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Compd.	Appearance	Yield/%	mp/℃	Purity/%^a	[α]$ {}_{\mathrm{D}}^{25\mathrm{ }\mathrm{℃}} $ (g·100 mL^-1, MeOH)	Configuration (J_1″-2″/Hz)
1b	Brown yellow solid	16.7	145-147	99.25	-103.80 (0.100)	β-(7.7)
2b	Brown yellow solid	29.5	187-189	99.38	-134.63 (0.100)	β-(7.5)
3b	Brown yellow solid	11.7	181-182	98.25	-109.36 (0.100)	β-(7.1)
4b	Brown yellow solid	11.2	213-215	98.13	-127.60 (0.100)	β-(7.0)
5b	Brown yellow solid	42.0	168-169	99.14	-310.80 (0.100)	β-(7.7)
6b	Brown yellow solid	42.5	198-200	99.03	-447.20 (0.100)	β-(7.3)
7b	Brown yellow solid	11.1	196-197	95.50	-193.33 (0.075)	β-(7.9)
8b	Brown yellow solid	8.4	225-227	98.12	-215.60 (0.100)	β-(7.6)
9b	Brown yellow solid	34.1	109-110	99.51	-15.80 (0.100)	β-(7.7)
10b	Brown yellow solid	34.0	122-123	99.23	7.00 (0.100)	β-(7.5)
11b	Brown yellow solid	16.0	167-170	99.39	-5.00 (0.100)	β-(7.7)
12b	Brown yellow solid	30.3	171-173	99.05	-71.20 (0.100)	β-(7.7)
13b	Brown yellow solid	14.7	280-282	97.60	-117.40 (0.100)	β-(7.7)
14b	Brown yellow solid	32.7	245-248	99.12	-301.20 (0.100)	β-(7.6)
15b	Brown yellow solid	23.6	289-291	99.21	-615.80 (0.100)	β-(7.4)
16b	Brown yellow solid	29.6	148-150	99.47	-31.74 (0.076)	β-(7.1)
17b	Brown yellow solid	21.4	170-172	99.74	-33.00 (0.100)	β-(7.1)
18b	Brown yellow solid	17.8	102-104	99.48	-42.43 (0.076)	β-(8.3)
19b	Brown yellow solid	27.0	170-172	98.59	-13.20 (0.100)	β-(7.7)
20b	Brown yellow solid	17.1	206-208	99.24	-21.00 (0.100)	β-(7.1)
21b	Brown yellow solid	11.3	198-200	95.20	-95.60 (0.100)	β-(7.6)
22b	Brown yellow solid	21.7	171-173	99.57	9.00 (0.100)	β-(7.8)
23b	Brown yellow solid	20.3	162-164	99.60	-20.46 (0.061)	β-(7.7)
24b	Brown yellow solid	28.2	198-200	99.54	-2.63 (0.069)	β-(7.7)
25b	Brown yellow solid	14.7	239-240	100.00	-0.20 (0.100)	β-(7.7)

Compd.	Appearance	Yield/%	mp/℃	Purity/%^a	[α]$ {}_{\mathrm{D}}^{25\mathrm{ }\mathrm{℃}} $ (g·100 mL^-1, MeOH)	Configuration (J_1″-2″/Hz)
1b	Brown yellow solid	16.7	145-147	99.25	-103.80 (0.100)	β-(7.7)
2b	Brown yellow solid	29.5	187-189	99.38	-134.63 (0.100)	β-(7.5)
3b	Brown yellow solid	11.7	181-182	98.25	-109.36 (0.100)	β-(7.1)
4b	Brown yellow solid	11.2	213-215	98.13	-127.60 (0.100)	β-(7.0)
5b	Brown yellow solid	42.0	168-169	99.14	-310.80 (0.100)	β-(7.7)
6b	Brown yellow solid	42.5	198-200	99.03	-447.20 (0.100)	β-(7.3)
7b	Brown yellow solid	11.1	196-197	95.50	-193.33 (0.075)	β-(7.9)
8b	Brown yellow solid	8.4	225-227	98.12	-215.60 (0.100)	β-(7.6)
9b	Brown yellow solid	34.1	109-110	99.51	-15.80 (0.100)	β-(7.7)
10b	Brown yellow solid	34.0	122-123	99.23	7.00 (0.100)	β-(7.5)
11b	Brown yellow solid	16.0	167-170	99.39	-5.00 (0.100)	β-(7.7)
12b	Brown yellow solid	30.3	171-173	99.05	-71.20 (0.100)	β-(7.7)
13b	Brown yellow solid	14.7	280-282	97.60	-117.40 (0.100)	β-(7.7)
14b	Brown yellow solid	32.7	245-248	99.12	-301.20 (0.100)	β-(7.6)
15b	Brown yellow solid	23.6	289-291	99.21	-615.80 (0.100)	β-(7.4)
16b	Brown yellow solid	29.6	148-150	99.47	-31.74 (0.076)	β-(7.1)
17b	Brown yellow solid	21.4	170-172	99.74	-33.00 (0.100)	β-(7.1)
18b	Brown yellow solid	17.8	102-104	99.48	-42.43 (0.076)	β-(8.3)
19b	Brown yellow solid	27.0	170-172	98.59	-13.20 (0.100)	β-(7.7)
20b	Brown yellow solid	17.1	206-208	99.24	-21.00 (0.100)	β-(7.1)
21b	Brown yellow solid	11.3	198-200	95.20	-95.60 (0.100)	β-(7.6)
22b	Brown yellow solid	21.7	171-173	99.57	9.00 (0.100)	β-(7.8)
23b	Brown yellow solid	20.3	162-164	99.60	-20.46 (0.061)	β-(7.7)
24b	Brown yellow solid	28.2	198-200	99.54	-2.63 (0.069)	β-(7.7)
25b	Brown yellow solid	14.7	239-240	100.00	-0.20 (0.100)	β-(7.7)

Compd.	K_D/mol·L^-1	Compd.	K_D/mol·L^-1
LF1	6.56×10^-4	10b	7.74×10^-5
LF2	4.82×10^-5	17b	1.65×10^-4
LF3	3.58×10^-5	18b	1.45×10^-4
LF4	1.37×10^-5	23b	2.69×10^-4
4b	7.91×10^-5	24b	3.71×10^-5
8b	8.70×10^-4	25b	2.16×10^-4

Compd.	K_D/mol·L^-1	Compd.	K_D/mol·L^-1
LF1	6.56×10^-4	10b	7.74×10^-5
LF2	4.82×10^-5	17b	1.65×10^-4
LF3	3.58×10^-5	18b	1.45×10^-4
LF4	1.37×10^-5	23b	2.69×10^-4
4b	7.91×10^-5	24b	3.71×10^-5
8b	8.70×10^-4	25b	2.16×10^-4

二苯甲酮类多酚的糖基化衍生、活性评价及与PDIA6的相互作用

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李蔷 ¹^,^# , 程宁宁 ¹^,^# , 冯秀娥 ¹^,²^,^* , 李青山 ¹^,²^,³^,^*

药学学报 | 研究论文 2024,59(6): 1706-1719

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药学学报 | 研究论文 2024, 59(6): 1706-1719

二苯甲酮类多酚的糖基化衍生、活性评价及与PDIA6的相互作用

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李蔷¹^,^#, 程宁宁¹^,^#, 冯秀娥¹^,²^,^*, 李青山¹^,²^,³^,^*

作者信息

1.山西医科大学药学院, 山西太原 030001

2.山西医科大学, 教育部慢性肾脏病医药基础研究创新中心, 山西太原 030001

3.山西中医药大学, 基于炎性反应的重大疾病创新药物山西省重点实验室, 山西晋中 030619

通讯作者:

^*冯秀娥, Tel: 13191058319, E-mail: xiuefeng@163.com;

李青山, Tel: 86-351-3985190, E-mail: sxlqs2012@163.com

Glycosylation derivatization, bioactivity evaluation of benzophenone polyphenols and their interaction with protein disulfide isomerase A6

Qiang LI¹, Ning-ning CHENG¹, Xiu-e FENG¹^,²^,^*, Qing-shan LI¹^,²^,³^,^*

Affiliations

1. School of Pharmaceutical Science, Shanxi Medical University, Taiyuan 030001, China

2. Medicinal Basic Research Innovation Center of Chronic Kidney Disease, Ministry of Education, Shanxi Medical University, Taiyuan 030001, China

3. Shanxi Key Laboratory of Innovative Drug for the Treatment of Serious Diseases Basing on the Chronic Inflammation, Shanxi University of Chinese Medicine, Jinzhong 030619, China

出版时间: 2024-06-12 doi: 10.16438/j.0513-4870.2023-1384

文章导航

摘要

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蛋白质二硫键异构酶A6 (PDIA6) 与炎症和内质网应激密切相关。为了获得靶向PDIA6且具有强抗炎作用的二苯甲酮类多酚糖基衍生物, 本文以不同基团取代的二苯甲酮与α-溴代乙酰糖为起始原料, 经傅克酰基化、脱乙酰基反应合成了25个目标糖基衍生物, 并采用生物膜干涉(BLI) 技术, 定量研究其与PDIA6的相互作用, 同时评价其体外抗炎作用。结果表明, 化合物4b、10b、17b、18b、25b既与PDIA6有较高的亲和力, 又具有很强的抗炎作用, 这些化合物可能是通过直接作用于PDIA6, 进而影响炎症相关的信号通路。尤其是, 该类化合物对IL-1β和IL-6的抑制作用最为显著, 推测其在治疗炎症性疾病方面有重要的开发前景。

关键词

二苯甲酮类多酚 / 合成 / 糖基化 / 蛋白质二硫键异构酶A6 / 抗炎

Abstract

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Protein disulfide isomerase A6 (PDIA6) is closely related to inflammation and endoplasmic reticulum stress. To obtain the glycosyl derivatives of benzophenone polyphenols targeting PDIA6 with strong anti-inflammatory effects, twenty-five target glycosyl derivatives were synthesized by Friedel-Crafts acylation and deacetylation reaction, starting from the substituted benzophenone and α-bromoacetyl saccharide, and their interactions with PDIA6 were quantitatively investigated by bio-layer interferometry (BLI) technique. Their in vitro anti-inflammatory properties were also evaluated. The results showed that target compounds 4b, 10b, 17b, 18b, and 25b not only exhibit high affinity with PDIA6, but also present strong anti-inflammatory abilities. Above results suggest that this class of compounds can affect the signaling pathways related to inflammation by directly acting on PDIA6. In particular, such compounds exhibit the strong inhibitory effects on IL-1β and IL-6 release, suggesting the potential development prospect in the treatment of inflammatory diseases.

