药学学报

No.	Inhibition ratio/%		No.	Inhibition ratio/%		No.	Inhibition ratio/%
2a	16.60		2h	15.20		2o	18.80
2b	36.70	2i	35.77	2p	63.38
2c	13.34	2j	39.62	2q	45.33
2d	40.08	2k	57.52	2r	16.63
2e	75.21	2l	63.22	2s	-2.93
2f	50.85	2m	77.28	2t	-5.34
2g	40.42	2n	43.05	Baicalein	66.93

No.

Inhibition ratio/%

No.

Inhibition ratio/%

No.

Inhibition ratio/%

16.60

15.20

18.80

36.70

35.77

63.38

13.34

39.62

45.33

40.08

57.52

16.63

75.21

63.22

-2.93

50.85

77.28

-5.34

40.42

43.05

Baicalein

66.93

No.	Inhibition ratio/%		No.	Inhibition ratio/%		No.	Inhibition ratio/%
2a	16.60		2h	15.20		2o	18.80
2b	36.70	2i	35.77	2p	63.38
2c	13.34	2j	39.62	2q	45.33
2d	40.08	2k	57.52	2r	16.63
2e	75.21	2l	63.22	2s	-2.93
2f	50.85	2m	77.28	2t	-5.34
2g	40.42	2n	43.05	Baicalein	66.93

No.

Inhibition ratio/%

No.

Inhibition ratio/%

No.

Inhibition ratio/%

16.60

15.20

18.80

36.70

35.77

63.38

13.34

39.62

45.33

40.08

57.52

16.63

75.21

63.22

-2.93

50.85

77.28

-5.34

40.42

43.05

Baicalein

66.93

No.	Docking score	LogP	H-bound acceptor	H-bound donor	Ligand binding site	IC₅₀ /μmol·L^-1
2e	-7.3	4.8	5	3	Cys145, Gly143, Leu141, His163, Glu166, His41, Met49, Met165	0.98
2m	-5.8	2.5	5	5	Cys145, Gly143, Leu141, His41, Met49	0.36
2p	-5.7	3.2	4	4	Cys145, Ser144, Leu141, Met49, His41, Met49	1.89
Baicalein	-7.0	2.8	5	3	Cys145, Gly143, Asn142, Leu141, Met165, Glu166, Met49, His44, Cys44	0.93

No.

Docking score

LogP

H-bound acceptor

H-bound donor

Ligand binding site

IC₅₀ /μmol·L^-1

-7.3

4.8

Cys145, Gly143, Leu141, His163, Glu166, His41, Met49, Met165

0.98

-5.8

2.5

Cys145, Gly143, Leu141, His41, Met49

0.36

-5.7

3.2

Cys145, Ser144, Leu141, Met49, His41, Met49

1.89

Baicalein

-7.0

2.8

Cys145, Gly143, Asn142, Leu141, Met165, Glu166, Met49, His44, Cys44

0.93

No.	Docking score	LogP	H-bound acceptor	H-bound donor	Ligand binding site	IC₅₀ /μmol·L^-1
2e	-7.3	4.8	5	3	Cys145, Gly143, Leu141, His163, Glu166, His41, Met49, Met165	0.98
2m	-5.8	2.5	5	5	Cys145, Gly143, Leu141, His41, Met49	0.36
2p	-5.7	3.2	4	4	Cys145, Ser144, Leu141, Met49, His41, Met49	1.89
Baicalein	-7.0	2.8	5	3	Cys145, Gly143, Asn142, Leu141, Met165, Glu166, Met49, His44, Cys44	0.93

No.

Docking score

LogP

H-bound acceptor

H-bound donor

Ligand binding site

IC₅₀ /μmol·L^-1

-7.3

4.8

Cys145, Gly143, Leu141, His163, Glu166, His41, Met49, Met165

0.98

-5.8

2.5

Cys145, Gly143, Leu141, His41, Met49

0.36

-5.7

3.2

Cys145, Ser144, Leu141, Met49, His41, Met49

1.89

Baicalein

-7.0

2.8

Cys145, Gly143, Asn142, Leu141, Met165, Glu166, Met49, His44, Cys44

0.93

靶向冠状病毒Mpro的邻苯三酚与金刚烷类衍生物设计与合成

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马超洁 , 敖选丽 , 何蕾 , 王淑冰 , 周丽珠 , 和润婷 , 李建秀 , 孙燕虹 , 张琴 , 张荣平 , 车彦云 ^* , 侯博 ^*

药学学报 | 研究论文 2026,61(4): 1267-1275

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药学学报 |研究论文 2026 , 61 (4) : 1267 -1275

靶向冠状病毒Mpro的邻苯三酚与金刚烷类衍生物设计与合成

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马超洁, 敖选丽, 何蕾, 王淑冰, 周丽珠, 和润婷, 李建秀, 孙燕虹, 张琴, 张荣平, 车彦云^*, 侯博^*

作者信息

云南中医药大学中药学院, 云南省南药可持续利用研究重点实验室, 云南昆明 650500

通讯作者:

车彦云, Tel: 18213538189, E-mail: checpu@163.com

侯博, Tel: 18787498859, E-mail: kib_drug@163.com

作者简介:

共同第一作者.

