新型石胆酸-3-肟酯衍生物及其蛋白酪氨酸磷酸酯酶1B抑制活性

引用本文: 侍术智, 梁志鹏, 孙建勇, 石玉军. 新型石胆酸-3-肟酯衍生物及其蛋白酪氨酸磷酸酯酶1B抑制活性[J]. 有机化学, 2019, 39(7): 2106-2116. doi: 10.6023/cjoc201811020 shu

Citation: Shi shuzhi, Liang zhipeng, Sun jiangyong, Shi Yujun. Novel Ester Derivatives of Lithocolic Acid-3-oxime and Their Inhibitory Activities against Protein Tyrosine Phosphatase 1B[J]. Chinese Journal of Organic Chemistry, 2019, 39(7): 2106-2116. doi: 10.6023/cjoc201811020 shu

新型石胆酸-3-肟酯衍生物及其蛋白酪氨酸磷酸酯酶1B抑制活性

侍术智 ^a, ,
梁志鹏 ^b, ,
孙建勇 ^a, ,
石玉军 ^{a,
,}

a.
南通大学化学化工学院南通 226019
b.
南通大学分析测试中心南通 226019

通讯作者: 石玉军, ntushyj@163.com

摘要: 蛋白酪氨酸磷酸酯酶1B（PTP1B）是治疗糖尿病的潜在靶标.内源性甾体化合物石胆酸具有温和的PTP1B抑制活性.将石胆酸3-OH氧化后，进一步修饰得到含有肉桂酸片段的石胆酸肟酯类衍生物，并通过¹H NMR、¹³C NMR和HRMS鉴定其结构.生物活性筛选结果表明：所得目标化合物多数具有较强的PTP1B抑制活性，其中化合物12b的IC₅₀达到0.79 μmol·L^-1，是先导化合物石胆酸活性的15倍左右，同时该化合物对高度同源的T细胞蛋白酪氨酸磷酸酶（TCPTP）的选择性达到4倍左右.

关键词:

PTP1B抑制剂
/ TCPTP
/ 石胆酸肟
/ 合成

English

Novel Ester Derivatives of Lithocolic Acid-3-oxime and Their Inhibitory Activities against Protein Tyrosine Phosphatase 1B

a.
College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019
b.
Analysis & Testing Center, Nantong University, Nantong 226019

Corresponding author: Shi, Yujun, E-mail: ntushyj@163.com

Received Date: 14 November 2018
Revised Date: 08 January 2019
Available Online: 25 July 2019

Abstract: Protein tyrosine phosphatase-1B (PTP1B) is recognized as a potent target for the therapy of diabetes. Lithocolic acid (LCA), a kind of endogenic steroid, was reported as a moderate PTP1B inhibitor. In this paper, 3-hydroxyl of LCA was oxidized, followed by oximating and splicing with cinnamoyl to afford a novel series of derivatives, which were characterized by ¹H NMR, ¹³C NMR and HRMS spectra. The results of bioassays exhibited that most of the titled compounds were active to PTP1B. Among them, compound 12b, the most potent one, has an IC₅₀ of 0.79 µmol•L^-1, about 15-fold more potent than the lead compound. Besides, it also has a selectivity of about 4-fold over T-cell protein tyrosine phosphatase (TCPTP).

Key words:

近年来糖尿病治疗药物及其作用靶标呈现多样化的趋势, 其中对胰岛素信号通路的调控是这些药物重要作用机制之一^[1~4].研究表明胰岛素信号通路中一个关键的调控机制是对胰岛素受体(IR)、胰岛素受体底物(IRS)以及其它下游分子的蛋白质酪氨酸磷酸化进行可逆调节^[5].蛋白酪氨酸磷酸酯酶1B (PTP1B)是第一个被分离出来的蛋白酪氨酸磷酸化酶(PTP), 其结构也早已通过晶体的X射线衍射阐明:它由435个氨基酸组成, 包含一个催化活性位点(酪氨酸磷酸结合口袋)、一个芳基磷酸结合口袋(第二活性位点)以及由两个螺旋结构α3和α6末端空腔构成的非保守的“变构位点”. PTP1B通过对IR以及IRS的脱磷酸化作用下调胰岛素信号, 同时可使与瘦素受体相关的激酶JAK2 (酪氨酸蛋白激酶2)去磷酸化失活, 导致瘦素受体不能对瘦素产生应答, 从而引起瘦素抵抗.因而发现和发展具有PTP1B抑制活性的小分子化合物成为降糖药物研究的热点之一^[5~7].迄今为止, 虽然有包含反义寡核苷酸ISIS-113715及小分子化合物Ertiprotafib在内的多个候选药物进入临床研究, 但迄今为止尚无以PTP1B为主要靶点的药物获得上市批准.天然产物是PTP1B抑制剂的重要来源, 如Genaera公司开发的PTP1B抑制药物Trodusquemine便是早年从角鲨科动物肝脏中提取得到的^[e].研究发现熊果酸(1)、山楂酸(2)、齐墩果酸(3)等五环三萜类天然产物也均具有一定的PTP1B抑制活性, 但对TCPTP(T细胞蛋白酪氨酸磷酸酶)等同源酶的选择性不佳.汤杰课题组曾通过向A环并入取代含氮杂环(吡唑、噁唑、异噁唑等)后制备得到一系列衍生物, 在提高活性的同时, 改善了它们对TCPTP的选择性.但该类化合物存在刚性过强, 溶解性差, 类药性不佳等缺点^[8~10].

文献报道对于患有多囊卵巢综合症相关的胰岛素抵抗的女性, 肉桂提取物可以改善她们的空腹血糖值、葡萄糖抵抗以及胰岛素敏感性.从肉桂树皮中提取得到的肉桂酸对PTP1B有剂量依赖性抑制作用, 时间进程研究实验表明, 其抑制作用也是时间依赖的, 说明肉桂酸是一种快速结合的抑制剂^[11]. Moran等^[12]发现在肉桂酸的对位添加一个三肽(Gly-Glu-Glu)能够有效地提高其对PTP1B的抑制活性. Pei等^[13]发现有相同取代的肉桂醛抑制活性也有所提高, 但其活性不及肉桂酸衍生物. Khan等^[14]发现羽扇豆醇的肉桂酸酯衍生物(4)也具有一定的PTP1B抑制活性, 但其IC₅₀仅为7.46 µmol• L^－1, 但该衍生化策略引起了我们的关注, 因其在羽扇豆醇3-位引入活性片段的同时未进一步增加化合物的结构刚性.内源性甾体化合物石胆酸(5, LCA)也具有温和的PTP1B抑制活性, 其IC₅₀为12.7 µmol•L^－1^[15], 我们课题组^[16]曾系统阐述了石胆酸3、4、5和6位取代基及构型与PTP1B抑制活性的关系, 确证了5-H的β构型和23-COOH对抑制活性至关重要.

本文中, 我们在前期工作基础上, 保留石胆酸的关键活性骨架, 同时引入另一具有PTP1B抑制作用的肉桂酸片段, 得到了系列结构新颖的石胆酸-3-肟酯衍生物(图 2所示), 通过¹H NMR、¹³C NMR和HRMS鉴定了它们结构.并测试了它们对PTP1B抑制活性以及对TCPTP的选择性.其合成方法如Scheme 1所示.

