WO2015055116A1 - Selaginella pulvinata extractive and preparation method and application thereof - Google Patents

Selaginella pulvinata extractive and preparation method and application thereof Download PDF

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WO2015055116A1
WO2015055116A1 PCT/CN2014/088611 CN2014088611W WO2015055116A1 WO 2015055116 A1 WO2015055116 A1 WO 2015055116A1 CN 2014088611 W CN2014088611 W CN 2014088611W WO 2015055116 A1 WO2015055116 A1 WO 2015055116A1
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ethynyl
dzjb
bis
indole
methoxyphenyl
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French (fr)
Chinese (zh)
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尹胜
罗海彬
刘信
黄仪有
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中山大学
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    • C07C39/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic, containing six-membered aromatic rings and other rings, with unsaturation outside the aromatic rings
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    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
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    • C07C47/56—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing hydroxy groups
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    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
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    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/19—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups having unsaturation outside the aromatic ring
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    • C07C65/28—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups having unsaturation outside the aromatic rings
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    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
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    • C07C69/12—Acetic acid esters
    • C07C69/16—Acetic acid esters of dihydroxylic compounds
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    • C07C69/12—Acetic acid esters
    • C07C69/21—Acetic acid esters of hydroxy compounds with more than three hydroxy groups
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    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • the present invention relates to natural extracts and modifications thereof, and more particularly to a Pleurotus ostreatus extract and a preparation method and application thereof.
  • Cyclic nucleotide phosphodiesterases are an important family of super-enzymes that regulate the concentration of cAMP and cGMP in cells by hydrolyzing cAMP and cGMP, thereby regulating the transmission of second messengers in the body. Biochemical effects. PDEs are widely distributed in mammalian tissues, and their diversity results in specific distribution of different PDE enzymes at the cellular and subcellular levels, which can selectively regulate a variety of cellular functions, and is a good drug design and therapeutic target.
  • Type 4 phosphodiesterase (PDE4), which is a PDE family enzyme that specifically catalyzes the hydrolysis of cAMP, is mainly distributed in inflammatory cells in humans.
  • cAMP plays a key negative regulatory role in the physiological processes such as the release of cytokines. Therefore, increasing the intracellular cAMP concentration by inhibiting PDE4 will help reduce the damage of the inflammatory response to the body.
  • PDE4 inhibitors have been developed into anti-inflammatory drugs, such as Roflumilast, etc., which are mainly used for the treatment of inflammation in the lungs, especially for the treatment of asthma and chronic obstructive pulmonary disease.
  • taking such drugs can cause adverse reactions such as diarrhea and nausea.
  • the present invention is a Pleurotus ostreatus extract, which is shown in Structural Formula I:
  • R 1 , R 4 is H, alkyl or acyl
  • R 2 , R 3 , R 5 , R 7 represent a halogen, an aldehyde group, a carboxyl group, a sulfonic acid group, a nitro group, a nitroso group or a hydrogen atom, respectively;
  • R 6 represents H, -CH 3 , -CH 2 OH, -CHO or -COOH.
  • the R 1 and R 4 are hydrogen, methyl, ethyl or acetyl.
  • the R 2 , R 3 , R 5 and R 7 are hydrogen.
  • the compound is preferably:
  • DZJB-1 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted)biphenol,
  • DZJB-2 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde,
  • DZJB-3 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted)biphenol,
  • DZJB-4 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-indole-9,9-disubstituted)biphenol,
  • DZJB-5 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxyphenyl)-9H-indol-2-ol,
  • DZJB-6 7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde,
  • DZJB-7 (7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indol-2-yl) Methanol,
  • DJB-8 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde,
  • DZJB-9 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-2-methyl-9H-indole,
  • DZJB-10 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole,
  • DZJB-11 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
  • DZJB-12 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
  • DZJB-13 (7-acetoxy-2-(acetoxymethyl)-1-((4-acetoxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) bis (4) , 1-phenylene) diacetate
  • DZJB-14 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-formyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Diacetate,
  • DZJB-15 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) bis(4,1-phenylene)diacetic acid ester,
  • DZJB-16 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Diacetate,
  • DZJB-17 4-((7-acetoxy-9,9-bis(4-methoxyphenyl)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester,
  • DZJB-18 4-((7-acetoxy-2-formyl-9,9-bis(4-methoxybenzene)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester.
  • the pad-shaped cypress whole grass is powdered, ultrasonically extracted with 95% ethanol for 3 times, and the solvent is recovered to obtain an extract;
  • the extracted extract obtained in the step S1 is sequentially extracted with petroleum ether and ethyl acetate to obtain two fractions respectively, and then the ethyl acetate extract is subjected to MCI column chromatography, respectively, using 30%, 50%, 70%, 90 respectively. %, 100% methanol-water gradient elution to obtain various stages of eluent;
  • the eluate obtained in the step S2 is obtained by silica gel column chromatography, gel column chromatography, ODS column chromatography and high performance liquid chromatography, respectively.
  • the Paeonia serrata extract was first applied to inhibit PDE4 activity and exhibited very good inhibitory activity and selectivity. It is expected to become a new drug for the treatment of inflammation such as asthma.
  • Figure 1 is a flow chart showing the separation of the compounds of the present invention.
  • reagents, devices, and methods employed in the present invention are conventionally commercially available reagents, equipment, and methods of routine use.
  • DZJB-1 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted) biphenol
  • Example 2 The title compound was obtained by the procedure of Example 2, which was purified by the gel (LH-20) eluent as anhydrous ethanol. Its structure is as follows:
  • UV (MeOH) ⁇ max (log ⁇ ) 208 (4.76) nm, 288 (4.36) nm, 299 (4.41) nm, 315 (4.18) nm.
  • the reference compound was prepared according to the method of Example 5 with a retention time of 9.2 min.
  • the structure is as follows:
  • UV (MeOH) ⁇ max (log ⁇ ) 205 (4.89) nm, 288 (4.52) nm, 298 (4.53) nm, 316 (4.35) nm.
  • UV (MeOH) ⁇ max (log ⁇ ) 209 (4.76) nm, 291 (4.47) nm, 298 (4.49) nm, 314 (4.30) nm.
  • DZJB-6 7-Hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde
  • DZJB-7 (7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indol-2-yl)methanol preparation
  • DZJB-8 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde
  • DZJB-9 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-2-methyl-9H-indole
  • DZJB -10 Preparation of 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole
  • Example 5 The title compound was prepared according to the methods of Example 5, Example 6 and Example 10.