Key words

benzophenone polyphenol / synthesis / glycosylation / protein disulfide-isomerase A6 / anti-inflammation

引用本文

李蔷, 程宁宁, 冯秀娥, 李青山. 二苯甲酮类多酚的糖基化衍生、活性评价及与PDIA6的相互作用. 药学学报, 2024 , 59 (6) : 1706 -1719 . DOI: 10.16438/j.0513-4870.2023-1384

Qiang LI, Ning-ning CHENG, Xiu-e FENG, Qing-shan LI. Glycosylation derivatization, bioactivity evaluation of benzophenone polyphenols and their interaction with protein disulfide isomerase A6[J]. Acta Pharmaceutica Sinica, 2024 , 59 (6) : 1706 -1719 . DOI: 10.16438/j.0513-4870.2023-1384

正文

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炎症是当机体受到内源性或外源性有害刺激时产生的复杂病理反应, 如果不能及时控制会导致肿瘤、肺炎^[1]、动脉粥样硬化^[2]、肾小球肾炎^[3]、类风湿性关节炎^[4]、代谢紊乱^[5]等系列疾病, 甚至危及生命。细胞因子是宿主对感染、免疫反应、炎症和创伤反应的调节剂, 其中, IL-1β、IL-6和TNF-α是促炎细胞因子, 参与炎症反应的早期应答和放大; IL-10具有强大的抑制机体炎症的能力^[6]。诱导型一氧化氮合酶是炎症发生的重要媒介, 它能诱导NO生成^[7]。因此, 通过抗炎途径治愈或减轻疾病, 开发新型成药性更好的抗炎药物具有重要意义。

课题组前期合成了系列二苯甲酮类多酚化合物, 通过系统的体内外活性筛选, 获得了具有高效抗炎和抗氧化活性的多酚化合物2, 4′, 5′-三羟基-5, 2′-二溴二苯甲酮(LF1)、2-羟基-5, 2′-二溴-4′, 5′-二甲氧基二苯甲酮(LF2)、4-叔丁基-3′, 4′-二羟基二苯甲酮(LF3) 和4-叔丁基-4′, 5′-二羟基-2′-溴二苯甲酮(LF4), 见图 1。苗头化合物LF1具有抗大鼠急性肾盂肾炎和心肌缺血再灌注损伤等多种药理活性, 其作用机制与抑制炎症和抗氧化应激密切相关^[8,9]。

课题组前期利用基于活性的蛋白质谱分析(activity-based protein profiling, ABPP) 技术, 在RAW264.7细胞上捕获到LF1抗炎的关键蛋白二硫键异构酶A6 (protein disulfide isomerase associated 6, PDIA6)^[10], 其主要定位于内质网, 参与内质网应激, 与炎症和氧化应激密切相关, 作为氧化还原酶催化二硫键的形成, 通过识别未折叠或部分折叠蛋白并与其结合来抑制错误折叠蛋白质的聚集^[11,12]。PDIA6的缺乏可能会导致小鼠生长迟缓、胰岛素合成受损和糖尿病^[13]。

由于细胞表面存在大量糖蛋白和糖脂类受体分子, 在脂溶性分子中引入糖基是提高水溶性、降低毒性、提高亲合性、增加生物利用度, 甚至提高靶向性最有效的途径^[14,15]。基于此, 本文以LF1、LF2、LF3和LF4为先导物, 通过对其进行糖基化结构修饰得到系列糖基化目标产物, 采用生物膜干涉(BLI) 技术定量研究其与PDIA6蛋白的相互作用, 并评价其体外抗炎活性, 以期获得靶向PDIA6且具有强抗炎活性的候选药物。

结果与讨论

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1 化合物的合成

目标化合物1b~15b的合成见合成路线1。先导化合物LF1、LF2、LF3、LF4在1 mol·L^-1 NaHCO₃/KCl和四丁基溴化铵(TBAB) 作用下与α-溴代乙酰糖(Br-Gl) 发生催化醚化反应, 得到乙酰化的中间体化合物1a~15a, 最后在甲醇钠或氢氧化钠作用下脱掉乙酰基, 得到目标产物1b~15b。目标化合物16b~25b的合成见合成路线2, LF1和LF4的邻位酚羟基在(Boc)₂O作用下, 其中一个酚羟基被保护, 然后在1 mol·L^-1 NaHCO₃/KCl和TBAB作用下与Br-Gl反应, 后经三氟乙酸脱掉Boc保护基, 得到中间体化合物16a~25a, 最后在甲醇钠或氢氧化钠作用下脱掉乙酰基, 得到目标产物16b~25b。目标化合物的理化性质和构象见表 1。

多酚化合物与α-溴代乙酰糖的醚化反应, 是在碱性条件下进行的水-有机两相反应, 属非均相反应, 因此, 需加入相转移催化剂TBAB, 在1 mol·L^-1 NaHCO₃/KCl条件下进行反应, 薄层色谱(TLC) 监测反应, 并用5%浓硫酸乙醇溶液浸湿薄层板, 在110 ℃下放置15 min, 通过碳化判断反应进程。先导物的酚羟基首先在碱性条件下形成氧负离子, 再与α-溴代乙酰糖发生亲核取代反应, 此类糖基供体引入的乙酰基, 室温条件下在甲醇钠/甲醇条件下可以脱除。其中葡糖糖醛酸甲酯类中间体化合物需要在氢氧化钠/甲醇条件下脱除甲基和乙酰基。由于目标产物含有多个醇羟基, 极易溶于水, 实验过程中发现分离提纯较难, 导致收率较低。

先导物LF1~LF4均为多酚化合物, 进行糖基化修饰时, 反应选择性较差, 为了确定反应位点, 结构表征时需结合二维图谱进一步确证。以目标化合物4b、8b、12b、13b、16b、17b为例(图 2), 具体如下:

化合物4b的一维氢谱和碳谱显示只形成了2个氧苷键, 目标化合物上糖苷键的位置可能有3种情况: 在4′-OH和5′-OH上形成氧苷键; 在2-OH和4′-OH上形成氧苷键; 在2-OH和5′-OH上形成氧苷键。如果是后两者, 则糖环上1″-C上的H只会与苯环上3′-H和6′-H中的一个位点相关, 而¹H-¹H NOESY显示两个糖环上1″-H位移分别是5.20 (d, J = 7.4 Hz, 1H), 5.01 (d, J = 7.0 Hz, 1H), 分别与苯环上位移是7.85 (s, 1H), 7.41 (s, 1H) 的6′-H和3′-H相关, 所以糖环分别连接在4′和5′位酚羟基上。

化合物8b与4b类似, ¹H-¹H NOESY显示两个糖环上1″-C-H位移分别是5.18 (d, J = 7.6 Hz, 1H) 和5.00 (d, J = 7.6 Hz, 1H), 分别与苯环上位移是7.77 (s, 1H) 和7.38 (s, 1H) 的6′-H和3′-H相关, 所以糖环分别连接在4′和5′位酚羟基上。

化合物12b的一维氢谱和碳谱显示只形成了1个氧苷键, 目标化合物可能在3′-OH或4′-OH上形成氧苷键, 苯环上2′-H与6′-H是间位偶合, 偶合常数小于5′-H与6′-H之间的邻位偶合常数, 所以5′-H的位移是7.21 (d, J = 8.5 Hz, 1H)。¹H-¹H NOESY显示糖环上1″-C-H位移是4.99 (d, J = 7.7 Hz, 1H), 与7.21 (d, J = 8.5 Hz, 1H) 相关, 所以判断出糖环连接在了4′位酚羟基。

化合物13b与12b类似, 5′-H位移是7.20 (d, J = 8.5 Hz, 1H)。¹H-¹H NOESY显示糖环上1″-C-H位移是4.99 (d, J = 7.7 Hz, 1H), 与7.20 (d, J = 8.5 Hz, 1H) 相关, 所以判断出糖环连接在了4′位酚羟基。

化合物16b的一维氢谱和碳谱显示只形成了1个氧苷键, 二维谱HMBC谱图显示苯环上与羰基碳相关的6′-H的位移是6.86 (s, 1H), 那么7.50 (s, 1H) 表示3′-H。¹H-¹H NOESY显示糖环上1″-C-H位移是4.95 (d, J = 7.1 Hz, 1H), 与位移是7.50 (s, 1H) 的3′-H相关, 因此, 确定糖环连接在4′位酚羟基。16b、18b、20b、22b、23b均经由关键中间体LF4-Boc合成, 16b结构的确证间接证实了LF4-Boc结构上羟基的位置, 也对化合物18b、20b、22b、23b的确证起到关键桥梁作用, 其形成糖苷键的位点均与16b一致。

化合物17b的一维氢谱和碳谱显示只形成了1个氧苷键, 二维谱HMBC显示与羰基碳远程相关的6′-H位移是7.48 (s, 1H), 苯环3′-H位移则是7.29 (s, 1H)。¹H-¹H NOESY显示糖环上1″-C-H位移是5.10 (d, J = 7.1 Hz, 1H), 与位移是7.29 (s, 1H) 的苯环3′-H相关, 因此, 确定糖环连接在4′位酚羟基。17b、19b、21b、24b、25b均经由关键中间体LF1-Boc合成, 17b结构的确证间接证实了LF1-Boc结构上羟基的位置, 也对化合物19b、21b、24b、25b的确证起到关键桥梁作用, 其形成糖苷键的位点均与17b一致。

此外, 糖苷类化合物有α和β两种构型, 常依据氢谱中糖的端基质子的偶合常数来判断糖苷键的构型^[16,17], 糖苷键为α构型时, J_1″-2″ = 2~4; 糖苷键为β构型时, J_1″-2″ = 6~8。而且, 糖的端基质子信号一般出现在δ 5.0附近, 且多呈现特征性的双峰。目标化合物表征结果显示: 糖基的J_1″-2″值在7.0~8.3 Hz之间(表 1), 以此判断其糖苷键为β构型。进一步分析其反应机制, 先导化合物结构上的酚羟基与α-溴代乙酰糖发生醚化反应时, 发生了S_N2亲核取代, 酚羟基首先在碱性条件下形成氧负离子, 从背面进攻α-溴代乙酰糖, 使糖环的端基碳发生Walden转化, 最终形成β型糖苷。

2 目标化合物与PDIA6亲和力评价

内质网参与许多生物过程, 包括蛋白质折叠和细胞运输, 蛋白质二硫键异构酶(PDI) 是蛋白质折叠的关键参与者^[18]。采用BLI技术考察目标化合物与PDIA6蛋白之间的直接相互作用, 并对二者的亲和力进行定量(表 2)。亲和力强弱通过结合解离平衡常数K_D表示, K_D越小, 表示化合物与PDIA6的亲和力越大, 结果显示, 在10^-4~10^-7 mol·L^-1内, 有8个目标化合物与PDIA6的结合有明确的浓度依赖性, 其亲和力顺序为24b > 10b > 4b > 18b > 17b > 25b > 23b > 8b, 其中4b、17b、24b、25b平衡解离常数(K_D) 值在2.16×10^-4~3.71×10^-5 mol·L^-1内, 亲和力较先导物LF1更强(K_D值为6.56×10^-4 mol·L^-1), 8b的K_D值为8.70×10^-4 mol·L^-1与先导物LF1相当; 10b的K_D值为7.74×10^-5 mol·L^-1, 与先导物LF2相当(K_D值为4.82×10^-5 mol·L^-1); 18b和23b的K_D值分别为1.45×10^-4和2.69×10^-4 mol·L^-1, 亲和性低于先导物LF4 (K_D值为1.37×10^-5 mol·L^-1)。图 3为与PDIA6有较强亲和力的化合物的结合动力学过程, 横坐标前60 s表示化合物与蛋白缓慢结合, 后60 s表示化合物与蛋白缓慢解离, 目标化合物与PDIA6蛋白的结合响应信号值随化合物浓度的升高而浓度依赖性的增强。

