WO2022151002A1 - 甘草次酸衍生物、其合成方法、其医药组合物及其用途 - Google Patents

甘草次酸衍生物、其合成方法、其医药组合物及其用途 Download PDF

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WO2022151002A1
WO2022151002A1 PCT/CN2021/071373 CN2021071373W WO2022151002A1 WO 2022151002 A1 WO2022151002 A1 WO 2022151002A1 CN 2021071373 W CN2021071373 W CN 2021071373W WO 2022151002 A1 WO2022151002 A1 WO 2022151002A1
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formula
acid derivative
glycyrrhetic acid
zika virus
pharmaceutical composition
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PCT/CN2021/071373
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French (fr)
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林振文
巴蒂娜·莉迪亚
刘亚祁
莉亚 巴蒂娜
康德拉丁科·里玛
尤努索夫·马拉特
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林振文
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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms

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  • the present invention relates to a glycyrrhetic acid derivative, in particular to a glycyrrhetic acid derivative that can treat and improve Zika virus, its synthesis method, its pharmaceutical composition and its use.
  • Zika virus belongs to the genus Flavivirus, which forms a cycle of viral transmission through vector mosquitoes of the genus Aedes and primates, and makes primates bitten by vector mosquitoes carrying Zika virus. Animals are infected with Zika virus.
  • the Zika virus has a wide geographical distribution, and records have been found in Central Africa, Southeast Asia, India, the Caroline Islands, and Central and South America. Among them, the Zika virus epidemic in Central and South America spread rapidly in 2015, and Brazil There have even been more than 4,100 cases of neonatal microcephaly, which has been shown to be associated with Zika virus, and infected individuals may also have neurological effects and complications such as Guillain-Barre Syndrome. .
  • Zika virus Although the disease caused by Zika virus is quite serious, it can only be improved by rest and symptomatic treatment. There is no drug or vaccine for Zika virus prevention or treatment. Therefore, a novel drug was developed. It is important to fight against such viruses.
  • One object of the present invention is to provide a glycyrrhetic acid derivative, its synthesis method, its pharmaceutical composition and its use, and to apply the glycyrrhetic acid derivative in the application of anti-Zika virus, by derivatizing the glycyrrhetic acid
  • the drug has the effect of inhibiting the virus to effectively treat the Zika virus.
  • One embodiment of the present invention provides a glycyrrhetic acid derivative, which has a structure as shown in formula (I):
  • R 1 is butoxy, methoxy, a group such as formula (MAI) or a group such as formula (MAII)
  • R 2 is acetoxy, such as a group represented by formula (MBI) or a group represented by formula (MBII)
  • R 2 is a group represented by formula (MBII), the sulfur atom of the group is attached to carbon No. 1.
  • glycyrrhetic acid derivative wherein the glycyrrhetic acid derivative may have formula (IA), formula (IB), formula (IC), formula (ID), formula (IE), formula (IF) or A structure shown by formula (IG):
  • Another embodiment of the present invention provides a method for synthesizing glycyrrhetinic acid derivatives as described above, which includes a step of providing a precursor, a step of chemical reaction, and a step of product purification.
  • the step of providing the precursor is to provide a precursor, the precursor is 3-oxo-triterpenoid, semicarbazone or 3-acetoxyglycyrrhetinic acid chloride (3-O -acetyl-GLA chloride).
  • the chemical reaction step is to carry out a chemical reaction in a reaction mode to obtain a product, wherein the reaction mode is stirring at room temperature or refluxing at high temperature.
  • the product purification step is to purify the product to obtain the glycyrrhetic acid derivative.
  • Yet another embodiment of the present invention is to provide a pharmaceutical composition for treating or improving Zika virus, which comprises an effective dose of the aforementioned glycyrrhetic acid derivative.
  • the effective dose may be 0.1 ⁇ M to 10 ⁇ M. Preferably, it can be 10 ⁇ M.
  • a pharmaceutically acceptable excipient or carrier can be further included.
  • Another embodiment of the present invention is to provide the use of the aforementioned glycyrrhetic acid derivative, which is used to prepare a medicine for treating or improving Zika virus.
  • the drug for treating or improving Zika virus may be a drug for reducing the infectivity of Zika virus to cells.
  • the drug for treating or improving Zika virus can be a drug for inhibiting the early replication and late replication of Zika virus.
  • the glycyrrhetic acid derivative of the present invention has been confirmed to have anti-Zika virus efficacy through experimental data, and can inhibit the cytopathic effect and virus infectivity of Zika virus in vitro.
  • the glycyrrhetic acid derivative of the present invention is suitable as a medicine for treating and improving Zika virus infection, or as a pharmaceutical composition for treating and improving Zika virus disease in combination with a pharmaceutically acceptable excipient or carrier , has the potential to be used in the biomedical market.
  • FIG. 1 is a flow chart showing the steps of a method for synthesizing a glycyrrhetic acid derivative according to another embodiment of the present invention
  • Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D and Fig. 2E are bar graphs of cell viability test results of SF268 cells according to an embodiment of the present invention.
  • Example 3 is a microscopic image of the inhibitory ability of Example 1 of the present invention on ZIKV-induced SF268 cytopathic changes;
  • Figure 4A, Figure 4B, Figure 4C, Figure 4D and Figure 4E are the results of the infection inhibition test on infected SF268 cells according to an embodiment of the present invention.