  • DZJB-12 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-carboxylic acid
  • DZJB-15 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) bis(4,1-phenylene)diacetic acid Preparation of ester
  • DZJB-13 (7-acetoxy-2-(acetoxymethyl)-1-((4-acetoxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) bis (4) , 1-phenylene) diacetate
  • DZJB-14 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-formyl-9H-indole-9,9 -disubstituted) bis(4,1-phenylene) diacetate
  • DZJB-16 (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-methyl- Preparation of 9H-fluorene-9,9-disubstituted) bis(4,1-phenylene) diacetate
  • Example 3 The title compound was prepared according to the methods of Example 3, Example 4, Example 5 and Example 14.
  • the molecule to be tested contains the recombinant PDE4D2 protein (this recombinant protein is prepared by us.
  • this recombinant protein is prepared by us.
  • 20 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 10 mM MgCl 2 and 20,000-30,000 cpm of 3 H-cAMP were incubated for 15 minutes at room temperature, then 0.2 M ZnSO 4 and Ba, respectively.
  • test data for the inhibitory activity of the compounds of the present invention against PDE4, PDE5, and PDE9 are shown in Table 1 (inhibitor concentration when the IC 50 value reached 50% inhibition rate).

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Abstract

An in-depth study is performed on selaginella pulvinata extractives in the present invention, and it is found that the selaginella pulvinata extractives have a significant anti-inflammatory activity. Multiple column chromatography separation technologies and a semi-preparation high-performance liquid chromatography separation method are used to extract and separate a class of compounds (DZJB1-6) with a 9,9-diphenyl-1-(phenylethynyl)-9H-fluorene new skeleton structure, named as selaginpulvilinsA-F (structural formula (I)). The six compounds extracted and separated from selaginella pulvinata are subjected to structural modification, and then a series of compounds are obtained. These compounds can significantly inhibit the activity of type-4 phosphodiesterase (PDE4), and can be used for preparing PDE4 inhibitors, and treating asthma, chronic obstructive pneumonia, inflammations, and the like. According to the present invention, a new structure is provided for the development of PDE4 inhibitors.

Description

一种垫状卷柏提取物及其制备方法和应用Padded Selaginella extract and preparation method and application thereof 技术领域Technical field
本发明涉及天然提取物及其修饰，更具体地，涉及一种垫状卷柏提取物及其制备方法和应用。The present invention relates to natural extracts and modifications thereof, and more particularly to a Pleurotus ostreatus extract and a preparation method and application thereof.
背景技术Background technique
环核苷酸磷酸二酯酶(Cyclic nucleotide phosphodiesterases,PDEs)是一类重要的超级酶家族，通过对cAMP和cGMP的水解，有效控制细胞内的cAMP和cGMP浓度，从而调节体内第二信使所传导的生化作用。PDEs在哺乳动物组织中分布广泛，其多样性致使不同的PDE酶在细胞和亚细胞水平有着特定的分布，可选择性调节多种细胞功能，是良好的药物设计与治疗靶点。Cyclic nucleotide phosphodiesterases (PDEs) are an important family of super-enzymes that regulate the concentration of cAMP and cGMP in cells by hydrolyzing cAMP and cGMP, thereby regulating the transmission of second messengers in the body. Biochemical effects. PDEs are widely distributed in mammalian tissues, and their diversity results in specific distribution of different PDE enzymes at the cellular and subcellular levels, which can selectively regulate a variety of cellular functions, and is a good drug design and therapeutic target.
4型磷酸二酯酶(PDE4)作为特异性催化水解cAMP的PDE家族酶，在人体内主要分布于炎症细胞内。在炎症反应中，cAMP对细胞因子的释放等生理过程起着关键的负调节作用，因此通过抑制PDE4提高细胞内cAMP浓度将有助于减轻炎症反应对机体的伤害。目前，PDE4抑制剂已被开发成抗炎的药物，如罗氟司特(Roflumilast)等，临床上主要用于治疗肺部的炎症，尤其是针对于哮喘和慢性阻塞性肺病的治疗上。但服用此类药物会引起腹泻、恶心等不良反应，因此如何克服这些PDE4抑制剂存在的不良反应，研究新型的特异性抑制剂就成为研究的热点之一。天然产物是寻找具有新型PDE4抑制剂的重要来源之一，对于开发新一代疗效强、副作用小的PDE4抑制剂具有重要的意义。 Type 4 phosphodiesterase (PDE4), which is a PDE family enzyme that specifically catalyzes the hydrolysis of cAMP, is mainly distributed in inflammatory cells in humans. In the inflammatory response, cAMP plays a key negative regulatory role in the physiological processes such as the release of cytokines. Therefore, increasing the intracellular cAMP concentration by inhibiting PDE4 will help reduce the damage of the inflammatory response to the body. At present, PDE4 inhibitors have been developed into anti-inflammatory drugs, such as Roflumilast, etc., which are mainly used for the treatment of inflammation in the lungs, especially for the treatment of asthma and chronic obstructive pulmonary disease. However, taking such drugs can cause adverse reactions such as diarrhea and nausea. Therefore, how to overcome the adverse reactions of these PDE4 inhibitors has become one of the research hotspots. Natural products are one of the important sources for the discovery of novel PDE4 inhibitors, and are of great significance for the development of a new generation of PDE4 inhibitors with high efficacy and low side effects.
目前垫状卷柏中只报道过炔酚selaginellin类化合物，且多用于抗菌及抗氧化，对于作为PDE4抑制剂尚无研究。本发明所述从垫状卷柏中发现的新颖的炔 酚类化合物为首次应用在抑制PDE4方面。At present, only acetylenic selaginellin compounds have been reported in the genus Papyrus, and they are mostly used for antibacterial and anti-oxidation, and have not been studied as PDE4 inhibitors. Novel alkyne found in the genus Corydalis Phenolic compounds are used for the first time in the inhibition of PDE4.
发明内容Summary of the invention
本发明为一种垫状卷柏提取物，所述的垫状卷柏提取物如结构式Ⅰ所示：The present invention is a Pleurotus ostreatus extract, which is shown in Structural Formula I:
Figure PCTCN2014088611-appb-000001
Figure PCTCN2014088611-appb-000001
其中：among them:
R1，R4为H，烷基或酰基；R 1 , R 4 is H, alkyl or acyl;
R2，R3，R5，R7分别代表卤素，醛基，羧基，磺酸基，硝基，亚硝基或氢；R 2 , R 3 , R 5 , R 7 represent a halogen, an aldehyde group, a carboxyl group, a sulfonic acid group, a nitro group, a nitroso group or a hydrogen atom, respectively;
R6代表H，-CH3，-CH2OH，-CHO或-COOH。R 6 represents H, -CH 3 , -CH 2 OH, -CHO or -COOH.