3 体外抗炎活性

内质网应激与退行性疾病、癌症、关节炎、糖尿病、心血管疾病等病理过程中的炎症反应密切关联, 会诱导细胞因子的产生, 从而导致炎症反应。PDI被证实是NF-κB的负调节因子, 并且可能作为炎症性疾病的靶标^[19]。目标化合物与PDIA6具有较高的亲和力, 表明存在直接结合, 但是否是特异性结合还需要更深入的分析验证。而且前期已发现PDIA6为先导物LF1抗炎的关键蛋白, 因此, 有必要进一步测试目标化合物的体外抗炎活性。

3.1 目标化合物对RAW264.7细胞活力的影响

目标化合物对小鼠巨噬细胞RAW264.7活力的影响表明, 与空白对照组相比, 浓度为10 μmol·L^-1的化合物作用24 h无明显毒性(P > 0.05), 所以选择浓度10 μmol·L^-1进行后续活性筛选实验。

3.2 体外抗炎活性评价

3.2.1 目标化合物对NO的抑制作用

采用LPS诱导的小鼠巨噬细胞RAW264.7构建炎症模型,以塞来昔布为阳性对照, 评价化合物对NO分泌的影响。图 4结果表明, 与空白对照组相比, LPS刺激后, RAW264.7细胞培养上清中NO释放量显著增加, 化合物17b、24b、25b可显著抑制NO的分泌(P < 0.01), 5b、7b、18b对NO有一定的抑制作用(P < 0.05)。

3.2.2 目标化合物对IL-1β、TNF-α、IL-6和IL-10的抑制作用

考虑化合物作用途径较多,采用ELISA法测定其对LPS诱导的RAW264.7细胞培养上清中IL-1β、TNF-α、IL-6和IL-10释放量的影响。图 5结果表明, 所有目标化合物对IL-1β均有显著的抑制作用(P < 0.01); 化合物4b~7b、9b、10b、12b、13b、15b、17b、18b、25b可显著抑制IL-6的分泌(P < 0.01), 14b、16b、19b对IL-6有一定的抑制作用(P < 0.05), 均优于先导物和阳性对照; 化合物4b、10b、12b可显著抑制TNF-α的分泌(P < 0.01), 5b、20b对TNF-α有一定的抑制作用(P < 0.05); 化合物2b、19b、23b~25b可以显著促进抑炎因子IL-10的分泌(P < 0.01), 22b可促进IL-10的分泌(P < 0.05)。

进一步分析发现: 化合物5b可同时抑制NO、IL-1β、IL-6、TNF-α的分泌; 化合物4b、10b、12b可同时抑制IL-1β、IL-6、TNF-α的分泌; 化合物17b、18b、25b可同时抑制NO、IL-1β、IL-6的分泌, 且25b还可同时促进IL-10的分泌。进一步测试了5b、12b和17b对IL-1β抑制作用的IC₅₀值, 分别为1.67、19.98和3.59 μmol·L^-1, 5b和17b对IL-1β的抑制活性均优于先导化合物LF1 (IC₅₀为13.18 μmol·L^-1)。

小结

收起

本文合成了25个二苯甲酮类多酚的糖基衍生物, 均为新化合物。8个化合物与PDIA6有直接相互作用, 其中, 化合物4b、10b、17b、18b、25b既与PDIA6有较高的亲和力, 又有较强的抗炎作用。推测这些化合物可能通过作用PDIA6后, 进而影响炎症相关的信号通路。此外, 化合物5b、12b、19b虽与PDIA6没有直接相互作用, 但也显示了很强的抗炎作用, 化合物5b、12b可同时抑制IL-1β、IL-6和TNF-α的释放, 化合物19b可同时抑制IL-1β、IL-6并促进IL-10的分泌。IL-1β是痛风炎症形成和骨质破坏的关键因子^[20], IL-6与许多炎症疾病相关^[21]。本文合成的二苯甲酮类多酚糖基化合物对IL-1β和IL-6的抑制作用最为明显, 推测其在防治痛风等炎症性疾病方面有重要的开发前景。

实验部分

收起

WFH-203三用紫外分析仪(上海精科实业有限公司), RE-52AA旋转蒸发器(上海亚荣生化仪器厂), AVANCE400MHZ超导核磁共振光谱仪(瑞士布鲁克公司), ZQ-2000质谱仪(美国Waters公司), SGW X-4显微熔点仪(上海仪电物理光学仪器有限公司), Autopol Ⅲ型自动旋光仪(美国鲁道夫公司), Waters Acquity Arc型高效液相色谱仪, Forte Bio Octet Red 96e型生物膜干涉仪, SpectraMax Plus 384全波长多功能酶标仪。

RAW264.7细胞由武汉博士德生物工程有限公司提供, 标准胎牛血清、DMEM高糖、NO测试盒由上海碧云天公司提供, IL-6、IL-1β、TNF-α和IL-10试剂盒均由武汉博士德生物工程有限公司提供, 主要试剂溴代四乙酰葡萄糖、溴代四乙酰半乳糖、乙酰溴-α-D-麦芽糖购自上海麦克林生化科技有限公司, 乙酰溴代纤维二糖、二碳酸二叔丁酯、4-二甲氨基吡啶(DMAP) 购自上海阿拉丁生化科技股份有限公司, 乙酰溴-α-D-葡萄糖酮酸甲基酯购自上海毕得医药科技股份有限公司, 四丁基溴化铵(TBAB)、三氟乙酸购自萨恩化学技术有限公司, 薄层色谱用硅胶GF254购于青岛海洋化工厂, 其他常规试剂均为国产分析纯或化学纯。

1 化学合成

1.1 4-叔丁基二苯甲酮-3′, 4′-二-O-β-葡萄糖苷(1b)

关键中间体1a: 将27 mg (0.1 mmol) 的化合物LF3溶解于3 mL含有等物质的量KCl的1 mol·L^-1 NaHCO₃溶液中, 再加入123 mg (0.3 mmol) 的乙酰溴-α-D-葡萄糖和96 mg (0.3 mmol) 的相转移催化剂TBAB, 室温反应12 h, TLC监控反应进程(正己烷-乙酸乙酯-冰醋酸, 3∶2∶0.02), 反应结束后, 旋蒸除去乙腈, 用8 mL乙酸乙酯复溶, 加入8 mL水, 分出有机相, 水相用乙酸乙酯再萃取两次(8 mL×2), 合并有机相, 无水硫酸钠干燥、减压浓缩, 薄层制备得到棕黄色油状物20 mg, 产率21.5%。中间体2a~15a均按照此法合成。

化合物1b: 将25 mg (0.021 mmol) 的1a用3 mL甲醇溶解, 加入0.5 mL的1% MeONa/MeOH溶液, 室温反应6 h。TLC监测(正己烷-乙酸乙酯-冰醋酸, 3∶2∶0.02) 反应结束, 减压除去溶剂, 加入3 mL水复溶, 以1%盐酸调pH至中性, 乙醚萃取两次(2 mL×2), 合并有机相, 旋蒸浓缩得到棕黄色固体10 mg, 产率78.0%。目标化合物2b~15b均按照此法合成。

1.2 4-叔丁基-5′-羟基-2′-溴二苯甲酮-4′-O-β-葡萄糖苷(16b)

中间体LF4-Boc: 将460 mg (1.32 mmol) 的LF4用6 mL乙腈溶解, 加入300 μL (1.32 mmol) 的(Boc)₂O和65 mg (0.5 mmol) 的DMAP, 室温搅拌6 h。TLC监测(石油醚-乙酸乙酯, 3∶1) 反应结束, 旋蒸除去乙腈, 用8 mL乙酸乙酯复溶, 加入8 mL水, 分出有机相, 水相用乙酸乙酯再萃取两次(8 mL×2), 合并有机相, 无水硫酸钠干燥、减压浓缩, 经柱层析(石油醚-乙酸乙酯, 3∶1) 纯化, 得到黄色油状物533 mg, 产率90.0%。中间体LF1-Boc按照此法合成。

中间体LF4-Boc-Glu: 采用化合物1a类似合成步骤, 薄层制备得到棕色油状物39 mg, 产率为50.0%。中间体LF1-Boc-Glu、LF4-Boc-Gal、LF1-Boc-Gal、LF4-Boc-GlcU、LF1-Boc-GlcU、LF4-Boc-maltose、LF1-Boc-maltose、LF4-Boc-cellobiose、LF1-Boc-cellobiose均按照此法合成。

关键中间体16a: 将39 mg (0.05 mmol) 的LF4-Boc-Glu用1 mL二氯甲烷溶解, 加入400 μL (0.005 mmol) 三氟乙酸, 室温反应6 h。TLC监测(正己烷-乙酸乙酯-冰醋酸= 3∶2∶0.02) 反应结束, 旋蒸除去溶剂, 加入5 mL乙酸乙酯复溶, 用饱和碳酸氢钠洗涤有机相, 分到有机相, 再用5 mL水洗涤后, 无水硫酸钠干燥、减压浓缩得到棕黄色油状物15 mg, 产率为44.2%。中间体17a~25a按照此法合成。

化合物16b: 采用化合物1b类似合成步骤得到棕黄色固体3 mg, 产率29.6%。目标化合物17b~25b均按照此法合成。

中间体化合物的理化性质和波谱数据如下:

LF1-Boc: 黄色油状液, 产率48.0%, mp 139~141 ℃。¹H NMR (600 MHz, CDCl₃) δ 11.78 (s, 1H), 7.54 (d, J = 2.5 Hz, 1H), 7.41 (d, J = 2.5 Hz, 1H), 7.27 (s, 1H), 7.24 (s, 1H), 6.95~6.93 (m, 1H), 1.52 (s, 9H); MS (ESI) m/z: 487.05 [M+H]⁺。

LF4-Boc: 黄色油状液, 产率90.0%, mp 169~171 ℃。¹H NMR (600 MHz, CDCl₃) δ 7.76 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.30 (s, 1H), 7.25 (s, 1H), 1.54 (s, 9H), 1.35 (s, 9H); MS (ESI) m/z: 447.29 [M-H]^-。

1a: 黄色油状液, 产率21.5%, mp 175~176 ℃。¹H NMR (600 MHz, CDCl₃) δ 7.72 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 2.0 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 8.4, 2.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 5.30 (m, 5H), 5.26 (d, J = 7.2 Hz, 1H), 5.23 (dd, J = 8.2, 2.4 Hz, 2H), 5.20 (d, J = 9.8 Hz, 1H), 5.18 (d, J = 9.9 Hz, 1H), 4.27 (m, 2H), 4.13 (m, 2H), 2.11~2.02 (m, 24H), 1.37 (s, 9H); MS (ESI) m/z: 953.49 [M+Na]⁺。

