WO2021218148A1 - 10-(s)-17-氢-7-去氢穿心莲内酯及其工业色谱制备方法和用途 - Google Patents

10-(s)-17-氢-7-去氢穿心莲内酯及其工业色谱制备方法和用途 Download PDF

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WO2021218148A1
WO2021218148A1 PCT/CN2020/132794 CN2020132794W WO2021218148A1 WO 2021218148 A1 WO2021218148 A1 WO 2021218148A1 CN 2020132794 W CN2020132794 W CN 2020132794W WO 2021218148 A1 WO2021218148 A1 WO 2021218148A1
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dehydroandrographolide
hydro
preparation
organic solvent
crude product
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French (fr)
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刘地发
郭正友
王章伟
张毅
刘尧奇
邓双炳
王振
方礼
刘芳芳
张功俊
钟仁清
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江西青峰药业有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D307/60—Two oxygen atoms, e.g. succinic anhydride
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
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  • the present invention relates to andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide, a preparation method thereof, and use of the andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide for preparing drugs for treating inflammation.
  • Andrographis paniculata (Burm.F.)Nees is extracted from the whole plant of Andrographis paniculata (Burm.F.)Nees. It has antibacterial, anti-inflammatory, detoxification and other effects. It is an important active natural product. Andrographolide and its derivatives have been made into a variety of dosage forms (tablets, dripping pills, capsules, etc.) and are widely used in clinical practice, such as Xiyanping injection, Lianbizhi injection, Yanhuning injection, and Andrographis paniculata Ester tablets, etc. have shown good clinical effects of clearing away heat, detoxifying, anti-bacterial and anti-inflammatory. Luvone et al.
  • nitric oxide is related to the occurrence of acute and chronic inflammation [Luvone T, Carnuccio R, Di RM. Modulation of granuloma formation endogenous nitric oxide [J]. Eur J Pharmacol, 1994, 265(1/2) ):89-92.]. Andrographolide can significantly down-regulate the expression of inflammatory factors NO, tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin (IL)-6 in mouse macrophages RAW264.7 induced by lipopolysaccharide, thereby inhibiting the inflammatory response.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin-6
  • Xiyanping injection which is widely used clinically, has the functions of clearing away heat, detoxifying, antibacterial and anti-inflammatory, but the active ingredient of Xiyanping injection is the sulfonated product of andrographolide, which is not a single structure.
  • the use of single-structure small molecule compounds to further clarify the mechanism is of great significance to the study of its pharmacodynamics and toxicology; although andrographolides have achieved some research results, there is still nothing new for many diseases, especially in the field of anti-inflammatory
  • the particularly effective andrographolide derivatives or analogues are used clinically. Therefore, the development of new andrographolide derivative monomer drugs with anti-inflammatory activity has important social significance.
  • the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide and its preparation method and application.
  • the first aspect of the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide formula (I),
  • the second aspect of the present invention provides a method for preparing a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide,
  • Macroporous adsorption resin column purification Dissolve andrographolide total sulfonate in water, load the sample on the macroporous adsorption resin column, use a mixed solvent of organic solvent and water for elution, and combine to contain 10-(S)-17- The eluent of hydrogen-7-dehydroandrographolide was concentrated to obtain crude product 1;
  • the macroporous adsorption resin is a styrene-type macroporous adsorption resin, more preferably, the styrene-type macroporous adsorption resin model HPD-100S, D101S or LX-1180.
  • the organic solvent is an alcohol solvent, such as methanol or ethanol.
  • the organic solvent and water mixed solvent are 0%-60% methanol aqueous solution (preferably 35%-55% methanol aqueous solution) or 0%-60% ethanol aqueous solution (preferably 30%) by volume. % ⁇ 50% ethanol aqueous solution).
  • the elution is a gradient elution, such as: eluting with a volume ratio of 35%, 45%, and 55% methanol-water solution in sequence, or using a volume ratio of 30%, 40% in sequence. , 50% ethanol-water solution for elution.
  • a gradient elution such as: eluting with a volume ratio of 35%, 45%, and 55% methanol-water solution in sequence, or using a volume ratio of 30%, 40% in sequence. , 50% ethanol-water solution for elution.
  • the concentration temperature is 40-50°C.