图 1

图 1. 已报道的PTP1B抑制剂

Figure 1. Reported PTP1B inhibitors

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图 2

图 2. 石胆酸-3-肟酯类化合物的设计

Figure 2. Design of the ester derivatives of lithocolic acid-3-oxime

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图式 1

图式 1. 石胆酸-3-肟酯类化合物的合成路线

Scheme 1. Synthesis of the ester derivatives of lithocolic acid-3-oxime

Reagents and conditions: (a) IBX, DMSO, r.t., 24 h, 65%; (b) NH₂OH·HCl, EtOH, AcONa, reflux, 12 h, 55%; (c) Ac₂O, AcOK, 150 ℃, 6 h, 40~60%; (d) DPE, 200 ℃, 3 h, 65%~75%; (e) SOCl₂, DMF, DCM, reflux; (f) Pyridine, DCM, 0 ℃~r.t., overnight, 47%~62%.

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1. 结果与讨论

1.1 目标化合物的合成与表征

本文设计的目标化合物12a~12w采用汇聚式合成策略制备得到, 如Scheme 1所示.化合物12a~12w均未见文献报道, 合成中间体及目标化合物结构经¹H NMR、¹³C NMR及HRMS (ESI)确证.合成的最后一步为肉桂酰氯石胆酸-3-肟缩合, 起初考虑到23-COOH可能同时与肉桂酰氯反应, 因而预先将羧基用甲基保护.然而, 无论在酸性或碱性条件下脱甲基保护均导致肟酯的水解, 因而尝试了Scheme 1所示的未加保护的策略, 令人惊喜的是反应条件下未观察到23-COOH与肉桂酰氯的缩合, 从而可以顺利得到目标产物.以IBX为氧化剂顺利将石胆酸(5)的3-位羟基氧化得到酮(6) ^[17], 随后与盐酸羟胺在醋酸钠存在下乙醇中回流反应制备得到关键中间体石胆酸-3-肟(7).部分肉桂酸直接从阿拉丁购得, 无市售的取代肉桂酸以取代苯甲醛为原料在乙酸酐/乙酸钾体系中通过Perkin反应制备^[18], 但得到得产物为Z/E混合物(9和10), 将该混合物在二苯醚中200 ℃回流3 h, 再经石油醚/乙酸乙酯重结晶可得到单一E构型的取代肉桂酸(10), 以化合物10b为例, 其双键氢(PhCH＝CHCOOH)相互间的偶合常数为16.1, 证实其为E构型.化合物10的酰氯化曾尝试过草酰氯和纯氯化亚砜, 但前者转化不完全, 后者导致原料分解, 因而采用了氯化亚砜的二氯甲烷溶液.最终中间体肟(7)与10的缩合在二氯甲烷中以吡啶为缚酸剂完成, 由于产物极性较大, 分离纯化较困难, 部分产物在柱层析后需要用80%的乙醇/水重结晶得到纯品.

1.2 生物活性测试

参照文献方法^[19], 首先在20 μg/mL浓度下对目标化合物进行PTP1B抑制活性初筛(在40 μg/mL下对TCPTP的抑制率进行初筛), 然后对于抑制率高于50%的化合物进行复筛, 计算得到IC₅₀值, 如表 1所示.苯环上无取代基的化合物12a即显示出较好的PTP1B抑制活性, IC₅₀达到2.72 µmol•L^－1, 相当于先导物石胆酸的4.5倍.苯环上不同位点引入氯原子取代, 活性差异显著, 其中4-氯取代的化合物12b的IC₅₀达到0.79 µmol•L^－1, 同时对TCPTP也显示了4.3倍的选择性.但2-位(12d)或3-位(12c)单氯原子取代产物相比之下活性均明显下降.氟原子取代的化合物12e~12g的抑制活性不佳, 甚至低于不含取代基的12a.电负性较弱的溴原子取代产物12h~12j活性则较相应的氟代化合物活性明显增强, 其中4-位溴代化合物12h对PTP1B的IC₅₀值达到1.33 µmol•L^－1, 与化合物12b活性接近, 但同样对TCPTP的选择性一般. 4-位引入弱给电子能力的甲基, 活性较12h略有下降, 可能意味着苯环4-位弱吸电性的疏水基团有利于化合物与酶的结合.意外的是2-甲基取代的产物12m保持了较高的活性, IC₅₀达到1.53 µmol•L^－1.苯环上引入甲氧基取代的情形去引入氯或溴原子的情形相似, 依然是4-位取代的(12n)活性显著优于2位(12p)或3位(12o)取代的化合物.苯环上引入双取代基并未显示比单取代更佳的抑制活性, 仅2, 4-二氯(12q)和3, 4-二氯(12s)保持了较高活性, IC₅₀分别达到0.93和1.71 µmol•L^－1.其它双取代化合物12t~12w的活性均急剧下降, 同时对TCPTP的抑制活性也几乎消失.

表 1

表 1 化合物对PTP1B的抑制活性

Table 1. Inhibitory activities of the titled compounds against PTP1B

下载: 导出CSV


Compd.	R	IC₅₀ (μmol•L^－1)^a		TCPTP/PTP1B^c
Compd.	R	PTP1B	TCPTP	TCPTP/PTP1B^c
12a	H	2.72±0.36	7.32±1.15	2.69
12b	4-Cl	0.79±0.21	3.41±0.45	4.31
12c	3-Cl	10.22±1.05	＞40^b	—^d
12d	2-Cl	5.15±1.17	＞40	—
12e	4-F	28.70±2.98	＞40	—
12f	3-F	10.94±1.52	＞40	—
12g	2-F	15.25±2.04	＞40	—
12h	4-Br	1.33±0.25	3.18±0.62	2.39
12i	3-Br	4.29±1.12	10.32±1.70	2.41
12j	2-Br	9.12±1.38	11.28±1.67	1.24
12k	4-Me	2.81±0.14	15.35±1.44	5.46
12l	3-Me	9.38±2.12	18.22±1.07	1.94
12m	2-Me	1.53±0.77	9.02±1.15	5.90
12n	4-OMe	1.63±0.23	12.06±1.83	7.39
12o	3-OMe	19.55±1.19	＞40	—
12p	2-OMe	15.62±0.78	＞40	—
12q	2, 4-Cl₂	0.93±0.20	5.93±0.88	6.37
12r	2, 3-Cl₂	12.40±1.99	16.51±1.01	1.33
12s	3, 4-Cl₂l	1.71±0.32	4.72±0.46	2.76
12t	2-F-4-Br	24.27±2.16	＞40	—
12u	3-Cl-4-F	17.37±1.77	＞40	—
12v	2, 4-F₂	26.80±1.53	＞40	—
12w	3, 4-OMe₂	14.27±1.81	＞40	—
LCA		12.21±1.72	＞40	—
OA^e		2.67±0.32	5.98±0.21	2.2
^a IC₅₀ values were determined by regression analyses and expressed as means±SD of three replications; ^b IC₅₀＞40 μmol/L, the inhibition (%) on TCPTP was between 5% and 50% at 40 µmol•L^－1; ^c TCPTP/PTP1B, the IC₅₀ ratio of TCPTP and PTP1B; ^d “—”, none accurate value was calculated; ^e Olea- nolic acid (OA) as positive control.

1.3 计算机模拟对接

为了进一步探究化合物与PTP1B的作用方式及对TCPTP产生选择性的可能原因, 选择了活性最高的化合物12b与PTP1B做了分子对接研究, 并以12n和12q分别与两个酶的结合模型解释了产生活性和选择性差异的原因.分子对接采用AutoDock vina软件, 以PTP1B与抑制剂sulfamic acid的复合物晶体结构(PDB编号: 2F71)作为靶标^[20], 将sulfamic acid周围20×22×20 Å³以内的残基定义为结合位点.

