CN113620967A - 一种α-倒捻子素衍生物及其制备方法和应用 - Google Patents

一种α-倒捻子素衍生物及其制备方法和应用 Download PDF

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CN113620967A
CN113620967A CN202110876312.9A CN202110876312A CN113620967A CN 113620967 A CN113620967 A CN 113620967A CN 202110876312 A CN202110876312 A CN 202110876312A CN 113620967 A CN113620967 A CN 113620967A
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何细新
黄仪有
邓金辉
梁津豪
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Abstract

本发明属于药物化学技术领域，具体涉及一种α‑倒捻子素衍生物及其制备方法和应用。本发明提供的α‑倒捻子素衍生物对PDE4具有良好的选择性抑制作用，效果明显强于阳性对照药物，同时还有较好的药代动力学和类药性，口服无呕吐等不良反应，安全性高，可应用于制备治疗PDE4相关疾病的药物中；并且在抗肺纤维化实验中，α‑倒捻子素衍生物显示出显著的改善肺功能参数，减少肺纤维化病变的效果，具有较好的开发潜力。

Description

一种α-倒捻子素衍生物及其制备方法和应用
技术领域
本发明属于药物化学技术领域。更具体地，涉及一种α-倒捻子素衍生物及其制备方法和应用。
背景技术
特发性肺纤维化(IPF)是一种慢性进行性疾病，会造成肺功能下降，导致呼吸衰竭并最终导致死亡。来自新冠肺炎大流行的新数据表明，在SARS-CoV-2感染后出现大量肺纤维化症状，SARS、MERS等冠状病毒感染也会出现类似的症状。尽管IPF的致病机制已被广泛研究，但仍缺乏对IPF的有效治疗方法；IPF病患者从诊断到治疗的中位生存期仅为2～4年，肺移植是唯一可以提高预期寿命的手术治疗方法，不仅费用高昂、配型难，还具有较高的风险。而药物方面，目前只有两种抗纤维化药物——吡非尼酮(Pirfenidone)和尼达尼布(Nintedanib)，且这两种药物均存在不同程度的不良反应发生。因此，迫切需要研发更多用于治疗IPF病的新型治疗药物。
研究发现，第二信使环磷酸腺苷(cAMP)在IPF的发展过程中可以抑制成纤维细胞增殖或分化为肌成纤维细胞。磷酸二酯酶4(PDE4)，肺成纤维细胞中主要的cAMP降解酶，在纤维化的进展中被上调，采用选择性PDE4抑制剂罗氟司特对IPF模型进行实验，可证明PDE4抑制剂具有体内和体外抗纤维化作用；另一方面，PDE4广泛参与炎症过程，该过程也在IPF的发病机制中起作用；这些证据均表明PDE4是一种潜在IPF治疗靶标。如中国专利申请CN103748073A公开了一种新的软PDE4抑制剂，但是其在实际的临床应用中存在剂量依赖性副作用，如呕吐和恶心等。因此，迫切需要开发副作用小、安全性高的新型PDE4抑制剂，用于治疗如IPF病等与PDE4相关的疾病。
发明内容
本发明要解决的技术问题是克服现有治疗IPF病药物非常少，PDE4抑制剂存在依赖性副作用的缺陷和不足，提供一种具有显著抑制PDE4效果的α-倒捻子素衍生物。发明人发现，特定结构的α-倒捻子素衍生物具有良好的PDE4抑制活性，可以用于PDE4代谢相关的疾病治疗中，特别是抗肺纤维化疾病的治疗中。
因此，本发明的目的是提供一种α-倒捻子素衍生物。
本发明另一目的是提供所述α-倒捻子素衍生物的制备方法。
本发明另一目的是提供所述α-倒捻子素衍生物在制备PDE4抑制剂中的应用，及一种含有所述α-倒捻子素衍生物的PDE4抑制剂。
本发明另一目的是提供所述α-倒捻子素衍生物在制备防治抗纤维化药物中的应用，及一种含有所述α-倒捻子素衍生物的防治抗纤维化药物。
本发明上述目的通过以下技术方案实现：
一种α-倒捻子素衍生物，其特征在于，具有式(I)结构：
Figure BDA0003190442680000021
其中，R1为氢、烷基、环烷基、烷酰基、苄基或取代苄基；
R2为氢、烷基、取代烷基、烯烃基、取代烯烃基、苄基、取代苄基；
所述取代烷基、取代烯烃基、取代苄基的取代基团为卤素、烷氧基、甲基磺酰基、酯基或羧基。
优选地，所述烷基包括甲基、乙基、丙基、丁基、异丙基；所述环烷基包括环丙基、环丁基、环戊基、环己基；所述羧基为甲酸、乙酸、丙酸、丁酸、戊酸、己酸、反式巴豆酸、顺式巴豆酸等。
进一步地，所述R1为氢、C1～5烷基、C1～5烷酰基、苄基或取代苄基；
所述R2为氢、C1～5烷基、取代C1～5烷基、C1～5烯烃基或取代C1～5烯烃基。
优选地，所述R1为氢、甲基、乙基、丙基、丁基、异丙基、乙酰基、叔丁氧羰基、苄基、3-氟-苄基、4-氟-苄基、3-甲氧基-苄基、3-甲基-苄基、4-甲基磺酰基-苄基；
所述R2为氢、-(CH2)n-COO-CH2CH3、-(CH2)m-COOH、-CH2CH＝CH-COO-CH2CH3、-CH2CH＝CH-COOH；
其中，n＝1～4，m＝1～4。
更优选地，所述R1为H、甲基、乙基、异丙基、苄基、3-氟-苄基、3-甲氧基-苄基、3-甲基-苄基、4-甲基磺酰基-苄基；
所述R2为-(CH2)3-COOH、-(CH2)4-COOH或-CH2CH＝CH-COOH。
另外的，本发明还提供了所述α-倒捻子素衍生物的制备方法，包括以下步骤：
Figure BDA0003190442680000031
以α-倒捻子素衍生物化合物1作为原料，在溶剂中与醋酐或二碳酸二叔丁酯反应，再在20～60℃、催化剂存在条件下与溴代烷基羧酸酯反应，脱去保护基和长链羧酸酯中酯基，即得；
或，以α-倒捻子素衍生物化合物1作为原料，在溶剂中与溴代R1反应，再在20～60℃、催化剂存在条件下与溴代烷基羧酸酯反应，脱去长链羧酸酯中酯基，即得。
进一步地，所述溶剂为丙酮或二甲酰胺。
更进一步地，所述催化剂为碳酸钾、碳酸铯或氢化钠。
另外的，本发明还提供了所述α-倒捻子素衍生物在制备PDE4抑制剂中的应用。
进一步地，本发明还提供了一种PDE4抑制剂，包含有效量的所述α-倒捻子素衍生物。
另外的，本发明还提供了所述α-倒捻子素衍生物在制备防治与PDE4相关疾病药物中的应用。
更进一步地，所述与PDE4相关疾病包括组织纤维化、银屑病、老年性痴呆症、慢性阻塞性肺疾病、炎症性肠病和哮喘。现有技术研究发现，PDE4与多种疾病密切相关，PDE4抑制剂可以作为相关疾病的靶点。
优选地，所述与PDE4相关疾病为组织纤维化。
另外的，本发明还提供了一种防治抗纤维化药物，包含有效量的所述α-倒捻子素衍生物。
本发明具有以下有益效果：
本发明通过实验证明，本发明提供的α-倒捻子素衍生物对PDE4具有良好的选择性抑制作用，效果明显强于阳性对照药物，同时还有较好的药代动力学和类药性，口服无呕吐等不良反应，安全性高，可应用于制备治疗PDE4相关疾病的药物中；并且在抗肺纤维化实验中，α-倒捻子素衍生物显示出显著的改善肺功能参数、减少肺纤维化病变的效果，具有较好的开发潜力。
附图说明
图1为本发明实施例8中化合物18a在口服1.5g/kg剂量下的急性毒性实验结果统计图。
图2为本发明实施例9中化合物18a抗IPF测定结果统计图；其中，图A-各组大鼠肺呼吸功能指标测定结果；图B-各组肺组织的苏木精-伊红染色切片：未染色区域为肺泡上皮癌，蓝色为细胞核，红色为细胞质；图C-各组肺组织的Masson染色切片：未染色区域为肺泡上皮癌，深蓝区域为胶原质，红色区域为细胞结构；图D-纤维连接蛋白和α-肌动蛋白表达水平相对于3-磷酸甘油醛脱氢酶(GAPDH)抗体的蛋白质印迹分析。
图3为本发明实施例10化合物18a抑制体外上皮间质转化结果统计图。
图中，p值通过配对试验计算，与对照组相比，#p<0.05，##p<0.01，###p<0.001，####p<0.0001，与模型组相比，*p<0.05，**p<0.01，***p<0.001，****p<0.0001。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
本发明合成路线及主要产物如下：
Figure BDA0003190442680000051
实施例1化合物12a和12b的合成
化合物1(0.49～0.73mmol)溶于5mL二氯甲烷，然后室温下加入醋酐(0.54mmol)或二碳酸二叔丁酯(0.81mmol)和催化量的(DMAP)；室温反应3h后，减压下蒸干溶剂得粗产物，再经硅胶柱层析，以石油醚/乙酸乙酯洗脱，得到黄色固体12a，12b。
5-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyra no[3,2-b]xanthen-9-yl acetate(12a).Yield:93％.1H NMR(400MHz,CDCl3)δ13.50(s,1H),7.10(s,1H),6.73(d,J＝10.1Hz,1H),6.25(s,1H),5.58(d,J＝10.0Hz,1H),5.27–5.18(m,1H),4.14(d,J＝6.4Hz,2H),3.77(s,3H),2.39(s,3H),1.83(s,3H),1.69(s,3H),1.47(s,6H).13C NMR(100MHz,CDCl3)δ182.1,168.1,160.4,158.0,156.3,153.8,148.9,146.6,138.9,132.2,127.3,122.8,116.9,115.6,110.6,104.6,104.0,94.2,78.2,61.7,28.4,26.4,25.9,20.9,18.2.ESI-MSm/z:451.2[M+H]+.
