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Article(id=1308841407683584257, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841198224240856, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2026-0423, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1776182400000, receivedDateStr=2026-04-15, revisedDate=1777219200000, revisedDateStr=2026-04-27, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981708689, onlineDateStr=2026-09-21, pubDate=1781193600000, pubDateStr=2026-06-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981708689, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981708689, creator=13701087609, updateTime=1789981708689, updator=13701087609, issue=Issue{id=1308841198224240856, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='6', pageStart='1881', pageEnd='2376', issueExtLink='null', onlineDate='null', pubDate='1781193600000', pubDateStr='2026-06-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981658750, creator='13701087609', updateTime=1789984187230, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851803500933401, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841198224240856, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851803500933402, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841198224240856, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2212, endPage=2220, ext={EN=ArticleExt(id=1308841407901688066, articleId=1308841407683584257, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Design, synthesis and bioactivity evaluation of small-molecule inhibitors against SAP2 of drug-resistant Candida albicans, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

Secreted aspartic protease 2 (SAP2) stands as one of the most pivotal virulence factors in Candida albicans (C. albicans). Consequently, the development of potent small-molecule inhibitors targeting SAP2 represents a critical stride toward the treatment of invasive candidiasis. In our previous studies, the lead compound SAP2-IN-1 was successfully identified and synthesized via integrated strategies of virtual screening and scaffold hopping. Preliminary structure-activity relationship (SAR) investigation was performed through rational design, synthesis, and biological evaluation of 10 novel indolin-2-one derivatives derived from the SAP2-IN-1 scaffold. Enzymatic inhibition and cell-based assays confirmed that compound A1 possesses favorable SAP2 inhibitory activity, with an IC50 of 0.87 μmol·L-1. Notably, when combined with fluconazole (FLC), compound A1 effectively suppressed the growth of fluconazole-resistant C. albicans 0304103, yielding fractional inhibitory concentration index (FICI) of 0.047. Furthermore, preliminary mechanistic studies revealed that A1 significantly impeded biofilm formation and hyphal transition in drug-resistant C. albicans. It possesses great potential as a novel antifungal lead compound and provides a new theoretical basis and research strategy for the development of virulence factor-targeted inhibitors to overcome the drug resistance of C. albicans.

, authors=Hua SUN, Jie SU, Shan-chao WU*, authorsList=Hua SUN, Jie SU, Shan-chao WU, authorCompany=null, correspAuthors=Shan-chao WU, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1308841412238598435, articleId=1308841407683584257, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=抗耐药白念珠菌SAP2小分子抑制剂的设计、合成和生物活性评价, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

分泌型天冬氨酸蛋白酶2 (secreted aspartic protease 2, SAP2) 是白念珠菌重要的毒力因子之一。因此, 开发针对SAP2的小分子抑制剂对白念珠菌病的治疗至关重要。前期课题组通过虚拟筛选及骨架跃迁策略, 成功发现了具有抗耐药白念珠菌活性的先导化合物SAP2-IN-1。作为初步的构效关系讨论, 本研究针对先导化合物SAP2-IN-1开展基于药物合理设计, 初步合成了10个新型吲哚啉-2-酮衍生物, 并对所有目标化合物开展了酶水平抑制活性及真菌细胞水平活性测试。其中, 化合物A1对SAP2具有良好的抑制作用(IC50 = 0.87 μmol·L-1), 联用氟康唑(fluconazole, FLC) 时能够有效抑制耐FLC白念珠菌(Candida albicans, C. albicans) 0304103的生长(FICI = 0.047)。初步作用机制研究表明, 化合物A1能够抑制耐药白念珠菌生物被膜与菌丝形成, 兼具开发为新型抗真菌先导化合物的潜在价值, 同时为靶向毒力因子研发抑制剂、克服白念珠菌耐药困境提供了新的理论依据与研究策略。

, authors=孙华, 苏杰, 武善超*, authorsList=孙华, 苏杰, 武善超, authorCompany=null, correspAuthors=武善超, authorNote=

#

共同第一作者.

作者贡献:

孙华负责化合物合成及文章的撰写; 苏杰负责化合物波谱数据解析及体外活性实验; 通讯作者武善超负责课题规划, 实验技术指导及论文的修改、校对。

, correspAuthorsNote=
武善超, Tel: 86-21-81871242, E-mail:
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Reagents and conditions: (a) K2CO3, Pd(PPh3)4, p-tolylboronic acid, 1, 4-dioxane∶H2O (10∶1, v/v), 100 ℃, N2, yield 68.5%; (b) Diethylamine, absolute ethanol, 78 ℃, yield 76%; (c) (ⅰ) NaH, various bromides, DMF, 0 ℃ (ⅱ) Pd/C, H2, MeOH, rt and (iii) TFA, DCM, rt, yield 35%-43%, figureFileSmall=ue664oR/M3LGYM2jMw2IDw==, figureFileBig=GvzvvcgrsWiwioxwU8WTKw==, tableContent=null), ArticleFig(id=1308841416328044870, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841407683584257, language=EN, label=null, caption=null, figureFileSmall=J5MBxHdyU5dIgEze5o9g2A==, figureFileBig=CmtAqjtprocluQX9ZO/+sA==, tableContent=null), ArticleFig(id=1308841416428708167, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308841407683584257, language=CN, label=Fig.3, caption=Inhibition of biofilm formation by different concentrations of compound SAP2-IN-1 and 1 μg·mL-1 FLC (A). 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Compd.RIC50/μmol·L-1
A1 0.87 ± 0.35
A2 1.13 ± 0.14
A3 0.53 ± 0.12
A4 0.21 ± 0.15
A5 0.81 ± 0.07
A6 2.22 ± 0.21
A7 0.44 ± 0.08
A8 1.35 ± 0.63
A9 1.06 ± 0.29
A10 0.92 ± 0.35
SAP2-IN-1-1.7 ± 0.52
PepA-0.023 ± 0.01
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SAP2 inhibitory activity of the target compound

