/ja
Priority to ES19751253T priority patent/ES2971079T3/es
Priority to AU2019218150A priority patent/AU2019218150B2/en
Priority to US16/968,206 priority patent/US11174270B2/en
Priority to EP19751253.6A priority patent/EP3753942B1/en
Priority to CN201980012560.XA priority patent/CN111741960B/zh
Priority to KR1020207025733A priority patent/KR102423393B1/ko
Publication of WO2019154192A1 publication Critical patent/WO2019154192A1/zh

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    • 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
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

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  • the present invention relates to a crystal form of a 3,4-dihydrothieno[3,2-d]pyrimidine compound and a process for the preparation thereof, and to the use of the crystal form for the preparation of a medicament for treating a disease associated with HCMV virus.
  • HCMV Human cytomegalovirus
  • GCV ganciclovir
  • VGCV valganciclovir
  • FOS sodium foscarnet
  • CDV cidofovir
  • ganciclovir against cytomegalovirus
  • Sodium foscarnet and cidofovir are the two most common alternatives, but both have nephrotoxicity. Mutations in viral DNA polymerase targeted by these agents may lead to drug resistance, so there is still a largely unmet clinical need in the clinic, and we urgently need new and safer anti-human cytomegalovirus drugs.
  • the present invention provides Form A of Compound 1, the X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 theta angles:
  • the X crystal form of the above Compound 1 has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 theta angles: 9.15 ⁇ 0.2 °, 11.06 ⁇ 0.2 °, 11.95 ⁇ 0.2 °, 17.69 ⁇ 0.2 °. 19.03 ⁇ 0.2°, 19.46 ⁇ 0.2°, 20.00 ⁇ 0.2°, 20.63 ⁇ 0.2°.
  • the X-form analysis data of the A crystal form of the above Compound 1 is shown in Table 1.
  • the crystalline form of Form A of Compound 1 above has a differential scanning calorimetry curve having an onset of endothermic peak at 214.47 °C ⁇ 2 °C.
  • the A crystal form of the above compound 1 has a DSC pattern as shown in FIG.
  • the A crystal form of the above compound 1 has a thermogravimetric analysis curve at a weight loss of 0.1206% at 120.00 ⁇ 2 °C.
  • the T crystal of Form A of Compound 1 above is shown in Figure 3.
  • the invention also provides a preparation method of the A crystal form, which comprises adding the compound 1 to a mixed solvent of an alcohol solvent and water, heating and dissolving, and then cooling and crystallization.
  • the method for producing the above A crystal form wherein the alcohol solvent is selected from the group consisting of methanol, ethanol, and isopropanol.
  • the method for producing the above A crystal form wherein the mixed solvent of the alcohol solvent and water is selected from the group consisting of a mixed solvent of ethanol and water.
  • the volume ratio of the alcohol solvent to water in the mixed solvent of the alcohol solvent and water is selected from 1:0.2 to 1.5.
  • the invention also provides the use of the crystalline form A of Compound 1 for the preparation of a medicament for anti-HCMV virus.
  • Compound A has stable crystal form properties, low hygroscopicity, and good pharmaceutical prospects.
  • Compound 1 exhibited a good inhibitory activity against human cytomegalovirus replication in vitro.
  • Compound 1 has a reasonable plasma protein binding rate in all three species of plasma, indicating that the test compound has a moderate ratio of free drug concentration in the plasma of the above three species, and has good drug-forming properties.
  • Compound 1 exhibited excellent in vivo efficacy. During the in vivo efficacy study, the mice were stable in weight and clinically observed without abnormalities, indicating that the series of compounds had no significant side effects on mice at the dose administered.
  • Compound 1 is mainly used to prevent human cytomegalovirus infection in patients undergoing allogeneic hematopoietic stem cell transplantation, kidney transplantation, lung transplantation, and pancreas transplantation. Compared with existing clinical drugs, it has less toxic side effects, better oral bioavailability, and lower risk of drug resistance.
  • intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, combinations thereof with other chemical synthesis methods, and those skilled in the art.
  • Well-known equivalents, preferred embodiments include, but are not limited to, embodiments of the invention.
  • the present invention employs the following abbreviations: DMF stands for dimethylformamide; MsOH stands for methanesulfonic acid; EtOH stands for ethanol; NaOH stands for sodium hydroxide; M stands for mol/L; NBS stands for.