将活性最高的化合物12b与PTP1B做了分子对接研究.如图 3A~3C所示:与酶结合时化合物末端的23-羧基插入了PTP1B的一个正电性口袋(催化活性区域), 与Ala217、Arg221形成了氢键作用, 石胆酸的甾体骨架与周围氨基酸Asp181、Phe182、Ser216、Ala217等存在疏水作用.此外, 化合物还与第二活性位点的Tyr46、Asp48等氨基酸存在显著的疏水作用.除氢键、疏水作用以外, 卤键也是一类重要的分子间相互作用^[21].卤素(Cl、Br和I)与小分子上的R形成共价键时, 沿着R—X共价键的轴向方向, 在卤素原子的顶端会带有正电荷.当附近有O、N、S等带有部分负电荷的原子或者芳香基团π电子(Y)存在时, R—X…Y就能形成类似于氢键的吸引作用, 称为卤键^[21].肉桂酸片段的苯环上引入4-Cl, 可与A27主链上的氧原子形成卤键, 因此活性提高.而氟原子无法形成卤键作用, 因此化合物12e~12g等活性较差.

图 3

图 3. (A)~(C)化合物12b与PTP1B的对接模型、(D)化合物12n、12q与PTP1B的对接模型、(E)化合物12n、12q与PTP1B/ TCPTP叠加结构对接模型

Figure 3. (A)~(C) compound 12b docked into PTP1B, (D) compounds 12n & 12q docked into PTP1B, and (E) compounds 12n & 12q docked into the stack of PTP1B with TCPTP

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将化合物12n和12q对接进入PTP1B的口袋, 发现其结合模式与12b类似, 末端的23-羧基插入了PTP1B的催化口袋, 而肉桂酸片段苯环上的取代基不同使得化合物产生了一定的选择性(图 3D).将TCPTP的结构(PDB编号: 1L8K)叠合到PTP1B的结构之后发现, TCPTP容纳肉桂酸苯环片段的口袋较小, 4-OMe或2, 4-二氯取代可与TCPTP产生空间位阻, 从而对TCPTP活性较弱(图 3E).

2. 结论

本文以内源性甾体化合物石胆酸为先导骨架, 制备得到关键中间体石胆酸-3-肟, 随后通过引入肉桂酰砌块制备得到一组新型的石胆酸-3-肟酯类衍生物.生物活性测试表明, 该类化合物大多对蛋白酪氨酸磷酸酯酶1B(PTP1B)具有较好的抑制活性, 其中苯环4-氯取代的化合物12b的IC₅₀达到0.79 µmol•L^－1, 同时该化合物显示了对同源酶TCPTP4.3倍的选择性, 双氯取代的化合物(12q)尽管活性略低, 但显示了更优异的选择性(TCPTP/PTP1B＝6.37).分子模拟对接研究阐述了化合物与酶可能的结合方式.该类新型石胆酸衍生物将为以PTP1B为靶标的抗糖尿病药物研究提供新的先导化合物.进一步的结构优化和初步代谢稳定性研究正在进行中, 将后续发表.

3. 实验部分

3.1 仪器与试剂

熔点用毛细管法测定, 温度计未经校正; ¹H NMR及¹³C NMR用BRUKER AV-400型核磁共振仪测定(TMS为内标, CDCl₃或DMSO-d₆为溶剂); HRMS数据来源于Aglient 1100 (HPLC)与MicrOTOF Ⅱ(MS)联用仪; 酶标仪为Bio-RAD 680型.本实验所用试剂均为AR级或CP级.

3.2 化合物的合成

3.2.1 石胆酸-3-肟(7)的合成

将石胆酸(1.88 g, 5 mmol)溶于30 mL DMSO, 室温下加入IBX (1.96 g, 7 mmol), 搅拌反应24 h后, 将反应液倾入100 mL水中, 析出大量白色固体, 过滤, 收集滤饼.将滤饼用30 mL乙酸乙酯溶解后, 过滤, 收集滤液, 减压浓缩至干, 得石胆酸-3-酮(6)粗品.将粗品加入10 mL甲醇, 热回流后冷却至室温, 放置3 h后过滤.收集滤饼, 得白色固体产物石胆酸-3-酮(6) 1.03 g.收率55%. m.p. 121~123 ℃(文献值^[22] m.p. 122~122 ℃).

将石胆酸-3-酮(6, 3.74 g, 10 mmol)、盐酸羟胺(0.7 g, 10 mmol)和乙酸钠(1.23 g, 15 mmol)溶于50 mL乙醇, 升温至回流反应12 h, 降至室温后减压浓缩至干, 加入30 mL水洗涤、红外干燥后得白色固体石胆酸-3-肟(7)^[24] 3.11 g, 收率80%. m.p. 195~198 ℃(文献值^[23] m.p. 195~197 ℃); ¹H NMR (400 MHz, DMSO-d₆) δ: 10.06 (brs, 1H, COOH), 3.03 (d, J＝21.8 Hz, 1H, 2-CH₂), 2.76 (dd, J＝15.1, 5.0 Hz, 1H, 2-CH₂), 2.25~2.08 (m, 2H, CH₂COOH), 2.07~1.78 (m, 6H), 1.69~1.00 (m, 18H), 0.91 (s, 3H, 18-CH₃), 0.87 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.4 (COOH), 158.2 (N＝C), 56.1, 56.0, 42.7, 42.4, 37.4, 35.6, 35.4, 35.2, 32.1, 31.2, 28.1, 26.8, 26.6, 25.9, 25.7, 25.0, 24.2, 23.3, 22.6, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃).

3.2.2 (E)-对氯肉桂酸(10b)的合成

将对氯苯甲醛(1.40 g, 10 mmol)、乙酸酐(20 mL)及无水乙酸钾(2.76 g, 20 mmol)混合, 油浴升温至150 ℃, 剧烈搅拌反应6 h.将反应液冷却至室温后, 加入200 g冰水中, 析出黄色固体, 过滤, 收集滤饼, 水洗至中性.用石油醚/乙酸乙酯(V:V＝1:1)重结晶后, 过滤、红外干燥得E/Z对氯肉桂酸的混合物1.10 g.

将上步所得1.1 g混合物加入二苯醚(10 mL)中, 升温至190~200 ℃反应3 h, 冷却至室温后向溶液中滴加石油醚至有大量固体析出.过滤, 石油醚洗涤、干燥后得黄色固体(E)-对氯肉桂酸(10b)^[25] 1.0 g, 两步收率55.0%. m.p. 245~247 ℃(文献值m.p. 247~249 ℃^[25]); ¹H NMR (500 MHz, DMSO-d₆) δ: 12.4 (brs, 1H, COOH), 7.68 (d, J＝8.6 Hz, 2H, PhH), 7.54 (d, J＝16.1 Hz, 1H, CH＝CHCO), 7.42 (d, J＝8.6 Hz, 2H, PhH), 6.51 (d, J＝16.1 Hz, 1H, CH＝CHCO); ¹³C NMR (126 MHz, DMSO- d₆) δ: 167.8 (COOH), 142.9 (CH＝CHCO), 135.1 (PhC), 133.6 ((Ph—C)), 130.3 (2C, (Ph—C)), 129.3 (2C, (Ph—C)), 120.5 (CH＝CH—CO).

中间体10c、10g、10n~10v参照制备中间体10b的方法合成得到, 其它中间体10直接商业购得.

3.2.3 肉桂酰石胆酸肟酯(12)的合成

(E)-取代肉桂酸10 (5 mmol)溶于二氯乙烷(10 mL), 加入氯化亚砜(1.79 g, 15 mmol)和DMF (1滴), 升温至回流反应过夜, 次日将反应液减压浓缩至干得取代肉桂酰氯(11), 不经纯化直接用于下一步.