作者贡献:

马超洁、敖选丽完成部分化学工作及文章撰写; 何蕾、和润婷完成部分化学工作; 李建秀、孙燕虹、张琴完成部分分子对接; 王淑冰、周丽珠完成部分活性测试工作; 车彦云、侯博、张荣平负责实验设计及文章修改。

Design and synthesis of pyrogallol and adamantane derivatives targeting coronavirus Mpro

Chao-jie MA, Xuan-li AO, Lei HE, Shu-bing WANG, Li-zhu ZHOU, Run-ting HE, Jian-xiu LI, Yan-hong SUN, Qin ZHANG, Rong-ping ZHANG, Yan-yun CHE^*, Bo HOU^*

Affiliations

School of Chinese Materia Medica & Yunnan, Key Laboratory of Southern Medicine Utilization, Yunnan University of Chinese Medicine, Kunming 650500, China

出版时间: 2026-04-12 doi: 10.16438/j.0513-4870.2025-1030

文章导航

摘要

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通过酰氯法和碳二亚胺缩合法, 设计、合成含金刚烷的邻苯三酚类化合物, 以期获得更优抗冠状病毒活性苗头化合物。所合成的20个目标化合物均通过质谱、¹H NMR和¹³C NMR技术完成结构表征, 并测试冠状病毒主蛋白酶(main protease, Mpro) 抑制活性与分子对接。对20个靶向Mpro的邻苯三酚类似物进行活性筛选, 发现化合物2e和2m的IC₅₀值分别为0.98和0.36 μmol·L^-1, 化合物2e与阳性对照(黄芩素) IC₅₀较为接近。化合物2m与黄芩素相比活性更优, IC₅₀值约是黄芩素的0.4倍, 分子对接结果显示它与SARS-CoV-2 Mpro主要通过氢键、疏水键相互作用。本研究所得苗头化合物2m为抗冠状病毒药物开发提供新的物质基础与研究思路。

关键词

邻苯三酚类似物 / 合成 / 金刚烷类 / 冠状病毒 / 主蛋白酶

Abstract

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The pyrogallol compounds containing adamantanes were designed and synthesized by acyl chloride method and carbodiimide condensation method in order to obtain better anti-coronavirus activity compounds. All the 20 target compounds were characterized by mass spectrometry, ¹H NMR and ¹³C NMR, and the coronavirus main protease (Mpro) inhibitory activity and molecular docking were tested. The activity of 20 pyrogallol analogues targeting Mpro was screened. The IC₅₀ values of compounds 2e and 2m were 0.98 and 0.36 μmol·L^-1, respectively. Compound 2e is close to the positive drug (baicalein) IC₅₀. Compared with baicalein, compound 2m has better activity, and its IC₅₀ value is about 0.4 times that of baicalein. The molecular docking results show that it interacts with SARS-CoV-2 Mpro mainly through hydrogen bonds and hydrophobic bonds. The hit compound 2m obtained in this study provides a new material basis and research ideas for the development of anti-coronavirus drugs.

Key words

pyrogallol analogue / synthesis / adamantanes / coronavirus / main protease

引用本文

马超洁, 敖选丽, 何蕾, 王淑冰, 周丽珠, 和润婷, 李建秀, 孙燕虹, 张琴, 张荣平, 车彦云, 侯博. 靶向冠状病毒Mpro的邻苯三酚与金刚烷类衍生物设计与合成. 药学学报, 2026 , 61 (4) : 1267 -1275 . DOI: 10.16438/j.0513-4870.2025-1030

Chao-jie MA, Xuan-li AO, Lei HE, Shu-bing WANG, Li-zhu ZHOU, Run-ting HE, Jian-xiu LI, Yan-hong SUN, Qin ZHANG, Rong-ping ZHANG, Yan-yun CHE, Bo HOU. Design and synthesis of pyrogallol and adamantane derivatives targeting coronavirus Mpro[J]. Acta Pharmaceutica Sinica, 2026 , 61 (4) : 1267 -1275 . DOI: 10.16438/j.0513-4870.2025-1030

正文

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冠状病毒是一类具有包膜的正股单链RNA病毒, 根据系统分类学可分为α、β、γ和δ 4个属, 其中β属冠状病毒对人类健康威胁最大^[1]。β属可再分为A、B、C型, 包括HCoV-OC43和HCoV-HKUl, 非典型性肺炎病毒(SARS-CoV) 和2019新型冠状病毒(2019-nCoV) 及中东呼吸综合征冠状病毒(MERS-CoV)^[2]。全球范围内由SARS-CoV-2引起的新冠肺炎疫情已导致数百万人死亡, 威胁着公共健康和安全。新冠病毒变异毒株已经发现超1 000种, 值得关注的新冠病毒变异株主要有α、β、γ、δ与ο 5种。冠状病毒的靶点主要有主蛋白酶(main protease, Mpro)、RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)、木瓜样蛋白酶(papain-like protease, PLpro)、S蛋白^[3]。Mpro又称为3C样蛋白酶(3C-like protease, 3CLpro) 作为冠状病毒复制转录的核心调控元件, 因其序列高度保守性, 并且在人体没有同源的蛋白, Mpro已成为抗新冠病毒药物研发的核心靶点之一^{[4, 5]}。Mpro抑制剂不仅能抑制已有冠状病毒增殖, 还具有抑制新型冠状毒株的潜力, 这使其成为开发广谱抗病毒药物的理想靶点。已上市的Mpro抑制剂有帕昔洛韦(奈玛特韦+ 利托那韦)^[6]、恩替利韦^[7]、先诺欣(先诺特韦+ 利托那韦)^[8]、来瑞特韦(leritrelvir)^{[9, 10]}和泰中定(阿泰特韦+ 利托那韦)^[11]等。奈玛特韦、先诺特韦、阿泰特韦、来瑞特韦分别运用氰基和α-酮酰胺基团作为亲电弹头与Mpro的Cys145残基共价结合, 抑制Mpro活性从而抑制新冠病毒增殖, 随着Mpro抑制剂的广泛使用, 其耐药问题值得关注。