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Compd.RIC50/μmol·L-1
A1 0.87 ± 0.35
A2 1.13 ± 0.14
A3 0.53 ± 0.12
A4 0.21 ± 0.15
A5 0.81 ± 0.07
A6 2.22 ± 0.21
A7 0.44 ± 0.08
A8 1.35 ± 0.63
A9 1.06 ± 0.29
A10 0.92 ± 0.35
SAP2-IN-1-1.7 ± 0.52
PepA-0.023 ± 0.01
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Compd.MIC80/μg·mL-1FICIa
AloneSynergetic
A1/FLC> 64/ > 642/10.047
A2/FLC> 64/ > 648/80.250
A3/FLC> 64/ > 6416/20.281
A4/FLC> 64/ > 648/80.250
A5/FLC> 64/ > 6432/20.531
A6/FLC> 64/ > 6432/321.000
A7/FLC> 64/ > 648/40.188
A8/FLC> 64/ > 6464/41.062
A9/FLC> 64/ > 6432/20.531
A10/FLC> 64/ > 648/20.156
SAP2-IN-1/FLC> 64/ > 6432/40.562
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Synergistic antifungal activity in vitro of compounds combined with FLC against C. albicans 0304103. aSynergism was defined as FICI ≤ 0.5. FLC: Fluconazole; FICI: Fractional inhibitory concentration index

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Compd.MIC80/μg·mL-1FICIa
AloneSynergetic
A1/FLC> 64/ > 642/10.047
A2/FLC> 64/ > 648/80.250
A3/FLC> 64/ > 6416/20.281
A4/FLC> 64/ > 648/80.250
A5/FLC> 64/ > 6432/20.531
A6/FLC> 64/ > 6432/321.000
A7/FLC> 64/ > 648/40.188
A8/FLC> 64/ > 6464/41.062
A9/FLC> 64/ > 6432/20.531
A10/FLC> 64/ > 648/20.156
SAP2-IN-1/FLC> 64/ > 6432/40.562
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抗耐药白念珠菌SAP2小分子抑制剂的设计、合成和生物活性评价
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孙华 , 苏杰 , 武善超 *
药学学报 | 研究论文 2026,61(6): 2212-2220
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药学学报 |研究论文 2026 , 61 (6) : 2212 -2220
抗耐药白念珠菌SAP2小分子抑制剂的设计、合成和生物活性评价
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孙华, 苏杰, 武善超*
作者信息
  • 中国人民解放军海军军医大学药学院, 上海 200433
通讯作者:
武善超, Tel: 86-21-81871242, E-mail:
作者简介:

#

共同第一作者.

作者贡献:

孙华负责化合物合成及文章的撰写; 苏杰负责化合物波谱数据解析及体外活性实验; 通讯作者武善超负责课题规划, 实验技术指导及论文的修改、校对。

Design, synthesis and bioactivity evaluation of small-molecule inhibitors against SAP2 of drug-resistant Candida albicans
Hua SUN, Jie SU, Shan-chao WU*
Affiliations
  • College of Pharmacy, Naval Medical University, Shanghai 200433, China
出版时间: 2026-06-12 doi: 10.16438/j.0513-4870.2026-0423
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分泌型天冬氨酸蛋白酶2 (secreted aspartic protease 2, SAP2) 是白念珠菌重要的毒力因子之一。因此, 开发针对SAP2的小分子抑制剂对白念珠菌病的治疗至关重要。前期课题组通过虚拟筛选及骨架跃迁策略, 成功发现了具有抗耐药白念珠菌活性的先导化合物SAP2-IN-1。作为初步的构效关系讨论, 本研究针对先导化合物SAP2-IN-1开展基于药物合理设计, 初步合成了10个新型吲哚啉-2-酮衍生物, 并对所有目标化合物开展了酶水平抑制活性及真菌细胞水平活性测试。其中, 化合物A1对SAP2具有良好的抑制作用(IC50 = 0.87 μmol·L-1), 联用氟康唑(fluconazole, FLC) 时能够有效抑制耐FLC白念珠菌(Candida albicans, C. albicans) 0304103的生长(FICI = 0.047)。初步作用机制研究表明, 化合物A1能够抑制耐药白念珠菌生物被膜与菌丝形成, 兼具开发为新型抗真菌先导化合物的潜在价值, 同时为靶向毒力因子研发抑制剂、克服白念珠菌耐药困境提供了新的理论依据与研究策略。

白念珠菌  /  耐药性  /  构效关系  /  小分子抑制剂  /  协同抗真菌

Secreted aspartic protease 2 (SAP2) stands as one of the most pivotal virulence factors in Candida albicans (C. albicans). Consequently, the development of potent small-molecule inhibitors targeting SAP2 represents a critical stride toward the treatment of invasive candidiasis. In our previous studies, the lead compound SAP2-IN-1 was successfully identified and synthesized via integrated strategies of virtual screening and scaffold hopping. Preliminary structure-activity relationship (SAR) investigation was performed through rational design, synthesis, and biological evaluation of 10 novel indolin-2-one derivatives derived from the SAP2-IN-1 scaffold. Enzymatic inhibition and cell-based assays confirmed that compound A1 possesses favorable SAP2 inhibitory activity, with an IC50 of 0.87 μmol·L-1. Notably, when combined with fluconazole (FLC), compound A1 effectively suppressed the growth of fluconazole-resistant C. albicans 0304103, yielding fractional inhibitory concentration index (FICI) of 0.047. Furthermore, preliminary mechanistic studies revealed that A1 significantly impeded biofilm formation and hyphal transition in drug-resistant C. albicans. It possesses great potential as a novel antifungal lead compound and provides a new theoretical basis and research strategy for the development of virulence factor-targeted inhibitors to overcome the drug resistance of C. albicans.