将上述浓缩物溶于二氯甲烷(20 mL), 冰水浴冷却下, 加入1.95 g (5 mmol)石胆酸-3-肟(7), 随后溶液中缓慢滴加吡啶(0.79 g, 10 mmol), 室温搅拌过夜.次日, 加入冰水淬灭反应液, 分去水相, 有机相用饱和食盐水洗涤后, 干燥、过滤、减压浓缩、柱层析[(V(石油醚)/V(乙酸乙酯)＝10/1]得产物肉桂酰石胆酸肟酯(12).如有必要, 可用80%乙醇-水重结晶纯化产物.

肉桂酰石胆酸肟酯(12a):白色固体, 收率62%. m.p. 123.2~125.4 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.93 (brs, 1H, COOH), 7.76~7.70 (m, 3H, CH＝CHCO, PhH), 7.45~7.40 (m, 3H, PhH), 6.77~6.71 (m, 1H, CH＝CHCO), 2.97 (d, J＝14.0 Hz, 1H, 2-CH₂), 2.70 (t, J＝13.9 Hz, 1H, 2-CH₂), 2.25~2.11 (m, 2H, CH₂COOH), 2.10~1.78 (m, 6H), 1.70~1.04 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.88 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.5 (CH＝CHCO), 164.5 (N＝C), 145.5 (CH＝CHCO), 134.4 (PhC), 131.0 (PhC), 129.3 (2C, PhC), 128.9 (2C, PhC), 116.8 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 36.3, 35.6, 35.3, 32.1, 31.2, 28.1, 27.4, 27.0, 26.6, 25.9, 24.2, 22.9, 22.3, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₆NO₄ [M+H]⁺ 520.3427, found 520.3491.

4-氯肉桂酰石胆酸肟酯(12b):白色固体, 收率52%. m.p. 105.4~107.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.0 (brs, 1H, COOH), 7.79~7.81 (m, 2H, PhH), 7.72 (d, J＝16.1 Hz, 1H, CH＝CHCO), 7.50 (d, J＝8.4 Hz, 2H, PhH), 6.78 (dd, J＝16.1, 7.2 Hz, 1H, CH＝CHCO), 2.96 (d, J＝13.9 Hz, 0.5H, 2-CH₂), 2.82 (d, J＝11.1 Hz, 0.5H, 2-CH₂), 2.71 (t, J＝13.6 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝13.9 Hz, 0.5H, 2-CH₂), 2.19~2.27 (m, 2H, CH₂COOH), 1.82~2.14 (m, 6H), 1.04~1.67 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.87 (d, J＝6.3 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 179.9 (CH＝CHCO), 170.1 (COOH), 164.9 (N＝C), 144.3 (CH＝CHCO), 136.4 (PhC), 132.8 (PhC), 129.4 (2C, PhC), 129.2 (2C, PhC), 116.7 (CH＝CHCO), 56.4, 55.9, 43.0, 42.8, 40.7, 40.0, 36.9, 35.6, 35.3, 35.3, 31.0, 30.7, 28.2, 27.6, 27.1, 26.6, 25.9, 24.2, 23.0, 21.1, 18.3 (19-CH₃), 12.1 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅ClNO₄ [M+H]⁺ 554.3037, found 554.3012.

3-氯肉桂酰石胆酸肟酯(12c):白色固体, 收率55%. m.p. 51.4~53.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.95 (brs, 1H, COOH), 7.9 (s, 1H, PhH), 7.73~7.69 (m, 2H, CH＝CHCO, PhH), 7.51~7.44 (m, 2H, PhH), 6.85 (dd, J＝9.3, 6.8 Hz, 1H, CH＝CHCO), 2.98 (d, J＝13.5 Hz, 0.5H, 2-CH₂), 2.84 (d, J＝11.3 Hz, 0.5H, 2-CH₂), 2.72 (t, J＝13.7 Hz, 0.5H, 2-CH₂), 2.43 (t, J＝14.7 Hz, 0.5H, 2-CH₂), 2.27~2.18 (m, 2H, CH₂COOH), 2.14~1.82 (m, 6H), 1.68~1.01 (m, 18H), 0.95 (s, 3H, 18-CH₃), 0.88 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.64 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.4 (CH＝CHCO), 164.2 (N＝C), 143.8 (CH＝CHCO), 136.7 (PhC), 134.2 (PhC), 131.1 (PhC), 130.6 (PhC), 128.4 (PhC), 127.5 (PhC), 118.7 (CH＝CHCO), 56.0, 56.0, 42.9, 42.7, 40.5, 35.5, 35.4, 35.3, 35.2, 31.2, 31.1, 29.4, 28.1, 26.9, 26.6, 24.2, 23.0, 22.9, 21.2, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅ClNO₄ [M+H]⁺ 554.3037, found 554.3051.

2-氯肉桂酰石胆酸肟酯(12d):白色固体, 收率49%. m.p. 49.9~51.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.96 (brs, 1H, COOH), 8.00~7.96 (m, 2H, PhH), 7.56 (d, J＝7.8 Hz, 1H, CH＝CHCO), 7.49~7.39 (m, 2H, PhH), 6.83 (dd, J＝9.1, 6.9 Hz, 1H, CH＝CHCO), 2.95 (d, J＝13.7 Hz, 0.5 H, 2-CH₂), 2.81 (dd, J＝11.0, 4.1 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.4 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝14.1 Hz, 0.5H, 2-CH₂), 2.26~2.18 (m, 2H, CH₂COOH), 2.13~1.84 (m, 6H), 1.80~1.06 (m, 18H), 0.93 (d, J＝2.4 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.0 Hz, 3H, 21-CH₃), 0.62 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 174.8 (COOH), 170.1 (CH＝CHCO), 163.6 (N＝C), 139.6 (CH＝CHCO), 133.7 (PhC), 132.0 (PhC), 131.5 (PhC), 129.9 (PhC), 128.3 (PhC), 127.7 (PhC), 119.5 (CH＝CHCO), 55.5, 55.5, 42.4, 42.2, 35.1, 34.9, 34.8, 34.8, 30.7, 30.6, 27.6, 26.2, 26.1, 25.4, 25.3, 23.7, 22.5, 22.3, 20.6, 18.1, 18.0 (19-CH₃), 11.8 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅ClNO₄ [M+H]⁺ 554.3037, found 554.3029.

4-氟肉桂酰石胆酸肟酯(12e):白色固体, 收率47%. m.p. 64.7~66.5 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 12.0 (brs, 1H, COOH), 7.91~7.87(m, 2H, PhH), 7.78 (d, J＝16.0 Hz, 1H, CH＝CHCO), 7.32 (t, J＝8.8 Hz, 2H, PhH), 6.76 (dd, J＝9.2, 6.8 Hz, 1H, CH＝CHCO), 3.02 (d, J＝13.8 Hz, 0.5 H, 2-CH₂), 2.88 (d, J＝10.6 Hz, 0.5H, 2-CH₂), 2.76 (t, J＝13.8 Hz, 0.5H, 2-CH₂), 2.47 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.33~2.24 (m, 2H, CH₂COOH), 2.18~1.85 (m, 6H), 1.77~1.10 (m, 18H), 1.00 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.93 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.69 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.5 (CH＝CHCO), 170.2 (N＝C), 165.0 (CH＝CHCO), 163.5 (d, ¹J_C—F＝194 Hz, PhC), 144.3 (PhC), 131.3 (d, ³J_C—F＝9 Hz, 2PhC), 116.7 (CH＝CHCO), 116.4 (d, ²J_C—F＝22 Hz, 2PhC), 56.0, 55.9, 44.2, 42.9, 42.7, 35.5, 35.3, 35.2, 31.1, 31.1, 28.1, 27.4, 26.9, 26.6, 25.9, 25.8, 24.2, 23.0, 22.9, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅FNO₄ [M+H]⁺ 538.3333, found 538.3356.