所述的R1和R4为氢、甲基、乙基或乙酰基。The R 1 and R 4 are hydrogen, methyl, ethyl or acetyl.
所述的R2，R3，R5，R7为氢。The R 2 , R 3 , R 5 and R 7 are hydrogen.
所述的化合物优选为：The compound is preferably:
DZJB-1：4,4'-(7-羟基-2-羟甲基-1-(4-羟基苯)乙炔基-9H-芴-9,9-二取代)联苯酚、DZJB-1: 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted)biphenol,
DZJB-2：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-醛、DZJB-2: 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde,
DZJB-3：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)联苯酚、DZJB-3: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted)biphenol,
DZJB-4：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-9H-芴-9,9-二取代)联苯酚、DZJB-4: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-indole-9,9-disubstituted)biphenol,
DZJB-5：8-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醇、DZJB-5: 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxyphenyl)-9H-indol-2-ol,
DZJB-6：7-羟基-1-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醛、DZJB-6: 7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde,
DZJB-7：(7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-基) 甲醇、DZJB-7: (7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indol-2-yl) Methanol,
DJB-8：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-醛、DJB-8: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde,
DZJB-9：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-2-甲基-9H-芴、DZJB-9: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-2-methyl-9H-indole,
DZJB-10：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴、DZJB-10: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole,
DZJB-11：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-羧酸、DZJB-11: 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
DZJB-12：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-羧酸、DZJB-12: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
DZJB-13：(7-乙酰氧基-2-(乙酰氧基甲基)-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-13: (7-acetoxy-2-(acetoxymethyl)-1-((4-acetoxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) bis (4) , 1-phenylene) diacetate,
DZJB-14：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲酰基-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-14: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-formyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Diacetate,
DZJB-15：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-15: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) bis(4,1-phenylene)diacetic acid ester,
DZJB-16：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-16: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Diacetate,
DZJB-17：4-((7-乙酰氧基-9,9-二(4-甲氧基苯)-9H-芴-1-基)乙炔基)乙酸苯酯、DZJB-17: 4-((7-acetoxy-9,9-bis(4-methoxyphenyl)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester,
或DZJB-18：4-((7-乙酰氧基-2-甲酰基-9,9-二(4-甲氧基苯)-9H-芴-1-基)乙炔基)乙酸苯酯。 Or DZJB-18: 4-((7-acetoxy-2-formyl-9,9-bis(4-methoxybenzene)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester.
更进一步提供一种制备上述的垫状卷柏提取物的方法，如图1所示，Still further provided is a method for preparing the above-mentioned P. sylvestris extract, as shown in FIG.
S1.将垫状卷柏全草粉粹，用95％乙醇超声提取3次，回收溶剂，得提取浸膏；S1. The pad-shaped cypress whole grass is powdered, ultrasonically extracted with 95% ethanol for 3 times, and the solvent is recovered to obtain an extract;
S2.将步骤S1得到的提取浸膏依次用石油醚和乙酸乙酯萃取，分别得到两个部分，随后乙酸乙酯萃取物经MCI柱层析，分别用30％、50％、70％、90％、100％甲醇-水梯度洗脱，得各种阶段的洗脱液；S2. The extracted extract obtained in the step S1 is sequentially extracted with petroleum ether and ethyl acetate to obtain two fractions respectively, and then the ethyl acetate extract is subjected to MCI column chromatography, respectively, using 30%, 50%, 70%, 90 respectively. %, 100% methanol-water gradient elution to obtain various stages of eluent;
S3.分别将取步骤S2所得洗脱液，通过硅胶柱层析、凝胶柱层析、ODS柱层析以及高效液相色谱等分离手段，即得。S3. The eluate obtained in the step S2 is obtained by silica gel column chromatography, gel column chromatography, ODS column chromatography and high performance liquid chromatography, respectively.
更进一步提供一种上述的垫状卷柏提取物在制备PDE4抑制剂中的应用。Still further provided is the use of the above-described P. sylvestris extract for the preparation of a PDE4 inhibitor.
本发明有以下优点：The invention has the following advantages:
1.从天然产物中发现了一类新骨架化合物，丰富了天然产物的种类。1. A new class of skeleton compounds has been discovered from natural products, enriching the types of natural products.
2.垫状卷柏提取物首次应用在抑制PDE4活性方面，且表现出非常好的抑制活性以及选择性。有望成为治疗炎症例如哮喘的新药物。2. The Paeonia serrata extract was first applied to inhibit PDE4 activity and exhibited very good inhibitory activity and selectivity. It is expected to become a new drug for the treatment of inflammation such as asthma.
附图说明DRAWINGS
图1为本发明所涉及的化合物的分离流程图。Figure 1 is a flow chart showing the separation of the compounds of the present invention.
具体实施方式detailed description
下面结合具体实施例进一步详细说明本发明。除非特别说明，本发明采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。The invention will now be described in further detail with reference to specific embodiments. Unless otherwise stated, the reagents, devices, and methods employed in the present invention are conventionally commercially available reagents, equipment, and methods of routine use.
现在参考下列说明性的实施例，进一步解释本发明，其中NMR波谱采用Bruker AM-400spectrometer记录，TMS内标。柱色谱硅胶(300～400目)：青岛海洋化工厂；GF254硅胶薄层色谱预制板：青岛海洋化工厂；MCI填料(CHP20P，75～150μm)：日本Mitsubishi公司；葡聚糖凝胶(SephadexLH-20)：美国GE公司；ODS填料(12nm，S-50μm)：日本YMC公司；其余溶剂和试剂：分析纯(AR)，天津市百世化工有限公司。The invention will now be further explained with reference to the following illustrative examples in which NMR spectra were recorded using a Bruker AM-400 spectrometer, TMS internal standard. Column chromatography silica gel (300 ~ 400 mesh): Qingdao Ocean Chemical Plant; GF 254 silica gel thin layer chromatography prefabricated plate: Qingdao Ocean Chemical Plant; MCI filler (CHP20P, 75 ~ 150μm): Japan Mitsubishi company; Sephadex LH (SephadexLH -20): American GE company; ODS packing (12nm, S-50μm): Japan YMC company; other solvents and reagents: analytical pure (AR), Tianjin Best Chemical Co., Ltd.
