新型[1,3]二氧杂环戊烯并[4,5-f]异吲哚酮衍生物的设计、合成与活性研究

引用本文

GONG Yong-xiang, ZHU Qi-feng, ZHONG Jin-qing, LIU Li-fei, LI Xu-fei, ZHENG Xiao-he, LUO Hong-ying, ZHAO Xu-yang. Design, synthesis and biological evaluation of novel [1,3]dioxolo[4,5-f]isoindolone derivatives[J]. Acta Pharm Sin, 2015, 50(2): 191-198. 复制到剪切板

龚永祥, 朱齐凤, 钟金清, 刘礼飞, 李旭飞, 郑晓鹤, 骆红英, 赵旭阳. 新型[1,3]二氧杂环戊烯并[4,5-f]异吲哚酮衍生物的设计、合成与活性研究[J]. 药学学报, 2015, 50(2): 191-198. 复制到剪切板

新型[1,3]二氧杂环戊烯并[4,5-f]异吲哚酮衍生物的设计、合成与活性研究

龚永祥, 朱齐凤 , 钟金清, 刘礼飞, 李旭飞, 郑晓鹤, 骆红英, 赵旭阳

浙江海正药业股份有限公司, 浙江台州 318000

收稿日期: 2014-08-27;修回日期: 2014-09-25.

* 通讯作者：qfzhu@hisunpharm.com;hisunxyzhao@126.com

摘要：本文设计、合成了一系列新型[1,3]二氧杂环戊烯并[4,5-f]异吲哚酮衍生物, 并对其进行了乙酰胆碱酯酶抑制活性以及由东莨菪碱致小鼠记忆障碍的改善作用测试。实验结果表明目标化合物对乙酰胆碱酯酶(AChE)均有抑制作用, IC₅₀值在微摩尔级, 其中化合物I₁(IC₅₀=0.086 μmol·L^-1)和I₂(IC₅₀=0.080 μmol·L^-1)对AChE的抑制活性较好, 与阳性药多奈哌齐(IC₅₀=0.094 μmol·L^-1)相当; 且化合物I₁～I₄均具有改善由东莨菪碱引起的小鼠记忆障碍的作用。

关键词： [1,3]二氧杂环戊烯并[4,5-f]异吲哚酮乙酰胆碱酯酶抑制剂记忆障碍阿尔茨海默症

Design, synthesis and biological evaluation of novel [1,3]dioxolo[4,5-f]isoindolone derivatives

GONG Yong-xiang, ZHU Qi-feng , ZHONG Jin-qing, LIU Li-fei, LI Xu-fei, ZHENG Xiao-he, LUO Hong-ying, ZHAO Xu-yang

Zhejiang Hisun Pharmaceutical Co., Ltd., Taizhou 318000, China

Abstract: A series of [1,3]dioxolo[4,5-f]isoindolone derivatives were designed, synthesized and evaluated as inhibitors of acetylcholinesterases (AChE). Furthermore, their effects on memory impairment of mice induced by scopolamine were investigated with step-through test. The results suggested that most of the target compounds exhibited potential inhibition on AChE with IC₅₀ values at micromolar range. Compounds I₁ (IC₅₀ value of 0.086 μmol·L^-1) and I₂ (IC₅₀ value of 0.080 μmol·L^-1) showed the strongest AChE inhibitory activity, which are equipotent to donepezil (IC₅₀ value of 0.094 μmol·L^-1). Moreover, compounds I₁-I₄ could improve the memory impairment induced by scopolamine in mice.

Key words: [1,3]dioxolo[4,5-f]isoindolone acetylcholinesterase inhibitor memory impairment Alz-heimer's disease

阿尔茨海默症 (Alzheimer’s disease,AD),是一种以渐进性记忆障碍和认知功能下降为特征的神经退化性疾病^[1]。AD严重危害着老年人的身心健康,已成为严重的社会问题^{[2, 3, 4]}。

目前,临床上用于治疗AD的药物主要是乙酰胆碱酯酶 (acetylcholinesterase,AChE) 抑制剂,如他克林、多奈哌齐、利斯的明、加兰他敏和石杉碱甲等^[5],它们通过抑制AChE的活性,提高患者脑内的乙酰胆碱在突触间隙的浓度而达到治疗AD的效果^[6]。然而临床上证实这些药物只能用于轻度至中度AD患者,只能改善AD病人的症状,改善作用仅能持续约6～12个月,且病人对这些药物的响应率也只有50% 左右,同时伴有外周胆碱能毒副作用等问题^{[7, 8]}。面对这些问题,许多研究者积极尝试对上市AChE抑制剂药物进行结构改造,期望设计得到新型AChE抑制剂^[9]。