Candida albicans  /  antifungal resistance  /  structure-activity relationship  /  small molecule inhibitor  /  synergistic antifungal
孙华, 苏杰, 武善超. 抗耐药白念珠菌SAP2小分子抑制剂的设计、合成和生物活性评价. 药学学报, 2026 , 61 (6) : 2212 -2220 . DOI: 10.16438/j.0513-4870.2026-0423
Hua SUN, Jie SU, Shan-chao WU. Design, synthesis and bioactivity evaluation of small-molecule inhibitors against SAP2 of drug-resistant Candida albicans[J]. Acta Pharmaceutica Sinica, 2026 , 61 (6) : 2212 -2220 . DOI: 10.16438/j.0513-4870.2026-0423
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侵袭性真菌感染日益严重, 其发病率和死亡率居高不下, 严重威胁人类的生命健康[1-3]。其中白念珠菌占比50%~60%, 是主要的致病菌之一[3-6]。白念珠菌作为典型条件致病性真菌, 在健康人体内通常以共生形式存在; 但当机体免疫功能降低或微生态失衡时, 易突破宿主防御屏障引发感染[7]。由于靶基因突变、药物外排泵高表达、代谢通路代偿及生物被膜屏障等多重因素, 目前临床常用的四类抗真菌药物(图1) 已出现严重的耐药问题[8-13], 导致临床治疗效果显著下降。因此亟需具有新机制和新靶点的抗真菌药物, 尤其是抗耐药白念珠菌的药物[6, 14-17]。
研究表明, 生物被膜形成是白念珠菌产生耐药性的主要原因[18]。分泌型天冬氨酸蛋白酶2 (secreted aspartic protease 2, SAP2) 作为关键酸性水解酶, 参与真菌宿主体内存活、黏膜定植、组织侵袭及免疫逃逸等多个核心致病过程[19, 20]; 多项研究证实, SAP2还是调控白念珠菌生物被膜形成与稳定的重要毒力因子[20-23]。因此, 靶向抑制SAP2活性可有效阻断真菌生长、侵袭及生物被膜介导的耐药, 开发SAP2小分子抑制剂为应对白念珠菌耐药难题提供了全新策略[24-27]。
课题组前期通过虚拟筛选及骨架跃迁, 成功合成具有抗耐药白念珠菌活性的先导化合物SAP2-IN-1[21]。然而, 其构效关系尚不明确, 抗真菌活性有待进一步提升。因此, 本研究针对SAP2并基于药物合理设计, 以SAP2-IN-1为先导化合物, 对其结构中的碳碳双键结构进行还原修饰得到化合物A1 (图2), 其SAP2活性有所提升, 验证了该策略的可行性, 据此设计并合成了10个目标化合物。经过体外酶活抑制实验与协同抗真菌实验, 初步阐明构效关系, 最终发现化合物A1具有显著的SAP2抑制活性及抗真菌活性。
结果与讨论
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1　目标化合物的合成及结构表征
1.1　化学合成
目标化合物的合成路线如合成路线1所示。首先, 以6-溴吲哚酮为起始原料, 与对甲苯基硼酸或硼酸衍生物经Suzuki偶联反应制得中间体1 (合成路线1); 随后, 在碱性条件下进行羟醛缩合反应, 将苯甲醛衍生物偶联至吲哚酮母核上, 得到中间体3 (合成路线1); 最后, 以碳酸铯(Cs2CO3) 为碱, 在吲哚酮的氮原子上引入多种取代基, 再经氢气还原结构中的双键并在酸性条件下脱去叔丁酯, 最终得到目标化合物A1~A10 (合成路线1)。
1.2　结构表征
所合成的吲哚酮衍生物A1~A10的结构经1H NMR、13C NMR和MS确认(结果见实验方法部分)。
2　目标化合物的SAP2抑制活性评价
首先, 采用分光光度法对所有化合物的白念珠菌SAP2抑制活性进行检测, 以肽类抑制剂pepstatin A (PepA) 作为阳性对照[28, 29]。结果如表1所示, 目标化合物对SAP2表现出良好至优异的抑制活性, 其IC50值介于0.21~2.22 μmol·L-1之间。初步构效关系分析发现, 将先导物SAP2-IN-1中的碳碳双键还原时, 所得化合物A1 (IC50 = 0.87 μmol·L-1) 的SAP2抑制活性有所提升。替换成无取代芳香基团A9 (IC50 = 1.06 μmol·L-1) 时, 相比较A1的SAP2抑制活性有所降低。用氯原子替换苯环上的甲基基团时, 其中4-氯苯基(A3)、3, 4-二氯苯基(A4)、3-氯苯基(A7) 的SAP2抑制活性均出现不同程度的提升, 其IC50值分别为0.53、0.21和0.44 μmol·L-1。与之相反, 2-氯苯基A2 (IC50 = 1.13 μmol·L-1) 与4-氟苯基A6 (IC50 = 2.22 μmol·L-1) 的活性有所下降。4-三氟甲氧基苯基A5 (IC50 = 0.81 μmol·L-1) 或4-乙氧酰基苯基A10 (IC50 = 0.92 μmol·L-1) 取代A1中的4-甲基苯基时, 目标化合物的SAP2抑制活性无明显变化, 而4-三氟甲基苯基A8 (IC50 = 1.35 μmol·L-1) 的SAP2抑制活性有所下降。上述结果表明, 还原先导物SAP2-IN-1结构中的碳碳双键, 所得化合物均对SAP2有抑制活性。其中以3, 4-二氯苯基(A4) 的SAP2抑制活性为最强。
3　目标化合物的体外协同抗真菌活性评价
采用美国临床和实验室标准协会(Clinical and Laboratory Standards Institute, CLSI, M27-A3) 制定棋盘微量稀释法[30]对目标化合物进行体外协同抗真菌的活性测定, 抗真菌活性以达到80%抑制所需的最低抑菌浓度(MIC80) 表示, 通过计算协同指数(fractional inhibitory concentration index, FICI) 评估协同效应(FICI ≤ 0.5时判定存在协同作用)。结果如表2所示, 与氟康唑(fluconazole, FLC) 联用时, 化合物A1对耐药C. albicans 0304103表现出优异的协同作用, 其FICI值为0.047。与先导物SAP2-IN-1 (FICI = 0.562) 相比有显著提升, 进一步证实了先导物结构中的碳碳双键为非必需基团。通过对目标化合物进一步的活性分析, 在吲哚啉-2-酮的氮(N) 原子上引入氯原子及疏水侧链的芳香取代基有较好的体外协同抗真菌活性, 例如2-氯苯基A2 (FICI = 0.250)、4-氯苯基A3 (FICI = 0.281)、3, 4-二氯苯基A4 (FICI = 0.250)、3-氯苯基A7 (FICI = 0.188)。4-乙氧酰基苯基A10 (FICI = 0.156)。与之相反, 4-氟苯基A6 (FICI = 1.000)、4-三氟甲氧基苯基A5 (FICI = 0.531) 与无取代苯基A9 (FICI = 0.531) 的体外协同抗真菌效果较差。综上所述, 结构较小的甲基疏水性侧链化合物A1与FLC联用时对C. albicans 0304103的协同效应最佳。