已有研究表明, 一些天然产物也具有抑制Mpro的潜在活性, 其中黄酮类化合物占据一定的比例。Hu等^[12]对有潜在活性的黄酮类化合物进行筛选表明, 其与SARS-CoV-2的关键Mpro、PLpro和RdRp等蛋白结合, 从而抑制冠状病毒的增殖。近年来已有研究表明, 不同类型黄酮类化合物对冠状病毒Mpro有潜在的抑制作用, 甲氧基黄酮类化合物^[13]: 高车前素、泽兰黄酮; 黄酮醇类化合物: 草质素(herbacetin)^[14]; 黄酮类: 木犀草素(luteolin)^[15]; 黄烷-3-醇类: (-)-gallocatechin-3-O-gallate^[16]; 黄酮苷类: 野漆树苷(rhoifolin)^[14]、柳穿鱼叶苷(pectolinarin)^[14]、槲皮素苷^[16]等(图1)。

通过荧光共振能量转移(fluorescence resonance energy transfer, FRET) 测定具有共价抑制作用的黄芩素(baicalein) 对Mpro的IC₅₀为0.94 μmol·L^-1 ^[17]。通过X-射线发现, 黄芩素的3个酚羟基与Mpro的Leu141/Gly143的主链以及Ser144/His163的侧链形成多个氢键^[17]。Su等^[18]通过Mpro与杨梅素(myricetin) 及其衍生物结合的晶体结构揭示了邻苯三酚基团作为亲电试剂与Mpro的催化位点Cys145进行共价结合, 并证明了邻苯三酚可以用作亲电弹头。基于上述研究, Wu等^[19]对二氢杨梅素(dihydromyricetin) 进行结构改造合成二氢杨梅素衍生物, 体外药理研究显示对Mpro表现出良好的抑制活性, 该类衍生物中以邻苯三酚基团为典型的共价片段。金刚烷是一种作用于甲型流感病毒M2蛋白的化合物, 因其耐药性和不良反应现很少用于抗甲型流感。在新冠疫情期间其抑制SARS-CoV-2的体外药理活性已被报道^[20-22], 金刚烷在人体代谢度低, 绝大部分以原形药物通过肾脏随尿液排出, 可在金刚烷片段的基础上引入的邻苯三酚基团有望提高药物代谢度。其次, 冠状病毒Mpro结构中S2位点对应的空腔较大, 与之对应的底物P2位置往往选择体积较大的疏水基团, 使其能充分地与空腔接触。因此, 将邻苯三酚作为能与S1′位点作用的亲电弹头, 金刚烷作为P2片段与蛋白S2位点产生疏水相互作用, 使目标化合物与Mpro形成稳定的蛋白-小分子复合物。综上, 开发具有广谱的冠状病毒Mpro抑制剂对于解决现有药物耐药、供需不均、高价等问题十分重要。本研究将邻苯三酚类化合物与金刚烷类片段通过酰胺键、酯键连接获得一系列邻苯三酚类似物, 以期筛选出具有成药价值的抗冠状病毒活性的苗头化合物。

结果与讨论

收起

1　化学合成

以化合物2和盐酸金刚烷乙胺作为模版底物进行反应条件筛选。选用二氯甲烷(dichloromethane, DCM) 作为反应溶剂, 分别采用酰氯法及1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDCI]/4-二甲氨基吡啶(4-dimethylaminopyridine, DMAP) 缩合法合成中间体1a, 并对两种方法的产率进行对比, 以筛选出最优合成工艺。采用酰氯法和缩合剂合成1a的产率分别为60.8%和45.39%, 因此主要运用酰氯法合成后续化合物。

化合物2a~2g的合成如合成路线1所示, 以没食子酸(1) 为起始原料, 用乙酰基保护酚羟基得到化合物2。再将化合物2制成酰氯得到3, 化合物3分别与7个金刚烷类化合物反应得到中间物1a~1g, 最后将乙酰基脱保护得到终产物2a~2g。

化合物2h~2n的合成如合成路线2所示, 将2, 3, 4-三甲氧基苯甲酸(4) 制成酰氯(5), 化合物5分别与7个金刚烷类化合物通过酸胺缩合与酯化反应得到中间体1h~1n, 最后将酚羟基上的甲基去保护得到终产物2h~2n。

由于3, 4, 5-三甲氧基苯乙酸(6) 所制成的酰氯属于脂肪酰氯, 相较于上述两种芳香酰氯更活泼, 使用该酰氯反而使反应体系副产物增多, 而采用EDCI和DMAP缩合, 反应体系副产物减少。因此, 3, 4, 5-三甲氧基苯乙酸(6) 与金刚烷类似物则使用EDCI和DMAP缩合, 再将中间体用三溴化硼去保护得到终产物2o~2t (合成路线3), 目标化合物2a~2t结构如图2所示。

N-(1-(金刚烷-1-基)乙基)-3, 4, 5-三羟基苯甲酰胺(2a), 淡灰色油状物, 分子式为C₁₉H₂₅NO₄, 产率为31.3%。¹H NMR (400 MHz, MeOD) δ_H: 6.83 (s, 2H), 3.81 (q, J = 6.9 Hz, 1H), 2.01~1.60 (m, 15H), 1.10 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.47, 146.61, 137.81, 126.91, 107.81, 54.66, 39.70, 38.21, 37.84, 29.91, 14.17; ESI (m/z): [M-H]^- 330.17。

N-(金刚烷-1-基)-3, 4, 5-三羟基苯甲酰胺(2b), 红色固体, 分子式为C₁₇H₂₁NO₄, 产率为83.37%。¹H NMR (400 MHz, MeOD) δ_H: 6.75 (s, 2H), 2.17~1.65 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.18, 146.50, 137.62, 127.89, 107.67, 53.22, 42.33, 37.53, 30.98; ESI (m/z): [M-H]^- 302.14。