  • A is one of petroleum ether or cyclohexane
  • B is one of ethyl acetate, dichloromethane, chloroform or acetone ;
  • the mixed organic solvent (A/B) is a petroleum ether/acetone solution
  • a petroleum ether/acetone solution with a volume ratio of 5:1 to 4:1 is used for dissolution; and/or a petroleum ether/acetone solution with a volume ratio of 5:1 to 1::1 is used for elution.
  • the elution is a gradient elution, for example, a petroleum ether-acetone solution with a volume ratio of 5:1, 3:1, 2:1, and 1:1 is used for elution in sequence.
  • a gradient elution for example, a petroleum ether-acetone solution with a volume ratio of 5:1, 3:1, 2:1, and 1:1 is used for elution in sequence.
  • the concentration temperature is 40-50°C.
  • the packing of the HPLC chromatographic column is a reversed-phase C18 packing, more preferably XAqua, ODS-A or ODS-AQ.
  • the detection wavelength of the ultraviolet on-line detector is 150-300 nm, more preferably 225 nm.
  • the organic solvent is ethyl acetate.
  • the number of recrystallization is greater than or equal to 2 times.
  • the drying method is vacuum drying.
  • the drying temperature is 40-50°C.
  • the third aspect of the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide as a pharmaceutical active ingredient of the drug formulation, including but not limited to injections, tablets, Capsules, dispersible tablets.
  • the fourth aspect of the present invention provides the use of the above-mentioned new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide or the above-mentioned pharmaceutical preparation in the preparation of a medicine for the treatment of inflammatory diseases.
  • the inflammatory disease is preferably pneumonia, and the pneumonia is preferably new coronavirus pneumonia (COVID-19).
  • the present invention provides a new andrographolide derivative 10-(S)-17-hydro-7-dehydroandrographolide compound of formula (I).
  • LPS lipopolysaccharide
  • NO nitric oxide
  • RAW264.7 macrophages have a significant inhibitory effect. Therefore, the compound can be used as an inhibitor of nitric oxide and made into an anti-inflammatory drug for the treatment of inflammatory diseases. Further, it can be used for treatment Pneumonia, such as the new coronavirus pneumonia (COVID-19).
  • the preparation method of 10-(S)-17-hydro-7-dehydroandrographolide provided by the present invention has convenient operation and high yield.
  • Industrial chromatography technology can be used to prepare the compound with high purity in large quantities, which is suitable for large-scale production. .
  • Fig. 1 10-(S)-17-hydro-7-dehydroandrographolide hydrogen nuclear magnetic resonance spectrum of Example 2;
  • FIG. 1 HPLC detection diagram of 10-(S)-17-hydro-7-dehydroandrographolide in Example 2;
  • FIG. 4 Example 2 of 10-(S)-17-hydro-7-dehydroandrographolide anti-inflammatory activity test.
  • Purification by macroporous adsorption resin column Take 200.00g of andrographolide total sulfonate, add appropriate amount of purified water to dissolve it, load the sample on D101S macroporous adsorption resin column, and use volume ratio 30%, 40%, 50% ethanol-water solution in turn Perform elution, combine the eluted fractions containing 10-(S)-17-hydro-7-dehydroandrographolide, and concentrate under reduced pressure at 40°C to obtain crude product 1;
  • the proton nuclear magnetic resonance spectrum 1 H-NMR (400MH Z ) and the carbon nuclear magnetic resonance spectrum 13 C-NMR (100MH Z ) are shown in Figure 1 and Figure 2, and the data is shown in Table 1.
  • Example 2 The compound obtained in Example 2 was applied to lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophages, and the level of NO in the culture supernatant was detected by the Griess reagent colorimetric method. The ability of the tested drug to inhibit the release of NO was used as Screen indicators to evaluate the anti-inflammatory activity of the compound.
  • LPS lipopolysaccharide
  • the monomer compound was dissolved in DMSO to prepare a solution of 1.564, 3.125, 6.250, 12.50, 25.00 ⁇ g/ml.