4　化合物A1与FLC联用抑制C. albicans 0304103生物被膜的形成
研究表明, 生物被膜的形成是导致白念珠菌耐药的重要因素[31], 而SAP2是白念珠菌感染过程中的关键毒力因子。据此本研究进一步评估了化合物A1对生物被膜形成的抑制效果。当化合物SAP2-IN-1与1 μg·mL-1高浓度FLC联合使用时, 未观察到协同抑制作用(图3A)。与之相反, 在FLC浓度为1 μg·mL-1时, 64 μg·mL-1的化合物A1对生物膜形成达到60%的抑制率(图3B)。当FLC质量浓度降至0.5 μg·mL-1时, 64 μg·mL-1的化合物A1对生物膜形成达到60%的抑制率(图3C)。其对生物膜形成的抑制效果与化合物A1的浓度呈正相关, 而受FLC浓度变化的影响较小。上述结果表明, 该联合用药方案可有效抑制FLC耐药白念珠菌的生物膜形成。
5　化合物A1与FLC联用抑制C. albicans 0304103菌丝的形成
菌丝有助于真菌的黏附和侵入宿主, 并且对生物膜的形成至关重要[32]。基于生物膜抑制活性, 化合物A1被选用来评估其对FLC耐药白念珠菌从酵母形态向菌丝形态转变的影响。如图4所示, 单独使用化合物A1或FLC (64 μg·mL-1) 时均能观察到菌丝形成。当化合物A1 (64 μg·mL-1) 与FLC (64 μg·mL-1) 联合使用时可完全抑制菌丝形成。当化合物A1与FLC均降至32 μg·mL-1时, 其协同抑制菌丝形成能力减弱。上述结果表明, 化合物A1可与FLC协同作用, 阻断C. albicans 0304103的菌丝形成, 并显著降低其侵袭能力。
6　结论
本研究以SAP2-IN-1作为先导化合物并基于药物合理设计合成了10个新型吲哚酮衍生物。其中, 优选化合物A1表现出显著的抗SAP2活性(IC50 = 0.87 μmol·L-1) 与体外协同抗真菌活性(FICI = 0.047)。化合物A1通过抑制C. albicans 0304103生物被膜与菌丝的形成, 阻碍其生长与侵袭。本研究不仅验证了SAP2抑制剂作为抗白念珠菌的潜力, 同时为抗白念珠菌药物研发提供了先导化合物, 为解决白念珠菌的耐药性提供了新思路。最后, 针对该系列化合物的结构优化、生物活性评价、作用机制研究及成药性评估将在本实验室深入开展。
实验部分
收起
化学合成部分使用的原料均采购自探索平台、毕得医药、超聪化工等公司平台, 纯度均为分析纯和化学纯, 可直接使用。BSC-1004IIA2生物安全柜(苏州安泰空气技术有限公司); 低温离心机(赛默飞世尔科技公司); MJ-150-Ⅰ霉菌培养箱(上海一恒科学仪器有限公司); Tecan Infinite M200多功能酶标仪[帝肯(上海) 实验器材有限公司]; 电喷雾离子化质谱仪(ESI-MS, 美国Agilent公司); 1H NMR和13C NMR谱由德国Bruker公司Bruker-AVANCEIII 600型核磁共振仪进行测试, 氘代试剂为DMSO-d6, 以三甲基氯硅烷为内参, 化学位移(δH) 和耦合常数(J) 分别用ppm和Hz表示。目标产物使用HPLC检测, 纯度均高于95%。
1　化学合成
1.1　(对甲苯基)吲哚啉-2-酮(1) 的合成
将6-溴吲哚啉-2-酮(5.00 g, 23.70 mmol) 溶于60 mL的1, 4-二氧六环∶水(10∶1, v/v) 溶液中, 随后加入对甲苯硼酸(4.19 g, 30.81 mmol)、K2CO3 (8.17 g, 59.25 mmol), 最后加入Pd(PPh3)4 (1.09 g, 0.95 mmol)。置于氮气氛围下, 于100 ℃回流反应8 h。反应结束后, 向体系中加入100 mL的乙酸乙酯(ethyl acetate, EA) 稀释, 并用200 mL的饱和食盐水洗涤3次。有机相经无水硫酸钠(Na2SO4) 干燥后过滤, 减压浓缩得到粗品。经硅胶柱层析纯化, 洗脱剂为石油醚(petroleum ether, PE) 与EA的混合液(PE∶EA = 2∶1, v/v), 得到棕色固体化合物1 (3.62 g, 收率68.5%)。1H NMR (400 MHz, DMSO-d6) δH: 10.45 (s, 1H), 7.50 (d, J = 7.9 Hz, 2H), 7.27 (d, J = 7.6 Hz, 3H), 7.19 (dd, J = 7.7, 1.6 Hz, 1H), 7.01 (d, J = 1.6 Hz, 1H), 3.51 (s, 2H), 2.35 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δC: 177.01, 144.92, 140.29, 138.04, 137.19, 130.02, 126.94, 125.23, 120.04, 107.61, 36.08, 21.14; ESI-MS m/z [M+H]+ 224.28。
1.2　2,2′-((4-甲酰基-1,3-亚苯基)双(氧基))二乙酸二叔丁酯(2) 的合成
将2,4-二羟基苯甲醛(10.00 g, 72.46 mmol) 溶于丙酮(250 mL) 中, 随后加入2-溴乙酸叔丁酯(26.46 g, 159.42 mmol)、碳酸钾(K2CO3, 40.00 g, 298.84 mmol) 及碘化钾(KI, 1.20 g, 7.25 mmol)。反应混合物于室温搅拌24 h后过滤, 滤液经减压浓缩。所得残留物通过硅胶柱层析纯化(洗脱剂: PE∶EA = 3∶1, v/v), 得到白色固体化合物2 (19.16 g, 收率85.3%)。1H NMR (600 MHz, DMSO-d6) δH: 10.29 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 6.70 (d, J = 2.2 Hz, 1H), 6.66 (dd, J = 8.7, 1.5 Hz, 1H), 4.88 (s, 2H), 4.80 (s, 2H), 1.45 (d, J = 2.5 Hz, 18H); 13C NMR (151 MHz, DMSO-d6) δC: 187.84, 167.79, 167.61, 164.66, 162.46, 129.92, 119.57, 108.60, 100.64, 82.19, 66.26, 65.75, 28.19; ESI-MS m/z [M+H]+ 367.53。
1.3　2,2′-((4-((1-(4-甲基苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A1) 的合成