2a: 黄色油状液, 产率34.7%, mp 198~200 ℃。¹H NMR (600 MHz, DMSO) δ 7.69 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.50 (s, 1H), 7.22 (s, 1H), 5.74 (d, J = 7.9 Hz, 1H), 5.61 (d, J = 8.0 Hz, 1H), 5.36 (t, J = 9.6 Hz, 1H), 5.29 (t, J = 9.6 Hz, 1H), 5.06~5.04 (m, 1H), 5.03~5.01 (m, 1H), 5.01~4.98 (m, 2H), 4.94 (t, J = 9.7 Hz, 1H), 4.28 (dd, J = 8.8, 7.4 Hz, 1H), 4.19 (dd, J = 12.3, 6.1 Hz, 1H), 4.14~4.10 (m, 2H), 4.08 (dd, J = 10.0, 4.0 Hz, 1H), 2.03 (d, J = 3.1 Hz, 9H), 2.01 (s, 3H), 1.98 (d, J = 2.0 Hz, 6H), 1.95 (s, 3H), 1.78 (s, 3H), 1.30 (s, 9H); MS (ESI) m/z: 1 009.14 [M+H]⁺。

3a: 黄色油状液, 产率14.5%, mp 109~111 ℃。¹H NMR (600 MHz, DMSO) δ 7.80 (dd, J = 8.9, 2.5 Hz, 1H), 7.52 (d, J = 2.5 Hz, 1H), 7.45 (s, 1H), 7.28 (s, 1H), 7.21 (d, J = 9.0 Hz, 1H), 5.81 (d, J = 7.9 Hz, 1H), 5.64 (d, J = 8.0 Hz, 1H), 5.51 (d, J = 7.9 Hz, 1H), 5.39~5.25 (m, 4H), 5.04~4.96 (m, 5H), 4.78 (s, 1H), 3.59 (dd, J = 5.5, 4.0 Hz, 2H), 3.52 (dd, J = 5.9, 3.6 Hz, 2H), 3.49~3.44 (m, 2H), 3.38 (t, J = 6.6 Hz, 2H), 2.03 (s, 3H), 2.02 (d, J = 2.2 Hz, 9H), 2.00 (d, J = 1.8 Hz, 6H), 1.98 (s, 3H), 1.97 (s, 3H), 1.95 (d, J = 3.6 Hz, 6H), 1.91 (s, 3H), 1.82 (s, 3H); MS (ESI) m/z: 1 378.31 [M-H]^-。

4a: 黄色油状液, 产率11.2%, mp 241~243 ℃。¹H NMR (600 MHz, DMSO) δ 8.24 (d, J = 2.5 Hz, 1H), 8.02 (dd, J = 8.9, 2.5 Hz, 1H), 7.89 (s, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.45 (s, 1H), 5.88 (d, J = 8.0 Hz, 1H), 5.72 (d, J = 7.9 Hz, 1H), 5.41 (dt, J = 28.5, 9.6 Hz, 2H), 5.11~4.98 (m, 4H), 4.39~4.31 (m, 2H), 4.21 (dt, J = 12.4, 6.2 Hz, 2H), 4.12~4.06 (m, 2H), 2.08 (s, 3H), 2.03 (dd, J = 9.0, 5.0 Hz, 12H), 2.01 (s, 3H), 1.98 (d, J = 6.0 Hz, 6H); MS (ESI) m/z: 1 068.50 [M+Na]⁺。

5a: 黄色油状液, 产率54.0%, mp 94~96 ℃。¹H NMR (600 MHz, DMSO) δ 7.71 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.48 (dd, J = 8.5, 1.9 Hz, 1H), 7.30 (d, J = 8.6 Hz, 1H), 5.68 (d, J = 7.6 Hz, 1H), 5.54 (d, J = 7.7 Hz, 1H), 5.36 (d, J = 3.1 Hz, 1H), 5.33 (d, J = 3.2 Hz, 1H), 5.27 (dd, J = 6.1, 3.5 Hz, 2H), 5.24 (d, J = 7.5 Hz, 1H), 4.44 (t, J = 6.4 Hz, 1H), 4.42~4.38 (m, 1H), 4.00 (d, J = 3.8 Hz, 1H), 3.60 (d, J = 4.8 Hz, 1H), 3.52 (d, J = 2.2 Hz, 1H), 3.47 (d, J = 5.3 Hz, 1H), 3.38 (t, J = 6.6 Hz, 1H), 2.14 (d, J = 4.8 Hz, 6H), 2.08 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H), 1.95 (d, J = 5.9 Hz, 6H), 1.90 (s, 3H), 1.33 (s, 9H); MS (ESI) m/z: 953.74 [M+Na]⁺。

6a: 黄色油状液, 产率50.0%, mp 82~83 ℃。¹H NMR (600 MHz, DMSO) δ 7.70 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.52 (s, 1H), 7.25 (s, 1H), 5.65 (d, J = 7.6 Hz, 1H), 5.54 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 3.1 Hz, 1H), 5.24 (d, J = 4.6 Hz, 2H), 5.22~5.18 (m, 1H), 5.16 (dd, J = 10.4, 3.3 Hz, 1H), 4.49~4.46 (m, 1H), 4.33~4.30 (m, 1H), 4.16~4.07 (m, 4H), 3.96~3.91 (m, 1H), 2.16 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.30 (s, 9H); MS (ESI) m/z: 1 031.32 [M+Na]⁺。

7a: 黄色油状液, 产率15.9%, mp 116~117 ℃。¹H NMR (600 MHz, DMSO) δ 7.80 (dd, J = 9.0, 2.6 Hz, 1H), 7.49 (d, J = 2.6 Hz, 1H), 7.46 (s, 1H), 7.35 (s, 1H), 7.22 (d, J = 9.0 Hz, 1H), 5.70 (d, J = 7.7 Hz, 1H), 5.56 (d, J = 8.0 Hz, 1H), 5.46 (d, J = 7.4 Hz, 1H), 5.31 (d, J = 3.4 Hz, 2H), 5.25~5.15 (m, 6H), 4.99 (dd, J = 10.3, 8.0 Hz, 1H), 4.49 (dd, J = 8.3, 4.3 Hz, 1H), 4.32 (t, J = 6.5 Hz, 1H), 4.06~4.03 (m, 2H), 3.91 (dd, J = 11.3, 7.0 Hz, 1H), 3.62~3.57 (m, 1H), 3.52 (d, J = 5.3 Hz, 1H), 3.47 (dd, J = 5.8, 3.6 Hz, 1H), 3.37 (d, J = 6.5 Hz, 1H), 2.14 (s, 3H), 2.13 (s, 3H), 2.13 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 1.89 (s, 3H), 1.84 (s, 3H); MS (ESI) m/z: 1 398.72 [M+Na]⁺。

8a: 黄色油状液, 产率8.4%, mp 190~192 ℃。¹H NMR (600 MHz, DMSO) δ 8.24 (d, J = 2.5 Hz, 1H), 8.02 (dd, J = 8.9, 2.5 Hz, 1H), 7.90 (s, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.48 (s, 1H), 5.82 (d, J = 7.4 Hz, 1H), 5.60 (d, J = 7.8 Hz, 1H), 5.39 (dd, J = 15.9, 3.3 Hz, 2H), 5.30~5.23 (m, 4H), 4.56~4.50 (m, 2H), 4.15~4.09 (m, 4H), 2.16 (d, J = 10.1 Hz, 6H), 2.10 (s, 3H), 2.06 (s, 3H), 2.04 (d, J = 6.8 Hz, 6H), 1.96 (d, J = 7.6 Hz, 6H); MS (ESI) m/z: 1 084.30 [M+K]⁺。

9a: 黄色油状液, 产率42.6%, mp 72~73 ℃。¹H NMR (600 MHz, DMSO) δ 7.78 (d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.22~7.18 (m, 2H), 7.03 (s, 1H), 5.63 (d, J = 7.9 Hz, 1H), 5.29 (t, J = 9.5 Hz, 1H), 4.91 (t, J = 9.6 Hz, 1H), 4.68 (t, J = 8.7 Hz, 1H), 4.25~4.16 (m, 2H), 4.05 (d, J = 11.8 Hz, 1H), 3.87 (s, 3H), 3.75 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.92 (s, 3H), 1.86 (s, 3H); MS (ESI) m/z: 767.46 [M+Na]⁺。

10a: 黄色油状液, 产率41.2%, mp 89~91 ℃。¹H NMR (600 MHz, DMSO) δ 7.79 (dd, J = 8.9, 2.5 Hz, 1H), 7.48 (d, J = 2.5 Hz, 1H), 7.21 (d, J = 9.0 Hz, 1H), 7.20 (s, 1H), 7.05 (s, 1H), 5.55 (d, J = 8.0 Hz, 1H), 5.30 (d, J = 3.4 Hz, 1H), 5.18 (dd, J = 10.3, 3.5 Hz, 1H), 4.90~4.86 (m, 1H), 4.40 (t, J = 6.4 Hz, 1H), 4.10~4.02 (m, 2H), 3.87 (s, 3H), 3.76 (s, 3H), 2.13 (s, 3H), 2.02 (s, 3H), 1.90 (d, J = 4.2 Hz, 6H); MS (ESI) m/z: 767.14 [M+Na]⁺。

11a: 黄色油状液, 产率20.0%, mp 106~108 ℃。¹H NMR (600 MHz, DMSO) δ 7.78 (dd, J = 8.9, 2.6 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 7.17 (s, 1H), 7.05 (s, 1H), 5.69 (d, J = 7.7 Hz, 1H), 5.34 (t, J = 9.6 Hz, 1H), 4.94 (d, J = 9.8 Hz, 1H), 4.66 (d, J = 10.0 Hz, 1H), 4.64~4.62 (m, 1H), 3.87 (s, 3H), 3.76 (s, 3H), 3.62 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H), 1.89 (s, 3H); MS (ESI) m/z: 753.56 [M+Na]⁺。

12a: 黄色油状液, 产率40.5%, mp 151~153 ℃。¹H NMR (600 MHz, DMSO-d₆) δ 9.68 (s, 1H), 7.65 (dd, J = 8.7, 2.2 Hz, 2H), 7.57 (dd, J = 8.4, 2.3 Hz, 2H), 7.26 (d, J = 2.0 Hz, 1H), 7.19 (dd, J = 8.2, 2.2 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.56 (d, J = 7.9 Hz, 1H), 5.36 (d, J = 3.8 Hz, 2H), 5.32 (d, J = 3.9 Hz, 1H), 5.27~5.22 (m, 4H), 4.98 (d, J = 8.2 Hz, 2H), 4.83 (dd, J = 10.3, 3.8 Hz, 3H), 4.38 (d, J = 3.6 Hz, 1H), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H), 1.33 (s, 9H); MS (ESI) m/z: 911.35 [M+Na]⁺。