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Abstract

提供了一种新的化合物10-(S)-17-氢-7-去氢穿心莲内酯；体外实验表明该化合物对脂多糖(LPS)诱导的RAW264.7巨噬细胞中一氧化氮(NO)的产生具有显著的抑制作用，因此该化合物可作为一氧化氮抑制剂，制成抗炎药物，可用于治疗新型冠状病毒肺炎(COVID-19)；还提供了10-(S)-17-氢-7-去氢穿心莲内酯的制备方法，操作方便，收率高，可采用工业色谱技术大量制备高纯度该化合物，适用于大规模生产。

Description

10-(S)-17-氢-7-去氢穿心莲内酯及其工业色谱制备方法和用途
本申请要求申请日为2020/4/30的中国专利申请202010362402.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯及其制备方法和其用于制备治疗炎症药物的用途。
背景技术
穿心莲内酯是从爵床科植物穿心莲[Andrographis paniculata(Burm.F.)Nees]全草中提取的半日花烷型二萜类化合物，具有抗菌消炎，解毒等功效，是一种重要的活性天然产物。穿心莲内酯及其衍生物已经被制成多种剂型(片剂、滴丸、胶囊等)广泛应用于临床，如喜炎平注射液、莲必治注射液、炎琥宁注射液、穿心莲内酯片等在临床上均表现出良好的清热解毒、抗菌消炎功效。Luvone等认为，一氧化氮(NO)与急慢性炎症的发生均相关[Luvone T,Carnuccio R,Di RM.Modulation of granuloma formation endogenous nitric oxide[J].Eur J Pharmacol,1994,265(1/2):89-92.]。穿心莲内酯能够明显下调脂多糖诱导的小鼠巨噬细胞RAW264.7中炎症因子NO、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6的表达，从而抑制炎症反应。
目前临床上广泛使用的喜炎平注射液，具有清热解毒、抗菌消炎等功能，但是喜炎平注射液的活性成分为穿心莲内酯的磺化产物，并非单一结构。采用单一结构的小分子化合物以进一步明确机理，对其药效及其毒理学研究具有重要意义；尽管穿心莲内酯类化合物已取得一些研究成果，但是对于多种疾病，尤其抗炎领域仍然没有新的特别有效的穿心莲内酯衍生物或类似物用于临床。因此，开发新的具有抗炎活性的穿心莲内酯衍生物单体药物具有重要的社会意义。
发明内容
为了克服现有技术的不足，本发明提供了一种新的穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯及其制备方法和应用。
本发明第一方面，提供一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯式(Ι)，
Figure PCTCN2020132794-appb-000001
本发明第二方面，提供一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯的制备方法，
Figure PCTCN2020132794-appb-000002
包括以下步骤：
(1)大孔吸附树脂柱纯化：将穿心莲内酯总磺化物用水溶解，上样于大孔吸附树脂柱，采用有机溶剂和水混合溶剂进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱液，浓缩，得粗品1；
(2)硅胶柱纯化：将粗品1用混合有机溶剂(A/B)溶解，上样于硅胶柱进行纯化，用混合有机溶剂(A/B)洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱液，浓缩，得粗品2；
(3)重结晶：将粗品2用有机溶剂溶解，挥发部分有机溶剂，析出固体，过滤，干 燥。
优选的，所述步骤(1)中，大孔吸附树脂为苯乙烯型大孔吸附树脂，更优选的，苯乙烯型大孔吸附树脂型号HPD-100S、D101S或LX-1180。
优选的，所述步骤(1)中，有机溶剂为醇类溶剂，如甲醇或乙醇。
优选的，所述步骤(1)中，有机溶剂和水混合溶剂为体积比0％～60％甲醇水溶液(优选35％～55％甲醇水溶液)或体积比0％～60％乙醇水溶液(优选30％～50％乙醇水溶液)。
优选的，所述步骤(1)中，洗脱为梯度洗脱，如：依次用体积比35％、45％、55％甲醇-水溶液进行洗脱，或，依次用体积比30％、40％、50％乙醇-水溶液进行洗脱。
优选的，所述步骤(1)中，浓缩温度为40～50℃。
优选的，所述步骤(2)中，混合有机溶剂(A/B)中A为石油醚或环己烷中的一种，B为乙酸乙酯、二氯甲烷、氯仿或丙酮中的一种；
更优选的，混合有机溶剂(A/B)为石油醚/丙酮溶液；