Tert-butyl(5-hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-9-yl)carbonate(12b).Yield:74％.1HNMR(400MHz,CDCl3)δ13.51(s,1H),7.16(s,1H),6.73(d,J＝10.1Hz,1H),6.25(s,1H),5.58(d,J＝10.0Hz,1H),5.26–5.17(m,1H),4.15(d,J＝6.5Hz,2H),3.79(s,3H),1.84(s,3H),1.68(s,3H),1.57(s,9H),1.47(s,6H).13CNMR(100MHz,CDCl3)δ182.2,160.3,158.0,156.4,153.8,150.3,149.2,146.7,138.8,132.1,127.3,122.9,116.7,115.6,110.0,104.6,104.0,94.2,84.6,78.1,61.7,28.4,27.6,26.4,25.9,18.2.ESI-MSm/z:509.2[M+H]+.
实施例2化合物14a～14i的合成
化合物1(0.34～0.73mmol)溶于丙酮(4～5mL)，加入K2CO3(0.74～0.98mmol)和烷基碘或溴苄或取代溴苄(0.49～0.88mmol)，室温搅拌3～24h；反应完毕经乙酸乙酯萃取，无水Na2SO4干燥，粗产物经硅胶柱层析(石油醚/乙酸乙酯洗脱)得黄色固体14a～14i。
5-Hydroxy-8,9-dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14a).Yield:77％.1H NMR(400MHz,CDCl3)δ13.75(s,1H),6.74(s,1H),6.73(d,J＝10.0Hz,1H),6.23(s,1H),5.57(d,J＝10.0Hz,1H),5.28-5.20(m,1H),4.12(d,J＝6.6Hz,2H),3.96(s,3H),3.79(s,3H),1.84(s,3H),1.68(s,3H),1.47(s,6H).13C NMR(100MHz,CDCl3)δ182.2,159.8,158.3,158.1,156.4,155.5,144.2,137.4,132.0,127.2,123.3,115.9,112.0,104.6,104.0,98.5,94.1,78.1,61.1,56.2,28.5,26.3,26.1,18.3.ESI-MS m/z:423.3[M+H]+.
9-Ethoxy-5-hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14b).Yield:90％.1H NMR(400MHz,CDCl3)δ13.77(s,1H),6.73(d,J＝10.0Hz,1H),6.71(s,1H),6.22(s,1H),5.56(d,J＝10.0Hz,1H),5.28-5.21(m,1H),4.16(q,J＝7.0Hz,2H),4.13-4.09(m,2H),3.80(s,3H),1.85(s,3H),1.68(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ182.2,159.8,158.1,157.6,156.4,155.5,144.3,137.2,131.9,127.2,123.3,115.9,111.8,104.6,104.0,99.0,94.1,78.0,64.7,60.9,28.4,26.3,26.1,18.3,14.7.ESI-MS m/z:437.1[M+H]+.
5-Hydroxy-9-isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14c).Yield:86％.1H NMR(400MHz,CDCl3)δ13.80(s,1H),6.74(d,J＝10.0Hz,1H),6.72(s,1H),6.23(s,1H),5.56(d,J＝10.0Hz,1H),5.29-5.21(m,1H),4.68(hept,J＝6.1Hz,1H),4.12(d,J＝6.6Hz,2H),3.79(s,3H),1.85(s,3H),1.68(s,3H),1.47(s,6H),1.46(d,J＝6.1Hz,6H).13C NMR(100MHz,CDCl3)δ182.2,159.8,158.2,156.6,156.4,155.5,144.8,137.4,131.9,127.2,123.3,115.9,111.6,104.6,104.0,99.8,94.1,78.0,71.3,60.8,28.5,26.4,26.1,22.0,18.3.ESI-MS m/z:473.1[M+Na]+ .
5-Hydroxy-8-methoxy-9-(benzyloxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14d).Yield:99％.1H NMR(400MHz,CDCl3)δ13.75(s,1H),7.51-7.47(m,1H),7.47-7.45(m,1H),7.45-7.42(m,1H),7.42-7.39(m,1H),7.39-7.34(m,1H),6.80(s,1H),6.73(d,J＝10.5Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.29-5.23(m,1H),5.19(s,2H),4.14(d,J＝6.6Hz,2H),3.83(s,3H),1.85(s,3H),1.69(s,3H),1.47(s,6H).13C NMR(100MHz,CDCl3)δ182.2,159.9,158.1,157.2,156.4,155.3,144.4,137.5,135.8,132.0,128.9,128.5,127.5,127.2,123.3,115.9,112.2,104.6,104.0,99.7,94.1,78.1,70.8,61.1,28.5,26.4,26.1,18.3.ESI-MS m/z:521.1[M+Na]+.
5-Hydroxy-8-methoxy-9-((3-fluorobenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14e).Yield:86％.1H NMR(400MHz,CDCl3)δ13.71(s,1H),7.39(td,J＝7.9,5.7Hz,1H),7.26–7.18(m,2H),7.06(tdd,J＝8.4,2.6,1.0Hz,1H),6.77(s,1H),6.73(d,J＝10.0Hz,1H),6.23(d,J＝0.7Hz,1H),5.57(d,J＝10.0Hz,1H),5.29–5.21(m,1H),5.19(s,2H),4.14(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ182.0,163.0(d,1JC-F＝246.9Hz),159.8,158.0,156.8,156.2,155.2,144.3,138.2(d,3JC-F＝7.4Hz),137.6,132.0,130.4(d,3JC-F＝8.3Hz),127.1,123.1,122.6(d,4JC-F＝2.9Hz),115.7,115.3(d,2JC-F＝21.2Hz),114.1(d,2JC-F＝22.3Hz),112.3,104.5,103.9,99.6,94.0,78.0,69.9(d,4JC-F＝2.1Hz),61.0,28.3,26.2,25.9,18.2.19F NMR(376MHz,CDCl3)δ-112.2.ESI-MS m/z:517.1[M+H]+.
5-Hydroxy-8-methoxy-2,2-dimethyl-9-((3-methylbenzyl)oxy)-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14f).Yield:76％.1HNMR(400MHz,CDCl3)δ13.75(s,1H),7.33–7.24(m,3H),7.21–7.15(m,1H),6.80(s,1H),6.73(d,J＝10.0Hz,1H),6.22(s,1H),5.56(d,J＝10.0Hz,1H),5.28–5.22(m,1H),5.16(s,2H),4.13(d,J＝6.6Hz,2H),3.82(s,3H),2.39(s,3H),1.85(s,3H),1.46(s,6H).13CNMR(100MHz,CDCl3)δ182.1,159.7,158.0,157.2,156.2,155.2,144.3,138.5,137.3,135.6,131.9,129.1,128.7,128.0,127.1,124.4,123.1,115.8,112.0,104.5,103.9,99.6,94.0,77.9,70.8,60.9,28.3,26.2,25.9,21.5,18.2.ESI-MSm/z:513.1[M+H]+.