N-(双环[1.1.1]戊-1-基)-3, 4, 5-三羟基苯甲酰胺(2c), 淡黄色固体, 分子式为C₁₂H₁₃NO₄, 产率为80.2%。¹H NMR (400 MHz, MeOD) δ_H: 6.82 (s, 2H), 2.44 (s, 1H), 2.14 (s, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.76, 146.57, 138.11, 126.29, 107.83, 53.69, 25.78; ESI (m/z): [M-H]^- 234.08。

N-(3, 5-二甲基金刚烷-1-基)-3, 4, 5-三羟基苯甲酰胺(2d), 白色固体, 分子式为C₁₉H₂₅NO₄, 产率为46.02%。¹H NMR (400 MHz, MeOD) δ_H: 6.74 (s, 1H), 1.86 (s, 13H), 0.86 (d, J = 12.4 Hz, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.26, 146.50, 137.63, 127.89, 107.68, 54.89, 51.80, 43.83, 40.79, 33.32, 31.66, 30.74, 23.77; ESI (m/z): [M-H]^- 330.17。

金刚烷-1-基甲基-3, 4, 5-三羟基苯甲酸酯(2e), 淡黄绿色结晶, 分子式为C₁₈H₂₂O₅, 产率为62.82%。¹H NMR (400 MHz, MeOD) δ_H: 7.08 (s, 2H), 3.80 (s, 2H), 2.08~1.58 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 168.60, 146.50, 139.69, 121.72, 109.97, 75.21, 40.46, 38.06, 34.56, 29.57; ESI (m/z): [M-H]^- 317.14。

N-(金刚烷-1-基甲基)-3, 4, 5-三羟基苯甲酰胺(2f), 棕红色固体, 分子式为C₁₈H₂₃NO₄, 产率为65.68%。¹H NMR (400 MHz, MeOD) δ_H: 6.85 (s, 2H), 3.02 (s, 2H), 1.96 (s, 3H), 1.79~1.49 (m, 13H); ¹³C NMR (101 MHz, MeOD) δ_C: 171.00, 146.62, 137.86, 126.69, 107.78, 52.18, 41.48, 38.08, 35.85, 29.81; ESI (m/z): [M-H]^- 316.16。

3, 4, 5-三羟基-N-(3-羟基金刚烷-1-基)苯甲酰胺(2g), 棕红色油状物, 分子式为C₁₇H₂₁NO₅, 产率为46.5%。¹H NMR (400 MHz, MeOD) δ_H: 6.74 (s, 1H), 2.30~1.53 (m, 14H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.38, 146.52, 137.74, 127.68, 107.75, 69.71, 55.71, 44.94, 41.10, 36.16, 32.12; ESI (m/z): [M-H]^- 318.14。

N-(1-(金刚烷-1-基)乙基)-2, 3, 4-三羟基苯甲酰胺(2h), 灰色粉末, 分子式为C₁₇H₂₁NO₄, 产率为40.57%。¹H NMR (400 MHz, MeOD) δ_H: 7.25 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 3.86 (q, J = 6.9 Hz, 1H), 2.03~1.59 (m, 15H), 1.12 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.46, 150.61, 133.95, 120.01, 109.94, 107.81, 54.28, 39.71, 38.20, 37.55, 29.92, 14.35; ESI (m/z): [M-H]^- 330.18。

N-(金刚烷-1-基)-2, 3, 4-三羟基苯甲酰胺(2i), 褐色粉末, 分子式为C₁₇H₂₁NO₄, 产率为69.31%。¹H NMR (400 MHz, MeOD) δ_H: 7.14 (d, J = 8.8 Hz, 1H), 6.34 (d, J = 8.8 Hz, 1H), 2.22~1.68 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.21, 146.51, 137.62, 127.92, 119.73, 107.68, 53.23, 42.35, 37.55, 31.00; ESI (m/z): [M-H]^- 302.16。

N-(双环[1.1.1]戊-1-基)-2, 3, 4-三羟基苯甲酰胺(2j), 棕色固体, 分子式为C₁₂H₁₃NO₄, 产率为39.3%。¹H NMR (400 MHz, MeOD) δ_H: 7.10 (d, J = 8.8 Hz, 1H), 6.33 (d, J = 8.8 Hz, 1H), 2.45 (s, 1H), 2.16 (s, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 172.57, 151.89, 150.72, 133.93, 119.24, 108.79, 107.72, 53.79, 25.87; ESI (m/z): [M-H]^- 234.07。

N-(3, 5-二甲基金刚烷-1-基)-2, 3, 4-三羟基苯甲酰胺(2k), 棕色固体, 分子式为C₁₉H₂₅NO₄, 产率为68.42%。¹H NMR (400 MHz, MeOD) δ_H: 7.14 (d, J = 8.8 Hz, 1H), 6.33 (d, J = 8.8 Hz, 1H), 2.04~1.08 (m, 13H), 0.88 (s, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 170.87, 150.85, 150.34, 133.88, 119.75, 110.43, 107.57, 55.02, 51.80, 43.83, 40.93, 33.36, 31.69, 30.73; ESI (m/z): [M-H]^- 330.18。

金刚烷-1-基甲基-2, 3, 4-三羟基苯甲酸酯(2l), 淡红色结晶, 分子式为C₁₈H₂₂O₅, 产率为11.83%。¹H NMR (400 MHz, MeOD) δ_H: 7.29 (d, J = 8.9 Hz, 1H), 6.41 (d, J = 8.8 Hz, 1H), 3.90 (s, 2H), 2.05~1.62 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 171.78, 152.61, 152.29, 133.71, 122.04, 108.56, 106.25, 75.18, 40.41, 34.59, 29.57; ESI (m/z): [M-H]^- 317.16。