13a: 黄色油状液, 产率33.9%, mp 235~237 ℃。¹H NMR (600 MHz, DMSO) δ 9.68 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.4, 1.9 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 5.86 (d, J = 8.3 Hz, 1H), 5.49 (d, J = 8.0 Hz, 1H), 5.26~5.21 (m, 2H), 5.02 (d, J = 8.2 Hz, 1H), 4.99 (d, J = 8.0 Hz, 3H), 4.82 (d, J = 8.0 Hz, 2H), 4.75~4.73 (m, 4H), 2.07 (s, 3H), 2.00 (d, J = 2.0 Hz, 6H), 1.96 (d, J = 2.8 Hz, 6H), 1.92 (d, J = 4.4 Hz, 6H), 1.33 (s, 9H); MS (ESI) m/z: 889.45 [M+H]⁺。

14a: 黄色油状液, 产率39.8%, mp 216~217 ℃。¹H NMR (600 MHz, DMSO-d₆) δ 7.70 (d, J = 8.4 Hz, 2H), 7.60~7.56 (m, 3H), 7.43 (dd, J = 8.5, 1.7 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 5.66 (d, J = 8.0 Hz, 1H), 5.52 (d, J = 8.0 Hz, 1H), 5.29~5.24 (m, 4H), 4.96~4.87 (m, 4H), 4.84 (d, J = 8.0 Hz, 2H), 4.69~4.63 (m, 2H), 4.35 (d, J = 10.9 Hz, 1H), 4.13~3.97 (m, 11H), 3.91~3.82 (m, 2H), 2.05 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.98~1.96 (m, 21H), 1.92 (s, 3H), 1.92 (s, 3H), 1.33 (s, 9H); MS (ESI) m/z: 1 529.55 [M+Na]⁺。

15a: 黄色油状液, 产率31.6%, mp 250~253 ℃。¹H NMR (600 MHz, DMSO-d₆) δ 7.68 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.46 (s, 1H), 7.18 (s, 1H), 5.64~5.62 (m, 2H), 5.52 (dd, J = 7.1, 1.8 Hz, 2H), 5.24 (dd, J = 20.7, 10.8 Hz, 6H), 5.15 (s, 2H), 4.96~4.86 (m, 9H), 4.70~4.60 (m, 7H), 4.37 (s, 3H), 2.08 (s, 3H), 2.06~1.86 (m, 33H), 1.77 (s, 3H), 1.30 (s, 9H); MS (ESI) m/z: 1 582.57 [M-H]^-。

16a: 黄色油状液, 产率44.2%, mp 144~146 ℃。¹H NMR (600 MHz, DMSO) δ 9.98 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.35 (s, 1H), 6.86 (s, 1H), 5.57 (d, J = 7.9 Hz, 1H), 5.38 (d, J = 9.6 Hz, 1H), 5.09 (dd, J = 9.7, 8.0 Hz, 1H), 5.00 (t, J = 9.7 Hz, 1H), 4.18 (d, J = 6.4 Hz, 1H), 4.10 (dd, J = 8.8, 4.0 Hz, 2H), 2.05 (s, 3H), 2.02 (s, 3H), 2.02 (s, 3H), 1.98 (s, 3H), 1.31 (s, 9H); MS (ESI) m/z: 679.48 [M+H]⁺。

17a: 黄色油状液, 产率34.0%, mp 220~221 ℃。¹H NMR (600 MHz, DMSO) δ 8.22 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.9, 2.6 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.53 (s, 1H), 7.33 (s, 1H), 5.76 (d, J = 7.9 Hz, 1H), 5.40 (t, J = 9.6 Hz, 1H), 5.16 (dd, J = 9.7, 8.0 Hz, 1H), 5.09~5.01 (m, 2H), 4.21~4.15 (m, 2H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H); MS (ESI) m/z: 738.79 [M+Na]⁺。

18a: 黄色油状液, 产率27.7%, mp 121~123 ℃。¹H NMR (600 MHz, DMSO) δ 9.98 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.36 (s, 1H), 6.86 (s, 1H), 5.47 (d, J = 7.8 Hz, 1H), 5.27~5.25 (m, 2H), 4.14~4.09 (m, 4H), 2.04 (s, 6H), 2.01 (s, 3H), 1.96 (s, 3H), 1.31 (s, 9H); MS (ESI) m/z: 701.06 [M+Na]⁺。

19a: 黄色油状液, 产率39.8%, mp 152~153 ℃。¹H NMR (600 MHz, DMSO) δ 10.03 (s, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.53 (s, 1H), 7.34 (s, 1H), 5.67 (d, J = 7.7 Hz, 1H), 5.39 (d, J = 3.3 Hz, 1H), 5.34~5.31 (m, 1H), 4.15 (d, J = 6.3 Hz, 1H), 4.09 (d, J = 4.8 Hz, 1H), 3.59 (d, J = 4.7 Hz, 1H), 3.52 (d, J = 5.3 Hz, 1H), 2.17 (s, 3H), 2.07 (s, 3H), 2.02 (d, J = 12.3 Hz, 6H); MS (ESI) m/z: 715.01 [M-H]^-。

20a: 黄色油状液, 产率20.1%, mp 203~205 ℃。¹H NMR (600 MHz, DMSO) δ 7.66 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.35 (s, 1H), 6.87 (s, 1H), 5.68 (d, J = 7.8 Hz, 1H), 5.43 (t, J = 9.6 Hz, 1H), 5.12 (dd, J = 16.6, 9.5 Hz, 2H), 4.71 (d, J = 9.9 Hz, 1H), 3.66 (s, 3H), 2.02 (s, 3H), 2.01 (s, 6H), 1.31 (s, 9H); MS (ESI) m/z: 687.18 [M+Na]⁺。

21a: 黄色油状液, 产率14.0%, mp 155~156 ℃。¹H NMR (600 MHz, DMSO) δ 10.08 (s, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.54 (s, 1H), 7.35 (s, 1H), 5.86 (d, J = 7.7 Hz, 1H), 5.45 (t, J = 9.6 Hz, 1H), 5.20 (d, J = 7.8 Hz, 1H), 5.13 (d, J = 9.8 Hz, 1H), 4.80 (d, J = 9.9 Hz, 1H), 3.65 (s, 3H), 2.01 (d, J = 1.9 Hz, 9H); MS (ESI) m/z: 701.01 [M-H]^-。

22a: 黄色油状液, 产率20.1%, mp 135~136 ℃。¹H NMR (600 MHz, DMSO) δ 7.66 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.36 (s, 1H), 6.87 (s, 1H), 6.17 (d, J = 4.0 Hz, 1H), 5.75 (d, J = 6.7 Hz, 1H), 5.55 (dd, J = 14.6, 3.4 Hz, 2H), 5.31~5.27 (m, 3H), 5.26~5.20 (m, 3H), 5.09 (d, J = 5.8 Hz, 1H), 5.00 (dd, J = 9.9, 2.7 Hz, 2H), 4.90 (d, J = 3.7 Hz, 1H), 2.08 (s, 3H), 2.04~1.96 (m, 18H), 1.31 (s, 9H); MS (ESI) m/z: 967.57 [M-H]^-。

23a: 黄色油状液, 产率23.5%, mp 186~188 ℃。¹H NMR (600 MHz, DMSO) δ 9.93 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 9.2 Hz, 2H), 7.32 (s, 1H), 6.84 (s, 1H), 5.49 (d, J = 8.2 Hz, 1H), 5.26 (d, J = 5.8 Hz, 1H), 4.92~4.86 (m, 4H), 4.28~4.25 (m, 2H), 4.19~4.11 (m, 6H), 2.13~1.89 (m, 21H), 1.30 (s, 9H); MS (ESI) m/z: 988.94 [M+Na]⁺。

24a: 黄色油状液, 产率36.9%, mp 241~244 ℃。¹H NMR (600 MHz, DMSO) δ 10.01 (s, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.29 (s, 1H), 5.74 (d, J = 7.9 Hz, 1H), 5.45 (d, J = 9.1 Hz, 1H), 5.32 (d, J = 3.8 Hz, 1H), 5.26 (d, J = 11.3 Hz, 2H), 5.07~5.04 (m, 1H), 4.92 (dd, J = 10.5, 3.8 Hz, 1H), 4.51~4.48 (m, 1H), 4.37~4.33 (m, 2H), 4.11~4.09 (m, 3H), 4.04 (d, J = 10.7 Hz, 1H), 2.01 (d, J = 4.5 Hz, 9H), 1.99~1.96 (m, 12H); MS (ESI) m/z: 1 004.63 [M+H]⁺。

25a: 黄色油状液, 产率32.3%, mp 189~192 ℃。¹H NMR (600 MHz, DMSO) δ 10.01 (s, 1H), 8.22 (d, J = 2.5 Hz, 1H), 7.98 (dd, J = 8.9, 2.5 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.29 (s, 1H), 5.67 (d, J = 8.0 Hz, 1H), 5.29~5.27 (m, 2H), 5.08 (d, J = 8.2 Hz, 1H), 4.90 (d, J = 7.9 Hz, 2H), 4.69 (d, J = 9.5 Hz, 1H), 4.44 (d, J = 10.7 Hz, 1H), 4.28~4.25 (m, 1H), 4.23 (d, J = 6.8 Hz, 1H), 4.11~4.08 (m, 2H), 4.03 (d, J = 12.9 Hz, 1H), 3.91 (d, J = 9.5 Hz, 1H), 2.14 (s, 3H), 2.03 (d, J = 3.1 Hz, 6H), 2.01 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.93 (s, 3H); MS (ESI) m/z: 1 043.14 [M+K]⁺。

目标化合物的波谱数据如下:

1b: ¹H NMR (600 MHz, MeOD) δ 7.74 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 2.1 Hz, 1H), 7.60 (d, J = 8.5 Hz, 2H), 7.53 (dd, J = 8.5, 2.1 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 5.05 (d, J = 7.7 Hz, 1H), 4.94 (d, J = 7.6 Hz, 1H), 3.91 (dd, J = 12.1, 2.1 Hz, 1H), 3.80 (dd, J = 12.1, 2.4 Hz, 1H), 3.73 (dt, J = 12.1, 4.5 Hz, 2H), 3.62~3.56 (m, 2H), 3.51~3.42 (m, 6H), 1.40 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 195.7, 156.2, 151.7, 146.8, 134.7, 132.4, 129.6, 126.3, 125.1, 121.2, 117.3, 102.5, 101.5, 77.0, 76.8, 76.3, 76.3, 73.6, 73.5, 69.9, 69.6, 61.0, 60.7, 34.6, 30.1; MS (ESI) m/z: 617.49 [M+Na]⁺。