  • Example 5 is a microscopic image of the inhibitory ability of the treatment test of Example 1 of the present invention on the induction of SF268 cytopathies by ZIKV during virus infection;
  • FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D and FIG. 6E are the results of the infection inhibition test of the infected SF268 cells in the treatment test of the embodiment of the present invention during virus infection;
  • FIG. 7 is a microscopic image of the inhibitory ability of Example 1 of the present invention on ZIKV-induced SF268 cytopathic changes in a post-virus treatment test.
  • FIG. 8A , FIG. 8B , FIG. 8C , FIG. 8D and FIG. 8E are the results of the infection inhibition test on infected SF268 cells in the post-virus infection treatment test according to the embodiment of the present invention.
  • One embodiment of the present invention provides a glycyrrhetic acid derivative, which has a structure as shown in formula (I):
  • R 1 in formula (I) is each independently butoxy, methoxy, a group shown in formula (MAI) or a group shown in formula (MAII), and R 2 is each independently acetoxy group, a group represented by formula (MBI) or a group represented by formula (MBII), Represent single or double bonds to satisfy all valences:
  • R 2 is a group represented by formula (MBII), the sulfur atom of the group is attached to carbon No. 1.
  • FIG. 1 is a flow chart illustrating the steps of the synthesis method 100 of the glycyrrhetic acid derivative of the present invention.
  • the synthesis method 100 of glycyrrhetic acid derivatives includes step 110 , step 120 and step 130 .
  • Step 110 is a step of providing a precursor, which is to provide a precursor to carry out a chemical reaction, and the precursor used can be 3-oxo-triterpenoid, semicarbazone or 3-acetyl Oxyglycyrrhetinic acid chloride (3-O-acetyl-GLA chloride), but the present invention is not limited to this.
  • Step 120 is a chemical reaction step, which is to perform a chemical reaction in a reaction manner to obtain a product.
  • the reaction mode can be stirring at room temperature or refluxing at high temperature.
  • Step 130 is a product purification step, which is to purify the product to obtain a glycyrrhetic acid derivative.
  • the method of purification can be thin layer chromatography (TLC), but the present invention is not limited thereto.
  • Example 1 of the present invention The synthesis method of Example 1 of the present invention is as follows: the chloride of 3-acetoxyglycyrrhetinic acid is dissolved in dichloromethane and reacted with hydrazine, and then reacted with aromatic aldehyde in ethanol and heated to boiling, After 6 hours of reaction, the glycyrrhetic acid derivative of Example 1 was obtained.
  • Example 2 of the present invention is as follows: 1 mmol of 3-carbonyl triterpene is dissolved in 50 ml of C 2 H 5 OH, a solution of hydrochloric acid hemisulfide (6 mmol) in 5 ml of water and NaOAc (6 mmol) is added, and The mixture was refluxed for 6 hours under TLC control, the reaction mixture was diluted with cold water (50 ml), the formed precipitate was filtered off and recrystallized from 70% C 2 H 5 OH to give the glycyrrhetic acid derivative of Example 2 .
  • Example 3 of the present invention is as follows: SOCl2 is added to a solution of hemicarbhydrazone (1 mmol) in 20 ml of dichloromethane, and the mixture is stirred at room temperature for 12 hours. The solvent was then evaporated and the residue was recrystallized from 70% C2H5OH to give the glycyrrhetic acid derivative of Example 3 .
  • Example 4 of the present invention is as follows: 2 mmol of 2-aminopyridine is added to a solution of 1 mmol of 3-acetoxyglycyrrhetinic acid chloride in 15 ml of dichloromethane, and stirred at room temperature for 8 hours . After evaporation of the solvent, the residue was diluted with 20 ml of dichloromethane and washed with 5% aqueous NaHCO 3 , saturated NaCl solution and water, and dried over MgSO 4 . The product was chromatographed on a silica gel column eluting with benzene using a TLC control. The fractions of TLC were combined and evaporated to give the glycyrrhetinic acid derivative of Example 4.
  • Embodiment 5 of the present invention is the same as that of Embodiment 3, and details are not repeated here.
  • infrared spectroscopy and nuclear magnetic resonance spectroscopy are used to perform infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis data as shown in Table 8 below.
  • the cell line used for cell viability test was human glioblastoma and astrocytoma cell (SF268).
  • SF268 cells were cultured in a 96-well plate overnight at a cell amount of 5 ⁇ 10 3 per well, and then the glycyrrhetic acid derivatives of Example 1 to Example 7 were added to make the drug concentrations 0.1 ⁇ M, 1 ⁇ M, and 10 ⁇ M. and 50 ⁇ M, and quadruplicate experiments were performed for each concentration.
  • FIGS. 2A , 2B, 2C, 2D and 2E are bar graphs of the cell viability test results of SF268 cells according to an embodiment of the present invention, and FIG. 2A is the result of Example 1.
  • FIG. 2B is the result of Example 2
  • FIG. 2C is the result of Example 3
  • FIG. 2D is the result of Example 4
  • FIG. 2E is the result of Example 5. It can be seen from the test results that even if the added drug concentration of Example 1 to Example 5 reaches 50 ⁇ M, it does not significantly cause cell death, indicating that the glycyrrhetic acid derivatives of the present invention have no toxic effect on SF268 cells (CC 50 ⁇ 50 ⁇ M) .