5-Hydroxy-8-methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14g).Yield:85％.1HNMR(400MHz,CDCl3)δ13.75(s,1H),7.33(t,J＝7.9Hz,1H),7.07–7.01(m,2H),6.90(ddd,J＝8.3,2.7,1.0Hz,1H),6.79(s,1H),6.73(d,J＝10.0Hz,1H),6.23(s,1H),5.57(d,J＝10.1Hz,1H),5.27–5.22(m,1H),5.18(s,2H),4.14(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).13CNMR(100MHz,CDCl3)δ182.1,159.9,159.8,158.0,157.1,156.2,155.2,144.3,137.4,137.3,131.9,129.9,127.1,123.2,119.4,115.8,113.7,112.8,112.1,104.5,103.9,99.6,94.0,77.9,70.5,61.0,55.3,28.3,26.2,25.9,18.2.ESI-MS m/z:529.1[M+H]+.
5-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methylsulfonyl)benzyl)oxy)-2H,6H-pyrano[3,2-b]xanthen-6-one(14h).Yield:88％.1HNMR(400MHz,CDCl3)δ13.67(s,1H),8.01(d,J＝8.3Hz,2H),7.69(d,J＝8.2Hz,2H),6.76(s,1H),6.72(d,J＝10.0Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.28(s,2H),5.27–5.22(m,1H),4.14(d,J＝6.6Hz,2H),3.84(s,3H),3.09(s,3H),1.86(s,3H),1.69(s,3H),1.47(s,6H).13CNMR(100MHz,CDCl3)δ181.9,159.9,157.9,156.5,156.2,155.1,144.2,142.0,140.5,137.9,132.1,128.0,127.8,127.2,123.0,115.7,112.5,104.6,103.9,99.6,94.0,78.0,69.5,61.1,44.5,28.3,26.2,25.9,18.2.ESI-MSm/z:577.1[M+H]+.
5-Hydroxy-8-methoxy-9-((4-fluorobenzyl)oxy)-2,2-dimethyl7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14i).Yield:89％.1HNMR(400MHz,CDCl3)δ13.72(s,1H),7.45(dd,J＝8.6,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.78(s,1H),6.73(d,J＝10.0Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.29–5.20(m,1H),5.14(s,2H),4.13(d,J＝6.6Hz,2H),3.80(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).13CNMR(100MHz,CDCl3)δ182.0,162.7(d,1JC-F＝247.3Hz),159.8,158.0,156.9,156.2,155.2,144.3,137.5,131.9,131.4(d,4JC-F＝3.1Hz),129.3(d,3JC-F＝8.3Hz),127.1,123.1,115.8(d,2JC-F＝21.7Hz),115.7,112.2,104.5,103.9,99.5,94.0,78.0,70.1,60.9,28.3,26.2,25.9,18.2.19F NMR(376MHz,CDCl3)δ-113.4.ESI-MSm/z:517.1[M+H]+.
实施例3化合物13a～13d的合成
将化合物12a或12b(0.18～0.20mmol)溶于(5mL)丙酮，加入Cs2CO3(0.36～0.40mmol)和溴代烷基羧酸乙酯或溴代巴豆酸乙酯(0.18～0.79mmol)，室温搅拌过夜；蒸干溶剂，加入水，乙酸乙酯萃取3次；收集有机相，无水Na2SO4干燥，移除溶剂得粗产物。硅胶柱层析分离，乙酸乙酯/石油醚洗脱，得白色固体13a～13d。
Ethyl2-((9-acetoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)acetate(13a).Yield:62％.1H NMR(400MHz,CDCl3)δ7.07(s,1H),7.04(d,J＝10.1Hz,1H),6.55(s,1H),5.69(d,J＝10.2Hz,1H),5.26–5.21(m,1H),4.69(s,2H),4.26(q,J＝7.2Hz,2H),4.11(d,J＝6.6Hz,2H),3.76(s,3H),2.38(s,3H),1.83(s,3H),1.67(s,3H),1.47(s,6H),1.30(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ176.4,169.5,168.3,158.8,157.5,154.0,152.9,148.0,146.5,138.7,131.9,129.8,123.3,119.3,116.8,112.2,110.2,110.1,100.1,77.9,71.3,61.6,61.1,28.4,26.1,25.8,20.9,18.2,14.2.ESI-MS m/z:537.1[M+H]+.
Ethyl4-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoate(13b).Yield:39％.1HNMR(400MHz,CDCl3)δ7.11(s,1H),6.70(d,J＝10.1Hz,1H),6.51(s,1H),5.67(d,J＝10.1Hz,1H),5.26–5.21(m,1H),4.21–4.09(m,4H),4.01(t,J＝6.2Hz,2H),3.79(s,3H),2.63(t,J＝7.5Hz,2H),2.23–2.15(m,2H),1.84(s,3H),1.66(s,3H),1.56(s,9H),1.46(s,6H),1.27(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.3,173.5,158.5,157.6,155.0,152.9,150.5,148.2,146.6,138.6,131.5,129.9,123.5,119.2,116.4,112.1,111.0,109.5,99.7,84.4,77.7,74.1,61.6,60.4,30.9,28.3,27.6,26.1,25.8,25.6,18.2,14.3.ESI-MSm/z:623.3[M+H]+.
Ethyl5-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)pentanoate(13c).Yield:18％.1HNMR(400MHz,CDCl3)δ7.11(s,1H),6.71(d,J＝10.1Hz,1H),6.51(s,1H),5.66(d,J＝10.1Hz,1H),5.27–5.21(m,1H),4.17–4.11(m,4H),3.99(t,J＝6.1Hz,2H),3.79(s,3H),2.42(t,J＝7.0Hz,2H),1.94–1.85(m,4H),1.83(s,3H),1.65(s,3H),1.56(s,9H),1.46(s,6H),1.26(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.3,173.6,158.5,157.7,155.2,152.9,150.5,148.2,146.5,138.6,131.5,129.8,123.5,119.3,116.5,112.1,111.0,109.5,99.6,84.4,77.7,74.9,61.6,60.3,34.1,29.7,28.3,27.6,26.1,25.8,21.6,18.2,14.3.ESI-MSm/z:637.3[M+H]+.
Ethyl(E)-4-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(13d).Yield:70％.1HNMR(400MHz,CDCl3)δ7.14(dt,J＝15.7,4.8Hz,1H),7.12(s,1H),6.68(d,J＝10.1Hz,1H),6.55(s,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.68(d,J＝10.1Hz,1H),5.25–5.17(m,1H),4.71(dd,J＝4.8,2.0Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.6Hz,2H),3.79(s,3H),1.82(s,3H),1.66(s,3H),1.57(s,9H),1.46(s,6H),1.31(t,J＝7.2Hz,3H).13CNMR(100MHz,CDCl3)δ176.3,166.3,158.6,157.6,154.1,152.9,150.5,148.3,146.6,143.0,138.7,131.7,130.3,123.3,122.1,119.2,116.2,112.3,111.1,109.6,100.2,84.4,77.8,73.1,61.6,60.5,28.3,27.6,26.2,25.8,18.2,14.3.ESI-MS m/z:643.3[M+Na]+.
实施例4化合物15a～15g和19b的合成
分别将化合物14a～14i(0.14～0.71mmol)溶于丙酮(5mL)或DMF(3mL)，加入K2CO3(0.28～1.07mmol)和(E)-4-溴巴豆酸乙酯(0.28～1.86mmol)，反应物回流过夜，减压移除溶剂，加入水，等体积乙酸乙酯萃取3次；有机相经无水Na2SO4干燥，蒸掉溶剂得粗产物；硅胶柱层析分离，石油醚/乙酸乙酯洗脱得白色固体15a～15g或19b。
Ethyl(E)-4-((8,9-dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15a).Yield:79％.1H NMR(400MHz,CDCl3)δ7.13(dt,J＝15.7,4.8Hz,1H),6.68(s,1H),6.66(d,J＝10.5Hz,1H),6.51(s,1H),6.23(dt,J＝15.7,1.8Hz,1H),5.65(d,J＝10.1Hz,1H),5.26–5.19(m,1H),4.70(dd,J＝4.7,1.8Hz,2H),4.20(q,J＝7.1Hz,2H),4.08(d,J＝6.7Hz,2H),3.91(s,3H),3.76(s,3H),1.81(s,3H),1.63(s,3H),1.43(s,6H),1.28(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ176.2,166.3,158.1,157.5,157.2,154.3,154.1,144.0,143.2,137.2,131.4,130.2,123.7,122.0,116.4,114.3,112.2,111.2,100.2,98.0,77.6,73.1,60.9,60.4,56.0,28.3,26.0,25.9,18.2,14.3.ESI-MS m/z:535.2[M+H]+.