N-(金刚烷-1-基甲基)-2, 3, 4-三羟基苯甲酰胺(2m), 淡黄色粉末, 分子式为C₁₈H₂₃NO₄, 产率为54.68%。¹H NMR (400 MHz, MeOD) δ_H: 7.20 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 3.06 (s, 2H), 2.02~1.90 (m, 3H), 1.83~1.53 (m, 12H); ¹³C NMR (101 MHz, MeOD) δ_C: 171.55, 150.99, 150.61, 133.98, 119.55, 109.49, 107.80, 51.72, 41.49, 38.07, 35.61, 29.83; ESI (m/z): [M-H]^- 316.16。

2, 3, 4-三羟基-N-(3-羟基金刚烷-1-基)苯甲酰胺(2n), 淡黄色针状结晶, 分子式为C₁₇H₂₁NO₅, 产率为40.49%。¹H NMR (400 MHz, MeOD) δ_H: 7.16 (d, J = 8.9 Hz, 1H), 6.34 (d, J = 8.8 Hz, 1H), 2.74 (s, 2H), 2.35~1.99 (m, 10H), 1.76~1.69 (m, 2H); ¹³C NMR (101 MHz, MeOD) δ_C: 171.02, 150.94, 150.53, 133.90, 119.86, 110.14, 107.64, 64.23, 55.96, 53.34, 40.67, 35.51, 33.93, 30.75; ESI (m/z): [M-H]^- 318.13。

N-(1-(金刚烷-1-基)乙基)-2-(3, 4, 5-三羟基苯基)乙酰胺(2o), 黄色油状物, 分子式为C₂₀H₂₇NO₄, 产率为89.75%。¹H NMR (400 MHz, MeOD) δ_H: 6.32 (s, 2H), 3.57 (q, J = 6.9 Hz, 1H), 3.32 (s, 2H), 1.96~1.45 (m, 15H), 1.01 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ_C: 174.04, 147.19, 127.80, 109.18, 54.39, 43.80, 39.39, 38.11, 37.36, 29.82, 14.30; ESI (m/z): [M-H]^- 344.21。

N-(金刚烷-1-基)-2-(3, 4, 5-三羟基苯基)乙酰胺(2p), 褐色粉末, 分子式为C₁₈H₂₃NO₄, 产率为86.08%。¹H NMR (400 MHz, MeOD) δ_H: 6.27 (s, 2H), 3.20 (s, 2H), 1.85 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 173.82, 147.07, 133.08, 128.13, 109.01, 52.85, 44.50, 42.27, 37.47, 30.90; ESI (m/z): [M-H]^- 316.18。

N-(双环[1.1.1]戊-1-基)-2-(3, 4, 5-三羟基苯基)乙酰胺(2q), 棕红色油状物, 分子式为C₁₃H₁₅NO₄, 产率为93.51%。¹H NMR (400 MHz, MeOD) δ_H: 3.23 (s, 2H), 2.40 (s, 1H), 2.07 (s, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 175.18, 146.93, 133.14, 126.96, 108.91, 53.45, 43.22, 41.25, 25.52; ESI (m/z): [M-H]^- 248.11。

N-(3, 5-二甲基金刚烷-1-基)-2-(3, 4, 5-三羟基苯基)乙酰胺(2r), 黄色固体, 分子式为: C₂₀H₂₇NO₄, 产率为58.33%。¹H NMR (400 MHz, MeOD) δ_H: 6.27 (s, 2H), 3.20 (s, 2H), 2.14~1.07 (m, 13H), 0.83 (s, 6H); ¹³C NMR (101 MHz, MeOD) δ_C: 173.83, 147.03, 133.05, 128.08, 109.00, 54.48, 51.70, 48.20, 44.47, 43.73, 40.68, 33.21, 31.53, 30.66; ESI (m/z): [M-H]^- 344.21。

金刚烷-1-基甲基-2-(3, 4, 5-三羟基苯基)乙酸酯(2s), 淡黄色粉末, 分子式为C₁₉H₂₄O₅, 产率为56.33%。¹H NMR (400 MHz, MeOD) δ_H: 6.29 (s, 2H), 3.66 (s, 2H), 3.38 (s, 2H), 2.03~1.42 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 174.20, 147.01, 126.49, 109.36, 75.17, 42.03, 40.17, 38.00, 34.53, 29.52; ESI (m/z): [M-H]^- 331.18。

N-((金刚烷-1-基)甲基)-2-(3, 4, 5-三羟基苯基)乙酰胺(2t), 淡黄色固体, 分子式为C₁₉H₂₅NO₄, 产率为69.87%。¹H NMR (400 MHz, MeOD) δ_H: 6.31 (s, 2H), 3.30 (s, 2H), 2.85 (s, 2H), 1.95~1.41 (m, 15H); ¹³C NMR (101 MHz, MeOD) δ_C: 174.93, 147.15, 133.19, 127.78, 109.20, 52.00, 43.77, 41.17, 37.99, 29.72; ESI (m/z): [M-H]^- 330.19。

2　Mpro抑制试验

在1 μmol·L^-1浓度下, 测定目标化合物2a~2t对SARS-CoV-2 Mpro的抑制活性结果见表1, 化合物2e和2m对Mpro的抑制率略高于阳性对照黄芩素, 其余化合物活性均低于阳性对照。

将活性较好的目标化合物2e、2m、2p与阳性对照分别连续稀释配制成8个浓度梯度8、4、2、1、0.5、0.25、0.125、0.062 5 μmol·L^-1, 对其进行SARS-CoV-2 Mpro抑制作用评价, 测定IC₅₀值。结果表明, 化合物2e、2m、2p均表现出较为明显的浓度依赖性抑制活性(图3)。化合物2e和2m的IC₅₀值分别为0.98和0.36 μmol·L^-1。化合物2e与阳性对照活性较为接近, 化合物2m显示出较好的抑制活性。从结构上看, 化合物2m与黄芩素的羰基都有邻近的酚羟基。化合物2m中将邻苯三酚片段和金刚烷片段间连接的碳原子有3个, 这一特点与黄芩素C₆-C₃-C₆的结构特点较为相似。因此, 该结构特点可能是化合物2m活性较好的原因, 而化合物2e与黄芩素相比活性略低, 推测结构中羰基邻近的酚羟基对抑制Mpro活性起关键作用。