  • XRPD X-ray powder diffractometer
  • Test method Approximately 10-20 mg samples were used for XRPD detection.
  • DSC Differential Scanning Calorimeter
  • Test method A sample ( ⁇ 1 mg) was placed in a DSC aluminum pan for testing, and the sample was heated from 30 ° C to 280 ° C at a heating rate of 10 ° C / min under 50 mL / min N 2 .
  • TGA Thermal Gravimetric Analyzer
  • Test method A sample (2 to 5 mg) was placed in a TGA platinum pot for testing, and the sample was heated from room temperature to 300 ° C at a heating rate of 10 ° C/min under a condition of 25 mL/min N 2 .
  • Figure 1 is an XRPD spectrum of Cu-K ⁇ radiation of Form A of Compound 1.
  • Figure 3 is a TGA spectrum of Form A of Compound 1.
  • Step 1 Synthesis of Compound BB-1-3
  • Trifluoroacetic acid (4.62 g, 6.91 eq) was added to a solution of BB- 1-3 (1.80 g, 1.00 eq) in dichloromethane (30.00 mL), and the mixture was stirred at 15 ° C for 12 hours. The reaction mixture was concentrated to give a crude compound BB-1 (4.5 g, TFA). MS (ESI) m/z: 208.0 [M+].
  • HCMV human cytomegalovirus
  • EC50 EC 50
  • the HCMV used in this experiment was inserted into the reporter-enhanced green fluorescent protein (EGFP) by gene recombination, and the intracellular replication of the virus can be reflected by the expression level of GFP.
  • the inhibitory activity of the compounds on HCMV GFP expression was evaluated by measuring the fluorescence intensity in the wells of different concentrations of the compounds using the high-content cell analysis platform Acumen eX3.
  • MRC5 cells were seeded into black 96-well cell culture plates at a density of 20,000 cells per well, and then cultured overnight at 37 ° C in a 5% CO 2 incubator.
  • the US3-6-EGFP-HCMV-AD169 virus was added to the cell culture well at a certain MOI (0.003-0.1) and cultured for 3 hours at 37 ° C in a 5% CO 2 incubator.
  • the virus-containing medium was aspirated and 200 ⁇ l of cell culture medium containing different concentrations of compound (4 fold dilution, 6 test concentration points) was added. The final concentration of DMSO in the medium was 1%.
  • Virus control wells (with DMSO added, no compound added) and control wells (high concentration of control compound added) were set up.
  • the cell plates were incubated at 37 ° C in a 5% CO 2 incubator for 10 days, and the cells were changed on days 4 and 7. After 10 days of culture, the fluorescence intensity was measured using a high content cell analysis platform Acumen eX3 (TTP LabTech). Raw data was used for compound antiviral activity calculations.
  • the percent inhibition were introduced into GraphPad Prism software for data processing corresponding to the dose of the compound obtained - response curves and derived EC 50 values of the test compound.
  • the protein binding rate of the test compound in CD-1 mice, SD rats, and human plasma was evaluated by equilibrium dialysis.
  • Compound 1 was separately diluted into the plasma of the above three species to prepare a sample having a final concentration of 2 ⁇ M, and then the sample was added to a 96-well equilibrium dialysis apparatus, and dialyzed against a phosphate buffer solution at 37 ° C for 4 hours.
  • Warfarin was used as a control compound in the experiment.
  • the concentration of the test compound and warfarin in plasma and buffer was determined by LC-MS/MS.
  • H stands for human
  • R stands for rat
  • M stands for mouse
  • Compound 1 has a reasonable plasma protein binding rate in the plasma of three species, indicating that the free drug concentration of the test compound is moderate in the plasma of the above three species, and has good drug-forming properties.
  • HCMV Human cytomegalovirus
  • a gelatin sponge containing HCMV was transplanted into mice, and after 9 days of continuous administration, a gelatin sponge was collected for plaque detection.
  • the anti-HCMV effect of the compounds in this model was evaluated by measuring the amount of HCMV in the gelatin sponge.
  • the animals selected for the experiment were NOD SCID mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.), 5 weeks, male. 5 animals per group.
  • the day when the mice were subjected to gelatin sponge transplantation was set to day 0.
  • the animals were anesthetized by intraperitoneal injection of pentobarbital sodium at 75 mg/kg (10 ml/kg). After the animals entered the deep anesthesia state, the treated gelatin sponge was subcutaneously transplanted into the back of the mice.

