药学学报


13a	H	CH₂	958		13j	OCH₃	CH₂	> 5 000
13b	H	(CH₂)₃	937	13k	OCH₃	(CH₂)₃	916
13c	H	(CH₂)₄	112	13l	OCH₃	(CH₂)₄	552
13d	H	(CH₂)₅	26	13m	OCH₃	(CH₂)₅	82
13e	H	(CH₂)₆	3.8	13n	OCH₃	(CH₂)₆	11
13f	H	(CH₂)₇	5.9	13o	OCH₃	(CH₂)₇	21
13g	H		173	13p	OCH₃		272
13h	H	2 651	13q	OCH₃	> 5 000
13i	H	507	13r	OCH₃	295
SAHA			13


13a	H	CH₂	958		13j	OCH₃	CH₂	> 5 000
13b	H	(CH₂)₃	937	13k	OCH₃	(CH₂)₃	916
13c	H	(CH₂)₄	112	13l	OCH₃	(CH₂)₄	552
13d	H	(CH₂)₅	26	13m	OCH₃	(CH₂)₅	82
13e	H	(CH₂)₆	3.8	13n	OCH₃	(CH₂)₆	11
13f	H	(CH₂)₇	5.9	13o	OCH₃	(CH₂)₇	21
13g	H		173	13p	OCH₃		272
13h	H	2 651	13q	OCH₃	> 5 000
13i	H	507	13r	OCH₃	295
SAHA			13

Compd.	A549 IC₅₀/μmol·L^-1	HCT116 IC₅₀/μmol·L^-1
13d	3.73 ± 0.16	1.18 ± 0.11
13e	1.74 ± 0.10	2.43 ± 0.16
13f	2.95 ± 0.3	3.46 ± 0.23
13n	2.26 ± 0.18	1.23 ± 0.08
13o	0.89 ± 0.07	0.49 ± 0.03
11a	6.42 ± 0.51	5.56 ± 0.38
11b	11.38 ± 1.05	7.49 ± 0.42
SAHA	1.33 ± 0.09	0.77 ± 0.06

Compd.	A549 IC₅₀/μmol·L^-1	HCT116 IC₅₀/μmol·L^-1
13d	3.73 ± 0.16	1.18 ± 0.11
13e	1.74 ± 0.10	2.43 ± 0.16
13f	2.95 ± 0.3	3.46 ± 0.23
13n	2.26 ± 0.18	1.23 ± 0.08
13o	0.89 ± 0.07	0.49 ± 0.03
11a	6.42 ± 0.51	5.56 ± 0.38
11b	11.38 ± 1.05	7.49 ± 0.42
SAHA	1.33 ± 0.09	0.77 ± 0.06

Compd.	HDAC6 IC₅₀/nmol·L^-1		Compd.	HDAC6 IC₅₀/nmol·L^-1
13e	7.4		13p	9.2
13n	9.3	13q	278
13o	14	SAHA	13

Compd.	HDAC6 IC₅₀/nmol·L^-1		Compd.	HDAC6 IC₅₀/nmol·L^-1
13e	7.4		13p	9.2
13n	9.3	13q	278
13o	14	SAHA	13

Compd.	HDAC1 IC₅₀/nmol·L^-1	HDAC6 IC₅₀/nmol·L^-1	HDAC8 IC₅₀/nmol·L^-1	HDAC11 IC₅₀/nmol·L^-1
13e	3.8	7.4	72	979
13o	21	14	298	1 150
13p	272	9.2	> 5 000	4 695
SAHA	13	13	161	> 5 000

Compd.	HDAC1 IC₅₀/nmol·L^-1	HDAC6 IC₅₀/nmol·L^-1	HDAC8 IC₅₀/nmol·L^-1	HDAC11 IC₅₀/nmol·L^-1
13e	3.8	7.4	72	979
13o	21	14	298	1 150
13p	272	9.2	> 5 000	4 695
SAHA	13	13	161	> 5 000

基于反式-β-芳基烯丙酰四氢异喹啉母核的HDAC抑制剂: 设计、合成及其抗肿瘤活性研究

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高鑫 ¹ , 韩维维 ¹ , 田诗意 ¹ , 方方 ¹^,²^,³^,^* , 马晓东 ¹^,²^,³ , 柴化怡 ¹ , 韩京晶 ¹

药学学报 | 研究论文 2023,58(2): 413-422

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药学学报 | 研究论文 2023, 58(2): 413-422

基于反式-β-芳基烯丙酰四氢异喹啉母核的HDAC抑制剂: 设计、合成及其抗肿瘤活性研究

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高鑫¹, 韩维维¹, 田诗意¹, 方方¹^,²^,³^,^*, 马晓东¹^,²^,³, 柴化怡¹, 韩京晶¹

作者信息

1.安徽中医药大学药学院, 安徽合肥 230012

2.安徽省中医药科学院药物化学研究所, 安徽合肥 230012

3.中药研究与开发安徽省重点实验室, 安徽合肥 230012

通讯作者:

*方方, Tel: 15056913802, E-mail: ffahtcm@126.com

Structurally novel HDAC inhibitors based on the trans-β-arylacryl tetrahydroisoquinoline scaffold: the design, synthesis, and anti-cancer activity

Xin GAO¹, Wei-wei HAN¹, Shi-yi TIAN¹, Fang FANG¹^,²^,³^,^*, Xiao-dong MA¹^,²^,³, Hua-yi CHAI¹, Jing-jing HAN¹

Affiliations

1. College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China

2. Department of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei 230012, China

3. Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Hefei 230012, China

出版时间: 2023-02-12 doi: 10.16438/j.0513-4870.2022-1083

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摘要

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本研究基于前期获得的抗肿瘤反式-β-芳基烯丙酰四氢异喹啉骨架, 设计合成了18个HDAC抑制剂(histone deacetylases inhibitors, HDACis), 并对其进行抗肿瘤活性评价。体外抑酶活性测试结果表明, 化合物13d~13f、13m~13o呈现出优异的HDAC1抑制活性, IC₅₀在个位数纳摩尔或亚纳摩尔级。体外抗增殖实验结果表明, 13o表现出显著的抗增殖活性(A549, IC₅₀ = 0.89 μmol·L^-1; HCT116, IC₅₀ = 0.49 μmol·L^-1), 优于阳性对照vorinostat (SAHA); 此外, 对HDAC1抑制活性最强的13e (IC₅₀ = 3.8 nmol·L^-1) 亦呈现出优良的抗增殖活性(A549, IC₅₀ = 1.74 μmol·L^-1; HCT116, IC₅₀ = 2.43 μmol·L^-1)。Western blot实验表明, 化合物13e可剂量依赖性地上调A549细胞内组蛋白H3和α- tubulin的乙酰化水平。这些结果表明, 化合物13e和13o具有进一步研究价值。