进一步优选的，溶解时采用体积比5:1～4:1的石油醚/丙酮溶液；和/或，洗脱时采用溶剂为体积比5:1～1:1的石油醚/丙酮溶液。
优选的，所述步骤(2)中，洗脱为梯度洗脱，如：依次用体积比为5:1、3:1、2:1、1:1的石油醚-丙酮溶液进行洗脱。
优选的，所述步骤(2)中，浓缩温度为40～50℃。
优选的，所述步骤(2)中，HPLC色谱柱的填料为反相C18填料，更优选为XAqua、ODS-A或ODS-AQ。
优选的，所述步骤(2)中，紫外在线检测器的检测波长为150～300nm，更优选为225nm。
优选的，所述步骤(3)中，有机溶剂为乙酸乙酯。
优选的，所述步骤(3)中，重结晶次数大于或等于2次。
优选的，所述步骤(3)中，干燥方法为减压干燥。
优选的，所述步骤(3)中，干燥温度40～50℃。
本发明的第三方面，提供一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯作为药物活性成分的药物制剂，包括但不限于注射剂、片剂、胶囊、分散片。
本发明的第四方面，提供上述的新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯或上述的药物制剂在制备治疗炎症疾病药物中的用途，所述炎症疾病优选肺炎，所述肺炎优选新型冠状病毒肺炎(COVID-19)。
本发明的有益技术效果
1.本发明提供一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯式(Ι)化合物，经体外实验表明该化合物对脂多糖(LPS)诱导的RAW264.7巨噬细胞中一氧化氮(NO)的产生具有显著的抑制作用，因此该化合物可作为一氧化氮抑制剂，制成抗炎药物，用于治疗炎症疾病，进一步的，可用于治疗肺炎，如新型冠状病毒肺炎(COVID-19)。
2.本发明提供的10-(S)-17-氢-7-去氢穿心莲内酯制备方法，操作方便，收率高，可采用工业色谱技术大量制备高纯度该化合物，适用于大规模生产。
附图说明
图1：实施例2的10-(S)-17-氢-7-去氢穿心莲内酯核磁共振氢谱图；
图2：实施例2的10-(S)-17-氢-7-去氢穿心莲内酯核磁共振碳谱图；
图3：实施例2的10-(S)-17-氢-7-去氢穿心莲内酯HPLC检测图；
图4：实施例2的10-(S)-17-氢-7-去氢穿心莲内酯抗炎活性测试。
具体实施方式
在如下的实施例中所指的10-(S)-17-氢-7-去氢穿心莲内酯的化学结构式(结构中的阿拉伯数字是化学结构中碳原子的标位)：
Figure PCTCN2020132794-appb-000003
实施例1：穿心莲内酯总磺化物的制备
取无水乙醇50L，置反应釜中，缓慢加入20L浓硫酸，搅拌均匀后，加入50.00kg穿心莲内酯，搅拌，常温放置72小时。控制温度加入50L 95％乙醇，搅拌，加入50％氢氧化钠溶液调pH至7.0，加入乙醇至含醇量85％，静置24小时，过滤，滤液回收乙醇，浓缩成稠膏，真空干燥即得穿心莲内酯总磺化物。
实施例2-5：10-(S)-17-氢-7-去氢穿心莲内酯的制备
实施例2
(1)大孔吸附树脂柱纯化：取穿心莲内酯总磺化物200.00g，加适量纯化水溶解后上样D101S大孔吸附树脂柱，依次用体积比30％、40％、50％乙醇-水溶液进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，40℃减压浓缩干，得粗品1；
(2)硅胶柱纯化：将粗品1用适量体积比5:1石油醚-丙酮溶解后上样硅胶柱，依次用体积比为5:1、3:1、2:1、1:1的石油醚-丙酮溶液进行洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，HPLC色谱柱填料为XAqua(反向C18填料)，紫外检测波长为225nm。根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，40℃减压浓缩干，得粗品2；
(3)重结晶：将粗品2加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体继续加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体40℃减压干燥，得10-(S)-17-氢-7-去氢穿心莲内酯4.53g，纯度96.33％。
经NMR、ESI-MS等现代光谱技术鉴定了化合物10-(S)-17-氢-7-去氢穿心莲内酯的化学结构，并通过X-射线单晶衍射确定了该化合物绝对构型，其理化性质如下：
白色粉末，分子式为:C 20H 30O 5；
高分辨质谱HRESIMS m/z 349.2021[M–H] –(cald.for.C 20H 29O 5,349.2015)。核磁共振氢谱 1H-NMR(400MH Z)及核磁共振碳谱 13C-NMR(100MH Z)，见图1和图2，数据见表1。