3　分子对接

将黄芩素、2e、2m、2p与SARS-CoV-2 Mpro (PDB: 6m2n) 进行分子对接, 结果如表2所示。从对接结果可以看出化合物2e比阳性配体(黄芩素) 得分更高, 表明2e与Mpro蛋白的活性口袋亲和力更高。预测化合物2m的疏水参数(LogP值) 为2.5在0~3内, 表明该化合物具有良好体内吸收的潜力。此外, 化合物2m也是这些化合物中氢键供体数和受体数最多的化合物。

将上述活性较好的化合物2m与SARS-CoV-2 Mpro进行对接后的可视化见图4, 该化合物中邻苯三酚片段通过氢键与蛋白相互作用, 金刚烷片段主要与蛋白发生疏水相互作用。由于Mpro的Cys145-His41催化二联体影响冠状病毒Mpro的催化活性和整体构象的折叠^[23], 因此目标化合物若能与上述两个关键位点形成特异性相互作用将会对Mpro活性产生影响。化合物2m的邻苯三酚片段可与Cys145、Leu141、Gly143等残基发生共价的氢键相互作用, 金刚烷基团与His41氨基酸通过疏水相互作用。阳性配体黄芩素能与Glu166、Met165、Leu141、Asn142、Gly143等残基发生氢键相互作用, A环和C环能与Cys145通过氢键和π-硫键发生共价相互作用, His41与B环形成π-π相互作用。化合物2m和黄芩素二者与Mpro的作用方式比较相似, 邻苯三酚片段均与Leu141、Gly143残基形成氢键, 且两者都能与Cys145形成疏水相互作用, 金刚烷片段和苯环都与关键残基His41分别形成Alkyl-π、π-π疏水作用, 这些作用力可以增强分子间结合力。不同的是化合物2m其中一个酚羟基能直接与Cys145产生氢键相互作用, 推测化合物2m不仅与Cys145形成共价结合模式, 金刚烷片段与His41等残基还形成有利疏水相互作用阻止底物进入活性口袋的S1′和S2位点, 进而达到增强抑制Mpro活性的效果。

4　讨论

Mpro是冠状病毒抑制剂开发重要的靶点, 许多含有邻苯三酚片段的天然产物具有潜在的抑制Mpro的活性, 因此基于该结构片段设计、合成邻苯三酚类似物, 并期望获得活性较好的苗头分子。对所合成的目标化合物进行Mpro的抑制活性测试及分子对接, 结果显示化合物2m活性高于黄芩素, 该化合物作用于Mpro的活性口袋的催化二联体Cys145-His41位点上。

实验部分

收起

所有试剂和溶剂源于商业购买。使用硅胶(200~300目) 进行柱层析, 采用薄层色谱硅胶板(青岛海洋化工厂)、紫外灯观察(上海嘉鹏科技有限公司) 监测反应, 在Bruker AM 400 MHz型核磁共振仪(德国Bruker公司) 上测试NMR谱, 并分析核磁数据。采用Agilent 1290型超高效液相色谱-Agilent 6530型四极杆飞行时间串联质谱(美国Agilent公司) 获得质谱数据。

1　合成方法

1.1　没食子酸乙酰化

将3, 4, 5-三羟基苯甲酸(化合物1, 10 g, 1 eq.) 加入乙酸酐溶液(40 mL, 7 eq.) 中, 搅拌使其充分溶解。随后缓慢滴加浓硫酸(100 μL, 0.1 eq.), 滴加完毕后, 先将反应混合物置于室温条件下搅拌15 min; 待室温搅拌阶段结束, 将反应体系转移至油浴环境中, 控制油浴温度为90 ℃, 继续搅拌反应15 min。反应完成后, 立即将反应液趁热倒入冰水中, 持续搅拌直至白色沉淀完全析出。最后通过抽滤收集析出的固体, 得到白色粉末状产物, 即化合物2。将化合物2与化合物1采用正己烷-乙酸乙酯(1∶2, v/v) 进行薄层色谱层析, 化合物2比1极性小。¹H NMR (400 MHz, MeOD) δ_H: 7.78 (s, 2H), 2.31 (s, 9H); ¹³C NMR (101 MHz, MeOD) δ_C: 169.44, 168.25, 167.40, 144.90, 140.03, 130.14, 123.27, 20.38, 20.07; ESI (m/z): [M-H]^- 295.05。

1.2　酰氯的制备

将化合物2 (1.2g, 1 eq.) 溶于DCM中, 缓慢滴加草酰氯(4.5 mL, 13 eq.), 最后缓慢滴加1~2滴DMF, 反应4 h后, 将反应溶剂蒸干得到化合物3, 用于后续反应。化合物4采用类似方法合成化合物5。

1.3　制备中间体

1.3.1　5-((1-(金刚烷-1-基)乙基)氨基甲酰基)苯-1, 2, 3-三乙酸酯(1a)

将化合物3 (200 mg, 1.1 eq.) 缓慢滴加到溶于DCM的盐酸金刚乙胺(187 mg, 1.5 eq.) 和三乙胺(200 μL, 2.5 eq.) 中, 反应过夜。待反应完全后用1 mol·L^-1 HCl和乙酸乙酯萃取, 无水硫酸钠干燥有机相, 减压浓缩得到粗产物。将粗产物用200~300目硅胶柱层析, 正己烷-乙酸乙酯(3∶1, v/v) 进行洗脱, 得到中间体1a, 产率60.8%。ESI (m/z): [M+H]⁺ 458.22。采用类似方法合成1b~1g。