关键词

HDAC抑制剂 / 四氢异喹啉 / 反式-β-芳基烯丙酰基 / 抗肿瘤

Abstract

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In this study, a series of 18 histone deacetylases inhibitors (HDACis), derived from our in-house anti-cancer trans-β-arylacryl 1, 2, 3, 4-tetrahydroisoquinoline-based scaffold, were designed, synthesized, and antitumor evaluated. HDAC1 inhibitory activity assay showed that compounds 13d-13f and 13m-13o demonstrated attractive enzymatic activity with IC₅₀ at single-digit nanomolar or subnanomolar level.In addition, 13o exerted superior anti-proliferative activity (A549, IC₅₀ = 0.89 μmol·L^-1; HCT116, IC₅₀ = 0.49 μmol·L^-1) to that of vorinostat (SAHA).Besides, 13e, with the most potent HDAC1 enzymatic activity (IC₅₀ = 3.8 nmol·L^-1), also displayed attractive cellular activity (A549, IC₅₀ = 1.74 μmol·L^-1; HCT116, IC₅₀ = 2.43 μmol·L^-1). The Western blot analysis illustrated that 13e treatment increased the acetylation of H3 and α-tubulin in a dose-dependent manner in A549 cells. In summary, 13e and 13o deserve further functional investigation.

Key words

HDAC inhibitor / tetrahydroisoquinoline / trans-β-arylacryl / anti-cancer

引用本文

高鑫, 韩维维, 田诗意, 方方, 马晓东, 柴化怡, 韩京晶. 基于反式-β-芳基烯丙酰四氢异喹啉母核的HDAC抑制剂: 设计、合成及其抗肿瘤活性研究. 药学学报, 2023 , 58 (2) : 413 -422 . DOI: 10.16438/j.0513-4870.2022-1083

Xin GAO, Wei-wei HAN, Shi-yi TIAN, Fang FANG, Xiao-dong MA, Hua-yi CHAI, Jing-jing HAN. Structurally novel HDAC inhibitors based on the trans-β-arylacryl tetrahydroisoquinoline scaffold: the design, synthesis, and anti-cancer activity[J]. Acta Pharmaceutica Sinica, 2023 , 58 (2) : 413 -422 . DOI: 10.16438/j.0513-4870.2022-1083

正文

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表观遗传失调与多种疾病的发生和发展密切相关^[1-3]。组蛋白乙酰化/去乙酰化作为最常见的表观遗传学调控方式, 在基因的翻译后修饰中起着关键作用, 其动态平衡由组蛋白乙酰化转移酶(histone acetyltransferases, HATs) 和组蛋白去乙酰化酶(histone deacetylases, HDACs) 共同调节^{[4, 5]}。在人类恶性肿瘤中, HDAC过度激活, 组蛋白去乙酰化作用显著增强, 促进组蛋白和非组蛋白的N末端赖氨酸残基脱乙酰化, 继而使DNA与组蛋白致密结合, 导致抑癌基因转录沉默^{[6, 7]}。由于其过度表达与癌症的发生发展密切相关, HDAC被认为是临床上重要的抗癌靶标之一^[8]。迄今为止, 已有5种HDAC抑制剂(histone deacetylases inhibitors, HDACis) 获批上市, 用于治疗皮肤T细胞淋巴瘤、外周T细胞淋巴瘤和多发性骨髓瘤, 即vorinostat (SAHA, 1)、romidepsin (2)、belinostat (3)、panobinostat (4) 和chidamide (5) (图 1A)^[9-12], 这充分证明了HDAC作为抗癌靶标的应用价值。尽管所公开的HDACis结构多样, 但它们通常包括3个结构单元: 与催化口袋内部Zn²⁺螯合的Zn²⁺结合基团(ZBG)、与催化口袋表面残基相互作用的表面识别基团(CAP), 以及连接CAP与ZBG并占据催化通道的Linker^[13-15]。

1, 2, 3, 4-四氢异喹啉(1, 2, 3, 4-tetrahydroisoquinoline, THIQ) 是一类重要的含氮杂环类化合物, 广泛存在于多种天然产物和合成活性分子中, 具有广泛的药理学作用, 如抗癌、抗抑郁、抗血栓、抗炎、抗病毒等^[16-18], 目前已经成为药物设计所常用的优势片段之一^{[19, 20]}。近年来, 研究人员发现了许多具有潜在抗肿瘤活性的以THIQ为CAP的HDACis, 如化合物6和7 (图 1B)^{[21, 22]}。

反式-β-芳基丙烯酸类化合物, 如肉桂酸、对羟基肉桂酸、阿魏酸等, 在诱导肿瘤细胞凋亡、分化和抑制肿瘤细胞迁移等方面具有应用价值, 被广泛用于药物分子设计^[23-25]。近年来, 药物学家们设计合成了许多具有潜在抗癌活性的含芳基烯丙酰基的THIQ衍生物, 如化合物8 (图 1B)^[26]。

本研究基于课题组化合物库中两种具有体外抗肿瘤活性的反式-β-芳基烯丙酰基THIQ衍生物11a (A549, IC₅₀ = 6.42 μmol·L^-1; HCT116, IC₅₀ = 5.56 μmol·L^-1) 和11b (A549, IC₅₀ = 11.38 μmol·L^-1; HCT116, IC₅₀ = 7.49 μmol·L^-1), 依据HDACis的结构特征, 采用骨架迁越策略, 将11a和11b通过不同Linker与异羟肟酸ZBG共价连接, 设计合成了18个新型HDACis (图 1B)。在测定抑酶活性的基础上, 选择代表性化合物评价其体外抗肿瘤效果, 并开展初步的作用机制研究, 以期能够获得具有进一步研究价值的新型HDACis。

结果与讨论

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1 化合物的合成

目标化合物13a~13r的合成路线如路线1所示。以6, 7-二甲氧基-1, 2, 3, 4-四氢异喹啉盐酸盐(9) 为原料, 与对羟基肉桂酸(10a) 或阿魏酸(10b) 缩合得到相应的反式-β-芳基烯丙酰四氢异喹啉衍生物(中间体11a或11b), 进一步与卤代脂肪酸酯或卤甲基取代的芳酸酯进行O-烷基化反应得到中间体12a~12r, 最后经羟胺解得到目标化合物13a~13r。目标化合物结构均经¹H NMR、¹³C NMR和ESI-HR-MS确证。