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Abstract

3,4-二氢噻吩并[3,2-d]嘧啶类化合物(1)的A晶型及其制备方法和在制备抗HCMV病毒药物中的应用。

Description

一种3,4-二氢噻吩并[3,2-d]嘧啶类化合物的晶型及其制备方法
相关申请的引用
本申请主张如下优先权：
CN201810130625.8，申请日2018-02-08；
技术领域
本发明涉及一种3,4-二氢噻吩并[3,2-d]嘧啶类化合物晶型及其制备方法，还包括所述晶型在制备治疗与HCMV病毒相关疾病的药物中的应用。
背景技术
人类巨细胞病毒(HCMV)是人类8类疱疹病毒之一，它有着世界范围的分布和较高的临床发现。尽管诊断和治疗在进步，但是在孕期和免疫低下相关的临床情况：如器官或骨髓移植，癌症和艾滋病，人类巨细胞病毒感染依然有着显著的并发症。目前批准的抗病毒的药品包括更昔洛韦(GCV)，其前药缬更昔洛韦(VGCV)膦甲酸钠(FOS)和西多福韦(CDV)，这些药物的靶标都是病毒DNA聚合酶抑制剂。虽然有效，但是这些药品因为严重的毒副作用、低口服生物利用度(VGCV除外)，以及发生耐药性而受到限制。更昔洛韦抗巨细胞病毒的疗效有限，并且存在毒性。膦甲酸钠和西多福韦是两个最常见的替代品，但两者都有肾毒性。这些试剂靶向的病毒DNA聚合酶的突变可能导致耐药性，因此临床上仍然存在着极大未被满足的临床需求，我们迫切需要全新的、更为安全的抗人巨细胞病毒药物。
发明内容
本发明提供了化合物1的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：
17.69±0.2°、20.00±0.2°、20.63±0.2°。
Figure PCTCN2019073700-appb-000001
本发明的一些方案中，上述化合物1的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：9.15±0.2°、11.06±0.2°、11.95±0.2°、17.69±0.2°、19.03±0.2°、19.46±0.2°、20.00±0.2°、20.63±0.2°。
本发明的一些方案中，上述化合物1的A晶型，其XRPD图谱如图1所示。
本发明的一些方案中，上述化合物1的A晶型，其XRPD图谱解析数据如表1所示。
表1：化合物1的A晶型XRPD图谱解析数据
  2θ d(埃) 背景 峰高 峰高％ 面积 面积％ 半峰宽
1 9.15 9.657 206 799 37.7 7076 28.8 0.148
2 10.327 8.5592 194 150 7.1 1295 5.3 0.145
3 11.061 7.9923 179 641 30.3 5819 23.7 0.152
4 11.952 7.3987 174 536 25.3 6262 25.5 0.196
5 12.188 7.2559 172 334 15.8 4122 16.8 0.207
6 13.081 6.7623 165 86 4.1 865 3.5 0.169
7 14.355 6.1648 164 417 19.7 3603 14.7 0.145
8 15.284 5.7923 164 72 3.4 888 3.6 0.207
9 15.972 5.5442 161 335 15.8 3297 13.4 0.165
10 17.687 5.0104 179 1240 58.5 18975 77.3 0.257
11 18.492 4.7941 185 100 4.7 983 4 0.165
12 19.031 4.6595 210 503 23.7 4482 18.3 0.149
13 19.464 4.5569 215 922 43.5 7869 32.1 0.143
14 19.996 4.4367 227 997 47.1 8550 34.8 0.144
15 20.626 4.3027 203 2119 100 24546 100 0.194
16 21.512 4.1275 176 113 5.3 1085 4.4 0.161
17 22.502 3.948 164 289 13.6 4048 16.5 0.235
18 23.092 3.8485 176 259 12.2 2535 10.3 0.164
19 23.979 3.708 162 392 18.5 5433 22.1 0.232