2b: ¹H NMR (600 MHz, MeOD) δ 7.72 (d, J = 8.5 Hz, 2H), 7.56~7.54 (m, 3H), 7.27 (s, 1H), 5.00 (d, J = 7.5 Hz, 1H), 4.88 (d, J = 7.6 Hz, 1H), 3.89 (dd, J = 12.2, 2.2 Hz, 1H), 3.74~3.70 (m, 2H), 3.63 (dd, J = 12.1, 5.2 Hz, 1H), 3.55~3.41 (m, 8H), 1.35 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 194.6, 157.8, 149.4, 146.6, 144.9, 135.0, 133.7, 130.1, 119.4, 112.1, 110.0, 102.1, 102.0, 77.3, 77.0, 76.5, 76.4, 73.7, 73.7, 70.0, 69.8, 64.0, 60.8, 35.0, 30.5; MS (ESI) m/z: 695.46 [M+Na]⁺。

3b: ¹H NMR (600 MHz, MeOD) δ 7.66 (dd, J = 8.9, 2.6 Hz, 1H), 7.64 (d, J = 2.5 Hz, 1H), 7.49 (s, 1H), 7.35 (s, 1H), 7.21 (d, J = 8.8 Hz, 1H), 5.02 (d, J = 7.1 Hz, 1H), 4.90 (d, J = 7.8 Hz, 1H), 4.87 (d, J = 7.5 Hz, 1H), 3.90~3.82 (m, 2H), 3.74~3.63 (m, 4H), 3.52~3.36 (m, 8H), 3.25 (t, J = 9.4 Hz, 2H), 2.96 (dd, J = 9.2, 7.7 Hz, 2H); ¹³C NMR (150 MHz, MeOD) δ 189.8, 177.0, 172.4, 166.2, 157.2, 156.3, 147.4, 146.1, 139.7, 136.3, 131.7, 128.5, 121.5, 113.2, 109.4, 100.5, 100.0, 86.4, 84.6, 82.9, 78.4, 76.9, 76.5, 76.2, 75.7, 73.5, 73.4, 71.0, 69.8, 69.8, 69.5; MS (ESI) m/z: 895.56 [M+Na]⁺。

4b: ¹H NMR (600 MHz, DMSO) δ 8.46 (s, 1H), 8.27 (d, J = 2.3 Hz, 1H), 7.98 (dd, J = 8.9, 2.2 Hz, 1H), 7.85 (s, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.41 (s, 1H), 5.20 (d, J = 7.4 Hz, 1H), 5.01 (d, J = 7.0 Hz, 1H), 3.62~3.57 (m, 5H), 3.47~3.40 (m, 5H), 3.32~3.29 (m, 2H); ¹³C NMR (150 MHz, DMSO) δ 179.4, 167.6, 154.5, 152.4, 144.4, 137.3, 127.8, 122.2, 120.5, 116.3, 114.8, 111.9, 104.3, 101.5, 100.4, 77.1, 77.0, 76.2, 76.1, 73.2, 73.0, 69.4, 69.4, 60.5, 60.4; MS (ESI) m/z: 711.14 [M+H]⁺。

5b: ¹H NMR (400 MHz, DMSO) δ 7.65 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.40 (dd, J = 6.8, 1.7 Hz, 1H), 7.28 (d, J = 10.2 Hz, 1H), 4.93 (d, J = 7.7 Hz, 1H), 4.82 (d, J = 7.7 Hz, 1H), 3.79~3.45 (m, 12H), 1.30 (s, 9H); ¹³C NMR (100 MHz, MeOD) δ 196.3, 157.3, 148.7, 144.7, 134.3, 131.0, 129.9, 125.2, 125.1, 119.4, 116.1, 108.6, 102.1, 75.8, 75.5, 73.3, 73.2, 71.1, 70.9, 68.8, 68.7, 61.3, 60.8, 34.7, 30.1; MS (ESI) m/z: 617.23 [M+Na]⁺。

6b: ¹H NMR (400 MHz, DMSO) δ 7.68 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.44 (s, 1H), 7.19 (s, 1H), 5.00 (d, J = 7.3 Hz, 1H), 4.89 (d, J = 7.5 Hz, 1H), 3.71~3.54 (m, 12H), 1.31 (s, 9H); ¹³C NMR (100 MHz, MeOD) δ 195.2, 157.8, 149.6, 146.7, 135.2, 133.5, 130.0, 125.4, 122.9, 119.8, 112.3, 102.8, 102.6, 80.2, 75.9, 75.6, 73.2, 71.0, 70.9, 68.7, 68.7, 61.0, 60.8, 34.7, 30.0; MS (ESI) m/z: 695.19 [M+Na]⁺。

7b: ¹H NMR (400 MHz, DMSO) δ 7.70 (dd, J = 9.1, 2.7 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.38 (s, 1H), 7.28 (s, 1H), 7.22 (d, J = 8.9 Hz, 1H), 5.00 (d, J = 7.9 Hz, 1H), 4.79 (dd, J = 7.4, 5.3 Hz, 2H), 3.72~3.56 (m, 18H); ¹³C NMR (100 MHz, MeOD) δ 192.9, 155.4, 150.6, 146.4, 136.0, 133.9, 132.3, 131.4, 121.8, 121.2, 119.0, 113.7, 113.1, 102.8, 102.0, 102.0, 76.1, 76.0, 75.5, 73.6, 73.3, 70.9, 70.7, 70.6, 68.4, 68.1, 68.1, 60.7, 60.5, 60.2, 60.0; MS (ESI) m/z: 897.07 [M+Na]⁺。

8b: ¹H NMR (600 MHz, DMSO) δ 8.38 (s, 1H), 8.23 (d, J = 2.4 Hz, 1H), 7.99 (dd, J = 8.8, 2.4 Hz, 1H), 7.77 (s, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.38 (s, 1H), 5.18 (d, J = 7.6 Hz, 1H), 5.00 (d, J = 7.6 Hz, 1H), 3.77~3.65 (m, 5H), 3.59 (dq, J = 16.2, 5.5, 5.0 Hz, 3H), 3.52~3.46 (m, 4H); ¹³C NMR (150 MHz, DMSO) δ 174.3, 155.0, 152.5, 148.9, 145.1, 136.0, 122.8, 121.2, 116.7, 115.1, 111.4, 104.4, 103.3, 101.9, 101.2, 79.3, 76.2, 75.9, 73.8, 70.9, 70.5, 68.3, 68.2, 60.6, 60.4; MS (ESI) m/z: 709.07 [M-H]^-。

9b: ¹H NMR (600 MHz, MeOD) δ 7.60 (dd, J = 8.9, 2.6 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 7.11 (s, 1H), 7.04 (s, 1H), 4.84 (d, J = 7.7 Hz, 1H), 3.84 (s, 3H), 3.82~3.78 (m, 1H), 3.75 (s, 3H), 3.61 (dd, J = 12.0, 5.8 Hz, 1H), 3.37~3.32 (m, 2H), 3.22 (t, J = 9.3 Hz, 1H), 3.03 (dd, J = 9.2, 7.7 Hz, 1H); ¹³C NMR (150 MHz, DMSO) δ 94.0, 155.1, 152.2, 148.3, 135.9, 132.6, 132.3, 131.4, 118.6, 116.2, 114.3, 114.0, 111.8, 101.3, 76.9, 76.4, 73.4, 69.8, 61.1, 55.6, 55.4; MS (ESI) m/z: 599.08 [M+Na]⁺。

10b: ¹H NMR (600 MHz, MeOD) δ 7.65 (dd, J = 8.9, 2.5 Hz, 1H), 7.51 (d, J = 2.5 Hz, 1H), 7.28 (d, J = 8.9 Hz, 1H), 7.16 (s, 1H), 7.11 (s, 1H), 4.84 (d, J = 7.5 Hz, 1H), 3.90 (s, 3H), 3.85 (d, J = 3.2 Hz, 1H), 3.82 (s, 3H), 3.77~3.70 (m, 2H), 3.66~3.63 (m, 1H), 3.53 (dd, J = 9.7, 3.3 Hz, 1H), 3.48 (dd, J = 9.7, 7.5 Hz, 1H); ¹³C NMR (150 MHz, DMSO) δ 193.4, 155.3, 151.9, 148.2, 136.0, 132.4, 132.3, 131.9, 118.9, 116.6, 114.5, 113.7, 111.4, 102.0, 76.1, 73.8, 70.6, 68.3, 60.6, 56.6, 56.2; MS (ESI) m/z: 599.06 [M+Na]⁺。

11b: ¹H NMR (600 MHz, MeOD) δ 11.50 (s, 1H), 7.56 (dd, J = 8.9, 2.7 Hz, 1H), 7.38 (d, J = 2.6 Hz, 1H), 7.23 (d, J = 8.9 Hz, 1H), 7.08 (s, 1H), 7.03 (s, 1H), 4.88 (d, J = 7.7 Hz, 1H), 3.80 (s, 3H), 3.72 (s, 3H), 3.58 (d, J = 1.8 Hz, 1H), 3.51~3.45 (m, 1H), 3.37 (dd, J = 9.6, 7.3 Hz, 2H); ¹³C NMR (150 MHz, MeOD) δ 194.1, 169.0, 155.3, 152.2, 148.4, 135.8, 132.6, 132.2, 131.5, 118.7, 116.2, 114.2, 114.0, 111.9, 102.1, 75.8, 73.4, 70.7, 68.6, 61.0, 55.5; MS (ESI) m/z: 613.02 [M+Na]⁺; HR-MS (ESI): m/z C₂₁H₂₀Br₂O₁₀ [M+H]⁺计算值590.949 6, 测量值590.947 3。

12b: ¹H NMR (600 MHz, DMSO) δ 9.20 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 2.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.16 (dd, J = 8.3, 1.9 Hz, 1H), 5.65 (d, J = 2.8 Hz, 1H), 5.43 (d, J = 6.2 Hz, 1H), 5.07 (d, J = 4.0 Hz, 1H), 4.99 (d, J = 7.7 Hz, 1H), 4.94 (d, J = 5.4 Hz, 1H), 4.92~4.91 (m, 1H), 4.62~4.59 (m, 1H), 4.54 (t, J = 5.7 Hz, 1H), 3.74 (dd, J = 14.1, 5.9 Hz, 2H), 3.64~3.59 (m, 4H), 1.33 (s, 9H); ¹³C NMR (150 MHz, DMSO) δ 177.9, 155.6, 149.5, 146.9, 135.4, 131.8, 130.0, 129.8, 125.7, 122.7, 117.4, 115.5, 101.2, 79.5, 76.1, 75.9, 74.0, 73.8, 73.3, 72.9, 70.4, 61.3, 60.7, 35.3, 31.4; MS (ESI) m/z: 593.17 [M-H]^-。

13b: ¹H NMR (600 MHz, DMSO) δ 8.33 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 1.9 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.15 (dd, J = 8.4, 1.9 Hz, 1H), 5.14 (d, J = 18.3 Hz, 2H), 4.99 (d, J = 7.7 Hz, 1H), 4.86 (s, 1H), 4.71 (d, J = 5.9 Hz, 2H), 4.34 (d, J = 7.9 Hz, 1H), 3.77 (d, J = 13.7 Hz, 2H), 3.73~3.65 (m, 4H), 3.63 (d, J = 10.0 Hz, 1H), 1.33 (s, 9H); ¹³C NMR (150 MHz, DMSO) δ 177.5, 150.1, 145.8, 142.7, 133.8, 129.5, 125.0, 123.1, 122.8, 117.2, 115.7, 106.5, 103.2, 76.7, 76.4, 75.5, 74.4, 73.1, 70.0, 61.0, 60.1, 58.3, 56.0, 34.6, 30.1; MS (ESI) m/z: 617.21 [M+Na]⁺。