  • the cell line for cytopathic inhibition test and immunofluorescence staining analysis was SF268 cell, and the virus strain was Zika virus (ZIKV PRVABC59, ZIKV).
  • SF268 cells were cultured in a 6-well dish at 1.5 ⁇ 10 5 cells per well, containing 10% FBS (Fetal bovine serum, ) of DMEM (Dulbecco's modified eagle medium, ) to a total volume of 2 mL and incubated overnight at 37°C in a 5% CO2 incubator. On the next day, the culture medium was removed and the 6-well plate was washed twice with Phosphate buffered saline (PBS), and then once with DMEM without FBS, with a multiplicity of infection (MOI) of 0.05.
  • PBS Phosphate buffered saline
  • SF268 cells were infected with ZIKV, and the glycyrrhetic acid derivatives of Example 1 to Example 7 were respectively added to make the drug concentrations 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M to treat the cells. After 96 hours of culture, the results of the cytopathic inhibition test can be obtained by observing the cytopathic state and taking pictures.
  • FIG. 3 is a microscopic image of the inhibitory ability of Example 1 of the present invention on ZIKV-induced SF268 cytopathic changes.
  • Example 1 inhibited the cytopathic effect induced by ZIKV in a concentration-dependent manner, and showed a significant inhibitory effect when the drug concentration was 10 ⁇ M.
  • the inhibitory effect shown in FIG. 3 is a significant inhibition (+++), and the inhibitory effect of the derivatives of each example under this test on the cytopathic effects of infected cells is listed in Table 9 below.
  • the results of the graphs show that the glycyrrhetinic acid derivatives of the present invention have a significant inhibitory effect on ZIKV-induced cytopathies.
  • ZIKV anti-NS1 protein (GeneTex, Inc.) antibody was used as the primary antibody, and then the cells were shaken in a cold room at 4°C overnight. After washing three times with PBS every other day, the cells were treated with Goat anti-rabbit IgG H&L (Alexa 555, ThermoFisher) antibody was used as the secondary antibody for staining, and the cells were shaken in a cold room at 4°C for 1 hour in the absence of light. Finally, the nuclei were stained with DAPI (4'6-diamidino-2-phenylindole) fluorescent dye to count the number of cells, and Image J software was used to determine the percentage of NS1-positive cells in the infected cells.
  • DAPI 4,6-diamidino-2-phenylindole
  • Infection inhibition was calculated as: (percent NS1 positive in infected cells treated without the example drug - NS1 positive percentage in infected cells treated with the example drug) / NS1 positive percentage in infected cells treated with the example drug.
  • IC 50 half-maximum inhibitory concentration
  • FIGS. 4A to 4E are the results of the infection inhibition test on infected SF268 cells according to the embodiment of the present invention, and FIG. 4A is the result of Example 1.
  • FIG. 4B is the result of Example 2
  • FIG. 4C is the result of Example 3
  • FIG. 4D is the result of Example 4
  • FIG. 4E is the result of Example 5.
  • Table 10 is the data of the infection inhibition ability and antiviral activity of Examples 1 to 7 of the present invention on infected cells.
  • each embodiment of the present invention significantly reduces the percentage of NS1 positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the infection of SF268 cells by ZIKV This indicates that the glycyrrhetic acid derivatives of the present invention can significantly reduce the infectivity of Zika virus to cells.
  • SF268 cells were cultured at 1.5 x 10 5 cells per well in a 6-well dish, prepared in DMEM containing 10% FBS to a total volume of 2 mL, and incubated at 37 °C in an incubator with 5% CO 2 Incubate overnight. On the next day, the culture medium was removed and the 6-well plate was washed twice with phosphate buffered solution, and then once with DMEM without FBS, and then SF268 cells were infected with ZIKV with a virus infection dose of 0.05.
  • the glycyrrhetic acid derivatives of Examples 1 to 5 were added (treatment test during virus infection), and another group of cells was added with the glycyrrhetic acid derivatives of Examples 1 to 5 (after virus infection) 1 hour after infection. Treatment test) to make the drug concentration reach 0.1 ⁇ M, 1 ⁇ M and 10 ⁇ M to treat the cells.
  • the glycyrrhetic acid derivatives of Example 1 to Example 5 were co-cultured with the virus for one hour, and then the drug was removed. Finally, DMEM containing 2% FBS was added to make the total volume reach 2 mL, and the cells were cultured for 96 hours to observe the cytopathic inhibition effect. Carry out immunofluorescence staining.
  • FIG. 5 is a microscopic image of the inhibitory ability of Example 1 of the present invention on the ZIKV-induced SF268 cytopathic change in the treatment test during virus infection.
  • 6A to 6E are the results of the infection inhibition test of the infected SF268 cells in the treatment test of the embodiment of the present invention, wherein FIG. 6A is the result of Example 1, FIG. 6B is the result of Example 2, and FIG. 6C Fig. 6D is the result of Example 4, Fig. 6E is the result of Example 5. Please refer to Table 11 below together.
  • Table 11 shows the antiviral activity data of Example 1 to Example 5 of the present invention against ZIKV at the cell entry stage.
  • the results of the graph show that each embodiment of the present invention significantly reduces the percentage of NS1 positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the infectivity of ZIKV to SF268 cells , which indicates that the glycyrrhetic acid derivatives of the present invention can significantly reduce the infectivity of ZIKV to cells and have a significant inhibitory effect on the early replication of ZIKV.