2 活性评价

2.1 体外HDAC1抑酶活性实验

在11个Zn²⁺依赖性HDAC亚型中, HDAC1被抑制可产生显著的抗增殖活性^{[27, 28]}。以上市药物SAHA为阳性对照, 测试目标化合物对HDAC1的抑酶效果。结果如表 1所示, 化合物13e~13f、13m~13o对HDAC1表现出良好的抑制活性, IC₅₀在个位数纳摩尔或亚纳摩尔水平。其中, 13e、13f、13n的IC₅₀值分别为3.8、5.9、11 nmol·L^-1, 优于阳性对照SAHA (IC₅₀ = 13 nmol·L^-1)。初步构效关系显示, 连接链的结构与链长对化合物的抑酶活性具有重要影响。与亚甲(杂) 芳基作为连接链的化合物(13g~13i、13p~13r) 相比, 以饱和脂肪碳链为连接链的化合物(13c~13f、13m~13o) 对HDAC1抑制活性更强。其中, 含六碳饱和脂肪链的化合物13e和13n的抑酶活性最佳, 连接链的延长或缩短均会导致活性下降, 这可能与HDAC1细长的催化通道有关, 碳链过长或过短都将不能很好的与活性位点底部的Zn²⁺螯合, 从而使活性减弱。此外, 在苯丙烯酰片段的苯环上引入甲氧基会导致化合物的抑酶活性降低(13m vs 13d; 13n vs 13e; 13o vs 13f)。

2.2 体外抗增殖活性实验

以SAHA为阳性对照, 选取对HDAC1具有良好抑制活性的化合物13d~13f、13n、13o (IC₅₀ < 30 nmol·L^-1), 采用四甲基偶氮唑盐法(MTT比色法) 测试它们对人非小细胞肺癌A549细胞和人结肠癌HCT116细胞的体外抗肿瘤活性。结果如表 2所示, 目标化合物对所测试肿瘤细胞均呈现出良好的增殖抑制作用, IC₅₀值在个位数微摩尔或亚微摩尔级。其中, 化合物13o对两种肿瘤细胞的增殖抑制作用最强, 其IC₅₀值分别为0.89、0.49 μmol·L^-1, 优于阳性对照SAHA。对HDAC1抑制活性最强的化合物13e亦呈现出优良的抗增殖活性, 其IC₅₀值分别为1.74、2.43 μmol·L^-1。

2.3 体外HDAC6抑酶活性实验

HDAC1和HDAC6的抑制活性的差异反映了HDAC亚型选择性^{[29, 30]}。为了初步评价目标化合物的HDAC亚型选择性, 选取以饱和脂肪碳链作为连接链的化合物13e、13n、13o, 以及亚甲基(杂) 芳基作为连接链的化合物13p、13q, 测试它们对HDAC6的抑制活性。结果如表 3所示, 饱和脂肪碳链作为连接链的化合物13e、13n、13o对HDAC1/HDAC6均呈现出显著的抑制活性(HDAC1, IC₅₀ < 21 nmol·L^-1; HDAC6, IC₅₀ < 14 nmol·L^-1), 与SAHA相当或略优。亚甲基(杂) 芳基作为连接链的化合物13p、13q对HDAC6呈现出选择性抑制作用, 与已报道的HDAC6选择性抑制剂结构特征一致^[31-35]。

2.4 体外HDAC8和HDAC11抑酶活性实验

为进一步验证目标化合物的亚型选择性, 以HDAC8、HDAC11分别作为Class I HDACs、Class IV HDACs的代表, 补充测试了代表性化合物13e、13o及13p对此两种HDAC亚型的抑制活性。结果如表 4所示, 化合物13e对HDAC1/HDAC6/HDAC8/HDAC11的抑制活性均强于SAHA, 表明其与SAHA同属于HDAC泛抑制剂。化合物13p对HDAC1/HDAC8/HDAC11无抑制活性或活性较弱, 但对HDAC6呈现出显著的抑制活性(IC₅₀ = 9.2 nmol·L^-1), 提示该化合物可能为HDAC6选择性抑制剂, 这为进一步基于13p设计新型选择性HDACis提供了基础。

2.5 Western blot实验

进一步选择化合物13e, 通过Western blot实验考察了其对A549细胞中HDAC的底物组蛋白H3和α-微管蛋白(α-tubulin) 乙酰化水平表达的影响。结果如图 2A、B所示, 在低至0.5 μmol·L^-1的浓度下, 13e即可显著上调H3和α-tubulin的乙酰化水平, 且具剂量依赖性。化合物对H3和α-tubulin的乙酰化水平的影响, 反映出其在细胞内作用于HDAC靶点。

3 分子对接实验

基于已报道的配体/HDAC1 (PDB ID: 5ICN) 共晶结构^[36]定义结合位点, 将目标化合物分别与其进行分子对接, 以深入讨论该系列化合物构效关系。对接结果表明, 以烷基链为连接链的13b~13f显示出类似的结合模式, 均能够较好地占据受体蛋白的活性口袋: 它们通过单齿配位, 螯合活性口袋底部的Zn²⁺辅因子; 以氢键与受体蛋白极性区的氨基酸残基His140、GLY149产生相互作用; 此外, 通过π-烷基作用与受体蛋白表面结合(图 3A)。其中, 化合物13e与HDAC1的亲和性最强(图 3B), 连接链的延长或缩短(13b~13d、13f) 均会导致与受体蛋白的亲和力下降, 其原因可能是13e的连接链长度更适于分子末端的异羟肟酸螯合Zn²⁺辅因子, 而碳链过长或过短则不利于化合物与Zn²⁺形成配位, 致使活性减弱, 这与HDAC1抑酶实验结果相一致。在本研究所采用的分子对接条件下, 连接链含苯基或杂芳基的化合物13g~13i均未得到结合构象。