20 25.535 3.4855 150 107 5 907 3.7 0.142
21 26.109 3.4101 131 181 8.5 3511 14.3 0.325
22 27.669 3.2213 118 227 10.7 3412 13.9 0.252
23 28.891 3.0878 121 203 9.6 1845 7.5 0.152
24 29.444 3.0311 117 95 4.5 1817 7.4 0.321
25 30.825 2.8984 101 134 6.3 2466 10 0.309
26 32.106 2.7856 86 98 4.6 2314 9.4 0.396
27 35.74 2.5102 75 74 3.5 1595 6.5 0.361
本发明的一些方案中，上述化合物1的A晶型，其差示扫描量热曲线在214.47℃±2℃处具有吸热峰的起始点。
本发明的一些方案中，上述化合物1的A晶型，其DSC图谱如图2所示。
本发明的一些方案中，上述化合物1的A晶型，热重分析曲线在120.00±2℃处失重达0.1206％。
本发明的一些方案中，上述化合物1的A晶型，其TGA图谱如图3所示。
本发明还提供了A晶型的制备方法，包括将化合物1加入到醇类溶剂与水的混合溶剂中，加热溶解，然后降温析晶制得。
本发明的一些方案中，上述A晶型的制备方法，其中，所述醇类溶剂选自甲醇、乙醇和异丙醇。
本发明的一些方案中，上述A晶型的制备方法，其中，醇类溶剂与水的混合溶剂选自乙醇和水的混合溶剂。
本发明的一些方案中，上述A晶型的制备方法，其中，所述醇类溶剂与水的混合溶剂中，醇类溶剂和水的体积比选自1：0.2～1.5。
本发明还提供了化合物1的A晶型在制备抗HCMV病毒药物中的应用。
技术效果
化合物1的A晶型性质稳定，吸湿性小，成药前景良好。
化合物1展示出良好的体外抑制人巨细胞病毒复制的抑制活性。
化合物1在三个种属血浆中均具有合理的的血浆蛋白结合率，预示在上述三个种属的血浆中，受试化合物的游离态药物浓度比例适中，具有良好的成药性质。
化合物1展示出了优良的体内药效，在体内药效研究试验过程中，小鼠体重稳定，临床观察无异常，表明该系列化合物在给药剂量下对小鼠无明显副作用。
化合物1主要用于预防异体造血干细胞移植、肾移植、肺移植及胰腺移植接受患者所出现的人巨细胞病毒感染。和现有临床用药相比，具有毒副作用更小、具有更佳的口服生物利用度，以及更低发生药物耐药的风险。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在含有下列含义。一个特定的短语或术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文出现商品名时，旨在指代其对应的商品或其活性成分。
本发明的中间体化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。
本发明所使用的所有溶剂是市售的，无需进一步纯化即可使用。
本发明采用下述缩略词：DMF代表二甲基甲酰胺；MsOH代表甲磺酸；EtOH代表乙醇；NaOH代表氢氧化钠；M代表mol/L；NBS代表。
化合物经手工或者
Figure PCTCN2019073700-appb-000002
软件命名，市售化合物采用供应商目录名称。
本发明粉末X-射线衍射(X-ray powder diffractometer,XRPD)方法
仪器型号：布鲁克D8 advance X-射线衍射仪
测试方法：大约10～20mg样品用于XRPD检测。
详细的XRPD参数如下：
光管：Cu,kα,
Figure PCTCN2019073700-appb-000003
光管电压：40kV，光管电流：40mA
发散狭缝：0.60mm
探测器狭缝：10.50mm
防散射狭缝：7.10mm
扫描范围：4-40deg
步径：0.02deg
步长：0.12秒
样品盘转速：15rpm
本发明差热分析(Differential Scanning Calorimeter,DSC)方法
仪器型号：TA Q2000差示扫描量热仪
测试方法：取样品(～1mg)置于DSC铝锅内进行测试，在50mL/min N 2条件下，以10℃/min的升温速率，加热样品从30℃到280℃。
本发明热重分析(Thermal Gravimetric Analyzer,TGA)方法
仪器型号：TA Q5000IR热重分析仪