表1 10-(S)-17-氢-7-去氢穿心莲内酯的氢谱和碳谱数据(400/100MHz,DMSO)
Figure PCTCN2020132794-appb-000004
Figure PCTCN2020132794-appb-000005
实施例3
(1)大孔吸附树脂柱纯化：取穿心莲内酯总磺化物200.08g，加适量纯化水溶解后上样HPD-100S大孔树脂柱，依次用体积比35％、45％、55％甲醇-水溶液进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，50℃减压浓缩干，得粗品1；
(2)硅胶柱纯化：将粗品1用适量体积比5:1石油醚/丙酮混合溶剂溶解后上样于硅胶柱进行纯化，依次用体积比为5:1、3:1、2:1、1:1石油醚-丙酮溶液进行洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，HPLC色谱柱填料为ODS-A(反向C18填料)，紫外检测波长为225nm。根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，50℃减压浓缩干，得粗品2；
(3)重结晶：将粗品2加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体继续加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体50℃减压干燥，得10-(S)-17-氢-7-去氢穿心莲内酯4.39g，纯度96.54％。
实施例4
(1)大孔吸附树脂柱纯化：取穿心莲内酯总磺化物200.15g，加适量纯化水溶解后上样LX-1180大孔树吸附脂柱，依次用体积比30％、40％、50％乙醇-水溶液进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，40℃减压浓缩干，得粗品1；
(2)硅胶柱纯化：将粗品1用适量体积比4:1石油醚/丙酮溶液溶解后上样于硅胶柱进行纯化，依次用体积比为5:1、3:1、2:1、1:1的石油醚-丙酮溶液进行洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，HPLC色谱柱填料为ODS-AQ(反向C18填料)，紫外检测波长为225nm。根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，40℃减压浓缩干，得粗品2；
(3)重结晶：将粗品2加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体继续加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体50℃减压干燥，得10-(S)-17-氢-7-去氢穿心莲内酯4.72g，纯度96.19％。
实施例5
(1)大孔吸附树脂柱纯化：取穿心莲内酯总磺化物20.15kg，加适量纯化水溶解后 上样HPD-100S大孔树脂柱，依次用体积比35％、45％、55％甲醇-水溶液进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，50℃减压浓缩干，得粗品1；
(2)工业色谱级硅胶柱纯化：将粗品1用适量体积比5:1石油醚/丙酮混合溶剂溶解后上样于工业色谱系统纯化(DAC-HB300动态轴向压缩柱，硅胶填料)，依次用体积比为5:1、3:1、2:1、1:1石油醚-丙酮溶液进行洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，HPLC色谱柱填料为ODS-A(反向C18填料)，紫外检测波长为225nm。根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱流份，40℃减压浓缩干，得粗品2；
(3)重结晶：将粗品2加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体继续加入适量乙酸乙酯溶解，放置通风橱挥发掉部分溶剂至大量固体析出，过滤，固体40℃减压干燥，得10-(S)-17-氢-7-去氢穿心莲内酯415.28g，纯度96.08％。
实施例6：10-(S)-17-氢-7-去氢穿心莲内酯抗炎活性测试
将实施例2所得的化合物作用于脂多糖(LPS)诱导的RAW264.7小鼠巨噬细胞，用Griess试剂显色法检测培养上清液中NO水平，以受试药物抑制NO释放的能力作为筛选指标，评价化合物的抗炎活性。
1.药物溶液的配制
将单体化合物用DMSO溶解配制成1.564、3.125、6.250、12.50、25.00μg/ml溶液。