  • FIG. 7 is a microscopic image of the inhibitory ability of Example 1 of the present invention on ZIKV-induced SF268 cytopathic changes in the post-virus infection treatment test.
  • 8A to 8E are the results of the infection inhibition test of the infected SF268 cells in the post-virus infection treatment test according to the embodiment of the present invention, wherein FIG. 8A is the result of Example 1, FIG. 8B is the result of Example 2, and FIG. 8C The results of Example 3 are shown in FIG. 8D , and the results of Example 5 are shown in FIG. 8E . Please refer to the following table 12 together.
  • Table 12 shows the data of the antiviral activity of Example 1 to Example 5 of the present invention against ZIKV after infecting cells.
  • the results of the graph show that each embodiment of the present invention significantly reduces the percentage of NS1 positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the replication of ZIKV after entering SF268 cells.
  • the glycyrrhetic acid derivatives of the present invention can significantly reduce the replication ability of ZIKV after entering cells and have a significant inhibitory effect on the late replication of ZIKV.
  • the present invention provides a glycyrrhetic acid derivative represented by formula (I), which can inhibit the cytopathic effect and virus infectivity of ZIKV in vitro, and can also block the steps of early and late replication of the virus, and
  • the half maximum inhibitory concentration of ZIKV is less than 10 ⁇ M, so in addition to reducing the half maximum inhibitory concentration, it can also improve the therapeutic index (Therapeutic Index, TI). ratio of inhibitory concentrations. Therefore, the glycyrrhetic acid derivative of the present invention has the effect of treating and improving Zika virus infection, and is suitable as a medicine for treating and improving Zika virus, or can be used in combination with a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical composition for treating and improving Zika virus disease can further expand its use as an anti-Zika virus drug, and has deep potential in the biomedical market.

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Abstract

式(I)所示的甘草次酸衍生物、其合成方法、其医药组合物及其用途，前述的甘草次酸衍生物具有如式(I)所示的一结构，式(I)中各符号如说明书中所定义者。其可抑制兹卡病毒，适用于作为治疗及改善兹卡病毒的药物。

Description

甘草次酸衍生物、其合成方法、其医药组合物及其用途 技术领域
本发明是有关于一种甘草次酸衍生物，特别是有关于一种可治疗及改善兹卡病毒的甘草次酸衍生物、其合成方法、其医药组合物及其用途。
背景技术