测试方法：取样品(2～5mg)置于TGA铂金锅内进行测试，在25mL/min N 2条件下，以10℃/min的升温速率，加热样品从室温到300℃。
附图说明
图1为化合物1的A晶型的Cu-Kα辐射的XRPD谱图。
图2为化合物1的A晶型的DSC谱图。
图3为化合物1的A晶型的TGA谱图。
具体实施方式
为了更好的理解本发明的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本发明的内容所做的限制。
参考例1：化合物BB-1的制备
Figure PCTCN2019073700-appb-000004
合成路线：
Figure PCTCN2019073700-appb-000005
步骤1：化合物BB-1-3的合成
在室温下向化合物BB-1-1(2.00g,1.00eq)和化合物BB-1-2(1.51g,1.05eq)的甲苯(40.00mL)中加入三叔丁基膦(1M甲苯溶液,0.01eq)，Pd 2(dba) 3(91.48mg,0.01eq)和叔丁醇钾(1.68g,1.50eq)，反应液在氮气保护下100℃搅拌12小时。化合物浓缩旋干，粗产品经硅胶柱(PE/EtOAc＝3/1)纯化得到BB-1-3(2.30g)。
步骤2：化合物BB-1的合成
向BB-1-3(1.80g,1.00eq)的二氯甲烷(30.00mL)溶液中加入三氟乙酸(4.62g,6.91eq)，反应液在15℃下搅拌12小时。反应液浓缩旋干得粗品化合物BB-1(4.5g,TFA)。MS(ESI)m/z:208.0[M+1]。
实施例1：化合物1的制备
Figure PCTCN2019073700-appb-000006
步骤1：化合物1-2的合成
在15℃下，向化合物1-1(10.00g,1.00eq)和叠氮磷酸二苯酯(25.77g,1.20eq)的甲苯(150.00mL)溶液中加入三乙胺(23.69g,3.00eq)。反应液在氮气保护下15℃反应1.5小时，然后加热到80℃反应7小时。2-甲氧基-5-三氟甲基苯胺(15.66g,1.05eq)加入到反应液中继续反应12小时。反应液减压浓缩，所得物倒入HCl(1M,300mL)，用乙酸乙酯(200mL)萃取二次，合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩，所得物用PE:EtOAc(2:1)洗涤得1-2(10.00g,24.41mmol,收率31.28％)。 1H NMR(400MHz,DMSO-d6)9.72(s,1H),8.58(s,1H),8.47(s,1H),7.47–7.48(m,1H),7.33–7.35(m,2H),7.20(d,J＝8.4Hz,2H),7.02(d,J＝5.2Hz,1H),3.92(s,3H)。
步骤2：化合物1-3的合成
在0℃下，向化合物1-2(10.00g,1.00eq)的二氯甲烷(150.00mL)溶液中加入化合物NBS(5.63g,1.00eq)。混合物在0℃搅拌2小时后。反应液过滤，滤饼干燥后得到1-3(6.80g,收率46.80％)。MS (ESI)m/z:397.0[M+1]。
步骤3：化合物1-4的合成
在室温下，向化合物1-3(6.80g,1.00eq)和(E)-3-(4,4,5,5-四甲基-1,3,2-二氧杂环戊硼烷-2-基)丙烯酸甲酯(7.30g,2.00eq)的四氢呋喃(80.00mL)和水(10.00mL)溶液中加入化合物Pd(dppf)Cl 2(1.26g,0.10eq)和碳酸钾(7.14g,3.00eq)。混合物在氮气保护下55℃搅拌12小时。反应旋干过柱(PE/EtOAc＝5/1)得2-4(2.60g,4.68mmol,收率27.17％)。MS(ESI)m/z:401.1[M+1]。
步骤4：化合物1-5的合成
在室温下，向化合物1-4(2.65g,1.00eq)的四氢呋喃(15.00mL)溶液中加入氢氧化锂(1M,6.62mL,1.00eq)水溶液。混合物在40℃搅拌12小时。反应旋干得粗产品1-5(2.80g)。
步骤5：化合物1-6的合成
在0℃下，向化合物1-5(6.80g,1.00eq)的甲醇(60.00mL)溶液中加入氯化亚砜(2.40g,3.00eq)。混合物在氮气保护下70℃搅拌3小时。反应液旋干过柱(PE/EtOAc＝3/1至3/2)得化合物1-6(800.00mg,1.58mmol,收率23.54％)。 1H NMR：(400MHz,CDCl 3)7.59(d,J＝7.2Hz,1H),7.16(d,J＝5.2Hz,1H),7.05(d,J＝8.8Hz,1H),6.57(d,J＝5.2Hz,1H),5.40–5.43(m,1H),.3.89(s,3H),3.57(s,3H),2.75–2.81(m,2H)。