14b: ¹H NMR (600 MHz, MeOD) δ 7.63 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 2.1 Hz, 1H), 7.51~7.47 (m, 2H), 7.44 (dd, J = 8.5, 2.0 Hz, 1H), 7.27 (d, J = 8.5 Hz, 1H), 5.01 (d, J = 7.6 Hz, 1H), 4.87~4.83 (m, 3H), 4.37 (dd, J = 7.9, 4.0 Hz, 2H), 3.81 (dd, J = 10.8, 5.1 Hz, 5H), 3.64~3.50 (m, 13H), 3.33~3.28 (m, 4H), 1.28 (s, 9H); ¹³C NMR (150 MHz, DMSO) δ 184.0, 170.2, 164.8, 142.0, 140.5, 129.6, 126.4, 125.1, 117.6, 116.7, 109.4, 108.9, 106.8, 103.7, 103.2, 90.1, 86.7, 76.7, 75.7, 75.3, 74.6, 73.5, 73.2, 70.4, 70.0, 69.8, 69.8, 69.4, 68.6, 68.6, 66.8, 61.0, 60.1, 59.9, 55.7, 31.8, 30.1; MS (ESI) m/z: 941.37 [M+Na]⁺。

15b: ¹H NMR (600 MHz, MeOD) δ 7.74 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.25 (s, 1H), 7.18 (s, 1H), 5.19 (d, J = 6.9 Hz, 1H), 5.00 (d, J = 7.4 Hz, 1H), 4.48 (d, J = 7.7 Hz, 1H), 4.44 (d, J = 7.9 Hz, 1H), 3.97~3.82 (m, 5H), 3.78~3.54 (m, 11H), 3.50~3.36 (m, 6H), 3.25~3.20 (m, 2H), 1.38 (s, 9H); ¹³C NMR (150 MHz, DMSO) δ 187.6, 166.4, 144.2, 143.0, 138.0, 131.1, 130.0, 125.3, 121.4, 120.6, 118.7, 113.4, 106.2, 103.1, 102.1, 79.7, 78.3, 78.3, 76.7, 76.4, 75.3, 75.2, 74.4, 73.5, 73.2, 70.0, 68.6, 65.7, 64.3, 61.4, 61.0, 59.8, 56.2, 53.6, 50.3, 34.7, 30.1; MS (ESI) m/z: 1 019.83 [M+Na]⁺。

16b: ¹H NMR (600 MHz, MeOD) δ 8.42 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 7.50 (s, 1H), 6.86 (s, 1H), 4.95 (d, J = 7.1 Hz, 1H), 3.93 (dd, J = 12.2, 2.3 Hz, 1H), 3.76 (dd, J = 12.1, 5.6 Hz, 1H), 3.58~3.51 (m, 3H), 3.48~3.43 (m, 1H), 1.37 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 195.7, 157.7, 147.1, 146.6, 135.4, 133.6, 129.9, 125.4, 121.3, 116.2, 107.7, 102.3, 77.1, 76.1, 73.4, 69.9, 60.9, 34.7, 30.0; MS (ESI) m/z: 509.20 [M-H]^-。

17b: ¹H NMR (600 MHz, DMSO) δ 9.52 (s, 1H), 9.39 (s, 1H), 8.21 (d, J = 2.5 Hz, 1H), 7.96 (dd, J = 8.9, 2.5 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.48 (s, 1H), 7.29 (s, 1H), 5.10 (d, J = 7.1 Hz, 1H), 3.73 (d, J = 10.2 Hz, 1H), 3.51 (dd, J = 12.8, 6.0 Hz, 3H), 3.25~3.19 (m, 2H); ¹³C NMR (150 MHz, DMSO) δ 174.2, 155.0, 153.2, 150.6, 145.8, 137.5, 128.2, 122.7, 121.2, 116.4, 115.7, 109.5, 103.9, 101.2, 77.7, 76.4, 73.5, 70.1, 61.2; MS (ESI) m/z: 587.12 [M+K]⁺。

18b: ¹H NMR (600 MHz, MeOD) δ 8.57 (s, 1H), 7.73 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.50 (s, 1H), 6.83 (s, 1H), 4.61 (d, J = 8.3 Hz, 1H), 3.92 (d, J = 33.0 Hz, 2H), 3.81 (d, J = 14.2 Hz, 2H), 3.68~3.62 (m, 2H), 1.38 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 196.0, 157.7, 151.2, 147.6, 135.4, 133.7, 129.9, 125.3, 121.4, 116.6, 106.6, 103.2, 75.9, 73.1, 70.8, 68.8, 61.0, 34.7, 30.0; MS (ESI) m/z: 509.33 [M-H]^-; HR-MS (ESI): m/z C₂₃H₂₇BrO₈ [M-H]^-计算值509.081 1, 测量值509.081 6。

19b: ¹H NMR (600 MHz, MeOD) δ 9.44 (s, 1H), 8.43 (s, 1H), 8.23 (d, J = 2.3 Hz, 1H), 7.80 (dd, J = 8.7, 2.1 Hz, 1H), 7.50 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 4.99 (d, J = 7.7 Hz, 1H), 3.89~3.83 (m, 2H), 3.77~3.71 (m, 3H), 3.60 (d, J = 8.2 Hz, 1H); ¹³C NMR (150 MHz, MeOD) δ 175.0, 161.6, 156.5, 155.2, 153.7, 148.7, 142.3, 137.2, 134.4, 128.1, 120.0, 116.3, 106.9, 101.9, 76.1, 72.9, 68.8, 61.0, 56.1; MS (ESI) m/z: 549.36 [M+H]⁺。

20b: ¹H NMR (600 MHz, MeOD) 11.70 (s, 1H), 8.45 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.42 (s, 1H), 6.73 (s, 1H), 4.82 (d, J = 7.1 Hz, 1H), 3.56 (s, 1H), 3.50 (d, J = 2.8 Hz, 1H), 3.47 (d, J = 4.9 Hz, 1H), 3.38 (d, J = 6.6 Hz, 1H), 1.26 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 195.8, 172.7, 164.9, 157.7, 147.5, 133.7, 129.9, 125.4, 122.4, 121.0, 116.6, 107.3, 103.0, 76.0, 73.2, 70.7, 69.8, 31.4, 30.0; MS (ESI) m/z: 523.41 [M-H]^-; HR-MS (ESI): m/z C₂₃H₂₅BrO₉ [M+H]⁺计算值525.075 4, 测量值525.074 9。

21b: ¹H NMR (600 MHz, MeOD) δ 11.30 (s, 1H), 8.35 (s, 1H), 8.26 (s, 1H), 8.21 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 8.9, 2.5 Hz, 1H), 7.49 (s, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.22 (s, 1H), 5.12 (d, J = 7.6 Hz, 1H), 4.06 (d, J = 9.4 Hz, 1H), 3.56 (p, J = 9.8 Hz, 3H); ¹³C NMR (150 MHz, MeOD) δ 176.0, 172.3, 161.4, 153.7, 152.6, 149.8, 142.0, 141.5, 132.4, 129.4, 121.5, 117.8, 110.9, 105.3, 102.5, 82.1, 76.9, 62.0, 57.5; MS (ESI) m/z: 563.04 [M+H]⁺。

22b: ¹H NMR (600 MHz, MeOD) δ 8.10 (s, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.49 (s, 1H), 6.87 (s, 1H), 5.25 (d, J = 3.7 Hz, 1H), 5.00 (d, J = 7.8 Hz, 1H), 3.97~3.94 (m, 1H), 3.92~3.86 (m, 2H), 3.81 (t, J = 9.1 Hz, 1H), 3.77~3.60 (m, 8H), 1.38 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 195.7, 157.8, 147.0, 146.6, 133.6, 129.9, 125.4, 121.3, 117.6, 116.3, 107.7, 102.1, 101.5, 79.3, 75.8, 75.6, 73.6, 73.5, 73.0, 72.7, 70.2, 61.3, 60.4, 34.7, 30.0; MS (ESI) m/z: 694.80 [M+Na]⁺。

23b: ¹H NMR (600 MHz, MeOD) δ 8.10 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.48 (s, 1H), 6.87 (s, 1H), 5.01 (d, J = 7.7 Hz, 1H), 4.49 (d, J = 7.9 Hz, 1H), 3.96 (d, J = 2.9 Hz, 2H), 3.93 (dd, J = 11.9, 2.1 Hz, 1H), 3.76~3.61 (m, 6H), 3.44~3.39 (m, 2H), 3.29 (d, J = 8.0 Hz, 1H), 1.38 (s, 9H); ¹³C NMR (150 MHz, MeOD) δ 195.7, 157.8, 146.9, 146.6, 135.5, 133.6, 129.9, 125.4, 121.3, 116.3, 107.7, 103.2, 101.9, 78.6, 76.7, 76.4, 75.5, 74.4, 73.5, 73.2, 70.0, 61.0, 60.1, 34.7, 30.0; MS (ESI) m/z: 694.75 [M+Na]⁺。

24b: ¹H NMR (600 MHz, MeOD) δ 10.03 (s, 1H), 9.61 (s, 1H), 8.34 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 8.9, 2.4 Hz, 1H), 7.61 (s, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.38 (s, 1H), 5.26 (d, J = 3.7 Hz, 1H), 5.18 (d, J = 7.7 Hz, 1H), 4.02~3.99 (m, 1H), 3.94~3.85 (m, 3H), 3.78~3.73 (m, 3H), 3.69 (dd, J = 8.8, 6.3 Hz, 3H), 3.52~3.49 (m, 2H); ¹³C NMR (150 MHz, MeOD) δ 175.4, 151.3, 138.6, 129.5, 127.9, 126.1, 121.4, 117.8, 114.7, 111.6, 108.6, 107.4, 104.5, 103.0, 102.4, 89.7, 88.2, 84.4, 80.7, 77.2, 74.9, 74.2, 65.9, 61.9, 60.6; MS (ESI) m/z: 711.32 [M+H]⁺。

25b: ¹H NMR (600 MHz, DMSO) δ 9.77 (s, 1H), 9.71 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.97 (dd, J = 8.9, 2.4 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.52 (s, 1H), 7.32 (s, 1H), 5.23 (d, J = 7.7 Hz, 1H), 5.18~5.17 (m, 1H), 5.13 (d, J = 4.9 Hz, 1H), 4.75 (dd, J = 20.1, 5.5 Hz, 2H), 4.36 (d, J = 7.9 Hz, 1H), 3.79 (dd, J = 9.7, 5.0 Hz, 1H), 3.71 (t, J = 8.9 Hz, 3H), 3.53~3.44 (m, 4H); ¹³C NMR (150 MHz, DMSO) δ 183.6, 174.2, 164.9, 155.5, 150.8, 140.6, 132.6, 127.7, 126.5, 122.3, 116.7, 115.7, 109.1, 105.0, 103.5, 99.8, 92.8, 84.2, 80.1, 77.0, 70.5, 65.5, 57.1, 53.2, 52.6; MS (ESI) m/z: 732.50 [M+Na]⁺; HR-MS (ESI): m/z C₂₅H₂₈Br₂O₁₄ [M-H]^-计算值708.976 8, 测量值708.975 2。