3-氟肉桂酰石胆酸肟酯(12f):白色固体, 收率50%. m.p. 69.6~71.8 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.93 (brs, 1H, COOH), 7.74~7.66 (m, 2H, CH＝CHCO, PhH), 7.59 (d, J＝7.8 Hz, 1H, PhH), 7.50~7.44 (m, 1H, PhH), 7.27 (dd, J＝8.4, 2.4 Hz, 1H, PhH), 6.82 (dd, J＝9.1, 6.9 Hz, 1H, CH＝CHCO), 2.97 (d, J＝14.1 Hz, 0.5H, 2-CH₂), 2.82 (d, J＝10.8 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.8 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.14~1.77 (m, 6H), 1.71~1.01 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.88 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.6 (CH＝CHCO), 170.4 (N＝C), 163.0 (d, ¹J_C—F＝265 Hz, PhC), 144.1 (CH＝CHCO), 137.1 (d, ³J_C—F＝8 Hz, PhC), 131.3 (d, ³J_C—F＝8 Hz, PhC), 125.3 (PhC), 118.6 (d, ⁴J_C—F＝4 Hz, PhC), 115.0 (d, ²J_C—F＝23 Hz, PhC), 99.9 (CH＝CHCO), 56.0, 56.0, 44.2, 42.9, 42.7, 40.0, 37.1, 35.5, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.9, 24.2, 23.0, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅FNO₄ [M+H]⁺ 538.3333, found 538.3327.

2-氟肉桂酰石胆酸肟酯(12g):白色固体, 收率57%. m.p. 182.4~184.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.94 (brs, 1H, COOH), 8.01~7.95 (m, 2H, PhH), 7.56 (d, J＝6.6 Hz, 1H, CH＝CHCO), 7.49~7.39 (m, 2H, PhH), 6.84 (dd, J＝9.0, 7.0 Hz, 1H, CH＝CHCO), 2.94 (d, J＝14.2 Hz, 0.5 H, 2-CH₂), 2.81 (dd, J＝10.6, 4.6 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.8 Hz, 0.5H, 2-CH₂), 2.43 (t, J＝14.1 Hz, 0.5H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.13~1.78 (m, 6H), 1.71~1.03 (m, 18H), 0.94 (d, J＝2.9 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH3); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.2 (COOH), 170.8 (CH＝CHCO), 170.6 (N＝C), 164.1 (PhC), 140.1 (CH＝CHCO), 134.2 (PhC), 132.5 (PhC), 132.0 (PhC), 129.7 (d, ¹J_C—F＝163 Hz, PhC), 128.2 (PhC), 120.0 (CH＝CHCO), 56.0, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.1, 31.2, 28.1, 27.5, 26.9, 26.7, 25.9, 24.2, 22.9, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅FNO₄ [M+H]⁺ 538.3333, found 538.3352.

4-溴肉桂酰石胆酸肟酯(12h):白色固体, 收率60%. m.p. 139.5~142.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.74~7.69 (m, 3H, PhH, CH＝CHCO), 7.63 (d, J＝6.8 Hz, 2H, PhH), 6.79 (dd, J＝12.9, 7.2 Hz, 1H, CH＝CHCO), 2.97 (d, J＝11.4 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝11.9, 3.0 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝11.1 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝11.3 Hz, 0.5H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.13~1.78 (m, 6H), 1.69~1.07 (m, 18H), 0.94 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.88 (d, J＝5.2 Hz, 3H, 21-CH₃), 0.64 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.2 (CH＝CHCO), 164.3 (N＝C), 144.2 (PhC), 133.8 (CH＝CHCO), 132.3 (PhC), 130.8 (PhC), 130.8 (PhC), 124.4 (CH＝CHCO), 117.8 (PhC), 117.7 (PhC), 79.6, 56.0, 46.1, 44.2, 42.7, 37.1, 36.3, 35.5, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅BrNO₄ [M+H]⁺ 598.2532, found 598.2521.

3-溴肉桂酰石胆酸肟酯(12i):白色固体, 收率47%. m.p. 90.3~92.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.96 (brs, 1H, COOH), 8.03~8.02 (m, 1H, PhH), 7.76 (d, 1H, J＝6.2 Hz, CH＝CHCO), 7.70 (d, 1H, J＝12.8 Hz, PhH), 7.62 (dd, J＝6.4, 1.4 Hz, 1H, PhH), 7.39 (t, J＝6.3 Hz, 1H, PhH), 6.84 (dd, J＝12.8, 6.8 Hz, 1H, CH＝CHCO), 2.98 (d, J＝12.2 Hz, 0.5H, 2-CH₂), 2.84 (dd, J＝11.2, 2.5 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝11.1 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝11.4 Hz, 0.5H, 2-CH₂), 2.27~2.20 (m, 2H, CH₂CO- OH), 2.13~1.78 (m, 6H), 1.70~1.05 (m, 18H), 0.94 (d, J＝2.5 Hz, 3H, 18-CH₃), 0.88 (d, J＝5.2 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.6 (CH＝CHCO), 164.2 (N＝C), 143.8 (CH＝CHCO), 137.0 (PhC), 133.5 (PhC), 131.4 (PhC), 131.3 (PhC), 127.8 (PhC), 122.7 (PhC), 118.6 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 36.3, 35.6, 35.3, 35.2, 32.1, 31.1, 28.1, 27.4, 26.9, 26.6, 25.9, 24.2, 22.8, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅BrNO₄ [M+H]⁺ 598.2532, found 598.2560.

2-溴肉桂酰石胆酸肟酯(12j):白色固体, 收率60%. m.p. 102.7~104.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.95 (brs, 1H, COOH), 7.98~7.93 (m, 2H, PhH), 7.72 (dd, J＝5.4, 1.0 Hz, 1H, CH＝CHCO), 7.45 (td, J＝6.1, 0.9 Hz, 1H, PhH), 7.38 (td, J＝6.2, 1.3 Hz, 1H, PhH), 6.79 (dd, J＝12.7, 7.0 Hz, 1H, CH＝CHCO), 2.95 (d, J＝11.2 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝11.8, 3.7 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝11.2 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝11.2 Hz, 0.5H, 2-CH₂), 2.27~2.18 (m, 2H, CH₂COOH), 2.13~1.77 (m, 6H), 1.70~1.04 (m, 18H), 0.93 (d, J＝3.0 Hz, 3H, 18-CH₃), 0.87 (d, J＝5.1 Hz, 3H, 21-CH₃), 0.62 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.5 (CH＝CHCO), 164.0 (N＝C), 142.7 (CH＝CHCO), 133.7 (PhC), 132.7 (PhC), 128.9 (PhC), 128.7 (PhC), 125.2 (PhC), 120.1 (PhC), 120.0 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.1, 31.1, 28.1, 26.9, 26.7, 25.8, 24.2, 22.9, 22.8, 21.1, 18.6 (19-CH₃), 12.2 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₅BrNO₄ [M+H]⁺ 598.2532, found 598.2541.