实施例1Example 1
垫状卷柏萃取物的制备Preparation of padded Selaginella extract
取垫状卷柏全草1kg,粉碎成粗粉。加入8倍体积量的95％乙醇，浸泡7天， 减压抽滤，减压回收乙醇，余液浓缩至相对密度为1.25的稠膏。重复两次以上浸泡提取步骤，最终得垫状卷柏乙醇提取物50g。垫状卷柏乙醇提取物用1L水溶解，依次用等量的石油醚、乙酸乙酯各萃取三次。合并乙酸乙酯萃取液，减压浓缩，得到乙酸乙酯萃取物39g。Take 1kg of padded cypress whole grass and smash it into coarse powder. Add 8 times the volume of 95% ethanol and soak for 7 days. The mixture was filtered under reduced pressure, and ethanol was evaporated under reduced pressure, and the residue was concentrated to a thick paste with a relative density of 1.25. The soaking extraction step was repeated twice more, and finally 50 g of the ethanolic extract of Paeonia serrata was obtained. The ethanolic extract of Paeonia serrata was dissolved in 1 L of water, and extracted three times with an equal amount of petroleum ether and ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to give ethyl acetate.
实施例2Example 2
单体化合物的分离Separation of monomeric compounds
按照实施例1，得到乙酸乙酯萃取物39g，乙酸乙酯萃取物应用MCI柱初步分段，以甲醇∶水＝3∶7～10∶0洗脱；然后采用200–300目柱色谱硅胶以石油醚:乙酸乙酯或石油醚:丙酮或氯仿:甲醇等溶剂体系梯度洗脱，再反复采用凝胶柱LH-20、ODS柱纯化，最后经半制备高效液相色谱手段在(乙腈∶水)或(甲醇∶水)条件下进一步纯化，最终得到6个单体化合物，具体分离流程参考图1。According to Example 1, 39 g of ethyl acetate extract was obtained, and the ethyl acetate extract was initially fractionated by MCI column, eluted with methanol:water = 3:7 to 10:0; then 200-300 mesh column chromatography silica gel was used. Petroleum ether: ethyl acetate or petroleum ether: acetone or chloroform: methanol and other solvent system gradient elution, and then repeated using gel column LH-20, ODS column purification, and finally by semi-preparative high performance liquid chromatography (acetonitrile: water) Further purification under (methanol: water) conditions, finally obtaining 6 monomer compounds, the specific separation procedure is referred to Figure 1.
实施例3Example 3
DZJB-1：4,4'-(7-羟基-2-羟甲基-1-(4-羟基苯)乙炔基-9H-芴-9,9-二取代)联苯酚的制备Preparation of DZJB-1: 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted) biphenol
按照实施例2的方法，最后通过凝胶(LH-20氯仿:甲醇＝1:1)纯化，重结晶，得到题述化合物,黄色块状晶体，其结构如下：Purified by the method of Example 2, and finally purified by a gel (LH-20 chloroform:methanol = 1:1), and recrystallized to give the title compound, yellow block crystals.
Figure PCTCN2014088611-appb-000002
Figure PCTCN2014088611-appb-000002
UV(MeOH)λmax(logε)204(4.84)nm,300(4.47)nm。UV (MeOH) λ max (log ε) 204 (4.84) nm, 300 (4.47) nm.
IR(KBr)νmax3312,2207,1607,1510,1449,1357,1241,1174and833cm-1。IR (KBr) ν max 3312, 2207, 1607, 1510, 1449, 1357, 1241, 1174 and 833 cm -1 .
Negative ESIMS m/z511.2[M-H]-,546.9[M+Cl]-,556.7[M+HCOO]-；HRESIMS m/z511.1543[M+H]+(calcd for C34H24O5,511.1551)。Negative ESIMS m/z 511.2 [MH] - , 546.9 [M+Cl] - , 556.7 [M+HCOO] - ; HRESIMS m/z 511.1543 [M+H] + (calcd for C 34 H 24 O 5 , 511.1551).
4,4'-(7-羟基-2-羟甲基-1-(4-羟基苯)乙炔基-9H-芴-9,9-二取代)联苯酚波谱数据(400MHz,Methanol-d4)4,4'-(7-Hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-fluorene-9,9-disubstituted) biphenol spectrum data (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000003
Figure PCTCN2014088611-appb-000003
Figure PCTCN2014088611-appb-000004
Figure PCTCN2014088611-appb-000004
实施例4Example 4
DZJB-2：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-醛的制备Preparation of DZJB-2: 7-Hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde
按照实施例2的方法，最后通过凝胶(LH-20)洗脱剂为无水乙醇，纯化得到题述化合物。其结构如下：The title compound was obtained by the procedure of Example 2, which was purified by the gel (LH-20) eluent as anhydrous ethanol. Its structure is as follows:
Figure PCTCN2014088611-appb-000005
Figure PCTCN2014088611-appb-000005
UV(MeOH)λmax(logε)208(4.70)nm,294(4.24)nm,305(4.26)nm,314(4.28)nm,360(4.21)nm。UV (MeOH) λ max (log ε) 208 (4.70) nm, 294 (4.24) nm, 305 (4.26) nm, 314 (4.28) nm, 360 (4.21) nm.
IR(KBr)νmax3284,2202,1679,1604,1557and1507cm-1。IR (KBr) ν max 3284, 2202, 1679, 1604, 1557 and 1507 cm -1 .
positive ESIMS m/z511.1[M+H]+,negative ESIMS m/z509.1[M-H]-；HRESIMS m/z509.1386[M-H]-(calcd for C34H22O5,509.1394)。positive ESIMS m / z511.1 [M + H] +, negative ESIMS m / z509.1 [MH] -; HRESIMS m / z509.1386 [MH] - (calcd for C 34 H 22 O 5, 509.1394).
7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-醛波谱数据(400MHz,Methanol-d4)7-Hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde spectrum data (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000006
Figure PCTCN2014088611-appb-000006
Figure PCTCN2014088611-appb-000007
Figure PCTCN2014088611-appb-000007
实施例5Example 5
DZJB-3：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)联苯酚的制备 Preparation of DZJB-3: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted)biphenol
按照实施例2的方法，最后通过半制备高效液相，洗脱剂乙腈：水6:4，保留时间为10.8min，纯化得到题述化合物。其结构如下：Following the procedure of Example 2, the title compound was purified by semi-preparation of a high-purity liquid, eluent acetonitrile:water 6:4, retention time 10.8 min. Its structure is as follows:
Figure PCTCN2014088611-appb-000008
Figure PCTCN2014088611-appb-000008
UV(MeOH)λmax(logε)208(4.76)nm,288(4.36)nm,299(4.41)nm,315(4.18)nm。UV (MeOH) λ max (log ε) 208 (4.76) nm, 288 (4.36) nm, 299 (4.41) nm, 315 (4.18) nm.
IR(KBr)νmax3375,2193,1604,1508and1447cm-1。IR (KBr) ν max 3375, 2193, 1604, 1508 and 1447 cm -1 .
positive ESIMS m/z497.2[M+H]+,negative ESIMS m/z495.2[M-H]-；HRESIMS m/z495.1594[M-H]-(calcd for C34H24O4,495.1602)。positive ESIMS m / z497.2 [M + H] +, negative ESIMS m / z495.2 [MH] -; HRESIMS m / z495.1594 [MH] - (calcd for C 34 H 24 O 4, 495.1602).