2 纯度测定

色谱条件: Diamonsil C18 (250 mm × 4.6 mm, 5 µm) 色谱柱, 甲醇/水为流动相进行线性梯度洗脱: 5∶95洗脱40 min, 95∶5洗脱10 min, 5∶95洗脱10 min; 化合物1b~25b的检测波长分别为274.0、268.8、300.0、246.3、275.9、268.0、314.2、300.0、254.0、254.0、289.0、271.2、247.5、272.3、267.6、266.0、244.0、266.0、246.3、284.8、284.8、266.4、267.6、244.0、244.0 nm, 流速1.0 mL·min^-1, 柱温30 ℃, 进样体积10 μL。

3 生物膜干涉技术

设置没有固化蛋白的对照传感器以消除背景信号, 将His-PDIA6蛋白包被于Ni-NTA传感器浸没在PBST缓冲液中以平衡基线。再将传感器分别浸入20~200 μmol·L^-1不同浓度的目标化合物溶液中, 结合、解离时间均为60 s。采用Forte Bio Data Analysis软件(v.11.1.0.4) 计算得到结合解离常数。

4 体外抗炎实验

采用CCK-8法检测化合物的RAW264.7细胞毒性, 取对数生长期的RAW264.7细胞接种至96孔板, 培养4 h待细胞贴壁后, 分为以下几组: 空白对照组、LPS造模组(1 mg·L^-1) 以及化合物(10 μmol·L^-1) 和LPS (1 mg·L^-1) 共同作用组, 继续培养24 h取上清。塞来昔布(10 μmol·L^-1) 作为阳性对照。采用Griess法检测细胞NO分泌水平, 在546 nm处测量吸光度值。按照ELISA试剂盒说明书测定细胞培养上清中IL-1β、TNF-α、IL-6和IL-10的释放量。按照公式: 抑制率(%) = [(OD_模型组-OD_样品组)/(OD_模型组-OD_空白组)]×100%, 计算不同药物浓度下IL-1β的抑制率, 从而计算IC₅₀值。

5 统计学分析

数据以至少3个独立实验的平均值±标准差表示, 结果采用GraphPad Prism 8.0软件进行统计分析, 组间比较采用单因素方差分析(one-way ANOVA), P < 0.05表明差异具有统计学意义。

作者贡献: 李蔷主要负责化合物的合成、抗炎活性筛选、数据分析以及论文撰写; 程宁宁负责化合物的设计、分子互作和纯度测定; 冯秀娥负责论文的整体设计、修改、构效关系分析; 李青山负责论文的整体构思。

利益冲突: 本文作者声明无任何利益冲突。

基金

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重大新药创制国家科技重大专项(2018ZX09711001-001-017)
山西省重点研发计划项目(202102130501005)
山西省自然科学基金项目(201901D111209)
基于炎性反应的重大疾病创新药物山西省重点实验室2021年度开放课题(2021sxcxyw06)

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2024年第59卷第6期

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doi: 10.16438/j.0513-4870.2023-1384

接收时间：2023-12-12
首发时间：2025-11-26
出版时间：2024-06-12

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收稿日期：2023-12-12
修回日期：2024-04-15

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重大新药创制国家科技重大专项(2018ZX09711001-001-017)

山西省重点研发计划项目(202102130501005)

山西省自然科学基金项目(201901D111209)

基于炎性反应的重大疾病创新药物山西省重点实验室2021年度开放课题(2021sxcxyw06)

作者信息

1.山西医科大学药学院, 山西太原 030001

2.山西医科大学, 教育部慢性肾脏病医药基础研究创新中心, 山西太原 030001

3.山西中医药大学, 基于炎性反应的重大疾病创新药物山西省重点实验室, 山西晋中 030619

通讯作者:

^*冯秀娥, Tel: 13191058319, E-mail: xiuefeng@163.com;

李青山, Tel: 86-351-3985190, E-mail: sxlqs2012@163.com

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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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Compd.	Appearance	Yield/%	mp/℃	Purity/%^a	[α]$ {}_{\mathrm{D}}^{25\mathrm{ }\mathrm{℃}} $ (g·100 mL^-1, MeOH)	Configuration (J_1″-2″/Hz)
1b	Brown yellow solid	16.7	145-147	99.25	-103.80 (0.100)	β-(7.7)
2b	Brown yellow solid	29.5	187-189	99.38	-134.63 (0.100)	β-(7.5)
3b	Brown yellow solid	11.7	181-182	98.25	-109.36 (0.100)	β-(7.1)
4b	Brown yellow solid	11.2	213-215	98.13	-127.60 (0.100)	β-(7.0)
5b	Brown yellow solid	42.0	168-169	99.14	-310.80 (0.100)	β-(7.7)
6b	Brown yellow solid	42.5	198-200	99.03	-447.20 (0.100)	β-(7.3)
7b	Brown yellow solid	11.1	196-197	95.50	-193.33 (0.075)	β-(7.9)
8b	Brown yellow solid	8.4	225-227	98.12	-215.60 (0.100)	β-(7.6)
9b	Brown yellow solid	34.1	109-110	99.51	-15.80 (0.100)	β-(7.7)
10b	Brown yellow solid	34.0	122-123	99.23	7.00 (0.100)	β-(7.5)
11b	Brown yellow solid	16.0	167-170	99.39	-5.00 (0.100)	β-(7.7)
12b	Brown yellow solid	30.3	171-173	99.05	-71.20 (0.100)	β-(7.7)
13b	Brown yellow solid	14.7	280-282	97.60	-117.40 (0.100)	β-(7.7)
14b	Brown yellow solid	32.7	245-248	99.12	-301.20 (0.100)	β-(7.6)
15b	Brown yellow solid	23.6	289-291	99.21	-615.80 (0.100)	β-(7.4)
16b	Brown yellow solid	29.6	148-150	99.47	-31.74 (0.076)	β-(7.1)
17b	Brown yellow solid	21.4	170-172	99.74	-33.00 (0.100)	β-(7.1)
18b	Brown yellow solid	17.8	102-104	99.48	-42.43 (0.076)	β-(8.3)
19b	Brown yellow solid	27.0	170-172	98.59	-13.20 (0.100)	β-(7.7)
20b	Brown yellow solid	17.1	206-208	99.24	-21.00 (0.100)	β-(7.1)
21b	Brown yellow solid	11.3	198-200	95.20	-95.60 (0.100)	β-(7.6)
22b	Brown yellow solid	21.7	171-173	99.57	9.00 (0.100)	β-(7.8)
23b	Brown yellow solid	20.3	162-164	99.60	-20.46 (0.061)	β-(7.7)
24b	Brown yellow solid	28.2	198-200	99.54	-2.63 (0.069)	β-(7.7)
25b	Brown yellow solid	14.7	239-240	100.00	-0.20 (0.100)	β-(7.7)

Compd.

Appearance

Yield/%

mp/℃

Purity/%^a

[α]

$ {}_{\mathrm{D}}^{25\mathrm{ }\mathrm{℃}} $

(g·100 mL^-1, MeOH)

Configuration (J_1″-2″/Hz)

Brown yellow solid

16.7

145-147

99.25

-103.80 (0.100)

β-(7.7)

Brown yellow solid

29.5

187-189

99.38

-134.63 (0.100)

β-(7.5)

Brown yellow solid

11.7

181-182

98.25

-109.36 (0.100)

β-(7.1)

Brown yellow solid

11.2

213-215

98.13

-127.60 (0.100)

β-(7.0)

Brown yellow solid

42.0

168-169

99.14

-310.80 (0.100)

β-(7.7)

Brown yellow solid

42.5

198-200

99.03

-447.20 (0.100)

β-(7.3)

Brown yellow solid

11.1

196-197

95.50

-193.33 (0.075)

β-(7.9)

Brown yellow solid

8.4

225-227

98.12

-215.60 (0.100)

β-(7.6)

Brown yellow solid

34.1

109-110

99.51

-15.80 (0.100)

β-(7.7)

10b

Brown yellow solid

34.0

122-123

99.23

7.00 (0.100)

β-(7.5)

11b

Brown yellow solid

16.0

167-170

99.39

-5.00 (0.100)

β-(7.7)

12b

Brown yellow solid

30.3

171-173

99.05

-71.20 (0.100)

β-(7.7)

13b

Brown yellow solid

14.7

280-282

97.60

-117.40 (0.100)

β-(7.7)

14b

Brown yellow solid

32.7

245-248

99.12

-301.20 (0.100)

β-(7.6)

15b

Brown yellow solid

23.6

289-291

99.21

-615.80 (0.100)

β-(7.4)

16b

Brown yellow solid

29.6

148-150

99.47

-31.74 (0.076)

β-(7.1)

17b

Brown yellow solid

21.4

170-172

99.74

-33.00 (0.100)

β-(7.1)

18b

Brown yellow solid

17.8

102-104

99.48

-42.43 (0.076)

β-(8.3)

19b

Brown yellow solid

27.0

170-172

98.59

-13.20 (0.100)

β-(7.7)

20b

Brown yellow solid

17.1

206-208

99.24

-21.00 (0.100)

β-(7.1)

21b

Brown yellow solid

11.3

198-200

95.20

-95.60 (0.100)

β-(7.6)

22b

Brown yellow solid

21.7

171-173

99.57

9.00 (0.100)

β-(7.8)

23b

Brown yellow solid

20.3

162-164

99.60

-20.46 (0.061)

β-(7.7)

24b

Brown yellow solid

28.2

198-200

99.54

-2.63 (0.069)

β-(7.7)

25b

Brown yellow solid

14.7

239-240

100.00

-0.20 (0.100)

β-(7.7)

Compd.	K_D/mol·L^-1	Compd.	K_D/mol·L^-1
LF1	6.56×10^-4	10b	7.74×10^-5
LF2	4.82×10^-5	17b	1.65×10^-4
LF3	3.58×10^-5	18b	1.45×10^-4
LF4	1.37×10^-5	23b	2.69×10^-4
4b	7.91×10^-5	24b	3.71×10^-5
8b	8.70×10^-4	25b	2.16×10^-4

Compd.

K_D/mol·L^-1

Compd.

K_D/mol·L^-1

LF1

6.56×10^-4

10b

7.74×10^-5

LF2

4.82×10^-5

17b

1.65×10^-4

LF3

3.58×10^-5

18b

1.45×10^-4

LF4

1.37×10^-5

23b

2.69×10^-4

7.91×10^-5

24b

3.71×10^-5

8.70×10^-4

25b

2.16×10^-4