兹卡病毒(Zika virus)属于黄病毒属(Flavivirus)，其是经由斑蚊属的病媒蚊和灵长类动物形成病毒传播的循环，并使受带有兹卡病毒的病媒蚊叮咬后的灵长类动物罹患兹卡病毒感染症。
兹卡病毒于地理上的分布范围甚广，在中非、东南亚、印度、加罗林群岛及中南美洲等均有发现的纪录，其中，中南美洲的兹卡病毒疫情在2015年快速扩散，巴西甚至出现超过4,100例新生儿小头畸形的案例，已被证实与兹卡病毒相关，此外受感染者的神经系统亦可能受影响并产生并发症，如格林-巴利症候群(Guillain-Barre Syndrome)。
虽兹卡病毒所导致的疾病具有相当的严重性，然时至今日仅可通过休息与症状治疗改善，尚无药物或疫苗可供兹卡病毒的预防或治疗，是故开发一种新颖的药物来对抗此类病毒有其重要性。
发明内容
本发明的一目的在于提供一种甘草次酸衍生物、其合成方法、其医药组合物及其用途，并将甘草次酸衍生物应用于抗兹卡病毒的用途中，藉由甘草次酸衍生物具有抑制病毒的效果，以有效对兹卡病毒进行治疗。
本发明的一实施方式提供一种甘草次酸衍生物，其具有如式(I)所示的一结构：
Figure PCTCN2021071373-appb-000001
其中式(I)中，R 1为丁氧基、甲氧基、如式(MAI)所示的一基团或如式(MAII)所示的一基团，R 2为乙酰氧基、如式(MBI)所示的一基团或如式(MBII)所示的一基团，
Figure PCTCN2021071373-appb-000002
表示单键或双键以便满足所有价数：
Figure PCTCN2021071373-appb-000003
其中当R 2为如式(MBII)所示的基团时，基团的硫原子与1号碳相接。
依据前述的甘草次酸衍生物，其中所述甘草次酸衍生物可具有如式(IA)、式(IB)、式(IC)、式(ID)、式(IE)、式(IF)或式(IG)所示的一结构：
Figure PCTCN2021071373-appb-000004
Figure PCTCN2021071373-appb-000005
Figure PCTCN2021071373-appb-000006
本发明的另一实施方式提供一种如前述的甘草次酸衍生物的合成方法，包含一前驱物提供步骤、一化学反应步骤以及一产物纯化步骤。前驱物提供步骤是提供一前驱物，所述前驱物为3-羰基三萜(3-oxo-triterpenoid)、半卡腙(semicarbazone)或3-乙酰氧基甘草次酸的氯化物(3-O-acetyl-GLA chloride)。化学反应步骤是以一反应方式进行一化学反应，以获得一产物，其中反应方式为 于室温搅拌或高温回流。产物纯化步骤是将所述产物进行纯化，以获得所述的甘草次酸衍生物。
本发明的再一实施方式为提供一种用于治疗或改善兹卡病毒的医药组合物，其包含有效剂量的如前述的甘草次酸衍生物。
依据前述的医药组合物，其中所述有效剂量可为0.1μM至10μM。较佳地，可为10μM。
依据前述的医药组合物，更可包含一医药上可接受的赋形剂或载体。
本发明的又一实施方式为提供一种前述的甘草次酸衍生物的用途，其是用于制备治疗或改善兹卡病毒的药物。
依据前述的甘草次酸衍生物的用途，其中治疗或改善兹卡病毒的药物可为降低兹卡病毒对于细胞感染性的药物。
依据前述的甘草次酸衍生物的用途，其中治疗或改善兹卡病毒的药物可为抑制兹卡病毒的早期复制及晚期复制的药物。
藉此，本发明的甘草次酸衍生物经由实验数据证实具有抗兹卡病毒的功效，能够在体外抑制兹卡病毒的细胞病变效应和病毒感染性。是以本发明的甘草次酸衍生物适于作为治疗及改善兹卡病毒感染症的药物，或与医药上可接受的赋形剂或载体搭配作为可治疗及改善兹卡病毒疾病的医药组合物，具有运用于生医市场的潜能。
附图说明
为让本发明的上述和其他目的、特征、优点与实施例能更明显易懂，所附附图的说明如下：
图1是绘示本发明另一实施方式的甘草次酸衍生物合成方法的步骤流程图；
图2A、图2B、图2C、图2D及图2E为本发明的实施例对SF268细胞的细胞存活率试验结果长条图；
图3为本发明的实施例1对ZIKV诱导SF268细胞病变的抑制能力的显微影像；
图4A、图4B、图4C、图4D及图4E为本发明的实施例对受感染的SF268细胞的感染抑制力试验结果；
图5为本发明的实施例1于病毒感染时处理试验对ZIKV诱导SF268细胞病变的抑制能力的显微影像；
图6A、图6B、图6C、图6D及图6E为本发明的实施例于病毒感染时处理试验对受感染的SF268细胞的感染抑制力试验结果；
图7为本发明的实施例1于病毒感染后处理试验对ZIKV诱导SF268细胞病变的抑制能力的显微影像；以及
图8A、图8B、图8C、图8D及图8E为本发明的实施例于病毒感染后处理试验对受感染的SF268细胞的感染抑制力试验结果。
其中，符号说明：
100:甘草次酸衍生物的合成方法
110:前驱物提供步骤
120:化学反应步骤
130:产物纯化步骤
具体实施方式
以下将参照附图说明本发明的多个实施例。为明确说明起见，许多实务上的细节将在以下叙述中一并说明。然而，应了解到，这些实务上的细节不应用以限制本发明。也就是说，在本发明部分实施例中，这些实务上的细节是非必要的。此外，为简化附图起见，一些习知惯用的结构与元件在附图中将以简单示意的方式绘示之；并且重复的元件将可能使用相同的编号表示之。
<甘草次酸衍生物>
本发明的一实施方式提供一种甘草次酸衍生物，其具有如式(I)所示的一结构：
Figure PCTCN2021071373-appb-000007
其中式(I)中R 1各自独立为丁氧基、甲氧基、如式(MAI)所示的一基团或如式(MAII)所示的一基团，R 2各自独立为乙酰氧基、如式(MBI)所示的一基团或如式(MBII)所示的一基团，
Figure PCTCN2021071373-appb-000008
表示单键或双键以便满足所有价数：
Figure PCTCN2021071373-appb-000009