为进一步揭示化合物与靶蛋白的可能结合模式, 选取配体/HDAC6 (PDB ID: 5EDU) 共晶结构^[37]作为受体蛋白, 将化合物13e进行进一步分子对接研究。对接结果表明, 13e的六碳饱和脂肪链连接链可顺利进入靶蛋白的催化通道, CAP单元可与HDAC6的疏水蛋白表面形成良好的堆积作用; 13e中羟肟酸基团亦可与HDAC6蛋白活性口袋底部的Zn⁺螯合, 并与His610、Gly619形成氢键相互作用, 且其Linker中O原子可与His651形成氢键相互作用(图 3C)。分子模拟对接结果显示, 化合物13e与HDAC6亦能较好结合, 与其对两种亚型均有显著抑制活性的抑酶实验结果相吻合。

4 小结

本研究基于前期获得的抗肿瘤反式-β-芳基烯丙酰THIQ骨架, 设计合成了18个新型HDACis, 并对其进行抗肿瘤活性评价。体外抑酶活性测试结果表明, 化合物13d~13f、13m~13o呈现出优异的HDAC1抑制活性, IC₅₀在个位数纳摩尔或亚纳摩尔级。体外抗增殖实验结果表明, 13o表现出显著的抗增殖活性, 优于阳性对照SAHA; 此外, 对HDAC1抑制活性最强的13e亦呈现出优良的抗增殖活性。Western blot实验表明, 化合物13e可剂量依赖性地上调A549细胞内组蛋白H3和α-tubulin的乙酰化水平。总之, 化合物13e和13o具有进一步研究价值, 这为今后研究和开发高效、低毒和良好选择性的HDACis奠定基础。

实验部分

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核磁共振波谱仪为Bruker 400 MHz (瑞士Bruker公司); 高分辨质谱为AB SCIEX x500r (美国SCIEX公司); 熔点仪为WRS-1B数字熔点仪(未校正, 上海仪电物理光学仪器有限公司); 实验所售试剂均为市售化学纯或分析纯产品, 反应溶剂经过干燥处理, 其余未经纯化直接使用。

1 化合物合成

1.1 中间体11a~11b的合成

将原料10a~10b (8.0 mmol)、1-乙基-(3-二甲氨基丙基)碳酰二亚胺盐酸盐(EDCI, 9.6 mmol)、1-羟基苯并三唑(HOBt, 9.6 mmol) 和40 mL二氯甲烷依次加入100 mL圆底烧瓶中, 室温搅拌0.5 h, 然后加入6, 7-二甲氧基-1, 2, 3, 4-四氢异喹啉盐酸盐(9, 8.0 mmol)、三乙胺(24.0 mmol), 搅拌反应过夜。TLC监测反应完全, 反应液依次用饱和NaHCO₃溶液、水、饱和食盐水洗涤, 无水硫酸钠干燥, 过滤, 滤液减压浓缩, 硅胶柱色谱纯化[V(石油醚)∶V(乙酸乙酯) = 2∶1], 得白色固体, 产率79%~83%。

1.2 中间体12a~12r的合成

将中间体11a~11b (1.1 mmol)、K₂CO₃ (2.7 mmol) 和DMF (15 mL) 依次加入100 mL三口烧瓶中, 110 ℃下搅拌反应0.5 h后, 加入溴乙酸乙酯(1.4 mmol) 和碘化钾(0.1 mmol), 110 ℃搅拌过夜。TLC监测反应完全, 过滤, 向滤液中倾入水, 二氯甲烷萃取, 合并有机相, 有机相采用饱和食盐水洗涤、无水硫酸钠干燥、抽滤, 滤液减压浓缩, 硅胶柱色谱纯化[V(石油醚)∶V(乙酸乙酯) = 2∶1], 得黄色油状物, 产率42%~61%。

1.3 化合物13a~13r的合成

羟胺甲醇溶液的制备: 在冰浴条件下, 将KOH (2.00 g, 35.7 mmol) 的甲醇溶液(5 mL) 逐滴加入到NH₂OH·HCl (1.65 g, 23.8 mmol) 的甲醇溶液(8.5 mL) 中, 搅拌0.5 h后, 过滤, 所得滤液即为羟胺甲醇溶液。将中间体12a~12r (0.4 mmol) 和新鲜羟胺溶液(3.5 mL) 加入到50 mL圆底烧瓶中, 室温搅拌4 h。TLC监测反应完全, 反应液减压浓缩, 用5%盐酸调节pH值至7~8, 析出固体, 抽滤, 滤饼用少量水洗涤, 收集滤饼, 硅胶柱色谱纯化[V(二氯甲烷)∶V(甲醇) = 50∶1], 产率37%~64%。

13a 淡红色固体, 产率46%, mp 153.1~155.0 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.97 (s, 1H), 9.03 (s, 1H), 7.70 (d, J = 7.8 Hz, 2H), 7.51~7.46 (m, 1H), 7.24~7.14 (m, 1H), 6.99 (d, J = 7.1 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H), 4.53 (s, 2H), 3.87 (s, 2H), 3.76 (s, 3H), 3.72 (s, 3H), 3.63 (t, J = 7.1 Hz, 2H), 2.80~2.65 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 165.06, 164.06, 158.92, 147.41, 141.20, 141.11, 129.60, 128.46, 126.16, 125.44, 116.16, 114.91, 111.94, 110.07, 65.87, 55.55, 55.52, 43.96, 42.90, 28.79; ESI-HR-MS: m/z C₂₂H₂₄N₂O₆ [M+H]⁺计算值413.171 2, 测量值413.170 0。

13b 白色固体, 产率55%, mp 162.3~164.3 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.43 (s, 1H), 8.72 (s, 1H), 7.68 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 15.2 Hz, 1H), 7.18 (d, J = 15.2 Hz, 1H), 6.96 (d, J = 7.9 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H), 4.81 (s, 1H), 4.63 (s, 1H), 4.00 (t, J = 5.8 Hz, 2H), 3.72 (s, 6H), 3.32 (m, 2H), 2.79~2.67 (m, 2H), 2.13 (t, J = 7.0 Hz, 2H), 1.97~1.92 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.06, 165.57, 160.21, 147.86, 147.79, 141.82, 130.16, 128.27, 126.92, 125.72, 115.16, 112.43, 110.53, 105.74, 67.45, 56.01, 55.48, 44.42, 43.32, 29.24, 29.15, 25.22;ESI-HR-MS: m/z C₂₄H₂₈N₂O₆ [M+H]⁺计算值441.202 5, 测量值441.201 4。