将化合物1 (1.00 g, 4.48 mmol) 溶于乙醇(100 mL) 中, 随后加入二乙胺(0.65 g, 8.96 mmol) 及化合物2 (1.97 g, 5.38 mmol)。反应混合物于78 ℃搅拌反应8 h, 真空浓缩除去乙醇。随后, 将残留物溶于DCM (20 mL) 中, 用PE稀释, 此时有固体析出, 过滤得到黄色粗品化合物3。随后取该粗品200 mg溶于20 mL的DMF中, 加入4-甲基苄溴(96.7 mg, 0.53 mmol) 与碳酸铯(284.6 mg, 0.88 mmol), 70 ℃搅4 h。随后用EA萃取, 硅胶柱层析纯化(洗脱剂: PE∶EA = 2∶1, v/v), 得黄色粗品并重新溶于50 mL的甲醇中, 加入20 mg Pd/C在H2下搅拌至黄色消失, 用硅藻土过滤除去Pd/C, 减压抽滤后复溶于20% TFA (v/v) 的DCM (15 mL) 中, 搅拌1 h后, 旋干并用制备HPLC分离(0.1% TFA (w/w) in H2O/MeCN = 50%~70%, 30 min)。旋干得白色固体A1, 85.1 mg, 收率43%。1H NMR (600 MHz, DMSO-d6) δH: 7.45 (d, J = 8.1 Hz, 2H), 7.25~7.20 (m, 4H), 7.14~7.08 (m, 4H), 7.03 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 7.7 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.02~4.89 (m, 2H), 4.73 (s, 2H), 4.66 (s, 2H), 4.11 (dd, J = 9.0, 5.5 Hz, 1H), 3.48~3.43 (m, 1H), 2.77 (dd, J = 13.8, 9.1 Hz, 1H), 2.32 (s, 3H), 2.25 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.47, 170.65, 170.59, 158.29, 157.24, 144.19, 140.18, 137.77, 137.28, 136.95, 134.08, 131.98, 129.94, 129.72, 128.11, 127.71, 126.96, 125.34, 120.22, 119.67, 107.37, 105.87, 100.41, 65.34, 65.23, 44.63, 42.72, 31.15, 21.11; HRMS (ESI) m/z Calcd. for C34H31NO7 [M+H]+ 566.217 3, found 566.211 8。
1.4　2,2′-((4-((1-(2-氯苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A2) 的合成
合成方法同化合物A1, 得白色固体73.8 mg, 收率36%。1H NMR (600 MHz, DMSO-d6) δH: 12.98 (s, 2H), 7.52 (dd, J = 8.0, 1.3 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.33~7.30 (m, 1H), 7.27~7.24 (m, 1H), 7.22 (d, J = 7.9 Hz, 2H), 7.15 (dd, J = 7.8, 1.6 Hz, 1H), 7.06~7.03 (m, 2H), 7.00 (d, J = 7.7 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H), 5.12~5.01 (m, 2H), 4.72 (s, 2H), 4.67 (s, 2H), 4.17 (dd, J = 8.9, 5.6 Hz, 1H), 3.47 (dd, J = 13.5, 5.4 Hz, 1H), 2.83 (dd, J = 13.7, 9.0 Hz, 1H), 2.31 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.53, 170.64, 170.61, 158.34, 157.26, 144.13, 140.33, 137.65, 137.32, 133.97, 132.50, 132.06, 130.04, 129.94, 129.52, 128.24, 128.05, 128.02, 126.94, 125.53, 120.45, 119.61, 107.11, 105.92, 100.44, 65.36, 65.24, 44.73, 41.10, 31.11, 21.11; HRMS (ESI) m/z Calcd. for C33H28ClNO7 [M+H]+ 586.162 7, found 586.157 4。
1.5　2,2′-((4-((1-(4-氯苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A3) 的合成
合成方法同A1, 88.2 mg, 收率43%。1H NMR (600 MHz, DMSO-d6) δH: 12.99 (s, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.14~7.11 (m, 2H), 7.03 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.03 (d, J = 15.8 Hz, 1H), 4.96 (d, J = 15.9 Hz, 1H), 4.71 (s, 2H), 4.66 (s, 2H), 4.12 (dd, J = 9.0, 5.5 Hz, 1H), 3.45 (dd, J = 13.7, 5.5 Hz, 1H), 2.78 (dd, J = 13.7, 9.1 Hz, 1H), 2.33 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.52, 170.62, 170.57, 158.30, 157.24, 144.01, 140.27, 137.70, 137.31, 136.23, 132.41, 131.99, 129.94, 129.62, 129.08, 128.09, 126.99, 125.44, 120.36, 119.62, 107.22, 105.91, 100.42, 65.35, 65.22, 44.65, 42.31, 31.10, 21.13; ESI-MS m/z [M+H]+ 586.36。
1.6　2,2′-((4-((1-(3, 4-二氯苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A4) 的合成