步骤6：化合物1-7的合成
在室温下，化合物1-6(350mg,1.00eq)溶于三氯氧磷(17.13g)中，在氮气保护下110℃搅拌12小时。N,N-二异丙基乙基胺(564.90mg,5.00eq)加入到该反应中并继续氮气保护下110℃搅拌3小时。反应液减压浓缩，所得物加入水(100mL)中，用碳酸氢钠调至中性，乙酸乙酯(80mL)萃取三次，合并有机相用饱和食盐水洗涤，无水硫酸钠干燥，过滤，减压浓缩得粗品化合物1-7(300.00mg)。MS(ESI)m/z:419.1[M+1]。
步骤7：化合物1-8的合成
在室温下，向化合物1-7(150.00mg,1.00eq)和BB-1(172.61mg,1.50eq,TFA)的乙腈(5.00mL)溶液中加入碳酸钾(495mg,10.00eq)。混合物在80℃搅拌12小时。反应过滤，滤液减压浓缩至干得粗品化合物1-8(250.00mg)。MS(ESI)m/z:590.2[M+1]。
步骤8：化合物1-9的合成
在室温下，向化合物1-8(600.00mg,1.00eq)的甲醇(3.00mL),四氢呋喃(3.00mL)和水(1.00mL)溶液中加入氢氧化钠(122.40mg,3.00eq)。混合物在15℃搅拌3小时。反应液旋干，所得物HPLC制备得化合物1-9(450.00mg，收率76.65％)。MS(ESI)m/z:576.1[M+1]。
步骤9：化合物1和2的合成
化合物1-9(450.00mg,1.00eq)经过SFC制备方法：[柱子型号(IC(250mm×30mm,10μm)，流动相(A：二氧化碳，B：0.1氨水甲醇，梯度：B 40％-40％)]，得化合物1(130.00mg,收率28.46％，保留时间：第一个峰)和化合物2(150.00mg,收率32.40％，保留时间：第二个峰)。
化合物1  1H NMR(400MHz,d 4-MeOH)δ7.75(br.s.,1H),7.55(d,J＝8.0Hz,1H),7.12-7.38(m,2H),6.98(d,J＝5.0Hz,1H),6.51(br.s.,1H),6.10(br.s.,1H),5.05(br.s.,1H),4.10(br.s.,1H),3.84(s,6H),3.63-3.72(m,2H),3.14-3.29(m,1H),2.78–3.11(m,3H),2.52-2.72(m,1H),0.50(br.s.,3H)，MS(ESI)m/z:576.1[M+1].
化合物2  1H NMR(400MHz,d 4-MeOH)δ7.76(d,J＝5.8Hz,1H),7.62(d,J＝8.5Hz,1H),7.14-7.41(m,2H),6.98(d,J＝5.3Hz,1H),6.43(d,J＝5.3Hz,1H),6.01(br.s.,1H),5.15(br.s.,1H),3.96(br.s.,1H),3.63-3.87(m,6H),3.54(br.s.,1H),2.82–3.12(m,2H),2.63(dd,J＝7.3,14.6Hz,1H),1.00-1.12(m,3H).MS(ESI)m/z:576.1[M+1].
实施例2：A晶型的制备
取化合物1(50mg)，加入混合溶剂(乙醇：水＝1:1，1mL)中，于25℃，700rpm搅拌36小时，所得悬浮液离心，固体于35℃真空干燥12小时，即得A晶型。
实验例1
抗人巨细胞病毒荧光衰减实验
通过测定化合物的半数有效浓度(EC 50)值来评价化合物对人巨细胞病毒(HCMV)的抗病毒活性。本实验中使用的HCMV通过基因重组插入报告基因增强型绿色荧光蛋白(EGFP)，病毒在细胞内的复制可以由GFP的表达水平来反映。使用高内涵细胞分析平台Acumen eX3测定不同浓度化合物孔中的荧光强度来评价化合物对HCMV GFP表达的抑制活性。
抗HCMV荧光衰减实验
将MRC5细胞以20,000细胞每孔的密度种入黑色96孔细胞培养板中，随后置于37℃，5％CO 2培养箱中培养过夜。US3-6-EGFP-HCMV-AD169病毒以一定MOI(0.003-0.1)加入细胞培养孔中，于37℃，5％CO 2培养箱中培养3小时。病毒吸附后将含有病毒的培养基吸出并加入200μl含有不同浓度化合物(4倍倍比稀释，6个测试浓度点)的细胞培养基。培养基中DMSO终浓度为1％。设置病毒对照孔(加入DMSO,不含化合物)和抑制对照孔(加入高浓度的对照化合物)。细胞板置于37℃，5％CO 2培养箱中培养10天，第4天和第7天换液。培养10天后用高内涵细胞分析平台Acumen eX3(TTP LabTech)检测荧光强度。原始数据用于化合物抗病毒活性计算。