13c 红色固体, 产率64%, mp 168.6~170.3 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.38 (s, 1H), 8.70 (s, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 15.3 Hz, 1H), 7.18 (d, J = 15.3 Hz, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H), 4.81 (s, 1H), 4.64 (s, 1H), 4.01 (t, J = 5.9 Hz, 2H), 3.94~3.84 (m, 2H), 3.73 (s, 6H), 2.81~2.72 (m, 2H), 2.03 (t, J = 7.0 Hz, 2H), 1.71~1.63 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.41, 165.45, 160.12, 147.81, 147.75, 141.90, 129.97, 128.15, 126.58, 125.86, 116.09, 115.10, 112.30, 110.42, 67.65, 55.93, 55.84, 44.41, 43.33, 32.35, 29.26, 28.58, 22.23; ESI-HR-MS: m/z C₂₅H₃₀N₂O₆ [M+H]⁺计算值455.218 2, 测量值455.207 4。

13d 红色固体, 产率51%, mp 176.9~179.1 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.35 (s, 1H), 8.67 (s, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.48 (d, J = 15.2 Hz, 1H), 7.18 (d, J = 15.2 Hz, 1H), 6.96 (d, J = 8.7 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H), 4.81 (s, 1H), 4.63 (s, 1H), 4.00 (t, J = 6.4 Hz, 2H), 3.73 (s, 6H), 3.34 (s, 2H), 2.80~2.67 (m, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.74~1.70 (m, 2H), 1.58~1.53 (m, 2H), 1.41~1.38 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.48, 165.56, 160.33, 147.82, 147.72, 141.89, 130.19, 128.13, 126.58, 125.87, 116.07, 115.07, 112.30, 110.42, 67.94, 55.93, 55.82, 44.41, 43.34, 32.68, 29.26, 28.85, 25.63, 25.38; ESI-HR-MS: m/z C₂₆H₃₂N₂O₆ [M+H]⁺计算值469.233 8, 测量值469.232 1。

13e 淡红色固体, 产率40%, mp 185.2~186.7 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.44 (s, 1H), 8.71 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.47 (d, J = 15.2 Hz, 1H), 7.17 (d, J = 15.2 Hz, 1H), 6.95 (d, J = 8.1 Hz, 2H), 6.81 (s, 1H), 6.75 (s, 1H), 4.81 (s, 1H), 4.63 (s, 1H), 3.99 (t, J = 6.2 Hz, 2H), 3.87 (d, J = 6.9 Hz, 2H), 3.72 (s, 6H), 2.81~2.70 (m, 2H), 1.97 (t, J = 7.1 Hz, 2H), 1.72~1.68 (m, 2H), 1.53~1.49 (m, 2H), 1.40~1.36 (m, 2H), 1.31~1.26 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.59, 165.51, 160.35, 148.30, 147.85, 141.92, 130.27, 128.11, 127.68, 125.84, 120.71, 115.09, 112.30, 110.65, 67.98, 55.94, 55.83, 44.38, 43.26, 32.67, 30.43, 28.99, 28.81, 25.71, 25.55;ESI-HR-MS: m/z C₂₇H₃₄N₂O₆ [M+H]⁺计算值483.249 5, 测量值483.247 5。

13f 红色固体, 产率51%, mp 190.9~192.6 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 8.39 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 15.3 Hz, 1H), 7.18 (d, J = 15.3 Hz, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.82 (s, 1H), 6.76 (s, 1H), 4.81 (s, 1H), 4.64 (s, 1H), 4.00 (t, J = 6.5 Hz, 2H), 3.88 (t, J = 6.8 Hz, 2H), 3.73 (s, 6H), 2.81~2.66 (m, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.79~1.63 (m, 2H), 1.58~1.45 (m, 2H), 1.43~1.36 (m, 2H), 1.35~1.29 (m, 2H), 1.28~1.22 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.58, 165.52, 160.36, 147.85, 147.78, 141.88, 130.19, 128.11, 126.85, 125.87, 116.06, 115.08, 112.31, 110.42, 68.01, 55.94, 44.40, 43.37, 32.70, 29.24, 29.06, 28.99, 28.95, 25.87, 25.53; ESI-HR-MS: m/z C₂₈H₃₆N₂O₆ [M+H]⁺计算值497.265 1, 测量值497.266 0。

13g 白色固体, 产率54%, mp 159.0~160.8 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 7.94 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.46 (s, 1H), 7.23~7.16 (m, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.4 Hz, 1H), 6.76 (s, 1H), 5.23 (s, 2H), 4.81 (s, 1H), 4.63 (s, 1H), 3.90 (s, 2H), 3.73 (s, 6H), 2.80~2.72 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 164.41, 159.76, 147.81, 147.75, 141.77, 140.47, 132.73, 130.32, 130.20, 128.63, 128.59, 127.87, 127.60, 126.80, 125.85, 116.45, 115.53, 112.30, 110.43, 69.13, 55.95, 46.78, 44.43, 29.26; ESI-HR-MS: m/z C₂₈H₂₈N₂O₆ [M+H]⁺计算值489.202 5, 测量值489.200 5。

13h 白色固体, 产率38%, mp 115.1~116.4 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 11.21 (s, 1H), 9.12 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.64 (d, J = 8.6 Hz, 1H), 7.49 (d, J =16.0 Hz, 1H), 7.21 (d, J = 16.0 Hz, 1H), 7.09 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 9.0 Hz, 1H), 6.83 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 6.62~6.40 (m, 1H), 5.16 (s, 2H), 4.81 (s, 1H), 4.63 (s, 1H), 3.76 (s, 3H), 3.72 (s, 3H), 3.67~3.57 (m, 2H), 2.79~2.67 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 165.06, 158.87, 156.14, 151.80, 147.41, 146.47, 141.18, 129.71, 128.40, 126.17, 125.44, 116.19, 114.97, 113.53, 112.14, 111.93, 110.05, 61.61, 55.54, 55.51, 43.95, 42.90, 28.78;ESI-HR-MS: m/z C₂₆H₂₆N₂O₇ [M-H]^-计算值477.166 2, 测量值477.165 1。

13i 淡红色固体, 产率42%, mp 148.5~150.2 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 11.27 (s, 1H), 9.17 (s, 1H), 7.71 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 6.3 Hz, 2H), 7.42~7.32 (m, 1H), 7.19 (d, J = 15.0 Hz, 1H), 7.08~7.04 (m, 1H), 7.00 (s, 1H), 6.80 (d, J = 8.4 Hz, 2H), 6.76 (s, 1H), 5.38 (d, J = 8.9 Hz, 2H), 4.81 (s, 1H), 4.63 (s, 1H), 3.72 (s, 6H), 3.68~3.54 (m, 2H), 2.80~2.67 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 165.18, 161.94, 153.33, 147.85, 147.45, 141.21, 132.41, 132.40, 129.82, 129.70, 129.12, 129.06, 127.82, 126.73, 120.30, 115.18, 111.91, 110.16, 64.46, 55.57, 55.52, 42.74, 40.98, 29.01; ESI-HR-MS: m/z C₂₆H₂₆N₂O₆S [M-H]^-计算值493.143 4, 测量值493.144 0。