其中当R 2为如式(MBII)所示的基团时，基团的硫原子与1号碳相接。
<甘草次酸衍生物的合成方法>
本发明的另一实施方式提供一种如前述的甘草次酸衍生物的合成方法。请参照图1，图1是绘示本发明的甘草次酸衍生物的合成方法100的步骤流程图。甘草次酸衍生物的合成方法100包含步骤110、步骤120及步骤130。
步骤110为前驱物提供步骤，其是提供一前驱物以进行一化学反应，所使用的前驱物可为3-羰基三萜(3-oxo-triterpenoid)、半卡腙(semicarbazone)或3-乙酰氧基甘草次酸的氯化物(3-O-acetyl-GLA chloride)，但本发明不以此为限。
步骤120为化学反应步骤，其是以一反应方式进行化学反应，以获得一产 物。所述反应方式可为于室温搅拌或高温回流。
步骤130为产物纯化步骤，其是将产物进行纯化，以获得甘草次酸衍生物。其中纯化的方式可为薄层层析法(Thin layer chromatography,TLC)，但本发明不以此为限。
兹以下列具体试验例进一步示范说明本发明，用以有利于本发明所属技术领域的通常知识者，可在不需过度解读的情形下完整利用并实践本发明，而不应将其视为对本发明范围的限制，但用于说明如何实施本发明的材料及方法。
<试验例>
一、本发明的甘草次酸衍生物的合成与结构鉴定
请参照下表一至表七，为本发明的甘草次酸衍生物的实施例化合物式(IA)至化合物式(IG)的结构式。
表一
Figure PCTCN2021071373-appb-000010
表二
Figure PCTCN2021071373-appb-000011
表三
Figure PCTCN2021071373-appb-000012
表四
Figure PCTCN2021071373-appb-000013
表五
Figure PCTCN2021071373-appb-000014
表六
Figure PCTCN2021071373-appb-000015
表七
Figure PCTCN2021071373-appb-000016
请参照上表一至表七，当式(I)中R 1为式(MAI)所示的一基团，R 2为乙酰氧基，可得如式(IA)所示的结构的甘草次酸衍生物(实施例1)；当式(I)中R 1为丁氧基，R 2为式(MBI)所示的一基团，可得如式(IB)所示的结构的甘草次酸衍生物(实施例2)；当式(I)中R 1为甲氧基，R 2为式(MBII)所示的一基团，且式(MBII)所示的一基团的硫原子与1号碳相接，可得如式(IC)所示的结构的甘草次酸衍生物(实施例3)；当式(I)中R 1为式(MAII)所示的一基团，R 2为乙酰氧基，可得如式(ID)所示的结构的甘草次酸衍生物(实施例4)；当式(I)中R 1为丁氧基，R 2 为式(MBII)所示的一基团，且式(MBII)所示的一基团的硫原子与1号碳相接，可得如式(IE)所示的结构的甘草次酸衍生物(实施例5)；当式(I)中R 1为甲氧基，R 2为式(MBI)所示的一基团，可得如式(IF)所示的结构的甘草次酸衍生物(实施例6)；当式(I)中R 1为甲氧基，R 2为式(MBI)所示的一基团，可得如式(IG)所示的结构的甘草次酸衍生物(实施例7)。
本发明的实施例1的合成方法如下：将3-乙酰氧基甘草次酸的氯化物溶于二氯甲烷中并与联胺进行反应后，并与芳香醛在乙醇中反应并加热至沸腾，反应6小时，得到实施例1的甘草次酸衍生物。
本发明的实施例2的合成方法如下：将1mmol的3-羰基三萜溶解在50ml的C 2H 5OH中，加入盐酸半硫化碳(6mmol)的5ml水和NaOAc(6mmol)的溶液，并在TLC控制下将混合物回流6小时，将反应混合物用冷水(50ml)稀释，滤出形成的沉淀，并从70％的C 2H 5OH中重新结晶，得到实施例2的甘草次酸衍生物。
本发明的实施例3的合成方法如下：半卡腙(1mmol)在20ml二氯甲烷中的溶液中添加SOCl 2，并将混合物在室温下搅拌12小时。然后蒸发溶剂，并将残余物从70％的C 2H 5OH中重新结晶，得到实施例3的甘草次酸衍生物。
本发明的实施例4的合成方法如下：向1mmol的3-乙酰氧基甘草次酸的氯化物在15ml的二氯甲烷中的溶液中加入2mmol的2-氨基吡啶，并在室温下搅拌8小时。蒸发溶剂后，残余物用20ml二氯甲烷稀释，并用5％的NaHCO 3水溶液、饱和NaCl溶液及水洗涤，并用MgSO 4干燥。产物在硅胶柱上色谱分离，用TLC对照用苯洗脱。合并TLC的各个部分并蒸发，得到实施例4的甘草次酸衍生物。
本发明的实施例5的合成方法同实施例3的合成方法，在此不再赘述。
本发明的实施例6的合成方法与实施例7的合成方法同实施例2的合成方法，在此不再赘述。
本发明的各实施例以红外光谱仪及核磁共振光谱仪，进行红外光谱及核磁共振光谱分析数据如下表八所示。
表八
Figure PCTCN2021071373-appb-000017
Figure PCTCN2021071373-appb-000018
Figure PCTCN2021071373-appb-000019
Figure PCTCN2021071373-appb-000020
二、本发明的甘草次酸衍生物的抗兹卡病毒试验
2-1：细胞存活率试验
进行细胞存活率试验的细胞株为人类胶质母细胞瘤星形细胞(Human glioblastoma and astrocytoma cell,SF268)。首先将SF268细胞以每孔5×10 3的细胞量培养在96孔盘中过夜，之后分别加入实施例1至实施例7的甘草次酸衍生物，使其药物浓度达0.1μM、1μM、10μM及50μM，而各浓度进行四重复试验。药物处理96小时后，于每孔中添加10μL的MTT溶液，避光反应4小时，然后加入100μL的Solution C
Figure PCTCN2021071373-appb-000021