Ethyl(E)-4-((9-ethoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15b).Yield:86％.1HNMR(400MHz,CDCl3)δ7.14(dt,J＝15.7,4.8Hz,1H),6.70(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.28-5.22(m,1H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.15(q,J＝7.0Hz,2H),4.10(d,J＝6.7Hz,2H),3.80(s,3H),1.84(s,3H),1.65(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ176.4,166.4,158.2,157.7,156.6,154.4,154.2,144.2,143.4,137.3,131.5,130.2,123.8,122.1,116.5,114.3,112.3,111.3,100.3,98.6,77.8,73.2,64.5,60.9,60.6,28.4,26.1,26.1,18.4,14.8,14.4.ESI-MS m/z:549.2[M+H]+.
Ethyl(E)-4-((9-isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15c).Yield:78％.1H NMR(400MHz,CDCl3)δ7.14(dt,J＝15.7,4.8Hz,1H),6.68(d,J＝10.1Hz,1H),6.66(s,1H),6.52(s,1H),6.24(dt,J＝15.6,1.9Hz,1H),5.65(d,J＝10.1Hz,1H),5.31-5.20(m,1H),4.72(dd,J＝4.8,1.9Hz,2H),4.65(p,J＝6.1Hz,1H),4.21(q,J＝7.1Hz,2H),4.09(d,J＝6.8Hz,2H),3.77(s,3H),1.83(s,3H),1.64(s,3H),1.44(s,6H),1.43(d,J＝6.1Hz,6H),1.29(t,J＝7.1Hz,3H).13C NMR(100MHz,CDCl3)δ176.3,166.3,158.1,157.6,155.5,154.3,154.2,144.7,143.3,137.4,131.3,130.2,123.8,122.1,116.5,114.1,112.2,111.2,100.2,99.4,77.7,73.1,71.0,60.7,60.5,28.3,26.1,26.0,21.9,18.3,14.3.ESI-MS m/z:563.2[M+H]+.
Ethyl(E)-4-((9-(benzyloxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15d).Yield:81％.1H NMR(400MHz,CDCl3)δ7.50-7.47(m,1H),7.47-7.45(m,1H),7.45-7.41(m,1H),7.41-7.38(m,1H),7.38-7.33(m,1H),7.15(dt,J＝15.7,4.8Hz,1H),6.78(s,1H),6.69(d,J＝10.1,1H),6.53(s,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29-5.23(m,1H),5.20(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.6Hz,2H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.4,166.4,158.2,157.6,156.3,154.2,144.4,143.3,137.5,136.0,131.5,130.3,128.9,128.4,127.4,123.8,122.2,116.5,114.7,112.3,111.3,100.3,99.3,77.8,73.2,70.7,61.0,60.6,28.4,26.2,26.1,18.4,14.4.ESI-MS m/z:611.2[M+H]+.
Ethyl(E)-4-((8-methoxy-2,2-dimethyl-9-((3-methylbenzyl)oxy)-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15e).Yield:80％.1HNMR(400MHz,CDCl3)δ7.34-7.23(m,3H),7.20-7.09(m,2H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.53(d,J＝0.7Hz,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29-5.21(m,1H),5.16(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.83(s,3H),2.39(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.3,166.3,158.1,157.5,156.3,154.1,144.3,143.2,138.5,137.4,135.8,131.4,130.1,129.0,128.6,128.0,124.4,123.7,122.1,116.4,114.6,112.2,111.2,100.2,99.2,77.6,73.1,70.7,60.9,60.4,28.3,26.0,25.9,21.5,18.2,14.3.ESI-MSm/z:625.2[M+H]+.
Ethyl(E)-4-((8-methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15f).Yield:84％.1HNMR(400MHz,CDCl3)δ7.33(t,J＝7.9Hz,1H),7.14(dt,J＝15.7,4.8Hz,1H),7.07–7.01(m,2H),6.89(ddd,J＝8.3,2.7,0.9Hz,1H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.3,166.3,159.9,158.1,157.5,156.1,154.1,144.3,143.2,137.5,137.5,131.5,130.2,129.8,123.6,122.0,119.4,116.4,114.6,113.6,112.8,112.2,111.2,100.2,99.3,77.6,73.1,70.5,60.9,60.5,55.3,28.3,26.0,25.9,18.2,14.3.ESI-MSm/z:641.2[M+H]+.
Ethyl(E)-4-((8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methyl-sulfonyl)benzyl)oxy)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15g).Yield:71％.1HNMR(400MHz,CDCl3)δ8.01(d,J＝8.3Hz,2H),7.70(d,J＝8.3Hz,2H),7.14(dt,J＝15.7,4.8Hz,1H),6.74(s,1H),6.68(d,J＝10.1Hz,1H),6.53(s,1H),6.25(dt,J＝15.7,1.8Hz,1H),5.68(d,J＝10.1Hz,1H),5.29(s,2H),5.27–5.21(m,1H),4.72(dd,J＝4.8,1.8Hz,2H),4.23(q,J＝7.1Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),3.08(s,3H),1.85(s,3H),1.67(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.1,166.3,158.2,157.4,155.5,154.1,154.0,144.2,143.1,142.2,140.4,137.9,131.6,130.3,127.9,127.7,123.4,122.1,116.3,115.1,112.3,111.2,100.1,99.3,77.7,73.1,69.5,61.1,60.5,44.5,28.3,26.0,25.9,18.2,14.3.ESI-MSm/z:689.1[M+H]+.
Ethyl(E)-4-((9-((3-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(19b).Yield:89％.1HNMR(400MHz,CDCl3)δ7.38(td,J＝7.9,5.7Hz,1H),7.26–7.11(m,3H),7.05(td,J＝8.6,2.7Hz,1H),6.74(s,1H),6.69(d,J＝10.1Hz,1H),6.53(s,1H),6.25(dt,J＝15.7,2.0Hz,1H),5.67(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.2,166.3,163.0(d,1JC-F＝246.8Hz),158.2,157.5,155.9,154.1,154.1,144.3,143.2,138.4(d,3JC-F＝7.2Hz),137.6,131.5,130.4(d,3JC-F＝8.3Hz),130.2,123.6,122.6(d,4JC-F＝2.9Hz),122.1,116.4,115.2(d,2JC-F＝21.0Hz),114.8,114.1(d,2JC-F＝22.2Hz),112.2,111.2,100.2,99.2,77.7,73.1,69.8(d,4JC-F＝2.0Hz),61.0,60.4,28.3,26.0,25.9,18.2,14.3.19FNMR(376MHz,CDCl3)δ-112.3.ESI-MSm/z:629.1[M+H]+.
实施例5化合物15h、15i和18d的合成
将化合物14i(0.19～4.3mmol)溶于(5～20mL)丙酮，加入Cs2CO3(0.38～8.5mmol)，(E)-4-溴代巴豆酸乙酯(8.6mmol)或4-溴丁酸乙酯(0.39mmol)，回流过夜，减压除掉溶剂，加入水，等体积的乙酸乙酯萃取3次；收集有机相，经无水Na2SO4干燥，硅胶柱层析，石油醚/乙酸乙酯洗脱得白色固体15h、15i和18d。
Ethyl4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoate(15h).Yield:88％.1HNMR(400MHz,CDCl3)δ7.45(dd,J＝8.5,5.5Hz,2H),7.10(t,J＝8.6Hz,2H),6.76(s,1H),6.71(d,J＝10.1Hz,1H),6.50(s,1H),5.66(d,J＝10.1Hz,1H),5.29–5.24(m,1H),5.14(s,2H),4.19–4.10(m,4H),4.02(t,J＝6.2Hz,2H),3.81(s,3H),2.63(t,J＝7.5Hz,2H),2.20(p,J＝6.6Hz,2H),1.85(s,3H),1.66(s,3H),1.46(s,6H),1.27(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.2,173.5,162.6(d,1JC-F＝246.9Hz),158.1,157.5,155.8,154.9,154.0,144.2,137.4,131.7(d,4JC-F＝3.2Hz),131.3,129.7,129.2(d,3JC-F＝8.2Hz),123.7,116.5,115.7(d,2JC-F＝21.6Hz),114.8,112.1,111.1,99.7,99.2,77.5,74.1,69.9,60.9,60.4,30.9,28.3,26.0,25.9,25.6,18.2,14.3.19FNMR(376MHz,CDCl3)δ-113.6.ESI-MSm/z:631.2[M+H]+.