13j 白色固体, 产率56%, mp 123.9~125.8 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.77 (s, 1H), 9.01 (s, 1H), 7.48 (d, J = 15.3 Hz, 1H), 7.41 (s, 1H), 7.23 (d, J = 9.7 Hz, 1H), 7.19 (d, J = 15.3 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 10.6 Hz, 1H), 6.77 (s, 1H), 4.82 (s, 1H), 4.65 (s, 1H), 4.47 (s, 2H), 3.86 (s, 3H), 3.74 (s, 3H), 3.73 (s, 3H), 3.31 (s, 2H), 2.84~2.70 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 165.57, 165.40, 164.58, 160.02, 149.66, 149.18, 147.90, 147.76, 142.22, 129.47, 126.59, 125.73, 122.54, 117.13, 113.87, 112.32, 111.31, 110.31, 66.92, 56.19, 55.93, 44.44, 43.20, 29.31; ESI-HR-MS: m/z C₂₃H₂₆N₂O₇ [M+H]⁺计算值443.181 8, 测量值443.182 5。

13k 白色固体, 产率42%, mp 132.4~133.7 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.43 (s, 1H), 8.71 (s, 1H), 7.48 (d, J = 15.3 Hz, 1H), 7.39 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 15.3 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.82 (s, 1H), 6.77 (s, 1H), 4.82 (s, 1H), 4.64 (s, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.85 (s, 3H), 3.74 (s, 3H), 3.73 (s, 3H), 3.31~3.27 (m, 2H), 2.82~2.72 (m, 2H), 2.15 (t, J = 7.4 Hz, 2H), 1.97~1.93 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.12, 165.61, 149.99, 149.60, 147.87, 142.35, 142.29, 128.62, 126.60, 125.86, 122.76, 116.26, 113.25, 112.38, 111.35, 110.50, 68.04, 56.30, 55.98, 55.38, 44.46, 43.32, 29.34, 29.18, 25.28; ESI-HR-MS: m/z C₂₅H₃₀N₂O₇ [M+H]⁺计算值471.212 3, 测量值471.211 9。

13l 红色固体, 产率40%, mp 136.9~138.5 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.40 (s, 1H), 8.71 (s, 1H), 7.51 (d, J = 14.5 Hz, 1H), 7.45 (s, 1H), 7.39 (d, J = 5.4 Hz, 1H), 7.22 (d, J = 7.4 Hz, 1H), 7.00 (d, J = 14.5 Hz, 1H), 6.82 (s, 1H), 6.77 (s, 1H), 4.82 (s, 1H), 4.64 (s, 1H), 4.01~3.97 (m, 2H), 3.84 (s, 3H), 3.73 (s, 6H), 3.34 (s, 2H), 2.82~2.72 (m, 2H), 2.03 (t, J = 6.9 Hz, 2H), 1.67 (dd, J = 12.9, 6.3 Hz, 4H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.35, 165.53, 150.07, 149.54, 147.76, 147.68, 142.45, 128.41, 126.54, 125.77, 122.83, 116.09, 112.94, 112.28, 111.13, 110.40, 68.21, 56.24, 55.96, 55.93, 44.43, 43.29, 32.38, 29.32, 28.64, 22.31; ESI-HR-MS: m/z C₂₆H₃₂N₂O₇ [M+H]⁺计算值485.228 8, 测量值485.223 2。

13m 红色固体, 产率46%, mp 143.8~145.1 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.36 (s, 1H), 8.68 (s, 1H), 7.51 (d, J = 14.8 Hz, 1H), 7.46 (s, 1H), 7.34 (d, J = 10.6 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 6.99 (d, J = 14.8 Hz, 1H), 6.80 (s, 1H), 6.77 (s, 1H), 4.82 (s, 1H), 4.64 (s, 1H), 4.01~3.96 (m, 2H), 3.86~3.81 (m, 3H), 3.79~3.73 (m, 6H), 3.31~3.28 (m, 2H), 2.75~2.66 (m, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.74~1.69 (m, 2H), 1.59~1.54 (m, 2H), 1.41~1.36 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.49, 165.61, 150.13, 150.04, 149.48, 147.78, 142.41, 128.37, 126.52, 125.82, 122.84, 116.51, 116.04, 112.98, 112.31, 111.17, 68.56, 56.21, 55.94, 55.91, 44.43, 43.33, 32.69, 29.49, 28.93, 25.64, 25.37; ESI-HR-MS: m/z C₂₇H₃₄N₂O₇ [M+H]⁺计算值499.244 4, 测量值499.243 2。

13n 淡红色固体, 产率37%, mp 150.6~152.1 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.34 (s, 1H), 8.67 (s, 1H), 7.50 (d, J = 13.8 Hz, 1H), 7.45 (s, 1H), 7.36 (d, J = 5.0 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 6.98 (d, J = 13.8 Hz, 1H), 6.82 (s, 1H), 6.76 (s, 1H), 4.82 (s, 1H), 4.63 (s, 1H), 4.00~3.96 (m, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.31 (s, 2H), 2.73~2.62 (m, 2H), 1.95 (t, J = 6.7 Hz, 2H), 1.73~1.68 (m, 2H), 1.53~1.49 (m, 2H), 1.40~1.37 (m, 2H), 1.30 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.57, 165.43, 149.50, 149.45, 148.15, 146.76, 141.77, 129.31, 128.24, 127.80, 122.70, 121.73, 120.75, 112.72, 111.94, 110.50, 68.53, 56.28, 56.01, 55.95, 43.29, 43.27, 32.68, 29.50, 29.01, 28.79, 25.71, 25.55; ESI-HR-MS: m/z C₂₈H₃₆N₂O₇ [M+Na]⁺计算值535.240 2, 测量值535.239 7。