4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)联苯酚波谱数据(400MHz,Methanol-d4)4,4'-(7-Hydroxy-1-((4-hydroxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted) biphenol spectrum data (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000009
Figure PCTCN2014088611-appb-000009
Figure PCTCN2014088611-appb-000010
Figure PCTCN2014088611-appb-000010
实施例6Example 6
DZJB-4：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-9H-芴-9,9-二取代)联苯酚的制备Preparation of DZJB-4: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-indole-9,9-disubstituted)biphenol
按照实施例5的方法，保留时间为9.2min，制备得到提述化合物。结构如下：The reference compound was prepared according to the method of Example 5 with a retention time of 9.2 min. The structure is as follows:
Figure PCTCN2014088611-appb-000011
Figure PCTCN2014088611-appb-000011
UV(MeOH)λmax(logε)205(4.89)nm,288(4.52)nm,298(4.53)nm,316(4.35)nm。UV (MeOH) λ max (log ε) 205 (4.89) nm, 288 (4.52) nm, 298 (4.53) nm, 316 (4.35) nm.
IR(KBr)νmax3367,2203,1563,1509,1468cm-1。IR (KBr) ν max 3367, 2203, 1563, 1509, 1468 cm -1 .
Positive ESIMS m/z483.2[M+H]+,negative ESIMS m/z481.2[M-H]-； HRESIMS m/z505.1417[M+Na]+(calcd for C33H22O4,505.1410).Positive ESIMS m/z 483.2 [M+H] + , negative ESIMS m/z 481.2 [MH] - ; HRESIMS m/z 505.1417 [M+Na] + (calcd for C 33 H 22 O 4 , 505.1410) .
4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-9H-芴-9,9-二取代)联苯酚波谱数据(400MHz,Methanol-d4)4,4'-(7-Hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) biphenol spectrum data (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000012
Figure PCTCN2014088611-appb-000012
实施例7Example 7
DZJB-5：8-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醇的制备Preparation of DZJB-5: 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indol-2-ol
按照实施例2的方法，最后通过半制备高效液相，洗脱剂乙腈：水8:2，保留时间为8.0min，纯化得到题述化合物。其结构如下：Following the procedure of Example 2, the title compound was purified by semi-preparation of a high-purity liquid, eluent acetonitrile:water 8:2, retention time 8.0 min. Its structure is as follows:
Figure PCTCN2014088611-appb-000013
Figure PCTCN2014088611-appb-000013
UV(MeOH)λmax(logε)209(4.76)nm,291(4.47)nm,298(4.49)nm,314(4.30)nm。UV (MeOH) λ max (log ε) 209 (4.76) nm, 291 (4.47) nm, 298 (4.49) nm, 314 (4.30) nm.
IR(KBr)νmax3691,2207,1601,1503and1455cm-1。IR (KBr) ν max 3691, 2207, 1601, 1503 and 1455 cm -1 .
positive ESIMS m/z511.2[M+H]+,negative ESIMS m/z509.2[M-H]-；HRESIMS m/z509.1729[M-H]-(calcd for C35H26O4,509.1758)。positive ESIMS m / z511.2 [M + H] +, negative ESIMS m / z509.2 [MH] -; HRESIMS m / z509.1729 [MH] - (calcd for C 35 H 26 O 4, 509.1758).
8-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醇波谱数据(400MHz,Methanol-d4)Spectra of 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indol-2-ol (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000014
Figure PCTCN2014088611-appb-000014
Figure PCTCN2014088611-appb-000015
Figure PCTCN2014088611-appb-000015
实施例8Example 8
DZJB-6：7-羟基-1-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醛的制备Preparation of DZJB-6: 7-Hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde
按照实施例2的方法，最后通过半制备高效液相，洗脱剂乙腈：水7:3，保留时间为8.5min，纯化得到题述化合物。其结构如下：Following the procedure of Example 2, the title compound was purified by semi-preparation of a high-purity liquid, eluent acetonitrile:water 7:3, retention time 8.5 min. Its structure is as follows:
Figure PCTCN2014088611-appb-000016
Figure PCTCN2014088611-appb-000016
UV(MeOH)λmax(logε)208(4.77)nm,302(4.30)nm,315(4.33)nm,358(4.29)nm。UV (MeOH) λ max (log ε) 208 (4.77) nm, 302 (4.30) nm, 315 (4.33) nm, 358 (4.29) nm.
IR(KBr)νmax3436,2204,1669,1607,1555and1509cm-1。IR (KBr) ν max 3436, 2204, 1669, 1607, 1555 and 1509 cm -1 .
Positive ESIMS m/z539.0[M+H]+,negative ESIMS m/z537.3[M-H]-； HRESIMS m/z537.1720[M-H]-(calcd for C36H26O5,537.1707)。Positive ESIMS m / z539.0 [M + H] +, negative ESIMS m / z537.3 [MH] -; HRESIMS m / z537.1720 [MH] - (calcd for C 36 H 26 O 5, 537.1707).
7-羟基-1-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醛波谱数据(400MHz,Methanol-d4)7-Hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde spectrum data (400MHz, Methanol-d4)
Figure PCTCN2014088611-appb-000017
Figure PCTCN2014088611-appb-000017
实施例9 Example 9
DZJB-7：(7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-基)甲醇的制备DZJB-7: (7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indol-2-yl)methanol preparation
按照实施例3的方法，得到4,4'-(7-羟基-2-羟甲基-1-(4-羟基苯)乙炔基-9H-芴-9,9-二取代)联苯酚，用丙酮溶解，加入适量无水K2CO3，搅拌1min后加入CH3I,室温搅拌10h，得到题述化合物，其结构和核磁数据如下：According to the method of Example 3, 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted) biphenol was obtained. The acetone was dissolved, and an appropriate amount of anhydrous K 2 CO 3 was added. After stirring for 1 min, CH 3 I was added and stirred at room temperature for 10 h to obtain the title compound. The structure and NMR data are as follows:
Figure PCTCN2014088611-appb-000018
Figure PCTCN2014088611-appb-000018
1H NMR(400MHz,Methanol-d4)δ7.73(d,J＝7.8Hz,1H),7.69(d,J＝8.4Hz,1H),7.52(d,J＝7.7Hz,1H),7.19(d,J＝8.7Hz,4H),6.95(d,J＝8.7Hz,2H),6.91(dd,J＝8.4,2.3Hz,1H),6.84(d,J＝8.7Hz,2H),6.78(d,J＝2.3Hz,1H),6.68(d,J＝8.7Hz,4H),3.79(s,3H),3.72(s,6H),3.70(s,3H). 1 H NMR (400MHz, Methanol- d 4) δ7.73 (d, J = 7.8Hz, 1H), 7.69 (d, J = 8.4Hz, 1H), 7.52 (d, J = 7.7Hz, 1H), 7.19 (d, J = 8.7 Hz, 4H), 6.95 (d, J = 8.7 Hz, 2H), 6.91 (dd, J = 8.4, 2.3 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 2.3 Hz, 1H), 6.68 (d, J = 8.7 Hz, 4H), 3.79 (s, 3H), 3.72 (s, 6H), 3.70 (s, 3H).