4-甲基肉桂酰石胆酸肟酯(12k):白色固体, 收率54%. m.p. 81.9~83.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.97 (brs, 1H, COOH), 7.70~7.53 (m, 3H, PhH, CH＝CHCO), 7.24 (d, J＝7.9 Hz, 2H, PhH), 6.67 (dd, J＝16.0, 7.1 Hz, 1H, CH＝CHCO), 2.96 (d, J＝14.1 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝10.6, 4.7 Hz, 0.5H, 2-CH₂), 2.69 (t, J＝14.3 Hz, 0.5H, 2-CH₂), 2.40 (t, J＝14.1 Hz, 0.5H, 2-CH₂), 2.33(s, 3H, PhCH₃), 2.27~2.19 (m, 2H, CH₂COOH), 2.14~1.76 (m, 6H), 1.71~1.01 (m, 18H), 0.94 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.3 (CH＝CHCO), 170.0 (N＝C), 164.6 (CH＝CHCO), 145.5 (PhC), 141.0 (PhC), 131.8 (PhC), 130.0 (PhC), 128.9 (PhC), 128.6 (PhC), 115.6 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 22.3, 21.5 (PhCH₃), 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₄ [M+H]⁺ 534.3583, found 534.3563.

3-甲基肉桂酰石胆酸肟酯(12l):白色固体, 收率48%. m.p. 79.1~81.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.94 (brs, 1H, COOH), 7.67 (d, 1H, J＝16.1 Hz, CH＝CHCO), 7.57 (s, 1H, PhH), 7.52(d, J＝7.7 Hz, 1H, PhH), 7.32 (t, J＝7.6 Hz, 1H, PhH), 7.25(d, J＝7.6 Hz, 1H, PhH), 6.71 (dd, J＝16.1, 6.8 Hz, 1H, CH＝CHCO), 2.97 (d, J＝14.0 Hz, 0.5H, 2-CH₂), 2.83 (dd, J＝10.8, 4.7 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝14.0 Hz, 0.5H, 2-CH₂), 2.41 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.33 (s, 3H, PhCH₃), 2.27~2.19 (m, 2H, CH₂COOH), 2.13~1.77 (m, 6H), 1.71~1.05 (m, 18H), 0.94 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.4 (CH＝CHCO), 170.1 (N＝C), 164.5 (CH＝CHCO), 145.6 (PhC), 138.6 (PhC), 134.4 (PhC), 131.7 (PhC), 129.2 (PhC), 126.2 (PhC), 116.6 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.6, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 22.3, 21.2 (PhCH₃), 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₄ [M+ H]⁺ 534.3583, found 534.3552.

2-甲基肉桂酰石胆酸肟酯(12m):白色固体, 收率50%. m.p. 90.0~91.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.94 (brs, 1H, COOH), 7.92 (d, J＝15.8 Hz, 1H, CH＝CHCO), 7.77~7.74(m, 1H, PhH), 7.35~7.22 (m, 3H, PhH), 6.63 (dd, J＝15.9, 7.5 Hz, 1H, CH＝CHCO), 2.95 (d, J＝14.2 Hz, 0.5H, 2-CH₂), 2.82 (d, J＝10.6 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.8 Hz, 0.5H, 2-CH₂), 2.45~2.38 (m, 3.5H, PhCH₃, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.14~1.79 (m, 6H), 1.71~1.04 (m, 18H), 0.94 (d, J＝2.8 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.5 (CH＝CHCO), 164.5 (N＝C), 142.6 (CH＝CHCO), 137.9 (PhC), 133.1 (PhC), 131.2 (PhC), 130.8 (Ph—C), 127.1 (PhC), 116.7 (PhC), 126.9 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.6, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 22.3, 21.1, 19.7 (PhCH₃), 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₄ [M+H]⁺ 534.3583, found 534.3559.

4-甲氧基肉桂酰石胆酸肟酯(12n):白色固体, 收率52%. m.p. 71.8~73.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.97 (brs, 1H, COOH), 7.72~7.62 (m, 2.8H, PhH, CH＝CHCO), 7.54 (d, J＝16.0 Hz, 0.2H, PhH), 7.00~6.96 (m, 2H, PhH), 6.58 (dd, J＝16.0, 7.0 Hz, 0.8H, CH＝CHCO), 6.37 (d, J＝16.0 Hz, 0.2H, CH＝CHCO), 3.80 (d, J＝4.2 Hz, 3H, OCH₃), 2.96 (d, J＝14.0 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝10.5, 4.5 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝14.6 Hz, 0.5H, 2-CH₂), 2.41 (t, J＝14.4 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂COOH), 2.14~1.78 (m, 6H), 1.71~1.03 (m, 18H), 0.94 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.88 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.64 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.2 (CH＝CHCO), 169.9 (N＝C), 164.8 (CH＝CHCO), 161.7 (PhC), 145.3 (PhC), 130.7 (PhC), 130.3 (PhC), 127.1 (PhC), 114.8 (PhC), 114.0 (CH＝CHCO), 56.0 (OCH₃), 55.8, 55.7, 44.2, 42.9, 42.7, 37.1, 35.6, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.9, 24.2, 23.0, 22.9, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₅ [M+H]⁺ 550.3532 found 550.3512.

3-甲氧基肉桂酰石胆酸肟酯(12o):白色固体, 收率57%. m.p. 83.5~85.6 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 7.69 (d, J＝12.8 Hz, 1H, CH＝CHCO), 7.35~7.29 (m, 3H, PhH), 7.00 (dd, J＝6.2, 1.9 Hz, 1H, PhH), 6.76 (dd, J＝12.9, 6.9 Hz, 1H, CH＝CHCO), 3.80 (s, 3H, OCH₃), 2.97 (d, J＝11.2 Hz, 0.5H, 2-CH₂), 2.83 (dd, J＝8.9, 3.16 Hz, 0.5H, 2-CH₂), 2.69 (t, J＝11.3 Hz, 0.5H, 2-CH₂), 2.41 (t, J＝11.5 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂CO- OH), 2.13~1.76 (m, 6H), 1.70~1.05 (m, 18H), 0.94 (d, J＝2.4 Hz, 3H, 18-CH₃), 0.87 (d, J＝5.2 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.4 (CH＝CHCO), 170.1 (N＝C), 164.5 (CH＝CHCO), 160.0 (PhC), 145.5 (PhC), 135.9 (PhC), 130.4 (PhC), 121.6 (PhC), 117.1 (PhC), 113.4 (CH＝CHCO), 79.6, 56.0 (OCH₃), 55.7, 44.2, 42.9, 42.7, 37.1, 36.2, 35.3, 35.2, 32.1, 31.1, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 22.3, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₅ [M+H]⁺ 550.3532 found 550.3526.

2-甲氧基肉桂酰石胆酸肟酯(12p):白色固体, 收率53%. m.p. 87.9~90.0 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.95 (brs, 1H, COOH), 7.93 (dd, J＝16.2, 3.2 Hz, 1H, CH＝CHCO), 7.76~7.73 (m, 1H, PhH), 7.43 (t, J＝8.5 Hz, 1H, PhH), 7.10 (d, J＝8.4 Hz, 1H, PhH), 6.99 (t, J＝7.4 Hz, 1H, PhH), 6.71 (dd, J＝16.2, 5.3 Hz, 1H, CH＝CHCO), 3.88 (s, 3H, OCH₃), 2.93 (d, J＝14.0 Hz, 0.5H, 2-CH₂), 2.79 (d, J＝11.3 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.9 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂COOH), 2.14~1.81 (m, 6H), 1.70~0.98 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.88 (d, J＝6.5 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.1 (CH＝CHCO), 164.9 (N＝C), 158.4(CH＝CHCO), 140.2 (PhC), 132.7 (PhC), 129.2 (PhC), 122.7 (PhC), 121.1 (PhC), 116.7 (PhC), 112.2 (CH＝CHCO), 79.6, 56.1 (OCH₃), 56.0, 44.2, 42.9, 42.7, 37.1, 35.6, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 22.3, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₄H₄₈NO₅ [M+H]⁺ 550.3532 found 550.3545.