将结晶紫打散，使用ELISA读取器分别测量背景值波长630nm和样品波长570nm的吸光度，并以检测到的样品波长570nm的吸光度减去背景值波长630nm的吸光度，对每孔处理的细胞进行评估，以没有加入甘草次酸衍生物处理的组别的吸光度(OD值)为100％，而推算出细胞存活率(Survival rate,％)及衍生物对SF268细胞的半毒杀剂量(CC 50)。
请参照图2A、图2B、图2C、图2D及图2E，图2A至图2E为本发明的实施例对SF268细胞的细胞存活率试验结果长条图，其中图2A为实施例1的结果，图2B为实施例2的结果，图2C为实施例3的结果，图2D为实施例4的结果，图2E为实施例5的结果。由试验结果可见，即使所添加的实施例1至实施例5的药物浓度达50μM，亦未明显导致细胞死亡，表明本发明的甘草次酸衍生物对SF268细胞无毒性作用(CC 50≥50μM)。
2-2：细胞病变抑制试验及免疫荧光染色分析
进行细胞病变抑制试验及免疫荧光染色分析的细胞株为SF268细胞，病毒株为兹卡病毒(ZIKV PRVABC59,ZIKV)。
首先将SF268细胞以每孔1.5×10 5的细胞量培养于6孔盘中，以含有10％FBS(Fetal bovine serum,
Figure PCTCN2021071373-appb-000022
)的DMEM(Dulbecco’s modified eagle medium,
Figure PCTCN2021071373-appb-000023
)配制至总体积达2mL，并于37℃及5％CO 2的培养箱中培养过夜。隔日，去除培养液并以磷酸盐缓冲溶液(Phosphate buffered saline,PBS)洗涤6孔盘两次，再使用不含FBS的DMEM清洗一次后，以病毒感染剂量(Multiplicity  of infection,MOI)为0.05的ZIKV感染SF268细胞，并分别加入实施例1至实施例7的甘草次酸衍生物，使其药物浓度达0.1μM、1μM及10μM，以对细胞进行处理。培养96小时后，藉由观察细胞病变状态并拍照即可得到细胞病变抑制试验的结果。
请参考图3，图3为本发明的实施例1对ZIKV诱导SF268细胞病变的抑制能力的显微影像。从图中可见，实施例1以浓度依赖的方式抑制了ZIKV诱导的细胞病变，且在药物浓度为10μM时，呈现显著抑制的效果。图3所显示的抑制效果为显著抑制(+++)，在此试验下的各实施例衍生物对于受感染细胞的细胞病变的抑制效果整理如下表九。图表的结果表明，本发明的甘草次酸衍生物对于ZIKV诱导的细胞病变具有显著的抑制效果。
表九
Figure PCTCN2021071373-appb-000024
承上(培养96小时后)，于每孔中加入1mL 4％的福马林将细胞固定，之后于震荡器上室温反应30分钟，接着以PBS清洗两次，而后于每孔细胞中加入1mL 50mM的NH 4Cl，之后于震荡器上室温反应15分钟，以去除自体荧光，接着再以PBS清洗一次，而后加入BSA：Triton-100为1000：1比例的封闭液，然后于4℃冷房震荡反应4小时，经PBS清洗后，再以抗体进行染色，详细而言，是以ZIKV anti-NS1 protein(GeneTex,Inc.)抗体做为一抗，然后于4℃冷房震荡过夜。隔日以PBS清洗三次后，以Goat anti-rabbit IgG H&L(Alexa
Figure PCTCN2021071373-appb-000025
555,ThermoFisher)抗体做为二抗进行染色，在避光的情况下，于4℃冷房震荡 1小时。最后，使用DAPI(4’6-diamidino-2-phenylindole)荧光染料，将细胞核染色，以计数细胞数，并使用Image J软体来确定受感染细胞中NS1阳性细胞的百分比。感染抑制力的计算方式为：(未加入实施例药物处理的感染细胞中NS1阳性百分比–加入实施例药物处理的感染细胞中NS1阳性百分比)/加入实施例药物处理的感染细胞中NS1阳性百分比。根据感染抑制力的结果，计算得到半数最大抑制浓度(IC 50)，即为本发明的甘草次酸衍生物对于ZIKV的抗病毒活性。
请参考图4A、图4B、图4C、图4D及图4E，图4A至图4E为本发明的实施例对受感染的SF268细胞的感染抑制力试验结果，其中图4A为实施例1的结果，图4B为实施例2的结果，图4C为实施例3的结果，图4D为实施例4的结果，图4E为实施例5的结果。同时请参照下表十，表十为本发明实施例1至实施例7对受感染细胞的感染抑制力及抗病毒活性数据。由图表的结果可知，本发明的各实施例皆以浓度依赖的方式，显著降低了受感染的细胞中NS1阳性细胞的百分比，亦即本发明的各实施例能够显著降低ZIKV对于SF268细胞的感染性，这表明了本发明的甘草次酸衍生物能够显著降低兹卡病毒对于细胞的感染性。
表十
Figure PCTCN2021071373-appb-000026
2-3：病毒感染时及病毒感染后处理试验
首先将两组SF268细胞以每孔1.5×10 5的细胞量培养于6孔盘中，以含有 10％FBS的DMEM配制至总体积达2mL，并于37℃及5％CO 2的培养箱中培养过夜。隔日，去除培养液并以磷酸盐缓冲溶液洗涤6孔盘两次，再使用不含FBS的DMEM清洗一次后，以病毒感染剂量为0.05的ZIKV感染SF268细胞，且一组细胞在病毒感染同时分别加入实施例1至实施例5的甘草次酸衍生物(病毒感染时处理试验)，另一组细胞在感染后1小时分别加入实施例1至实施例5的甘草次酸衍生物(病毒感染后处理试验)，使其药物浓度达0.1μM、1μM及10μM，以对细胞进行处理。分别将实施例1至实施例5的甘草次酸衍生物与病毒共同培养一小时后去除药物，最后加入含2％FBS的DMEM让总体积达2mL，培养96小时以观察细胞病变抑制效果，并进行免疫荧光染色，免疫荧光染色的过程已于前段提及，在此不再赘述，利用荧光显微镜下观察NS1病毒蛋白和DAPI荧光数量并拍照，再利用Image J软体计算NS1病毒蛋白和DAPI量，计算出病毒感染的比率。