实施例10Example 10
DZJB-8：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-醛的制备Preparation of DZJB-8: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde
按照实施例4的方法，得到7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-醛，用丙酮溶解，加入适量无水K2CO3，搅拌1min后加入CH3I，室温搅拌10h，得到题述化合物，其结构和核磁数据如下：According to the method of Example 4, 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde was obtained, dissolved in acetone, and added. Appropriate amount of anhydrous K 2 CO 3 , stirring for 1 min, adding CH 3 I, stirring at room temperature for 10 h, the title compound was obtained, the structure and nuclear magnetic data are as follows:
Figure PCTCN2014088611-appb-000019
Figure PCTCN2014088611-appb-000019
1H NMR(400MHz,Methanol-d4)δ7.90(d,J＝8.0Hz,1H),7.75(d,J＝8.0Hz,1H),7.69(d,J＝8.4Hz,1H),7.19(d,J＝8.7Hz,4H),6.95(d,J＝8.7Hz,2H),6.91 (dd,J＝8.4,2.3Hz,1H),6.84(d,J＝8.7Hz,2H),6.78(d,J＝2.3Hz,1H),6.68(d,J＝8.7Hz,4H),3.80(s,3H),3.70(s,6H),3.69(s,3H). 1 H NMR (400 MHz, Methanol-d 4 ) δ 7.90 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.19 ( d, J = 8.7 Hz, 4H), 6.95 (d, J = 8.7 Hz, 2H), 6.91 (dd, J = 8.4, 2.3 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 6.78 ( d, J = 2.3 Hz, 1H), 6.68 (d, J = 8.7 Hz, 4H), 3.80 (s, 3H), 3.70 (s, 6H), 3.69 (s, 3H).
实施例11Example 11
DZJB-9：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-2-甲基-9H-芴,以及DZJB-10：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴的制备DZJB-9: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-2-methyl-9H-indole, and DZJB -10: Preparation of 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole
按照实施例5、实施例6以及实施例10的方法可制备题述化合物。The title compound was prepared according to the methods of Example 5, Example 6 and Example 10.
实施例12Example 12
DZJB-12：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-羧酸的制备Preparation of DZJB-12: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-carboxylic acid
按照实施例10的方法，得到7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-醛，加入二甲基亚砜溶解，在冰浴中加入1.2eq碱性高锰酸钾，室温下搅拌2～3h，反应得到题述化合物，其结构和核磁数据如下：According to the method of Example 10, 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde was obtained. Add dimethyl sulfoxide to dissolve, add 1.2 eq of basic potassium permanganate in an ice bath, stir at room temperature for 2 to 3 h, and obtain the title compound. The structure and nuclear magnetic data are as follows:
Figure PCTCN2014088611-appb-000020
Figure PCTCN2014088611-appb-000020
1H NMR(400MHz,Methanol-d4)δ8.21(d,J＝8.4Hz,1H),8.01,(d,J＝8.4Hz,1H),7.76(d,J＝8.2Hz,1H),7.48(d,J＝8.7Hz,2H),7.12(d,J＝8.6Hz,4H),7.06(s,1H),6.95(d,J＝8.7Hz,2H),6.89(d,J＝8.2Hz,1H),6.87(d,J＝8.6Hz,4H),3.85(s,3H),3.83(s,6H),3.82(s,3H). 1 H NMR (400MHz, Methanol- d 4) δ8.21 (d, J = 8.4Hz, 1H), 8.01, (d, J = 8.4Hz, 1H), 7.76 (d, J = 8.2Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.6 Hz, 4H), 7.06 (s, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.2) Hz, 1H), 6.87 (d, J = 8.6 Hz, 4H), 3.85 (s, 3H), 3.83 (s, 6H), 3.82 (s, 3H).
实施例13Example 13
DZJB-11：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-羧酸的制备Preparation of DZJB-11: 7-Hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-carboxylic acid
按照实施例12的方法，得到7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯) 乙炔基)-9H-芴-2-羧酸，加入二氯甲烷溶解，在-40℃环境下中加入三溴化硼，搅拌20～30h，反应得到题述化合物，其结构和核磁数据如下：According to the method of Example 12, 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxybenzene) was obtained. Acetylene)-9H-indole-2-carboxylic acid, dissolved in dichloromethane, added boron tribromide at -40 ° C, stirred for 20 ~ 30h, the reaction to obtain the title compound, its structure and nuclear magnetic data are as follows:
Figure PCTCN2014088611-appb-000021
Figure PCTCN2014088611-appb-000021
1H NMR(400MHz,Methanol-d4)δ8.21(d,J＝8.0Hz,1H),8.01(d,J＝8.0Hz,1H),7.67(d,J＝8.3Hz,1H),7.15(d,J＝8.7Hz,4H),6.89(d,J＝8.6Hz,2H),6.82(dd,J＝8.3,2.1Hz,1H),6.77(d,J＝2.1Hz,1H),6.72(d,J＝8.6Hz,2H),6.64(d,J＝8.7Hz,4H). 1 H NMR (400MHz, Methanol- d 4) δ8.21 (d, J = 8.0Hz, 1H), 8.01 (d, J = 8.0Hz, 1H), 7.67 (d, J = 8.3Hz, 1H), 7.15 (d, J = 8.7 Hz, 4H), 6.89 (d, J = 8.6 Hz, 2H), 6.82 (dd, J = 8.3, 2.1 Hz, 1H), 6.77 (d, J = 2.1 Hz, 1H), 6.72 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.7 Hz, 4H).