合成方法同化合物A1, 得白色固体84.6 mg, 收率39%。1H NMR (600 MHz, DMSO-d6) δH: 7.66 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 8.1 Hz, 2H), 7.24 (t, J = 8.8 Hz, 3H), 7.18 (d, J = 1.6 Hz, 1H), 7.14 (dd, J = 7.7, 1.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.01 (q, J = 16.1 Hz, 2H), 4.72 (s, 2H), 4.66 (s, 2H), 4.14 (dd, J = 8.8, 5.7 Hz, 1H), 3.45 (dd, J = 13.6, 5.7 Hz, 1H), 2.80 (dd, J = 13.8, 8.9 Hz, 1H), 2.32 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.64, 170.63, 170.58, 158.29, 157.23, 143.87, 140.34, 138.46, 137.64, 137.34, 131.91, 131.66, 131.33, 130.50, 129.97, 129.92, 128.09, 127.95, 127.01, 125.49, 120.48, 119.59, 107.14, 105.88, 100.42, 65.35, 65.21, 44.67, 41.95, 30.99, 21.11; HRMS (ESI) m/z Calcd. for C33H27Cl2NO7 [M+H]+ 620.123 7, found 620.118 4。
1.7　2,2′-((4-((2-氧代-6-(对甲苯基)-1-(4-(三氟甲氧基)苄基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A5) 的合成
合成方法同化合物A1, 得白色固体93.4 mg, 收率42%。1H NMR (600 MHz, DMSO-d6) δH: 7.48~7.42 (m, 4H), 7.33 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.15~7.12 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H), 5.10~4.99 (m, 2H), 4.72 (s, 2H), 4.66 (s, 2H), 4.13 (dd, J = 8.9, 5.6 Hz, 1H), 3.46 (dd, J = 13.5, 5.5 Hz, 1H), 2.80 (dd, J = 13.7, 9.1 Hz, 1H), 2.32 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.55, 170.64, 170.57, 158.31, 157.25, 148.00, 144.04, 140.31, 137.70, 137.32, 136.69, 132.02, 129.92, 129.60, 128.11, 127.00, 125.47, 121.68, 120.40, 119.62, 107.19, 105.89, 100.41, 65.34, 65.20, 44.66, 42.28, 31.12, 21.10; HRMS (ESI) m/z Calcd. for C34H28F3NO8 [M+H]+ 636.184 0, found 636.179 1。
1.8　2,2′-((4-((1-(4-氟苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A6) 的合成
合成方法同化合物A1, 得白色固体73.7 mg, 收率37%。1H NMR (600 MHz, DMSO-d6) δH: 7.47 (d, J = 8.1 Hz, 2H), 7.38~7.35 (m, 2H), 7.24 (d, J = 7.9 Hz, 2H), 7.18~7.14 (m, 2H), 7.12 (d, J = 7.6 Hz, 2H), 7.03 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.04~4.93 (m, 2H), 4.72 (s, 2H), 4.66 (s, 2H), 4.12 (dd, J = 9.0, 5.5 Hz, 1H), 3.45 (dd, J = 13.7, 5.5 Hz, 1H), 2.78 (dd, J = 13.8, 9.1 Hz, 1H), 2.33 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.51, 170.63, 170.59, 162.69, 161.08, 158.29, 157.23, 144.05, 140.25, 137.72, 137.30, 133.39, 131.98, 129.94, 129.82, 129.76, 128.11, 126.99, 125.41, 120.32, 119.63, 115.96, 115.82, 107.26, 105.89, 100.41, 65.34, 65.22, 44.64, 42.24, 31.08, 21.13; HRMS (ESI) m/z Calcd. for C33H28FNO7 [M+H]+ 570.192 3, found 570.187 5。
1.9　2,2′-((4-((1-(3-氯苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A7) 的合成
合成方法同化合物A1, 得白色固体71.7 mg, 收率35%。1H NMR (600 MHz, DMSO-d6) δH: 7.49~7.45 (m, 3H), 7.38~7.32 (m, 2H), 7.26~7.23 (m, 3H), 7.16 (d, J = 1.6 Hz, 1H), 7.13 (dd, J = 7.8, 1.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.54 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.4, 2.4 Hz, 1H), 5.06~4.98 (m, 2H), 4.73 (s, 2H), 4.66 (s, 2H), 4.14 (dd, J = 8.8, 5.6 Hz, 1H), 3.46 (dd, J = 13.7, 5.5 Hz, 1H), 2.79 (dd, J = 13.8, 9.0 Hz, 1H), 2.33 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.62, 170.65, 158.29, 157.23, 144.00, 140.28, 139.79, 137.67, 137.34, 133.71, 131.90, 131.04, 129.94, 128.09, 127.84, 127.71, 126.99, 126.29, 125.44, 120.41, 119.61, 107.21, 105.87, 100.42, 65.36, 65.22, 44.64, 42.45, 30.99, 21.12; HRMS (ESI) m/z Calcd. for C33H28ClNO7 [M+H]+ 586.162 7, found 586.157 8。