13o 红色固体, 产率57%, mp 157.5~158.9 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 10.34 (s, 1H), 8.66 (s, 1H), 7.50 (d, J = 12.8 Hz, 1H), 7.45 (s, 1H), 7.36 (d, J = 6.7 Hz, 1H), 7.21 (d, J = 6.3 Hz, 1H), 6.99 (d, J = 12.8 Hz, 1H), 6.82 (s, 1H), 6.76 (s, 1H), 4.82 (s, 1H), 4.63 (s, 1H), 4.01~3.97 (m, 2H), 3.87 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.68 (s, 2H), 2.78~2.66 (m, 2H), 1.94 (t, J = 6.8 Hz, 2H), 1.70 (s, 2H), 1.57 (s, 2H), 1.51~1.47 (m, 2H), 1.40 (s, 4H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.19, 165.15, 149.13, 149.09, 147.86, 147.55, 142.05, 135.36, 127.84, 127.49, 120.30, 115.98, 112.48, 111.29, 110.57, 110.21, 68.15, 55.93, 55.51, 55.46, 42.88, 82.84, 32.26, 28.76, 28.65, 28.56, 28.50, 25.44, 25.11; ESI-HR-MS: m/z C₂₉H₃₈N₂O₇ [M+H]⁺计算值527.275 7, 测量值527.274 1。

13p 白色固体, 产率55%, mp 129.7~130.8 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 11.28 (s, 1H), 9.14 (s, 1H), 7.78 (d, J = 7.4 Hz, 2H), 7.51 (d, J = 7.3 Hz, 2H), 7.48~7.44 (m, 1H), 7.42 (s, 1H), 7.22 (d, J = 9.0 Hz, 1H), 7.07~7.00 (m, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.82 (s, 1H), 6.76 (s, 1H), 5.19 (s, 2H), 4.82 (s, 1H), 4.64 (s, 1H), 3.86 (s, 3H), 3.73 (s, 6H), 3.50~3.46 (m, 2H), 2.81~2.67 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 165.50, 164.15, 149.67, 149.39, 147.82, 147.73, 142.27, 140.43, 132.72, 128.93, 127.98, 127.52, 126.53, 125.76, 122.58, 116.39, 113.67, 112.27, 112.20, 110.40, 69.68, 56.18, 56.06, 55.82, 44.29, 43.17, 29.25; ESI-HR-MS: m/z C₂₉H₃₀N₂O₇ [M+H]⁺计算值519.213 1, 测量值519.211 9。

13q 白色固体, 产率58%, mp 101.4~103.0 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 11.26 (s, 1H), 9.14 (s, 1H), 7.51 (d, J = 16.2 Hz, 1H), 7.40 (d, J = 3.4 Hz, 1H), 7.36 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 16.2 Hz, 1H), 7.07 (s, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.72 (s, 1H), 6.60~6.44 (m, 1H), 5.11 (s, 2H), 4.83 (s, 1H), 4.64 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 3.66~3.62 (m, 2H), 2.81~2.67 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 169.56, 164.49, 156.11, 153.32, 151.82, 149.20, 147.86, 146.45, 141.70, 135.38, 128.74, 128.39, 122.71, 120.99, 120.19, 113.56, 112.42, 112.33, 110.60, 110.25, 62.10, 55.94, 55.60, 55.54, 44.42, 43.41, 29.01; ESI-HR-MS: m/z C₂₇H₂₈N₂O₈ [M+H]⁺计算值509.192 4, 测量值509.193 5。

13r 淡白色固体, 产率48%, mp 111.9~113.4 ℃。¹H NMR (400 MHz, DMSO-d₆): δ 11.27 (s, 1H), 9.15 (s, 1H), 7.63 (s, 1H), 7.53~7.49 (m, 1H), 7.46 (s, 1H), 7.41 (d, J = 6.3 Hz, 1H), 7.24 (d, J = 4.6 Hz, 1H), 7.21~7.18 (m, 1H), 7.11 (s, 1H), 6.81 (d, J = 7.0 Hz, 1H), 6.76 (s, 1H), 5.33 (s, 2H), 4.82 (s, 1H), 4.64 (s, 1H), 3.85 (s, 3H), 3.73 (s, 6H), 3.66~3.54 (m, 2H), 2.81~2.67 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆): δ 172.36, 172.23, 159.01, 157.80, 155.50, 155.48, 155.38, 142.81, 134.13, 133.57, 131.65, 129.64, 128.42, 128.08, 127.27, 125.50, 123.19, 122.61, 122.48, 119.26, 63.07, 52.98, 52.95, 52.92, 44.65, 44.41, 16.96; ESI-HR-MS: m/z C₂₇H₂₈N₂O₇S [M+H]⁺计算值525.169 5, 测量值525.167 4。

2 化合物生物活性测试

2.1 体外HDAC1/HDAC6/HDAC8/HDAC11抑酶活性实验

HDAC抑酶活性实验采用基于荧光信号的检测方法测定。配制化合物的DMSO溶液、酶缓冲液、含有底物和胰蛋白酶的混合溶液。实验中阳性对照化合物SAHA测试终浓度为3 μmol·L^-1起始, 3倍稀释, 10个浓度。其余待测化合物测试终浓度为5 μmol·L^-1起始, 5倍稀释, 7个浓度, 均单孔测试。将待测化合物的DMSO溶液和酶缓冲溶液分别加入到384孔板中, 1 000 r·min^-1离心60 s, 振荡混匀后室温孵育15 min。而后加入含有底物和胰蛋白酶的混合溶液, 起始反应。将反应板1 000 r·min^-1离心1 min, 使用Synergy连续读取荧光信号, 并计算抑制率。最后通过分析软件GraphPad Prism 9计算IC₅₀值。

2.2 体外抗增殖活性实验

以SAHA为阳性对照, 采用MTT法评价目标化合物在体外对A549、HCT116细胞的增殖抑制作用。将对数生长期的细胞以1×10⁵细胞/孔接种于96孔板, 置于37 ℃、5% CO₂条件下培养, 直至细胞90%融合后, 用无血清培养基孵育2 h使细胞同步化。随后, 弃去上清, 分别加入含有各化合物(浓度为: 0.03、0.1、0.3、1、3、10、30 μmol·L^-1) 的培养基孵育72 h, 孵育结束前4 h, 每孔加入20 μL MTT溶液(5 mg·mL^-1)。孵育结束后, 将96孔板以1.5×10³ r·min^-1离心3 min, 弃去各孔上清液, 每孔加入150 μL DMSO, 细胞振荡仪上振荡10 min, 待结晶物充分溶解后用酶标仪测定OD₅₇₀, 计算抑制率, 并通过Graphpad Prism 9计算IC₅₀值。