实施例14Example 14
DZJB-15：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯的制备DZJB-15: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) bis(4,1-phenylene)diacetic acid Preparation of ester
按照实施例6的方法，得到4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-9H-芴-9,9-二取代)联苯酚，加入吡啶溶解，加入1.1eq乙酸酐，室温下搅拌反应10～15h，得到题述化合物，其结构和核磁数据如下：According to the method of Example 6, 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) biphenol was obtained, dissolved in pyridine, and added. 1.1 eq acetic anhydride, stirring reaction at room temperature for 10-15 h, the title compound was obtained, the structure and nuclear magnetic data are as follows:
Figure PCTCN2014088611-appb-000022
Figure PCTCN2014088611-appb-000022
1H NMR(400MHz,Methanol-d4)δ7.84(d,J＝8.5Hz,1H),7.80(d,J＝8.0Hz,1H),7.56(d,J＝8.6Hz,2H)),7.48(d,J＝8.0Hz,1H),7.36(s,1H),7.34(t,J＝8.0Hz,1H),7.25(d,J＝8.6Hz,2H),7.20(d,J＝8.7Hz,4H),7.19(d,J＝8.5Hz,1H), 7.17(d,J＝8.7Hz,4H),2.30(s,3H),2.28(s,6H),2.27(s,3H). 1 H NMR (400MHz, Methanol- d 4) δ7.84 (d, J = 8.5Hz, 1H), 7.80 (d, J = 8.0Hz, 1H), 7.56 (d, J = 8.6Hz, 2H)), 7.48 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.7) Hz, 4H), 7.19 (d, J = 8.5 Hz, 1H), 7.17 (d, J = 8.7 Hz, 4H), 2.30 (s, 3H), 2.28 (s, 6H), 2.27 (s, 3H).
实施例15Example 15
DZJB-13：(7-乙酰氧基-2-(乙酰氧基甲基)-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-14：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲酰基-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯以及DZJB-16：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯的制备DZJB-13: (7-acetoxy-2-(acetoxymethyl)-1-((4-acetoxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) bis (4) , 1-phenylene) diacetate, DZJB-14: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-formyl-9H-indole-9,9 -disubstituted) bis(4,1-phenylene) diacetate and DZJB-16: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-methyl- Preparation of 9H-fluorene-9,9-disubstituted) bis(4,1-phenylene) diacetate
按照实施例3、实施例4、实施例5以及实施例14的方法可制备题述化合物。The title compound was prepared according to the methods of Example 3, Example 4, Example 5 and Example 14.
实施例16Example 16
DZJB-17：4-((7-乙酰氧基-9,9-二(4-甲氧基苯)-9H-芴-1-基)乙炔基)乙酸苯酯的制备Preparation of DZJB-17: 4-((7-Acetoxy-9,9-bis(4-methoxyphenyl)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester
按照实施例7的方法，得到8-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醇，加入吡啶溶解，加入1.1eq乙酸酐，室温下搅拌反应10～15h，得到题述化合物，其结构和核磁数据如下：According to the method of Example 7, 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxyphenyl)-9H-indol-2-ol was obtained, dissolved in pyridine, and 1.1 eq was added. Acetic anhydride is stirred at room temperature for 10-15 hours to obtain the title compound. The structure and nuclear magnetic data are as follows:
Figure PCTCN2014088611-appb-000023
Figure PCTCN2014088611-appb-000023
1H NMR(400MHz,Methanol-d4)δ7.84(d,J＝8.5Hz,1H),7.80(d,J＝8.0Hz,1H),7.56(d,J＝8.6Hz,2H)),7.48(d,J＝8.0Hz,1H),7.36(s,1H),7.34(t,J＝8.0Hz,1H),7.25(d,J＝8.6Hz,2H),7.19(d,J＝8.5Hz,1H),7.12(d,J＝8.7Hz,4H),6.87(d,J＝8.7Hz,4H),3.83(s,6H),2.28(s,6H). 1 H NMR (400MHz, Methanol- d 4) δ7.84 (d, J = 8.5Hz, 1H), 7.80 (d, J = 8.0Hz, 1H), 7.56 (d, J = 8.6Hz, 2H)), 7.48 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 7.19 (d, J = 8.5) Hz, 1H), 7.12 (d, J = 8.7 Hz, 4H), 6.87 (d, J = 8.7 Hz, 4H), 3.83 (s, 6H), 2.28 (s, 6H).
实施例17Example 17
垫状卷柏提取物对PDE4酶的抑制作用及对PDE酶家族的选择性Inhibition of PDE4 enzyme by P. serrata extract and its selectivity to PDE family
待测分子与含有重组PDE4D2蛋白(该重组蛋白是我们制备得到，详细制备方法可参考我们已发表且含有制备该重组蛋白的文献：Bioorganic & Medicinal Chemistry Letters，2012年，22卷，页码：3261–3264)，20mMTris-HCl,pH7.5,2mM二硫苏糖醇(dithiothreitol),10mMMgCl2以及20,000–30,000cpm的3H-cAMP在室温下孵育15分钟，然后分别用0.2M ZnSO4and Ba(OH)2中止反应，然后利用PerkinElmer2910计数仪测量上清液中未反应的3H-cGMP，每个分子至少测量三次，对PDE4D2蛋白活性抑制的IC50值通过浓度测试及非线性回归，计算获得。The molecule to be tested contains the recombinant PDE4D2 protein (this recombinant protein is prepared by us. For detailed preparation methods, please refer to our published literature and the preparation of the recombinant protein: Bioorganic & Medicinal Chemistry Letters, 2012, Vol. 22, page 3261 – 3264), 20 mM Tris-HCl, pH 7.5, 2 mM dithiothreitol, 10 mM MgCl 2 and 20,000-30,000 cpm of 3 H-cAMP were incubated for 15 minutes at room temperature, then 0.2 M ZnSO 4 and Ba, respectively. OH) 2 was stopped, and then the unreacted 3 H-cGMP in the supernatant was measured using a PerkinElmer 2910 counter, and each molecule was measured at least three times. The IC 50 value for inhibition of PDE4D2 protein activity was calculated by concentration test and nonlinear regression. .
同时也测试了垫状卷柏提取物对PDE4酶、PDE5酶、PDE9酶的选择性.The selectivity of PDE4, PDE5 and PDE9 enzymes was also tested.
本发明化合物对PDE4、PDE5、PDE9的抑制活性测试数据如表1(IC50值为抑制率达到50％时的抑制剂浓度)。The test data for the inhibitory activity of the compounds of the present invention against PDE4, PDE5, and PDE9 are shown in Table 1 (inhibitor concentration when the IC 50 value reached 50% inhibition rate).