Ethyl(Z)-4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15i).Compound15i was isolated from the crude product of compound 18d.1HNMR(400MHz,CDCl3)δ7.45(dd,J＝8.5,5.4Hz,2H),7.11(t,J＝8.7Hz,2H),6.83(dt,J＝11.7,4.8Hz,1H),6.76(s,1H),6.71(d,J＝10.1Hz,1H),6.53(s,1H),5.88(dt,J＝11.7,2.4Hz,1H),5.66(d,J＝10.1Hz,1H),5.31–5.25(m,1H),5.15(s,2H),5.13(dd,J＝4.7,2.4Hz,2H),4.13(q,J＝7.2Hz,4H),3.80(s,3H),1.83(s,3H),1.64(s,3H),1.46(s,6H),1.25(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.2,166.0,162.6(d,1JC-F＝246.8Hz),158.1,157.5,155.9,154.5,154.1,147.7,144.1,137.5,131.6(d,4JC-F＝3.1Hz),131.3,129.9,129.2(d,3JC-F＝8.2Hz),123.6,119.1,116.5,115.7(d,2JC-F＝21.6Hz),114.7,112.2,111.1,100.0,99.2,77.6,73.5,69.9,60.9,60.2,28.3,26.1,25.8,18.2,14.2.19FNMR(376MHz,CDCl3)δ-113.6.ESI-MSm/z:629.2[M+H]+.
Ethyl(E)-4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(18d).Yield：86％.1HNMR(400MHz,CDCl3)δ7.45(dd,J＝8.5,5.5Hz,2H),7.18–7.07(m,3H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29–5.20(m,1H),5.15(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.81(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).13CNMR(100MHz,CDCl3)δ176.2,166.3,162.7(d,1JC-F＝247.0Hz),158.1,157.5,156.0,154.11,154.08,144.3,143.2,137.6,131.6(d,4JC-F＝3.1Hz),131.5,130.2,129.2(d,3JC-F＝8.3Hz),123.6,122.0,116.4,115.7(d,2JC-F＝21.6Hz),114.7,112.2,111.2,100.1,99.2,77.7,73.1,70.0,60.9,60.4,28.3,26.0,25.9,18.2,14.3.19FNMR(376MHz,CDCl3)δ-113.6.ESI-MS m/z:629.1[M+H]+.
实施例6化合物16a～16c、17a～17e、18a～18c、19a和20a～20c的合成
将化合物13a～13d,15a～15i,18d和19b(0.04～0.56mmol)分别溶于丙酮/水(v/v,1:4)，加入NaOH(0.22～2.81mmol)，60℃反应2～4小时，反应完毕倒入10mL水中，5％HCl酸化调pH 1，乙酸乙酯萃取；有机相经无水Na2SO4干燥，浓缩得粗产物；硅胶层析分离，二氯甲烷/甲醇洗脱，得目标化合物(白色固体)。
2-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)acetic acid(16a).Yield:58％.1H NMR(400MHz,DMSO-d6)δ6.91(d,J＝10.1Hz,1H),6.75(s,1H),6.64(s,1H),5.85(d,J＝10.1Hz,1H),5.17–5.09(m,1H),4.57(s,2H),3.97(d,J＝6.7Hz,2H),3.69(s,3H),1.77(s,3H),1.61(s,3H),1.42(s,6H).13C NMR(100MHz,DMSO-d6)δ175.9,170.8,158.1,157.3,156.3,154.0,153.9,143.8,136.7,130.9,130.7,124.3,116.6,113.1,111.9,110.3,101.8,99.9,78.2,71.1,60.6,28.3,26.0,25.8,18.4.ESI-HRMS m/z:[M+H]+(calcd for C26H26O8 467.1700,found 467.1696).
4-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoic acid(16b).Yield:73％.1H NMR(400MHz,Methanol-d4)δ6.74–6.69(m,2H),6.51(s,1H),5.78(d,J＝10.1Hz,1H),5.24–5.17(m,1H),4.06(d,J＝6.5Hz,2H),3.97(t,J＝6.3Hz,2H),3.76(s,3H),2.59(t,J＝7.4Hz,2H),2.19–2.10(m,2H),1.82(s,3H),1.66(s,3H),1.45(s,6H).13C NMR(100MHz,Methanol-d4)δ178.2,177.2,159.8,159.0,157.2,156.1,155.8,145.0,138.4,131.9,131.4,125.3,117.1,114.7,113.3,111.9,102.5,100.7,78.8,75.5,61.3,31.6,28.5,26.9,26.7,26.0,18.4.ESI-HRMS m/z:[M+H]+(calcd for C28H30O8 495.2013,found495.2009).
5-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)pentanoic acid(16c).Yield:77％.1H NMR(400MHz,CDCl3)δ6.80(s,1H),6.71(d,J＝10.1Hz,1H),6.52(s,1H),5.66(d,J＝10.1Hz,1H),5.30–5.21(m,1H),4.08(d,J＝6.4Hz,2H),3.99(t,J＝5.8Hz,2H),3.80(s,3H),2.50(t,J＝6.8Hz,2H),1.98–1.87(m,4H),1.82(s,3H),1.65(s,3H),1.45(s,6H).13C NMR(100MHz,CDCl3)δ178.8,176.4,158.2,157.6,155.1,154.7,153.5,142.5,136.9,131.6,129.8,123.6,116.6,114.7,112.0,111.0,101.2,99.7,77.5,74.7,62.0,33.7,29.5,28.3,26.3,25.8,21.4,18.2.ESI-HRMS m/z:[M+H]+(calcd for C29H32O8 509.2170,found509.2166).
(E)-4-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17a).Yield:71％.1HNMR(400MHz,MeOD+CDCl3)δ7.13(dt,J＝15.6,4.9Hz,1H),6.72(s,1H),6.69(d,J＝10.1Hz,1H),6.57(s,1H),6.18(dt,J＝15.7,1.9Hz,1H),5.81(d,J＝10.1Hz,1H),5.20(d,J＝6.5Hz,1H),4.66(dd,J＝4.8,1.5Hz,2H),4.06(d,J＝6.3Hz,2H),3.76(s,3H),1.82(s,3H),1.65(s,3H),1.46(s,6H).13C NMR(100MHz,MeOD+CDCl3)δ178.1,169.5,159.7,158.9,157.3,155.8,155.2,145.0,144.7,138.4,132.0,131.7,125.1,123.5,116.9,114.6,113.4,111.9,102.4,101.1,78.9,74.3,61.3,28.5,26.9,26.0,18.4.ESI-HRMSm/z:[M+H]+(calcd for C28H28O8 493.1857,found 493.1857).
(E)-4-((8,9-Dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17b).Yield:44％.1H NMR(400MHz,CDCl3)δ7.26(dt,J＝15.6,4.5Hz,1H),6.72(s,1H),6.68(d,J＝10.1Hz,1H),6.55(s,1H),6.30(dt,J＝15.6,1.8Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.21(m,1H),4.77(dd,J＝4.5,1.9Hz,2H),4.11(d,J＝6.6Hz,2H),3.95(s,3H),3.79(s,3H),1.83(s,3H),1.65(s,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ176.4,171.3,158.2,157.6,157.4,154.5,154.1,146.1,144.1,137.4,131.7,130.4,123.7,121.1,116.4,114.5,112.3,111.3,100.3,98.1,77.8,73.0,61.1,56.1,28.4,26.1,26.1,18.3.ESI-HRMS m/z:[M+H]+(calcd for C29H30O8 507.2013,found 507.2017).
(E)-4-((9-Ethoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17c).Yield:64％.1HNMR(400MHz,CDCl3)δ7.29-7.22(m,1H),6.69(s,1H),6.68(d,J＝10.1Hz,1H),6.54(s,1H),6.29(dt,J＝15.6,2.0Hz,1H),5.68(d,J＝10.1Hz,1H),5.28-5.21(m,1H),4.77(dd,J＝4.2,1.5Hz,2H),4.22-4.12(m,2H),4.11(d,J＝7.1Hz,2H),3.80(s,3H),1.84(s,3H),1.66(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ176.4,171.3,158.2,157.7,156.7,154.4,154.1,146.2,144.2,137.3,131.5,130.4,123.7,121.1,116.4,114.3,112.2,111.3,100.3,98.6,77.8,73.0,64.5,60.9,28.4,26.1,26.1,18.3,14.7.ESI-HRMS m/z:[M+H]+(calcd for C30H32O8 521.2170,found 521.2166).