1.3.2　5-((金刚烷-1-基)氨基甲酰基)苯-1, 2, 3-三乙酸酯(1b)

以1-金刚烷胺为起始原料, 采用1a合成方法, 得到中间体1b, 产率56.27%。ESI (m/z): [M+H]⁺ 430.19。

1.3.3　5-(双环[1.1.1]戊-1-基氨基甲酰基)苯-1, 2, 3-三乙酸酯(1c)

以双环[1.1.1]戊烷-1-胺盐酸盐为起始原料, 采用1a合成方法, 得到中间体1c, 产率62.26%。ESI (m/z): [M+H]⁺ 362.12。

1.3.4　5-((3, 5-二甲基金刚烷-1-基)氨基甲酰基)苯- 1, 2, 3-三乙酸酯(1d)

以3, 5-二甲基金刚烷胺为起始原料, 采用1a合成方法, 得到中间体1d, 产率25%。ESI (m/z): [M+H]⁺ 458.22。

1.3.5　5-((金刚烷-1-基甲氧基)羰基)苯-1, 2, 3-三乙酸酯(1e)

以1-金刚烷甲醇为起始原料, 采用1a合成方法, 得到中间体1e, 产率38.94%。¹H NMR (400 MHz, MeOD) δ_H: 7.08 (s, 1H), 3.65 (s, 2H), 2.07~1.90 (m, 12H), 1.81~1.64 (m, 12H); ¹³C NMR (101 MHz, MeOD) δ_C: 173.39, 173.13, 168.60, 146.53, 139.78, 121.65, 110.01, 109.95, 75.17, 40.06, 38.02, 34.17, 29.52, 20.68, 20.49; ESI (m/z): [M+H]⁺ 445.19。

1.3.6　5-((金刚烷-1-基甲基)氨基甲酰基)苯-1, 2, 3-三乙酸酯(1f)

以1-金刚烷甲胺为起始原料, 采用1a合成方法, 得到中间体1f, 产率39.02%。ESI (m/z): [M+H]⁺ 444.20。

1.3.7　5-((3-羟基金刚烷-1-基)氨基甲酰基)苯-1, 2, 3-三乙酸酯(1g)

以3-氨基-1-金刚烷醇为起始原料, 采用1a合成方法, 得到中间体1g, 产率27.24%。ESI (m/z): [M+Na]⁺ 468.16。

1.3.8　 N-(1-(金刚烷-1-基)乙基)-2, 3, 4-三甲氧基苯甲酰胺(1h)

化合物5 (200 mg, 1.1 eq.) 缓慢滴加到溶于DCM的盐酸金刚乙胺(255 mg, 1.5 eq.) 和三乙胺(255 μL, 2.5 eq.) 中, 反应过夜。待反应完全后, 用1 mol·L^-1 HCl和乙酸乙酯萃取, 无水硫酸钠干燥有机相, 减压浓缩得到粗产物。将粗产物用200~300目硅胶柱层析, 正己烷-乙酸乙酯(3∶1, v/v) 进行洗脱, 得到中间体1h, 产率33.96%。ESI (m/z): [M+H]⁺ 374.23。采用类似方法合成1i~1n。

1.3.9　 N-(金刚烷-1-基)-2, 3, 4-三甲氧基苯甲酰胺(1i)

以1-金刚烷胺为起始原料, 采用1h合成方法, 得到中间体1i, 产率73.45%。ESI (m/z): [M+H]⁺ 346.20。

1.3.10　 N-(双环[1.1.1]戊-1-基)-2, 3, 4-三甲氧基苯甲酰胺(1j)

以双环[1.1.1]戊烷-1-胺盐酸盐为起始原料, 采用1h合成方法, 得到中间体1j, 产率64.95%。ESI (m/z): [M+H]⁺ 278.14。

1.3.11　 N-(3, 5-二甲基金刚烷-1-基)-2, 3, 4-三甲氧基苯甲酰胺(1k)

以3, 5-二甲基金刚烷胺为起始原料, 采用1h合成方法, 得到中间体1k, 产率81.51%。ESI (m/z): [M+H]⁺ 374.23。

1.3.12　金刚烷-1-基甲基-2, 3, 4-三甲氧基苯甲酸酯(1l)

以1-金刚烷甲醇为起始原料, 采用1h合成方法, 得到中间体1l, 产率83.74%。ESI (m/z): [M+H]⁺ 361.20。

1.3.13　 N-(金刚烷-1-基甲基)-2, 3, 4-三甲氧基苯甲酰胺(1m)

以1-金刚烷甲胺为起始原料, 采用1h合成方法, 得到中间体1m, 产率84.7%。¹H NMR (400 MHz, CDCl₃) δ_H: 7.91 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 9.0 Hz, 1H), 4.00 (s, 3H), 3.89 (d, J = 7.8 Hz, 6H), 3.16 (d, J = 6.0 Hz, 2H), 2.01~1.97 (m, 4H), 1.77~1.60 (m, 9H), 1.50~1.45 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ_C: 165.18, 156.34, 152.37, 141.80, 126.92, 119.26, 107.65, 61.96, 61.10, 56.17, 51.44, 40.59, 40.03, 37.18, 37.14, 37.04, 33.88, 28.42, 28.32; ESI (m/z): [M+H]⁺ 360.22。