Figure PCTCN2014088611-appb-000024
Figure PCTCN2014088611-appb-000024
Figure PCTCN2014088611-appb-000025
Figure PCTCN2014088611-appb-000025

Claims (6)

  1. 一种垫状卷柏提取物，其特征在于，所述的垫状卷柏提取物如结构式Ⅰ所示：A padded cypress extract, characterized in that the scutellaria extract is of the formula I:
    Figure PCTCN2014088611-appb-100001
    Figure PCTCN2014088611-appb-100001
    其中：among them:
    R1，R4为H，烷基或酰基；R 1 , R 4 is H, alkyl or acyl;
    R2，R3，R5，R7为卤素，醛基，羧基，磺酸基，硝基，亚硝基或氢；R 2 , R 3 , R 5 , R 7 are halogen, aldehyde, carboxyl, sulfonic acid, nitro, nitroso or hydrogen;
    R6为H，-CH3，-CH2OH，-CHO或-COOH。R 6 is H, -CH 3 , -CH 2 OH, -CHO or -COOH.
  2. 根据权利要求1所述的垫状卷柏提取物，其特征在于，所述的R1和R4为氢、甲基、乙基或乙酰基。The Ascacia platensis extract according to claim 1, wherein said R 1 and R 4 are hydrogen, methyl, ethyl or acetyl.
  3. 根据权利要求1所述的垫状卷柏提取物，其特征在于，所述的R2，R3，R5，R7为氢。The Pleurotus ostreatus extract according to claim 1, wherein said R 2 , R 3 , R 5 and R 7 are hydrogen.
  4. 根据权利要求1所述的垫状卷柏提取物，其特征在于，所述的化合物为The Pleurotus ostreatus extract according to claim 1, wherein the compound is
    DZJB-1：4,4'-(7-羟基-2-羟甲基-1-(4-羟基苯)乙炔基-9H-芴-9,9-二取代)联苯酚、DZJB-1: 4,4'-(7-hydroxy-2-hydroxymethyl-1-(4-hydroxyphenyl)ethynyl-9H-indole-9,9-disubstituted)biphenol,
    DZJB-2：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-醛、DZJB-2: 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-aldehyde,
    DZJB-3：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)联苯酚、DZJB-3: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted)biphenol,
    DZJB-4：4,4'-(7-羟基-1-((4-羟基苯)乙炔基)-9H-芴-9,9-二取代)联苯酚、DZJB-4: 4,4'-(7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9H-indole-9,9-disubstituted)biphenol,
    DZJB-5：8-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醇、DZJB-5: 8-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxyphenyl)-9H-indol-2-ol,
    DZJB-6：7-羟基-1-((4-羟基苯)乙炔基)-9,9-二(4-甲氧基苯)-9H-芴-2-醛、DZJB-6: 7-hydroxy-1-((4-hydroxyphenyl)ethynyl)-9,9-bis(4-methoxybenzene)-9H-indole-2-aldehyde,
    DZJB-7：(7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-基)甲醇、DZJB-7: (7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indol-2-yl)methanol,
    DJB-8：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-醛、DJB-8: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-aldehyde,
    DZJB-9：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-2-甲基-9H-芴、DZJB-9: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-2-methyl-9H-indole,
    DZJB-10：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴、DZJB-10: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole,
    DZJB-11：7-羟基-9,9-二(4-羟基苯)-1-((4-羟基苯)乙炔基)-9H-芴-2-羧酸、DZJB-11: 7-hydroxy-9,9-bis(4-hydroxyphenyl)-1-((4-hydroxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
    DZJB-12：7-甲氧基-9,9-二(4-甲氧基苯)-1-((4-甲氧基苯)乙炔基)-9H-芴-2-羧酸、DZJB-12: 7-methoxy-9,9-bis(4-methoxyphenyl)-1-((4-methoxyphenyl)ethynyl)-9H-indole-2-carboxylic acid,
    DZJB-13：(7-乙酰氧基-2-(乙酰氧基甲基)-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-13: (7-acetoxy-2-(acetoxymethyl)-1-((4-acetoxyphenyl)ethynyl)-9H-indole-9,9-disubstituted) bis (4) , 1-phenylene) diacetate,
    DZJB-14：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲酰基-9H-芴-9,9-二取代)二(4,1-亚苯基) 二乙酸酯、DZJB-14: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-formyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Base) Diacetate,
    DZJB-15：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-15: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-9H-fluorene-9,9-disubstituted) bis(4,1-phenylene)diacetic acid ester,
    DZJB-16：(7-乙酰氧基-1-((4-乙酰氧基苯)乙炔基)-2-甲基-9H-芴-9,9-二取代)二(4,1-亚苯基)二乙酸酯、DZJB-16: (7-acetoxy-1-((4-acetoxyphenyl)ethynyl)-2-methyl-9H-indole-9,9-disubstituted) bis(4,1-phenylene) Diacetate,
    DZJB-17：4-((7-乙酰氧基-9,9-二(4-甲氧基苯)-9H-芴-1-基)乙炔基)乙酸苯酯、DZJB-17: 4-((7-acetoxy-9,9-bis(4-methoxyphenyl)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester,
    或DZJB-18：4-((7-乙酰氧基-2-甲酰基-9,9-二(4-甲氧基苯)-9H-芴-1-基)乙炔基)乙酸苯酯。Or DZJB-18: 4-((7-acetoxy-2-formyl-9,9-bis(4-methoxybenzene)-9H-indol-1-yl)ethynyl)acetic acid phenyl ester.
  5. 一种制备权利要求1所述的垫状卷柏提取物的方法，其特征在于，A method of preparing the P. sylvestris extract according to claim 1, wherein
    S1.将垫状卷柏全草粉粹，用95％乙醇超声提取3次，回收溶剂，得提取浸膏；S1. The pad-shaped cypress whole grass is powdered, ultrasonically extracted with 95% ethanol for 3 times, and the solvent is recovered to obtain an extract;
    S2.将步骤S1得到的提取浸膏依次用石油醚和乙酸乙酯萃取，分别得到两个部分，随后乙酸乙酯萃取物经MCI柱层析，分别用30％、50％、70％、90％、100％甲醇-水梯度洗脱，得各种阶段的洗脱液；S2. The extracted extract obtained in the step S1 is sequentially extracted with petroleum ether and ethyl acetate to obtain two fractions respectively, and then the ethyl acetate extract is subjected to MCI column chromatography, respectively, using 30%, 50%, 70%, 90 respectively. %, 100% methanol-water gradient elution to obtain various stages of eluent;
    S3.分别将取步骤S2所得洗脱液，通过硅胶柱层析、凝胶柱层析、ODS柱层析以及高效液相色谱等分离手段，即得。S3. The eluate obtained in the step S2 is obtained by silica gel column chromatography, gel column chromatography, ODS column chromatography and high performance liquid chromatography, respectively.
  6. 一种根据权利要求1所述的垫状卷柏提取物在制备PDE4抑制剂中的应用。 Use of a P. sylvestris extract according to claim 1 for the preparation of a PDE4 inhibitor.
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