(E)-4-((9-Isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17d).Yield:63％.1H NMR(400MHz,CDCl3)δ7.29-7.21(m,2H),6.70(s,1H),6.68(d,J＝10.0Hz,1H),6.55(s,1H),6.29(dt,J＝15.7,1.9Hz,1H),5.68(d,J＝10.1Hz,1H),5.28-5.22(m,1H),4.77(dd,J＝4.5,2.0Hz,2H),4.67(hept,J＝6.1Hz,1H),4.10(d,J＝6.7Hz,2H),3.79(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.46(s,3H),1.44(s,3H).13C NMR(100MHz,CDCl3)δ176.4,170.8,158.2,157.7,155.6,154.4,154.1,146.1,144.8,137.5,131.5,130.4,123.8,121.0,116.4,114.2,112.2,111.3,100.3,99.5,77.8,73.0,71.1,60.8,28.4,26.2,26.1,22.0,18.3.ESI-HRMS m/z:[M+H]+(calcd for C31H34O8 535.2326,found 535.2321).
(E)-4-((9-(Benzyloxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17e).Yield:55％.1H NMR(400MHz,CDCl3)δ7.52-7.47(m,1H),7.47-7.45(m,1H),7.45-7.42(m,1H),7.42-7.38(m,1H),7.38-7.33(m,1H),7.29-7.22(m,1H),6.78(s,1H),6.68(d,J＝10.0Hz,1H),6.54(s,1H),6.36-6.24(m,1H),5.69(d,J＝10.1Hz,1H),5.25(t,J＝6.1Hz,1H),5.20(s,2H),4.82-4.70(m,2H),4.12(d,J＝6.4Hz,2H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).13CNMR(100MHz,CDCl3)δ176.4,171.0,158.3,157.6,156.3,154.3,154.1,146.1,144.4,137.6,136.0,131.6,130.4,128.9,128.4,127.4,123.7,121.1,116.4,114.7,112.3,111.3,100.4,99.4,77.8,73.0,70.7,61.1,28.4,26.2,26.1,18.4.ESI-HRMSm/z:[M+H]+(calcd for C35H33FO8 583.2326,found 583.2321).
(E)-4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoicacid(18a).Yield:78％.1H NMR(400MHz,CDCl3)δ7.45(dd,J＝8.6,5.4Hz,2H),7.25(dt,J＝15.6,4.5Hz,1H),7.11(t,J＝8.7Hz,2H),6.77(s,1H),6.68(d,J＝10.2Hz,1H),6.55(s,1H),6.30(dt,J＝15.6,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.21(m,1H),5.15(s,2H),4.77(dd,J＝4.5,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.81(s,3H),1.84(s,3H),1.66(s,3H),1.47(s,6H).13C NMR(100MHz,CDCl3)δ176.3,170.6,162.7(d,1JC-F＝246.9Hz),158.2,157.5,156.0,154.1,154.0,146.0,144.3,137.6,131.6(d,4JC-F＝3.2Hz),131.55,130.3,129.3(d,3JC-F＝8.2Hz),123.5,120.9,116.2,115.7(d,2JC-F＝21.7Hz),114.7,112.2,111.2,100.2,99.2,77.7,72.8,70.0,60.9,28.3,26.0,25.9,18.2.19FNMR(376MHz,CDCl3)δ-113.5.ESI-HRMS m/z:[M+H]+(calcd for C35H33FO8 601.2232,found 601.2232).
4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoic acid(18b).Yield:87％.1HNMR(400MHz,CDCl3)δ7.45(dd,J＝8.6,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.77(s,1H),6.68(d,J＝10.1Hz,1H),6.53(s,1H),5.68(d,J＝10.1Hz,1H),5.28–5.23(m,1H),4.11(d,J＝6.6Hz,2H),4.02(t,J＝5.9Hz,2H),3.81(s,3H),2.76(t,J＝7.1Hz,2H),2.21(p,J＝6.7Hz,2H),1.85(s,3H),1.66(s,3H),1.46(s,6H).13CNMR(100MHz,CDCl3)δ177.7,176.7,162.7(d,1JC-F＝246.9Hz),158.4,157.6,156.1,154.7,154.2,144.3,137.5,131.6(d,4JC-F＝3.2Hz),131.5,130.0,129.3(d,3JC-F＝8.3Hz),123.6,116.3,115.7(d,2JC-F＝21.6Hz),114.6,112.2,111.0,99.9,99.2,77.7,74.1,70.0,60.9,31.4,28.3,26.1,25.9,25.7,18.2.19FNMR(376MHz,CDCl3)δ-113.5.ESI-HRMS m/z:[M+H]+(calcd for C35H35FO8603.2389,found 603.2373).
(Z)-4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoicacid(18c).Yield:52％.1HNMR(400MHz,Chloroform-d)δ7.45(dd,J＝8.5,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.92(dt,J＝11.8,4.6Hz,1H),6.76(s,1H),6.67(d,J＝10.1Hz,1H),6.52(s,1H),5.89(dt,J＝11.8,2.4Hz,1H),5.66(d,J＝10.1Hz,1H),5.26–5.21(m,1H),5.14(s,2H),5.06(dd,J＝4.6,2.5Hz,2H),4.09(d,J＝6.8Hz,2H),3.79(s,3H),1.80(s,3H),1.61(s,3H),1.45(s,6H).13CNMR(101MHz,CDCl3)δ176.2,170.4,162.6(d,1JC-F＝247.0Hz),158.2,157.5,156.0,154.2,154.1,150.2,144.2,137.6,131.6(d,4JC-F＝3.3Hz),131.4,130.1,129.2(d,3JC-F＝8.2Hz),123.5,118.3,116.3,115.7(d,2JC-F＝21.6Hz),114.6,112.1,111.0,100.1,99.2,77.7,73.4,69.9,60.9,28.3,26.0,25.8,18.2.19FNMR(376MHz,CDCl3)δ-113.6.ESI-HRMS m/z:[M+H]+(calcd for C35H33FO8601.2232,found 601.2222).
(E)-4-((9-((3-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoicacid(19a).Yield:87％.1HNMR(400MHz,CDCl3)δ7.39(td,J＝8.0,5.8Hz,1H),7.29–7.18(m,3H),7.05(tdd,J＝8.4,2.6,1.0Hz,1H),6.75(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.30(dt,J＝15.7,1.9Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.22(m,1H),5.19(s,2H),4.77(dd,J＝4.5,2.0Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.67(s,3H),1.47(s,6H).13CNMR(100MHz,CDCl3)δ176.2,170.8,163.0(d,1JC-F＝246.7Hz),158.2,157.5,155.9,154.1,154.0,146.0,144.3,138.4(d,3JC-F＝7.4Hz),137.7,131.6,130.4(d,3JC-F＝8.4Hz),130.3,123.5,122.6(d,4JC-F＝2.8Hz),120.9,116.2,115.2(d,2JC-F＝21.3Hz),114.8,114.1(d,2JC-F＝22.4Hz),112.2,111.2,100.2,99.2,77.7,72.8,69.8(d,4JC-F＝1.9Hz),61.0,28.3,26.0,25.9,18.2.19FNMR(376MHz,CDCl3)δ-112.3.ESI-HRMSm/z:[M+H]+(calcdforC35H33FO8 601.2232,found 601.2236).
(E)-4-((8-Methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoicacid(20a).Yield:79％.1H NMR(400MHz,CDCl3)δ7.33–7.21(m,4H),7.19–7.15(m,1H),6.78(s,1H),6.69(d,J＝10.1Hz,1H),6.55(s,1H),6.30(dt,J＝15.7,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.29–5.22(m,1H),5.16(s,2H),4.77(dd,J＝4.6,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.83(s,3H),2.39(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ176.3,171.0,158.1,157.5,156.3,154.2,154.0,146.0,144.3,138.5,137.4,135.8,131.5,130.3,129.0,128.6,128.0,124.4,123.6,121.0,116.3,114.5,112.2,111.2,100.3,99.2,77.7,72.9,70.7,60.9,28.3,26.0,25.9,21.5,18.2.ESI-HRMSm/z:[M+H]+(calcd for C36H36O8 597.2483,found 597.2473).
(E)-4-((8-Methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoicacid(20b).Yield:75％.1HNMR(400MHz,CDCl3)δ7.33(t,J＝7.9Hz,1H),7.25(dt,J＝15.7,4.5Hz,1H),7.08–7.01(m,2H),6.89(dd,J＝8.1,2.6Hz,1H),6.77(s,1H),6.68(d,J＝10.1Hz,1H),6.54(s,1H),6.30(dt,J＝15.7,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.77(dd,J＝4.6,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).13C NMR(100MHz,CDCl3)δ176.3,170.4,159.9,158.2,157.5,156.2,154.1,154.0,145.9,144.3,137.5,131.5,130.3,129.8,123.6,120.9,119.4,116.3,114.6,113.6,112.8,112.2,111.2,100.3,99.3,77.7,72.9,70.5,61.0,55.3,28.3,26.0,25.9,18.2.ESI-HRMS m/z:[M+H]+(calcd for C36H36O9613.2432,found 613.2421).