2, 4-二氯肉桂酰石胆酸肟酯(12q):白色固体, 收率60%. m.p. 78.1~80.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.95 (brs, 1H, COOH), 8.02 (dd, J＝8.5, 3.6 Hz, 1H, CH＝CHCO), 7.90 (d, J＝16.0 Hz, 1H, CH＝CHCO), 7.74 (s, 1H, PhH), 7.50 (dd, J＝8.6, 2.1 Hz, 1H, PhH), 6.87 (dd, J＝16.0, 7.2 Hz, 1H, CH＝CHCO), 2.93 (d, J＝14.2 Hz, 0.5H, 2-CH₂), 2.80 (dd, J＝10.8, 4.6 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.8 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝14.1 Hz, 0.5H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.13~1.78 (m, 6H), 1.70~1.03 (m, 18H), 0.93 (d, J＝3.1 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.62 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.6 (CH＝CHCO), 164.0 (N＝C), 138.9 (CH＝CHCO), 136.1 (PhC), 135.0 (PhC), 131.1 (PhC), 130.1 (PhC), 129.9 (PhC), 128.5 (PhC), 120.7 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.0, 31.1, 28.1, 27.5, 26.9, 26.7, 25.8, 24.2, 22.9, 22.3, 21.1 (19-CH₃), 18.6 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₄Cl₂NO₄ [M+H]⁺ 588.2647 found 588.2665.

2, 3-二氯肉桂酰石胆酸肟酯(12r):白色固体, 收率55%. m.p. 99.7~102.3 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.96 (brs, 1H, COOH), 8.00~7.94 (m, 2H, PhH), 7.72 (dd, J＝6.4, 1.2 Hz, 1H, CH＝CH—CO), 7.43 (t, J＝6.4 Hz, 1H, PhH), 6.86 (dd, J＝12.8, 7.2 Hz, 1H, CH＝CHCO), 2.94 (d, J＝11.2 Hz, 0.5 H, 2-CH₂), 2.81 (dd, J＝8.4, 3.7 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝11.3 Hz, 0.5H, 2-CH₂), 2.43 (t, J＝11.2 Hz, 0.5H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.13~1.75 (m, 6H), 1.68~1.02 (m, 18H), 0.93 (d, J＝3.0 Hz, 3H, 18-CH₃), 0.87 (d, J＝5.2 Hz, 3H, 21-CH₃), 0.62 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.7 (CH＝CHCO), 163.9 (N＝C), 140.0 (CH＝CHCO), 134.6 (PhC), 133.0 (PhC), 132.5 (PhC), 132.0 (PhC), 129.0 (PhC), 127.5 (PhC), 121.5 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.0, 31.1, 28.1, 27.5, 26.9, 26.7, 25.8, 24.2, 22.9, 22.3, 21.1 (19-CH₃), 18.6 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₄Cl₂NO₄ [M+H]⁺ 588.2647 found 588.2658.

3, 4-二氯肉桂酰石胆酸肟酯(12s):白色固体, 收率52%. m.p. 145.4~147.9 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.94 (brs, 1H, COOH), 8.10 (s, 1H, PhH), 7.78~7.67 (m, 3H, PhH, CH＝CHCO), 6.87 (dd, J＝16.2, 6.9 Hz, 1H, CH＝CHCO), 2.97 (d, J＝14.0 Hz, 0.5H, 2-CH₂), 2.83 (dd, J＝10.7, 4.7 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝14.0 Hz, 0.5H, 2-CH₂), 2.41 (t, J＝14.4 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂COOH), 2.13~1.78 (m, 6H), 1.71~1.00 (m, 18H), 0.94 (d, J＝2.6 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.4 (CH＝CHCO), 164.1 (N＝C), 142.7 (CH＝CHCO), 135.4 (PhC), 133.2 (PhC), 132.2 (PhC), 131.4 (PhC), 130.6 (PhC), 128.9 (PhC), 119.2 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.6, 35.3, 35.2, 32.1, 31.2, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 22.8, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₄- Cl₂NO₄ [M+H]⁺ 588.2647 found 588.2633.

2-氟-4-溴肉桂酰石胆酸肟酯(12t):白色固体, 收率58%. m.p. 160.1~162.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.94 (brs, 1H, COOH), 7.89~7.84 (m, 1H, PhH), 7.73~7.67 (m, 1H, PhH), 7.50 (dd, J＝8.4, 1.9 Hz, 1H, CH＝CHCO), 6.83 (dd, J＝16.2, 7.0 Hz, 1H, CH＝CHCO), 2.94 (d, J＝14.0 Hz, 0.5H, 2-CH₂), 2.80 (dd, J＝10.3, 4.6 Hz, 0.5H, 2-CH₂), 2.70 (t, J＝13.9 Hz, 0.5H, 2-CH₂), 2.42 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂COOH), 2.14~1.75 (m, 6H), 1.71~1.01 (m, 18H), 0.93 (d, J＝2.8 Hz, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.8 (COOH), 170.6 (CH＝CHCO), 164.1 (N＝C), 160.8 (d, ¹J_C—F＝257 Hz, PhC), 136.2 (CH＝CHCO), 131.3 (PhC), 128.7 (Ph—C), 124.8 (PhC), 121.6 (d, ²J_C—F＝10 Hz, PhC), 120.0 (d, ²J_C—F＝11 Hz, PhC), 100.0 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.0, 31.1, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 22.9, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C33H44BrFNO4 [M+H]⁺ 616.2438 found 616.2454.

3-氯-4-氟肉桂酰石胆酸肟酯(12u):白色固体, 收率53%. m.p. 144.9~147.1 ℃; δ 11.98 (brs, 1H, COOH), 8.10~8.07 (m, 1H, PhH), 7.82~7.79 (m, 1H, PhH), 7.70 (d, J＝12.8 Hz, 1H, CH＝CHCO), 7.48 (t, J＝7.1 Hz, 1H, CH＝CHCO), 6.81 (dd, J＝12.9, 6.7 Hz, 1H, CH＝CHCO), 2.97(d, J＝11.1 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝8.4, 3.7 Hz, 0.5H, 2-CH₂), 2.69 (t, J＝11.2 Hz, 0.5H, 2-CH₂), 2.41 (t, J＝11.5 Hz, 0.5H, 2-CH₂), 2.27~2.19 (m, 2H, CH₂COOH), 2.13~1.76 (m, 6H), 1.70~1.03 (m, 18H), 0.94 (d, J＝2.5 Hz, 3H, 18-CH₃), 0.87 (d, J＝5.2 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (126 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.5 (d, J＝35 Hz, CH＝CHCO), 164.2 (N＝C), 158.7 (d, ¹J_C—F＝252 Hz, PhC), 143.0 (CH＝CHCO), 132.7, 130.9 (d, ³J_C—F＝6 Hz, PhC), 129.9 (t, ³J_C—F＝8 Hz, PhC), 120.8 (d, ²J_C—F＝18 Hz, PhC), 118.4 (d, ⁴J_C—F＝3 Hz, CH＝CHCO), 117.9 (d, ²J_C—F＝21 Hz, PhC), 79.6, 56.0, 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.1, 31.1, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 22.9, 21.1, 18.6 (19-CH₃), 12.2 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₄ClFNO₄ [M+H]⁺572.2943 found 572.2977.