请参照图5、图6A、图6B、图6C、图6D及图6E，图5为本发明的实施例1于病毒感染时处理试验对ZIKV诱导SF268细胞病变的抑制能力的显微影像，图6A至图6E为本发明的实施例于病毒感染时处理试验对受感染的SF268细胞的感染抑制力试验结果，其中图6A为实施例1的结果，图6B为实施例2的结果，图6C为实施例3的结果，图6D为实施例4的结果，图6E为实施例5的结果。请一并参照下表十一，表十一为本发明实施例1至实施例5对ZIKV于进入细胞阶段的抗病毒活性数据。图表的结果表明，本发明的各实施例皆以浓度依赖的方式，显著降低了受感染的细胞中NS1阳性细胞的百分比，亦即本发明的各实施例能够显著降低ZIKV对于SF268细胞的感染性，这表明了本发明的甘草次酸衍生物能够显著降低ZIKV对于细胞的感染性并对于ZIKV的早期复制具有显著的抑制效果。
表十一
Figure PCTCN2021071373-appb-000027
请参照图7、图8A、图8B、图8C、图8D及图8E，图7为本发明的实施例1于病毒感染后处理试验对ZIKV诱导SF268细胞病变的抑制能力的显微影像，图8A至图8E为本发明的实施例于病毒感染后处理试验对受感染的SF268细胞的感染抑制力试验结果，其中图8A为实施例1的结果，图8B为实施例2的结果，图8C为实施例3的结果，图8D为实施例4的结果，图8E为实施例5的结果。请一并参照下表十二，表十二为本发明实施例1至实施例5对ZIKV于感染细胞后的抗病毒活性数据。图表的结果表明，本发明的各实施例皆以浓度依赖的方式，显著降低了受感染的细胞中NS1阳性细胞的百分比，亦即本发明的各实施例能够显著降低ZIKV进入SF268细胞后的复制能力，这表明了本发明的甘草次酸衍生物能够显著降低ZIKV进入细胞后复制能力并对于ZIKV的晚期复制具有显著的抑制效果。
表十二
Figure PCTCN2021071373-appb-000028
综上所述，本发明提供如式(I)所示的甘草次酸衍生物，可在体外抑制ZIKV的细胞病变效应和病毒感染性，亦能阻断病毒的早期和晚期复制的步骤，且其对ZIKV的半数最大抑制浓度皆小于10μM，故除降低了半数最大抑制浓度外，还能提高治疗指数(Therapeutic Index,TI)，详细来说，所述治疗指数为半毒杀剂量与半数最大抑制浓度的比值。因此，本发明的甘草次酸衍生物，具有治疗及改善兹卡病毒感染症的效果，适于作为治疗及改善兹卡病毒的药物，或与医药上可接受的赋形剂或载体搭配作为可治疗及改善兹卡病毒疾病的医药组合物，更能进一步扩大作为抗兹卡病毒药物的用途，深具生医市场的潜能。
本发明已以实施方式揭露如上，然其并非用以限定本发明，任何熟习此技艺者，在不脱离本发明的精神和范围内，当可作各种的更动与润饰，因此本发明的保护范围当视后附的申请专利范围所界定者为准。

Claims (10)

  1. 一种甘草次酸衍生物，其特征在于，该甘草次酸衍生物具有如式(I)所示的结构：
    Figure PCTCN2021071373-appb-100001
    其中该式(I)中，R 1为丁氧基、甲氧基、如式(MAI)所示的基团或如式(MAII)所示的基团，R 2为乙酰氧基、如式(MBI)所示的基团或如式(MBII)所示的基团，
    Figure PCTCN2021071373-appb-100002
    表示单键或双键以便满足所有价数：
    Figure PCTCN2021071373-appb-100003
    其中当R 2为如式(MBII)所示的基团时，该基团的硫原子与1号碳相接。
  2. 如权利要求1所述的甘草次酸衍生物，其中该甘草次酸衍生物具有如式(IA)、式(IB)、式(IC)、式(ID)、式(IE)、式(IF)或式(IG)所示的结构：
    Figure PCTCN2021071373-appb-100004
    Figure PCTCN2021071373-appb-100005
    Figure PCTCN2021071373-appb-100006
  3. 一种如权利要求1所述的甘草次酸衍生物的合成方法，其特征在于，包含：
    前驱物提供步骤，是提供前驱物，该前驱物为3-羰基三萜(3-oxo-triterpenoid)、半卡腙(semicarbazone)或3-乙酰氧基甘草次酸的氯化物(3-O-acetyl-GLA chloride)；
    化学反应步骤，是以一反应方式进行化学反应，以获得产物，其中该反应方式为于室温搅拌或高温回流；以及
    产物纯化步骤，是将该产物进行纯化，以获得该甘草次酸衍生物。
  4. 一种用于治疗或改善兹卡病毒的医药组合物，其特征在于，该用于治疗或改善兹卡病毒的医药组合物包含有效剂量的如权利要求1所述的甘草次酸衍生物。
  5. 如权利要求4所述的医药组合物，其中该有效剂量为0.1μM至10μM。
  6. 如权利要求5所述的医药组合物，其中该有效剂量为10μM。
  7. 如权利要求4所述的医药组合物，更包含医药上可接受的赋形剂或载体。
  8. 一种如权利要求1所述的甘草次酸衍生物的用途，其特征在于，其是用于制备治疗或改善兹卡病毒的药物。
  9. 如权利要求8所述的甘草次酸衍生物的用途，其中该治疗或改善兹卡病毒的药物为降低兹卡病毒对于细胞感染性的药物。
  10. 如权利要求8所述的甘草次酸衍生物的用途，其中该治疗或改善兹卡病毒的药物为抑制兹卡病毒的早期复制及晚期复制的药物。
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