(E)-4-((8-Methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methylsulfonyl)benzyl)oxy)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(20c).Yield:67％.1HNMR(400MHz,DMSO-d6)δ8.00(d,J＝8.3Hz,2H),7.77(d,J＝8.3Hz,2H),7.12(s,1H),7.02(dt,J＝15.7,4.8Hz,1H),6.66(s,1H),6.61(d,J＝10.2Hz,1H),6.11(dt,J＝15.7,1.9Hz,1H),5.90(d,J＝10.1Hz,1H),5.44(s,2H),5.17–5.10(m,1H),4.65(dd,J＝4.8,1.9Hz,2H),4.01(d,J＝6.7Hz,2H),3.76(s,3H),3.24(s,3H),1.77(s,3H),1.61(s,3H),1.43(s,6H).13C NMR(100MHz,DMSO-d6)δ175.6,167.3,158.2,157.3,156.1,154.0,153.9,144.2,143.6,142.6,140.9,136.5,131.5,131.0,128.5,127.8,124.1,122.8,115.8,114.2,112.3,111.1,100.3,100.1,78.3,73.3,69.7,61.0,43.9,28.3,26.1,25.8,18.5.ESI-HRMS m/z:[M+H]+(calcd for C36H36O10S 661.2102,found661.2090).
实施例7化合物对PDE4抑制活性测定
测定化合物对PDE4D2的抑制活性，以化合物18a为代表，测定其对PDEs亚型选择性，其他化合物效果与化合物18a类似。
实验方法：
先将表达PDEs质粒转化入大肠杆菌(E.coli)菌株BL21(codonplus)中，携带重组质粒的大肠杆菌(E.coli)细胞在LB培养基中在37℃下生长至A600＝0.7，然后加入0.1mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达，并在15℃培养20h。重组的PDEs催化结构域通过Ni-NTA亲和柱(Qiagen)、Q柱(GE Healthcare)和Superdex 100柱(GE Healthcare)纯化。
以3H-环磷酸鸟苷(cGMP)或3H-环磷酸腺苷(cAMP)为底物，测定磷酸二酯酶(PDEs)活性；检测缓冲液含有20mM Tris-HCl(pH 7.5)、10mM MgC l2或4mM MnCl2、1mM DTT和10～30nM 3H-cGMP或3H-cAMP(20,000-30,000CPM/检测)；反应在室温下进行15min，然后加入0.2M硫酸锌终止；反应产物(3H-环磷酸鸟苷cGMP或3H-环磷酸腺苷cAMP)用0.2N的氢氧化钡沉淀，而未反应的(3H-环磷酸鸟苷cAMP或3H-环磷酸腺苷cGMP)保留在上清液中，PerkinElmer 2910液体闪烁计数器测量，通过非线性回归计算IC50值。
实验结果参见表1～2。
Figure BDA0003190442680000221
表1化合物对PDE4D2抑制活性
Figure BDA0003190442680000222
Figure BDA0003190442680000231
由表可见，本发明提供的化合物对PDE4D2具有显著的抑制活性，大部分化合物IC50显著小于阳性对照组咯利普兰(Rolipram)；其中，化合物16a、18d、19b抑制效果较差，IC50>1000。
表2化合物18a对PDEs亚型选择性
PDEs IC<sub>50</sub>(nM) 选择性指数
PDE4D2(86-413) 4.2±0.5 -
PDE4B2(152-487) 140±18 33
PDE1C(147-531) 440±92 104
PDE2A(580-919) 940±79 223
PDE3A(679-1087) >10000 >2300
PDE5A1(535-860) 93±20 22
PDE7A1(130-482) 298±9 70
PDE8A1(480-828) 710±65 169
PDE9A2(181-506) >10000 >2300
PDE10A2(449-770) 260±6 61
由表可见，化合物18a对PDE3A和PDE9A的抑制作用非常弱，IC50值超过10μM，选择性倍数超过2000；化合物18a对PDE4D与对PDE1C、PDE2A和PDE8A相比，显示出>100倍的选择性，与PDE5A、PDE7A和PDE10A相比也显示出>22倍的选择性；化合物18a还显示出33倍于PDE4B的中等选择性。上述结果表明，化合物18a是一种选择性PDE4D抑制剂。
实施例8化合物18a的药代动力学及类药性测定
以化合物18a为代表，测定化合物18a的药代动力学及类药性，其他化合物效果与化合物18a类似。
实验方法：
(1)药代动力学实验：选取SD大鼠6只(3只静脉注射，3只口服)，10个时间点，给药后不同时间点采集血浆，用HPLC-MS/MS的方法测定血浆中化合物的浓度，分析药代动力学参数AUC(0-t)、AUC(0-∞)t1/2、Tmax、Cmax等。
(2)体外肝微粒体稳定性测试
配制0.5mM供试品或者阳性对照品(酮色林)；1.5μM供试品或者阳性对照品在0.75mg/mL肝微粒体混悬液中；配制6mM NADPH在PBS中；按照时间点(0、5、15、30、45和60min)在96孔板中分装30μL含1.5μM化合物或者阳性对照品的0.75mg/mL肝微粒体混悬液；在0点加入150μL含内标的ACN后加入15μL配制好的NADPH(6mM)，反应板封装好；96孔反应板在37℃水浴中预热5min，加入15μL配制好的NADPH(6mM)起始反应(化合物与阳性对照品在反应体系中最终浓度均为1μM)；在5、15、30、45和60min加入150μL含内标的ACN终止反应；将反应板震荡5min，样品保存于-80℃冰箱直至测样；测样前样品4000rpm离心15min，取上清80μL加入80μL超纯水混合均匀，LC-MS/MS进样分析。
(3)小鼠的急性毒性实验：取成年清洁级昆明小鼠30只，每组10只，共3组，随机分为空白对照组(溶媒)、正常组、1500mg/kg。药物悬浮于0.5％CM C-Na，按照1ml/kg口服给药。给药后立即观察动物的反应情况，持续观察12小时后，每天观察一次，连续观察14天，评价化合物的急性毒性。
实验结果参见表3～4和图1。
表3化合物18a在SD大鼠上的药代动力学特征(口服给药5mg/kg)
Figure BDA0003190442680000241
Figure BDA0003190442680000251
由表可见，SD大鼠口服5mg/kg化合物18a后，t1/2、Cmax和AUC的药代动力学参数分别为1.02h、1067ng/mL和2106h*ng/mL。
表4化合物18a的类药性测定
Figure BDA0003190442680000252
由表可见，化合物18a对人细胞色素P450酶CYP1A2、2B6、2D6和3A4亚型的IC50值大于25μM，对CYP2C9的IC50值为1.7μM，表明化合物18a一般不会诱导药物-药物相互作用；并且化合物18a对hERG钾离子通道的IC50值大于30μM，心脏毒性风险较低；另外，评价了1.5g/kg的剂量下对小鼠的急性毒性，结果参见图1，显示化合物18a在口服1.5g/kg剂量下无急毒性展现了良好的安全性。
综上所述，化合物18a具有合理的选择性和理化性质，适合后续的药效学研究。其他化合物也具有类似的性质。
实施例9化合物18a抗IPF测定
实验方法：40只雄性SD大鼠(5～6周，200～220g，中山大学动物研究机构伦理委员会(SYSU-IAUC-2021-000184)的批准，并于中山大学实验动物中心饲养)在24±1℃，60～70％的相对湿度，光照12h(8:00～20:00)培养条件下，将大鼠随机分为对照组、模型组、化合物18a(10mg/kg)组和阳性对照组(PFD，150mg/kg)。在实验之前，老鼠可以自由获取食物和水，然后，对照组用生理盐水治疗，其余的用BLM进行单次气管内滴注，剂量为5mg/kg；治疗从第二天开始，用药物载体(对照组)、化合物18a(10.0mg/kg)或PFD(150mg/kg)治疗4周：将化合物18a和PFD溶解在0.5％羧甲基纤维素钠溶液中，并以0.4mL/100g的剂量口服给药。在整个实验间隔期间观察并记录重量、毛发和呼吸状态的变化。口服28天后，测量各组的呼吸水平。