2.3 Western blot实验

将对数生长期的A549细胞消化后, 吹打成单细胞悬液, 每孔4×10⁵个细胞接种于12孔板, 于给药各孔中分别加入不同浓度的化合物13e和SAHA溶液, 设置空白对照孔。孵育72 h后, 以PBS洗涤细胞, RIPA裂解液裂解, 离心, 收集上清液即为蛋白提取液。使用BCA蛋白浓度测定试剂盒测定样品蛋白浓度。通过15% SDS-PAGE胶分离蛋白, 湿法将蛋白转移至PVDF膜, 分别与一抗anti-Ac-H3、anti-H3、anti-Ac-α-tubulin、anti-α-tubulin、anti-GAPDH以及二抗羊抗兔IgG-HRP孵育, 将配好的ECL显色液加到PVDF膜进行曝光成像。

2.4 分子对接研究

作图软件使用Pymol, 分子对接使用Discovery Studio (version 2.5; Accelrys, San Diego, CA, USA, 2019)。使用蛋白质晶体结构为HDAC1 (PDB ID: 5ICN)、HDAC6 (PDB ID: 5EDU)。对接实验步骤如下: 将小分子导入软件, 并对其结构优化, 得到预处理的小分子配体。去除蛋白分子内部小分子配体和溶剂分子, 对蛋白加氢, 附CHARMM力场, 得到预处理的分子对接受体。应用Discovery Studio 2019软件中的CDOCKER模块将小分子配体与蛋白受体进行对接, 综合打分函数和相互作用模式选取对接构象。

作者贡献: 高鑫负责完成相关实验研究, 并完成论文撰写等工作; 方方负责选题、实验设计和指导、稿件修改及稿件定稿等工作; 马晓东协助指导化学实验、稿件修改等工作; 韩维维和田诗意协助化合物的合成及结构鉴定工作; 柴化怡和韩京晶协助活性测定实验。

利益冲突: 作者声明本文不存在任何利益冲突。

基金

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安徽省高等学校自然科学研究重点项目资助(KJ2019A1004)

参考文献

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2023年第58卷第2期

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doi: 10.16438/j.0513-4870.2022-1083

接收时间：2022-09-29
首发时间：2025-11-21
出版时间：2023-02-12

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收稿日期：2022-09-29
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安徽省高等学校自然科学研究重点项目资助(KJ2019A1004)

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1.安徽中医药大学药学院, 安徽合肥 230012

2.安徽省中医药科学院药物化学研究所, 安徽合肥 230012

3.中药研究与开发安徽省重点实验室, 安徽合肥 230012

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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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13a	H	CH₂	958		13j	OCH₃	CH₂	> 5 000
13b	H	(CH₂)₃	937	13k	OCH₃	(CH₂)₃	916
13c	H	(CH₂)₄	112	13l	OCH₃	(CH₂)₄	552
13d	H	(CH₂)₅	26	13m	OCH₃	(CH₂)₅	82
13e	H	(CH₂)₆	3.8	13n	OCH₃	(CH₂)₆	11
13f	H	(CH₂)₇	5.9	13o	OCH₃	(CH₂)₇	21
13g	H		173	13p	OCH₃		272
13h	H	2 651	13q	OCH₃	> 5 000
13i	H	507	13r	OCH₃	295
SAHA			13

Compd.

R₂

Linker

HDAC1
IC₅₀/nmol·L^-1

Compd.

R₂

Linker

HDAC1
IC₅₀/nmol·L^-1

13a

CH₂

958

13j

OCH₃

CH₂

> 5 000

13b

(CH₂)₃

937

13k

OCH₃

(CH₂)₃

916

13c

(CH₂)₄

112

13l

OCH₃

(CH₂)₄

552

13d

(CH₂)₅

13m

OCH₃

(CH₂)₅

13e

(CH₂)₆

3.8

13n

OCH₃

(CH₂)₆

13f

(CH₂)₇

5.9

13o

OCH₃

(CH₂)₇

13g

173

13p

OCH₃

272

13h

2 651

13q

OCH₃

> 5 000

13i

507

13r

OCH₃

295

SAHA

Compd.	A549 IC₅₀/μmol·L^-1	HCT116 IC₅₀/μmol·L^-1
13d	3.73 ± 0.16	1.18 ± 0.11
13e	1.74 ± 0.10	2.43 ± 0.16
13f	2.95 ± 0.3	3.46 ± 0.23
13n	2.26 ± 0.18	1.23 ± 0.08
13o	0.89 ± 0.07	0.49 ± 0.03
11a	6.42 ± 0.51	5.56 ± 0.38
11b	11.38 ± 1.05	7.49 ± 0.42
SAHA	1.33 ± 0.09	0.77 ± 0.06

Compd.

A549
IC₅₀/μmol·L^-1

HCT116
IC₅₀/μmol·L^-1

13d

3.73 ± 0.16

1.18 ± 0.11

13e

1.74 ± 0.10

2.43 ± 0.16

13f

2.95 ± 0.3

3.46 ± 0.23

13n

2.26 ± 0.18

1.23 ± 0.08

13o

0.89 ± 0.07

0.49 ± 0.03

11a

6.42 ± 0.51

5.56 ± 0.38

11b

11.38 ± 1.05

7.49 ± 0.42

SAHA

1.33 ± 0.09

0.77 ± 0.06

Compd.	HDAC6 IC₅₀/nmol·L^-1		Compd.	HDAC6 IC₅₀/nmol·L^-1
13e	7.4		13p	9.2
13n	9.3	13q	278
13o	14	SAHA	13

Compd.

HDAC6
IC₅₀/nmol·L^-1

Compd.

HDAC6
IC₅₀/nmol·L^-1

13e

7.4

13p

9.2

13n

9.3

13q

278

13o

SAHA

Compd.	HDAC1 IC₅₀/nmol·L^-1	HDAC6 IC₅₀/nmol·L^-1	HDAC8 IC₅₀/nmol·L^-1	HDAC11 IC₅₀/nmol·L^-1
13e	3.8	7.4	72	979
13o	21	14	298	1 150
13p	272	9.2	> 5 000	4 695
SAHA	13	13	161	> 5 000

Compd.

HDAC1
IC₅₀/nmol·L^-1

HDAC6
IC₅₀/nmol·L^-1

HDAC8
IC₅₀/nmol·L^-1

HDAC11
IC₅₀/nmol·L^-1

13e

3.8

7.4

979

13o

298

1 150

13p

272

9.2

> 5 000

4 695

SAHA

161

> 5 000