1.10　2,2′-((4-((2-氧代-6-(对甲苯基)-1-(4-(三氟甲基)苄基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A8) 的合成
合成方法同化合物A1, 得白色固体91.2 mg, 收率42%。1H NMR (600 MHz, DMSO-d6) δH: 13.00 (s, 2H), 7.71 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.15~7.13 (m, 2H), 7.04 (d, J = 8.2 Hz, 1H), 6.81~6.79 (m, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 8.3, 2.4 Hz, 1H), 5.14 (d, J = 16.2 Hz, 1H), 5.07 (d, J = 16.2 Hz, 1H), 4.72 (s, 2H), 4.66 (d, J = 3.2 Hz, 2H), 4.15 (dd, J = 8.9, 5.6 Hz, 1H), 3.46 (dd, J = 13.6, 5.4 Hz, 1H), 2.81 (dd, J = 13.7, 9.0 Hz, 1H), 2.32 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.57, 170.60, 170.53, 158.32, 157.27, 144.02, 142.09, 140.33, 137.67, 137.31, 132.02, 129.91, 128.40, 128.10, 127.00, 126.01, 125.98, 125.50, 120.43, 119.63, 107.14, 105.96, 100.45, 65.39, 65.24, 44.69, 42.61, 31.13, 21.11; ESI-MS m/z [M+H]+ 620.41。
1.11　2,2′-((4-((1-苄基-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A9) 的合成
合成方法同化合物A1, 得白色固体79.3 mg, 收率41%。1H NMR (600 MHz, DMSO-d6) δH: 13.00 (s, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.36~7.31 (m, 4H), 7.28~7.25 (m, 1H), 7.23 (d, J = 7.9 Hz, 2H), 7.11 (dd, J = 7.6, 1.7 Hz, 1H), 7.10 (s, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.04 (d, J = 15.8 Hz, 1H), 4.97 (d, J = 15.8 Hz, 1H), 4.73 (d, J = 2.0 Hz, 2H), 4.67 (s, 2H), 4.13 (dd, J = 9.0, 5.5 Hz, 1H), 3.47 (dd, J = 13.7, 5.5 Hz, 1H), 2.78 (dd, J = 13.8, 9.1 Hz, 1H), 2.32 (s, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.51, 170.62, 170.57, 158.31, 157.25, 144.21, 140.22, 137.76, 137.28, 137.14, 131.98, 129.93, 129.10, 128.12, 127.77, 127.68, 126.96, 125.36, 120.27, 119.70, 107.33, 105.93, 100.44, 65.38, 65.27, 44.65, 42.99, 31.13, 21.12; ESI-MS m/z [M+H]+ 552.38。
1.12　2,2′-((4-((1-(4-乙氧羰基苄基)-2-氧代-6-(对甲苯基)吲哚啉-3-基)甲基)-1,3-亚苯基)双(氧基))二乙酸(A10) 的合成
合成方法同化合物A1, 得白色固体83.1 mg, 收率38%。1H NMR (600 MHz, DMSO-d6) δH: 12.98 (s, 2H), 7.92 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 7.13 (dd, J = 7.7, 1.6 Hz, 1H), 7.08 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.44 (dd, J = 8.3, 2.4 Hz, 1H), 5.12 (d, J = 16.3 Hz, 1H), 5.05 (d, J = 16.2 Hz, 1H), 4.71 (s, 2H), 4.66 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 4.14 (dd, J = 8.9, 5.5 Hz, 1H), 3.46 (dd, J = 13.7, 5.5 Hz, 1H), 2.81 (dd, J = 13.7, 9.0 Hz, 1H), 2.32 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δC: 177.54, 170.61, 170.52, 165.94, 158.30, 157.26, 144.03, 142.60, 140.28, 137.68, 137.30, 132.06, 129.96, 129.92, 129.46, 128.08, 127.85, 126.97, 125.48, 120.38, 119.63, 107.20, 105.92, 100.45, 65.38, 65.23, 61.16, 44.72, 42.78, 31.11, 21.11, 14.62; ESI-MS m/z [M+H]+ 624.47。
2　体外协同抗真菌活性测试
2.1　底物
H2N-Dabcyl-Arg-Lys-Pro-Ala-Leu-Phe-Phe-Arg-Leu-Glu(EDANS)-Arg-CO2H [生工生物工程(上海) 股份有限公司定制]; 阳性药: PepA (Sigma)、SAP2酶(上海陶术生物技术有限公司原核表达)。
2.2　SAP2酶的使用浓度的确定
① 底物用DMSO配置成18.75 μmol·L-1浓度的待用溶液; ② SAP2酶用pH 4.5柠檬酸钠缓冲液稀释10倍, 室温活化2 h。酶的使用浓度通过水解18.75 μmol·L-1浓度的底物, 当荧光强度变化量为100 U·min-1时, 此浓度下的酶的浓度即为酶的使用浓度。(比如酶与底物作用10 min, 荧光变化1 000 U选定此时的浓度)。