1.3.14　 N-(3-羟基金刚烷-1-基)-2, 3, 4-三甲氧基苯甲酰胺(1n)

以3-氨基-1-金刚烷醇为起始原料, 采用1h合成方法, 得到中间体1n, 产率34.8%。ESI (m/z): [M+Na]⁺ 384.18。

1.3.15　 N-(1-(金刚烷-1-基)乙基)-2-(3, 4, 5-三甲氧基苯基)乙酰胺(1o)

将2, 3, 4–三甲氧基苯乙酸(100 mg, 1.1 eq)、盐酸金刚乙胺(87 mg, 1 eq.)、EDCI (92 mg, 1.2 eq.) 和DMAP (59 mg, 1.2 eq.) 溶于反应溶剂DCM中, 反应过夜。将上述反应用1 mol·L^-1 HCl和乙酸乙酯萃取3次, 无水硫酸钠干燥有机相, 减压浓缩得到粗产物。将粗产物用200~300目硅胶柱层析, 正己烷-乙酸乙酯(3∶1, v/v) 进行洗脱, 得到对应中间体1o, 产率81.15%。ESI (m/z): [M+H]⁺ 388.25。采用类似合成方法合成1p~1t。

1.3.16　 N-(金刚烷-1-基)-2-(3, 4, 5-三甲氧基苯基)乙酰胺(1p)

以1-金刚烷胺为起始原料, 采用1o合成方法, 得到中间体1p, 产率75.57%。¹H NMR (400 MHz, CDCl₃) δ_H: 6.45 (s, 2H), 3.85 (d, J = 6.6 Hz, 9H), 3.41 (s, 2H), 1.95~1.93 (m, 5H), 1.68~1.63 (m, 6H), 1.58 (s, 4H); ¹³C NMR (101 MHz, CDCl₃) δ_C: 170.05, 153.59, 149.45, 131.32, 106.35, 61.04, 56.31, 52.13, 45.43, 41.68, 36.43, 29.53; ESI (m/z): [M+H]⁺ 360.22。

1.3.17　 N-(双环[1.1.1]戊-1-基)-2-(3, 4, 5-三甲氧基苯基)乙酰胺(1q)

以双环[1.1.1]戊烷-1-胺盐酸盐为起始原料, 采用1o合成方法, 得到中间体1q, 产率49.88%; ESI (m/z): [M+H]⁺ 292.15。

1.3.18　 N-(3, 5-二甲基金刚烷-1-基)-2-(3, 4, 5-三甲氧基苯基)乙酰胺(1r)

以3, 5-二甲基金刚烷胺为起始原料, 采用1o合成方法, 得到中间体1r, 产率86.6%。ESI (m/z): [M+H]⁺ 388.25。

1.3.19　金刚烷-1-基甲基2-(3, 4, 5-三甲氧基苯基)乙酸酯(1s)

以1-金刚烷甲醇为起始原料, 采用1o合成方法, 得到中间体1s, 产率97.69%。ESI (m/z): [M+H]⁺ 375.22。

1.3.20　 N-((金刚烷-1-基)甲基)-2-(3, 4, 5-三甲氧基苯基)乙酰胺(1t)

以1-金刚烷甲胺为起始原料, 采用1o合成方法, 得到中间体1t, 产率79.29%。ESI (m/z): [M+H]⁺ 374.23。

1.4　酚羟基上的乙酰基和甲基去保护

将酸胺缩合与酯化反应得到的中间体(1a~1g) 用水合肼对酚羟基上的乙酰基脱保护。采用乙酰基保护的中间体(30 mg, 1 eq.) 溶于DCM反应溶剂中, 加入水合肼(7 eq.), 室温反应1 h后旋干反应。用1 mol·L^-1 HCl和乙酸乙酯萃取, 无水硫酸钠干燥有机相, 减压浓缩得到终产物。

将甲基保护酚羟基的中间体(1h~1t) 采用路易斯酸脱保护。在0 ℃下, 将中间体(30 mg, 1 eq.) 溶于DCM中, 缓慢滴加三溴化硼溶液(11 eq.), 15 min后逐渐恢复至室温反应10 h。用冰水淬灭反应, 用乙酸乙酯萃取3次, 无水硫酸钠干燥有机相, 减压浓缩得到终产物。

1.5　Mpro抑制试验

运用FRET蛋白酶分析来测量化合物对SARS-CoV-2 Mpro的抑制活性, 荧光底物MCA-AVLQSGFR-Lys (Dnp)-Lys-NH₂ (Beyotime)。基于FRET的蛋白酶试验如下。将5 μL的SARS-CoV-2 Mpro (浓度为4 μmol·L^-1) 与155 μL缓冲液(50 mmol·L^-1 Tris-HCl, pH 7.2, 1 mmol·L^-1 EDTA) 混合, 加入20 μL不同浓度梯度的化合物, 并于36.9 ℃恒温孵育10 min。再添加20 μL (终浓度为20 μmol·L^-1) 荧光底物来引发反应。使用Bio-Tek SynergyH1 (多功能微孔板检测仪) 平板阅读器在激发波长320 nm和发射波长405 nm的荧光信号下进行测量, 获取荧光强度。并与阴性对照组比较, 计算抑制率。

1.6　分子对接

所有对接和评分计算均使用AutoDockTools 1.5.7进行^[24]。将化合物使用Open Babel GUl能量最小化后导入AutoDockTools, 并对其进行调整电荷、判定化合物的root、选择可扭转键后, 保存为pdbqt格式。从蛋白质数据库中检索SARS-CoV-2 Mpro (PDB: 6m2n), 进行去除水分、加氢、计算电荷、添加原子类型处理完蛋白后保存为pdbqt文件, 并设置对接盒子(center x = -47.06; center y = 0.85; center z = -7.35), 将处理完的化合物和蛋白文件进行对接。

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2026年第61卷第4期

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doi: 10.16438/j.0513-4870.2025-1030

接收时间：2025-08-29
首发时间：2026-09-21
出版时间：2026-04-12

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收稿日期：2025-08-29
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云南中医药大学中药学院, 云南省南药可持续利用研究重点实验室, 云南昆明 650500

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车彦云, Tel: 18213538189, E-mail: checpu@163.com

侯博, Tel: 18787498859, E-mail: kib_drug@163.com

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https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2025-1030

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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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