2, 4-二氟肉桂酰石胆酸肟酯(12v):白色固体, 收率56%. m.p. 157.1~159.2 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 11.95 (brs, 1H, COOH), 8.03~7.97 (m, 1H, PhH), 7.71 (d, J＝16.2 Hz, 1H, CH＝CHCO), 7.41~7.35 (m, 1H, PhH), 7.22~7.17 (m, 1H, PhH), 6.78 (dd, J＝16.2, 6.7 Hz, 1H, CH＝CHCO), 2.94 (d, J＝14.0 Hz, 0.2H, 2-CH₂), 2.80 (dd, J＝10.4, 4.6 Hz, 0.8H, 2-CH₂), 2.70 (t, J＝13.8 Hz, 0.2H, 2-CH₂), 2.42 (t, J＝14.1 Hz, 0.8H, 2-CH₂), 2.28~2.19 (m, 2H, CH₂COOH), 2.13~1.77 (m, 6H), 1.68~1.01 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 170.8 (CH＝CHCO), 170.5 (N＝C), 164.3 (CH＝CHCO), 164.0 (dd, ¹J_C—F＝252 Hz, ²J_C—F＝13 Hz, Ph—C), 161.4 (dd, ¹J＝255 Hz, ²J_C—F＝13 Hz, PhC), 136.3 (PhC), 131.6 (d, ³J_C—F＝10 Hz, PhC), 119.2, 113.0 (d, ²J＝22 Hz, PhC), 105.1 (CH＝CHCO), 79.6, 56.0, 44.2, 42.9, 42.7, 37.0, 35.5, 35.3, 35.2, 32.0, 31.1, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 22.9, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₃H₄₄F₂- NO₄ [M+H]⁺ 556.3238 found 556.3257.

3, 4-二甲氧基肉桂酰石胆酸肟酯(12w):白色固体, 收率52%. m.p. 85.7~87.8 ℃; ¹H NMR (400 MHz, DMSO- d₆) δ: 11.95 (brs, 1H, COOH), 7.65 (d, J＝16.0 Hz, 1H, CH＝CHCO), 7.38 (s, 1H, PhH), 7.26 (d, J＝8.3 Hz, 1H, PhH), 7.26 (d, J＝8.3 Hz, 1H, PhH), 7.48 (d, J＝8.4 Hz, 1H, PhH), 6.63 (dd, J＝15.9, 6.2 Hz, 1H, CH＝CHCO), 3.80 (d, J＝5.8 Hz, 6 H, 2-OCH₃), 2.97 (d, J＝13.9 Hz, 0.5H, 2-CH₂), 2.82 (dd, J＝10.5, 4.6 Hz, 0.5H, 2-CH₂), 2.69 (t, J＝14.0 Hz, 0.5H, 2-CH₂), 2.40 (t, J＝14.2 Hz, 0.5H, 2-CH₂), 2.26~2.19 (m, 2H, CH₂COOH), 2.13~1.78 (m, 6H), 1.70~1.06 (m, 18H), 0.94 (s, 3H, 18-CH₃), 0.87 (d, J＝6.4 Hz, 3H, 21-CH₃), 0.63 (s, 3H, 19-CH₃); ¹³C NMR (100 MHz, DMSO-d₆) δ: 175.3 (COOH), 169.7 (CH＝CHCO), 164.8 (N＝C), 151.5 (CH＝CHCO), 149.4 (PhC), 145.8 (PhC), 127.3 (PhC), 123.7 (PhC), 114.1 (PhC), 111.9 (PhC), 110.7 (CH＝CHCO), 79.6, 56.0, 56.0 (2C, OCH₃), 44.2, 42.9, 42.7, 37.1, 35.5, 35.3, 35.2, 32.1, 31.1, 28.1, 27.4, 26.9, 26.6, 25.8, 24.2, 23.0, 21.1, 18.6 (19-CH₃), 12.3 (18-CH₃); HRMS (ESI) calcd for C₃₅H₅₀- NO₆ [M+H]⁺ 580.3638 found 580.3683.

3.3 PTP1B及TCPTP抑制活性的测试

用于筛选的PTP1B和TCPTP酶均是从大肠杆菌中表达并纯化的GST融合蛋白. PTP1B和TCPTP的水解底物对硝基苯磷酸二钠(pNPP)的磷酯得到的产物在405 nm处有很强的光吸收, 因而可以通过检测405 nm处吸光度测试反应酶的活性以及化合物对酶的作用.测试的具体步骤和方法参照文献进行^[19].

辅助材料(Supporting Information) 目标化合物12a~12w的¹H NMR和¹³C NMR图谱.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

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图 1 已报道的PTP1B抑制剂

Figure 1 Reported PTP1B inhibitors

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图 2 石胆酸-3-肟酯类化合物的设计

Figure 2 Design of the ester derivatives of lithocolic acid-3-oxime

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图式 1 石胆酸-3-肟酯类化合物的合成路线

Scheme 1 Synthesis of the ester derivatives of lithocolic acid-3-oxime

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图 3 (A)~(C)化合物12b与PTP1B的对接模型、(D)化合物12n、12q与PTP1B的对接模型、(E)化合物12n、12q与PTP1B/ TCPTP叠加结构对接模型

Figure 3 (A)~(C) compound 12b docked into PTP1B, (D) compounds 12n & 12q docked into PTP1B, and (E) compounds 12n & 12q docked into the stack of PTP1B with TCPTP

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表 1 化合物对PTP1B的抑制活性

Table 1. Inhibitory activities of the titled compounds against PTP1B


Compd.	R	IC₅₀ (μmol•L^－1)^a		TCPTP/PTP1B^c
Compd.	R	PTP1B	TCPTP	TCPTP/PTP1B^c
12a	H	2.72±0.36	7.32±1.15	2.69
12b	4-Cl	0.79±0.21	3.41±0.45	4.31
12c	3-Cl	10.22±1.05	＞40^b	—^d
12d	2-Cl	5.15±1.17	＞40	—
12e	4-F	28.70±2.98	＞40	—
12f	3-F	10.94±1.52	＞40	—
12g	2-F	15.25±2.04	＞40	—
12h	4-Br	1.33±0.25	3.18±0.62	2.39
12i	3-Br	4.29±1.12	10.32±1.70	2.41
12j	2-Br	9.12±1.38	11.28±1.67	1.24
12k	4-Me	2.81±0.14	15.35±1.44	5.46
12l	3-Me	9.38±2.12	18.22±1.07	1.94
12m	2-Me	1.53±0.77	9.02±1.15	5.90
12n	4-OMe	1.63±0.23	12.06±1.83	7.39
12o	3-OMe	19.55±1.19	＞40	—
12p	2-OMe	15.62±0.78	＞40	—
12q	2, 4-Cl₂	0.93±0.20	5.93±0.88	6.37
12r	2, 3-Cl₂	12.40±1.99	16.51±1.01	1.33
12s	3, 4-Cl₂l	1.71±0.32	4.72±0.46	2.76
12t	2-F-4-Br	24.27±2.16	＞40	—
12u	3-Cl-4-F	17.37±1.77	＞40	—
12v	2, 4-F₂	26.80±1.53	＞40	—
12w	3, 4-OMe₂	14.27±1.81	＞40	—
LCA		12.21±1.72	＞40	—
OA^e		2.67±0.32	5.98±0.21	2.2
^a IC₅₀ values were determined by regression analyses and expressed as means±SD of three replications; ^b IC₅₀＞40 μmol/L, the inhibition (%) on TCPTP was between 5% and 50% at 40 µmol•L^－1; ^c TCPTP/PTP1B, the IC₅₀ ratio of TCPTP and PTP1B; ^d “—”, none accurate value was calculated; ^e Olea- nolic acid (OA) as positive control.

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