Figure PCTCN2019073700-appb-000007
将抑制百分数分别导入GraphPad Prism软件进行数据处理得出化合物对应的剂量-效应曲线并得出测试化合物的EC 50值。
结果见表2：
表2
化合物 EC 50(μM)
化合物1 0.0002
结论：化合物1展示出良好的体外抑制人巨细胞病毒复制的抑制活性。
实验例2
化合物的血浆蛋白结合率测试
待测化合物在CD-1小鼠、SD大鼠和人血浆中的蛋白结合率采用平衡透析法进行了评估。将化合物1分别稀释到上述三个物种的血浆中，配制成终浓度为2μM的样品，然后将样品加入到96孔平衡透析装置中，在37℃下用磷酸盐缓冲溶液透析4小时。实验采用华法林(warfarin)作为对照化合物。血浆和缓冲液中受试化合物与warfarin的浓度用LC-MS/MS法进行测定。
结果显示如表-3。
表-3
Figure PCTCN2019073700-appb-000008
注：H代表人，R代表大鼠，M代表小鼠
结论：化合物1在三个种属血浆中均具有合理的的血浆蛋白结合率，预示在上述三个种属的血浆中，受试化合物的游离态药物浓度比例适中，具有良好的成药性质。
实验例3
人巨细胞病毒(HCMV)小鼠移植模型
将含有HCMV的明胶海绵移植到小鼠体内，连续给药9天后，收集明胶海绵用于空斑检测。通过检测明胶海绵中的HCMV数量评价化合物在该模型中的抗HCMV作用。
实验选用的动物为NOD SCID小鼠(购自上海斯莱克实验动物有限公司)，5周，雄性。每组5只动物。将小鼠进行明胶海绵移植当天设为第0天。事先用HCMV病毒(毒株：GFP-AD169)感染人包皮成纤维细胞(HFF，MOI＝0.03)，再将感染HCMV的HFF细胞加到1cm 2大小的明胶海绵内孵育待用。动物经75mg/kg(10ml/kg)的戊巴比妥钠腹腔注射麻醉，待动物进入深麻状态后，将处理好的明胶海绵皮下移植到小鼠的背部。从第1天至第9天，每天口服给予8mg/kg(10ml/kg)的化合物1，每天一次。第9天给药后4小时，将明胶海绵取出，消化后进行空斑检测。试验数据显示，化合物1对HCMV病毒载量的下降分别为2.38log PFU/ml，展示出了优良的体内药效，在体内药效研究试验过程中，小鼠体重稳定，临床观察无异常，表明该系列化合物在给药剂量下对小鼠无明显副作用。

Claims (12)

  1. 化合物1的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：17.69±0.2°、20.00±0.2°、20.63±0.2°。
    Figure PCTCN2019073700-appb-100001
  2. 根据权利要求1所述化合物1的A晶型，其X射线粉末衍射图谱在下列2θ角处具有特征衍射峰：9.15±0.2°，11.06±0.2°，11.95±0.2°，17.69±0.2°，19.03±0.2°，19.46±0.2°，20.00±0.2°，20.63±0.2°。
  3. 根据权利要求2所述化合物1的A晶型，其XRPD图谱如图1所示。
  4. 根据权利要求1～3任意一项所述化合物1的A晶型，其差示扫描量热曲线在214.47℃±2℃处具有吸热峰的起始点。
  5. 根据权利要求4所述化合物1的A晶型，其DSC图谱如图2所示。
  6. 根据权利要求1～3任意一项所述化合物1的A晶型，热重分析曲线在120.00±2℃处失重达0.1206％。
  7. 根据权利要求6所述化合物1的A晶型，其TGA图谱如图3所示。
  8. A晶型的制备方法，包括将化合物1加入到醇类溶剂与水的混合溶剂中，加热溶解，然后降温析晶制得。
  9. 根据权利要求8所述A晶型的制备方法，其中，所述醇类溶剂选自甲醇、乙醇和异丙醇。
  10. 根据权利要求8所述A晶型的制备方法，其中，醇类溶剂与水的混合溶剂选自乙醇和水的混合溶剂。
  11. 根据权利要求8所述A晶型的制备方法，其中，所述醇类溶剂与水的混合溶剂中，醇类溶剂和水的体积比选自1：0.2～1.5。
  12. 根据权利要求1～7任意一项所述化合物1的A晶型在制备抗HCMV病毒药物中的应用。
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