检测结束，给大鼠腹腔注射4％戊巴比妥钠，从腹主动脉抽取10mL血液，在2800转/min和4℃下离心10min后，收集血清并储存在-80℃的冰箱中。左下肺骨折在安乐死后取出，在室温下将组织浸入4％缓冲聚甲醛中，并包埋在石蜡中，制作5μm厚的切片，安装在载玻片上，进行苏木精-伊红和马森(Masson)染色。
各组的肺组织在添加1％蛋白酶抑制剂混合物和1mM苯基甲磺酰氟的裂解缓冲液中匀浆，裂解物在12,000克和4℃下离心10min，收集上清液用于后续用于免疫印迹分析。
上述每个上清液样品的20μg用于蛋白质印迹：蛋白质样品通过SDS-PAGE分离，然后转移到聚偏氟乙烯膜(美国密理博公司)，在室温下，用5％脱脂奶粉在Tris-缓冲盐水Tween 20(TBS-T)中封闭膜1.5h，连续在4℃下用如下初级抗体探测膜过夜：抗FN抗体(1:5000，Abcam，ab2413，美国)、抗α-SMA抗体(1:300，Abcam，ab7817，美国)和抗GAPDH抗体(1μg/mL，Abcam，ab9484，美国)；然后，在室温下用相应的山羊抗小鼠lgG二级抗体(Boster，中国)将膜染色1小时，最后，使用ECL试剂(美国)对信号进行可视化，并用Bio-Rad QualityOne软件对免疫印迹带进行密度定量。
上述实验结果参见图2，由图2A可见，与对照组(Con)相比，模型组(Mod)大鼠的肺功能参数，包括呼气中流速(EF50)、呼气峰流速(PEF)、呼吸频率(n)和增强停顿(金边)显著增加，表明BLM诱导的IPF模型成功建立；在相同条件下，连续28天后，口服剂量为10mg/kg的化合物18a比口服剂量为150mg/kg的市售药物吡非尼酮(PFD)具有更好的抗IPF效应。并且，化合物18a组中与肺功能相关的参数如EF50、PEF和金边指数恢复到与对照组接近的水平，这表明PDE4抑制剂化合物18a在改善IPF大鼠的肺功能方面具有显著的效果。
由图2B、2C可见，苏木精-伊红染色显示模型组肺泡紊乱，肺泡壁破裂融合，有明显的炎性细胞浸润；化合物18a(10mg/kg)和PFD(150mg/kg)都可以有效地减轻肺的结构损伤，减少纤维化病变。此外，IPF的病理特征之一胶原沉积的表达大大增加，如Masson染色切片所示，模型组气管周围有大面积的胶原沉积和细胞增生5C，而化合物18a和PFD都能几乎完全抑制胶原沉积至正常水平。
由图2D可见，通过蛋白质印迹，IPF肺组织中的标记蛋白α-平滑肌肌动蛋白(α-SMA)和纤连蛋白(Fibronectin)的表达水平相对于模型组中的表达水平上调，化合物18a和PFD能显著降低α-SMA和纤连蛋白的水平。
上述结果均表明，PDE4抑制剂化合物18a改善了博莱霉素(BLM)诱导的肺纤维化。
实施例10化合物18a抑制体外上皮细胞间质转化(EMT)
上皮间质转化是IPF病的一个重要发病机制，可由纤维化前细胞因子TGF-β(转化生长因子-β)诱导，在此过程中，纤维连接蛋白(Fibronectin)、N-钙粘蛋白(N-cadherin)和波形纤维蛋白(vimentin)等霍尔标记物的表达水平发生改变。本实施例通过免疫印迹法(western bolt)分析进一步评估化合物18a在TGF-β诱导的A549细胞模型中的抗IPF潜能。
实验方法：A549细胞(购自中国科学院上海细胞库)在含有10％胎牛血清的杜尔贝科改良鹰培养基(DMEM)中培养，并置于含5％CO2的37℃恒温培养箱中；A549细胞以5×105个/mL接种在细胞培养皿中，培养过夜；将细胞分为空白对照组、模型组和给药组，除空白组外，均加入10ng/mL TGF-β，给药组加入最终浓度为20μM和10μM的化合物18a；孵育48h后，将细胞皿置于0℃下，弃去培养基，随后用4℃的PBS溶液洗涤三次；用RIPA裂解液提取细胞总蛋白，用BCA法定量蛋白：使用N-钙粘蛋白(1:1000，CST，13116，美国)、波形纤维蛋白(1:1000，CST，5741，美国)和FN(1:5000，Abcam，ab2413，美国)的抗体通过Western印迹法测定各组样品中的蛋白质水平。所有结果均以平均扫描电镜给出。使用单向方差分析与邦费罗尼(Bonferroni)多重比较法进行分析，p值小于0.05被认为具有统计学意义。结果参见图3。
由图可见，在TGF-β-诱导48h后，A549细胞中纤维连接蛋白(Fibronectin)、N-钙粘蛋白和波形纤维蛋白的表达水平显著上调；化合物18a治疗能以剂量依赖性方式(10～20μmol/L)显著降低N-钙粘蛋白、波形蛋白纤维和纤维连接蛋白的高表达，表明PDE4抑制剂化合物18a能预防TGF-β诱导的EMT过程。
实施例11化合物18a比格犬催吐试验
实验方法：将比格犬(8只雄性比格犬购自广州医药研究总院有限公司，IAC UC编号:SYSUIAUC-2021-000130)随机分成2组，实验前对比格犬进行12h禁食，将药物以1mg/mL悬浮于0.5％羧甲基纤维素钠溶液中，口服给药(化合物18a 10mg/kg，咯利普兰1mg/kg)，然后用15mL水冲洗灌洗管，确保所有化合物进入狗的胃；持续观察动物180分钟，观察其干呕、过度流涎和呕吐的行为。结果参见表5。
表5化合物18a比格犬催吐试验结果
药物 剂量(mg/kg,p.o.) 呕吐动物数/测试动物数
化合物18a 10 0/4
咯利普兰 1 4/4
由表可见，本发明化合物18a相比较于咯利普兰，口服时不会引起呕吐，顺应性好。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种α-倒捻子素衍生物，其特征在于，具有式(I)结构：
Figure FDA0003190442670000011
其中，R1为氢、烷基、环烷基、烷酰基、苄基或取代苄基；
R2为氢、烷基、取代烷基、烯烃基、取代烯烃基、苄基或取代苄基；
所述取代烷基、取代烯烃基、取代苄基的取代基团为卤素、烷氧基、甲基磺酰基、酯基或羧基。
2.根据权利要求1所述α-倒捻子素衍生物，其特征在于，所述R1为氢、C1～5烷基、C1～5烷酰基、苄基或取代苄基；
所述R2为氢、C1～5烷基、取代C1～5烷基、C1～5烯烃基或取代C1～5烯烃基。
3.根据权利要求2所述α-倒捻子素衍生物，其特征在于，所述R1为氢、甲基、乙基、丙基、丁基、异丙基、乙酰基、苄基、3-氟-苄基、4-氟-苄基、3-甲氧基-苄基、3-甲基-苄基、4-甲基磺酰基-苄基；
所述R2为氢、-(CH2)n-COO-CH2CH3、-(CH2)m-COOH、-CH2CH＝CH-COO-CH2CH3、-CH2CH＝CH-COOH；
其中，n＝1～4，m＝1～4。
4.根据权利要求3所述α-倒捻子素衍生物，其特征在于，所述R1为H、甲基、乙基、异丙基、苄基、3-氟-苄基、3-甲氧基-苄基、3-甲基-苄基、4-甲基磺酰基-苄基；
所述R2为-(CH2)3-COOH、-(CH2)4-COOH或-CH2CH＝CH-COOH。
5.权利要求1～4任一所述α-倒捻子素衍生物的制备方法，其特征在于，包括以下步骤：
Figure FDA0003190442670000012
以α-倒捻子素衍生物化合物1作为原料，在溶剂中与醋酐或二碳酸二叔丁酯反应，再在20～60℃、催化剂存在条件下与溴代烷基羧酸酯反应，脱去保护基和长链羧酸酯中酯基，即得；
或，以α-倒捻子素衍生物化合物1作为原料，在溶剂中与溴代R1反应，再在20～60℃、催化剂存在条件下与溴代烷基羧酸酯反应，脱去长链羧酸酯中酯基，即得。
6.权利要求1～4任一所述α-倒捻子素衍生物在制备PDE4抑制剂中的应用。
7.一种PDE4抑制剂，其特征在于，包含有效量的权利要求1～4任一所述α-倒捻子素衍生物。
8.权利要求1～4任一所述α-倒捻子素衍生物在制备防治与PDE4相关疾病药物中的应用。
9.根据权利要求8所述应用，其特征在于，所述与PDE4相关疾病包括组织纤维化、银屑病、老年性痴呆症、慢性阻塞性肺疾病、炎症性肠病和哮喘。
10.一种防治抗纤维化药物，其特征在于，包含有效量的权利要求1～4任一所述α-倒捻子素衍生物。
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