2.试验方法
(1)单个小鼠巨噬细胞悬液用含10％的胎牛血清培养液配制而成，取对数期的小鼠巨噬细胞RAW264.7接种到96孔板中，每孔10 5细胞数，设置3个复孔。
(2)经过24小时培养，每孔加入不同浓度的受试药物同时加入1ug/mL LPS。
(3)37℃培养24小时后，每孔加Griess溶液100ul继续培养5分钟，停止培养。
(4)用酶联免疫检测仪测定570nm波长处的光密度(OD)值，并计算NO抑制率。
3.实验结果
10-(S)-17-氢-7-去氢穿心莲内酯化合物对RAW264.7细胞中的NO抑制作用IC50为 9.184μg/ml，具体参见图4。
结果表明10-(S)-17-氢-7-去氢穿心莲内酯化合物对脂多糖(LPS)诱导的小鼠巨噬细胞RAW264.7NO的生成有显著抑制作用，可作为一氧化氮抑制剂，表明本发明所述化合物具有明显的抗炎活性；因此可以用于制备新的抗炎活性药物；进一步的，可用于治疗肺炎，如新型冠状病毒肺炎(COVID-19)。

Claims (10)

  1. 一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯，结构为式(Ι)，
    Figure PCTCN2020132794-appb-100001
  2. 一种新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯的制备方法，
    Figure PCTCN2020132794-appb-100002
    其特征在于，包括以下步骤：
    (1)大孔吸附树脂柱纯化：将穿心莲内酯总磺化物用水溶解，上样于大孔吸附树脂柱，采用有机溶剂和水混合溶剂进行洗脱，合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱液，浓缩，得粗品1；
    (2)硅胶柱纯化：将粗品1用混合有机溶剂(A/B)溶解，上样于硅胶柱进行纯化，用混合有机溶剂(A/B)洗脱，用紫外在线检测器检测分离情况，根据出峰时间及色谱峰高度，确定洗脱液收集起止时间，收集洗脱液进行HPLC检测，根据检测结果合并含有10-(S)-17-氢-7-去氢穿心莲内酯的洗脱液，浓缩，得粗品2；
    (3)重结晶：将粗品2用有机溶剂溶解，挥发部分有机溶剂，析出固体，过滤，干燥。
  3. 根据权利要求2所述的制备方法，其特征在于：所述步骤(1)中，大孔吸附树脂为苯乙烯型大孔吸附树脂，优选树脂型号HPD-100S、D101S或LX-1180。
  4. 根据权利要求2所述的制备方法，其特征在于：所述步骤(1)中，有机溶剂为醇类溶剂；和/或，有机溶剂和水混合溶剂为体积比0％～60％甲醇-水溶液或体积比0％～60％乙醇-水溶液；和/或，浓缩温度为40～50℃。
  5. 根据权利要求2所述的制备方法，其特征在于：所述步骤(2)中，
    混合有机溶剂(A/B)中A为石油醚或环己烷，B为乙酸乙酯、二氯甲烷、氯仿或丙酮；
    优选的，混合有机溶剂(A/B)为石油醚/丙酮溶液；
    更优选的，溶解时采用体积比5:1～4:1的石油醚/丙酮溶液；和/或，洗脱时采用溶剂为体积比5:1～1:1的石油醚/丙酮溶液。
  6. 根据权利要求2所述的制备方法，其特征在于：所述步骤(2)中，HPLC色谱柱的填料为反相C18填料，优选为XAqua、ODS-A或ODS-AQ。
  7. 根据权利要求2所述的制备方法，其特征在于：所述步骤(2)中，紫外在线检测器的检测波长为150～300nm；和/或，浓缩温度为40～50℃。
  8. 根据权利要求2所述的制备方法，其特征在于：所述步骤(3)中，有机溶剂为乙酸乙酯；和/或，重结晶次数大于或等于2次；和/或，干燥方法为减压干燥。
  9. 一种以如权利要求1所述的如式(I)所示的新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯或权利要求2-8任一项所述方法制得的新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯作为药物活性成分的药物制剂，所述的药物制剂优选地包括但不限于注射剂、片剂、胶囊、分散片。
  10. 权利要求1所述的新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯或权利要求2-8任一项所述方法制得的新穿心莲内酯衍生物10-(S)-17-氢-7-去氢穿心莲内酯或权利要求9所述的药物制剂在制备治疗炎症疾病药物中的用途，所述炎症疾病优选肺炎，所述肺炎优选新型冠状病毒肺炎(COVID-19)。
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