2.3　实验方法
阳性药PepA用DMSO配制成10 μmol·L-1的溶液, 其他待测样品用DMSO溶解, 配制成100 μmol·L-1的溶液。①将185 µL柠檬酸钠缓冲液、5 µL SAP2酶、5 µL抑制剂依次加入黑色96孔板, 设置双复孔, PepA做阳性对照, 未加入抑制剂的DMSO做空白。溶液体系充分混合振荡, 30 ºC孵育30 min; ②加入18.75 μmol·L-1底物5 µL, 振板混合均匀, 37 ℃孵育1 h后测定化合物荧光发光(λex = 340/30 nm, λem = 485/20 nm)。根据单位时间荧光发光的增加程度, 计算出每个浓度下抑制剂的抑制率; ③采用GraphPad Prism 10软件进行非线性回归分析, 绘制剂量-效应曲线并计算半数抑制浓度(IC50)。
3　体外协同抗真菌活性测试
将目标化合物与阳性药FLC均配制成2 mg·mL-1的DMSO溶液。
3.1　活化待测菌株
于-80 ℃低温冰箱中取出冻存的待测菌株(C. albicans 0304103, 由海军军医大学附属长征医院提供), 吸取10 μL菌液装入含1 mL YEPD培养基的15 mL摇菌管中, 30 ℃气浴恒温振荡培养箱中, 200 r·min-1培养24 h后, 取10 μL菌液置于新的含1 mL YEPD培养基的15 mL摇菌管中, 继续振荡孵育16 h。
3.2　配制菌悬液
将活化后的菌液置于1.5 mL的离心管中, 离心1 min (3 000 r·min-1)。吸弃上清液, 用1 mL PBS缓冲液洗涤菌株, 离心, 重复洗涤3次。用生物显微镜计数, 最后用RPMI 1640培养基配成(1×103 CFU·mL-1) 的菌悬液。
3.3　FICI的测定
将配制好的菌悬液涡旋后转移至6孔板中, 第一孔加2.6 mL的菌悬液, 其余孔加1.3 mL, 接着在第一孔里加83.2 μL的待测样品(此时待测样品浓度为64 μg·mL-1), 依次对半稀释。将在6孔板中配制好的含药菌悬液按化合物浓度从高到低(64~2 μg·mL-1) 依次转移至96孔细胞培养板的A~F行, 第1列加200 μL, 2~10列加100 μL。第11列的A11~F11孔和第G行的G1~G10孔分别加100 μL的未加药的菌悬液作为阴性对照。将配制好的分别加入第一列的A1~G1孔中分别加入6.4 μL的FLC溶液使其浓度为64 μg·mL-1, 对半稀释至第9列。第12列和H行分别加入100 μL的RPMI 1640培养基作为空白对照组。将96孔板置于35 ℃恒温培养箱孵育48 h后, 使用酶标仪测定每孔的A630值。根据公式(1) 计算出抑菌率及MIC80, 再根据公式(2) 计算协同指数FICI。
$\begin{aligned}\text {抑菌率 }= & \left(A_{630 \text { 阴性对照组 }}-A_{630 \text { 药物组 }}\right) /\left(A_{630 \text { 阳性对照组 }}-\right. \\& \left.A_{630 \text { 药物组 }}\right) \times 100 \%\end{aligned}$
$\begin{aligned}\mathrm{FICI}= & \mathrm{MIC}_{80 \text { 化合物 (联用) } / \mathrm{MIC}_{80 \text { 化合物 (单用) }}+} \\& \mathrm{MIC}_{80 \text { FLC (联用) }} / \mathrm{MIC}_{80 \text { FLC }(\text { 单用) }}\end{aligned}$
4　生物膜形成抑制实验
将活化后的C. albicans 0304103配制成(1×106 CFU·mL-1) 的RPMI 1640的菌悬液。随后将配制好的菌悬液涡旋后转移至96孔板中, 每孔100 μL。置于37 ℃恒温培养箱孵育1.5 h, 使菌株完成黏附并初步形成生物被膜。接着加入100 μL的含有不同浓度的待测化合物的RPMI 1640, 以FLC为阳性对照。于37 ℃恒温培养箱孵育24 h。最后吸弃RPMI 1640并用PBS清洗两次后, 加入120 μL的XTT/甲萘醌溶液(含0.5 mg·mL-1 XTT钠盐和1 μmol·L-1甲萘醌的PBS溶液), 继续孵育3 h (避光)。最后利用酶标仪测定492 nm下的各孔的A492值。根据公式(3) 计算生物被膜形成率。
$\begin{aligned}\text { 生物被膜形成率 }= & \left(A_{492 \text { 药物组 }}-A_{492 \text { 空白组 }}\right) / \\& \left(A_{492 \text { 阳性对照组 }}-A_{492 \text { 空白组 }}\right) \times 100 \%\end{aligned}$
5　菌丝形成抑制实验
将活化后的C. albicans 0304103配制成(1×105 CFU·mL-1) 的RPMI 1640的菌悬液。将配制好的菌悬液涡旋后转移至6孔板中, 每孔4 mL。接着加入不同浓度的待测化合物, 以FLC为阳性对照, DMSO为空白对照, 将6孔板置于37 ℃恒温培养箱孵育3~4 h后, 置于倒置荧光显微镜下, 观察并拍照记录。
6　统计学方法
对每组数据平行测试3次, 平均值±标准差表示, 使用GraphPad Prism 10软件作图和分析, 通过单因素分析计算显著性差异, P < 0.05表示差异有统计学意义。

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2026年第61卷第6期
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doi: 10.16438/j.0513-4870.2026-0423
  • 接收时间：2026-04-15
  • 首发时间：2026-09-21
  • 出版时间：2026-06-12
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  • 收稿日期：2026-04-15
  • 修回日期：2026-04-27
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    中国人民解放军海军军医大学药学院, 上海 200433

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武善超, Tel: 86-21-81871242, E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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