多奈哌齐是第二个获美国FDA批准用于治疗AD的乙酰胆碱酯酶 (acetylcholinesterase,AChE) 抑制剂。它与TcAChE蛋白复合物的X-衍射晶体结构 (Protein Data Bank,代码为1EVE) (图 1) 显示^{[9, 10]}: 苄基部分作用于AChE的活性中心,与Trp84的吲哚环产生π-π堆积作用,哌啶环质子化的氮原子与中央部位的Phe330的苯环产生阳离子-π作用,茚酮部分延伸至外周阴离子位点,与Trp279形成π-π堆积作用。因此,对多奈哌齐的结构改造主要集中在对茚酮环^{[11, 12, 13, 14]}、引入卤素^[15,16]、苄基哌啶环^[14,17]等部位。本文尝试对多奈哌齐进行结构改造 (图 2),将A部分的邻二酚醚通过亚甲基连接起来变成 ^[1,3] 二氧杂环戊烯,将B部分茚酮换成了异吲哚啉酮结构,同时将C部分延长了碳链,将D部分苄基变换成取代苄基,从而得到20个全新结构类型的^[1,3]二氧杂环戊烯并[4,5-f]异吲哚酮衍生物。

Figure 1 Interaction of donepezil with some selected residues of the AChE gorge

Figure 2 Rational design of target compounds

目标化合物的合成见合成路线1和2,其中,原料1参照文献^{[18, 19]}方法合成,原料2参照文献^[20]方法合成。

Scheme 1 Synthentic route of compounds 3-7. Reagents and conditions: i) KOH,DMSO; ii) a. NaBH₄,MeOH/THF; b. Ac₂O,Et₃N; c. H₂,Pd/C; iii,iv) MeI,LDA/THF; v) a. MeMgI,HCl; b. Et₂Zn/TFA/CH₂I₂

Scheme 2 Synthetic routes of target compounds (I₁-I₄,II₁-II₄,III₁-III₄,IV₁-IV₄,V₁-V₄)

目标化合物活性测定,首先采用改良的Ellman法^[21]对其进行了体外乙酰胆碱酯酶抑制活性筛选,然后采用避暗实验进一步考察具有较好AChE抑制活性的化合物对东莨菪碱所致小鼠学习记忆障碍的改善作用。结果与讨论 1 目标化合物的合成

中间体3的合成以1和2为起始原料,碱性条件下加热反应得到。中间体4的合成由3经过三步反应生成,先将3用硼氢化钠还原成醇,然后醇与乙酸酐发生酯化、再用钯炭还原得到。4与碘甲烷单取代得产物5,5进一步取代得双取代产物6。中间体3与甲基碘化镁发生格氏加成同时脱水得烯,然后与卡宾 (Et₂Zn/THF/CH₂I₂) 发生环丙烷化反应得到中间体7。中间体3～7在酸性条件下脱除氨基保护基 (Boc) 得到8～12。化合物8～12再与不同的苄氯反应得到目标化合物。目标化合物I～V的结构经¹H NMR、ESI-MS及元素分析得以确证,产物的结构、收率和熔点见表 1,波谱数据见表 2。

Table 1 Structures and physical properties of target compounds I-V

Compd.	R¹	R²	R³	Yield/%	mp/℃
I₁	=O		H	60.4	136.5-138.5
I₂	=O		2-F	66.1	140.0-141.6
I₃	=O		3-F	58.3	166.5-167.8
I₄	=O		4-F	49.9	140.3-142.1
II₁	H	H	H	53.4	134.3-136.3
II₂	H	H	2-F	67.2	123.4-125.5
II₃	H	H	3-F	70.2	105.8-108.1
II₄	H	H	4-F	65.1	130.7-133.4
III₁	H	CH₃	H	47.4	123.6-125.9
III₂	H	CH₃	2-F	65.4	127.4-129.0
III₃	H	CH₃	3-F	66.3	139.1-141.3
III₄	H	CH₃	4-F	58.3	117.2-119.2
IV₁	CH₃	CH₃	H	67.7	95.0-96.2
IV₂	CH₃	CH₃	2-F	72.5	122.6-124.2
IV₃	CH₃	CH₃	3-F	69.1	107.0-109.3
IV₄	CH₃	CH₃	4-F	48.6	96.7-98.5
V₁	-CH₂CH₂-		H	65.2	150.0-152.7
V₂	-CH₂CH₂-		2-F	56.3	155.1-157.4
V₃	-CH₂CH₂-		3-F	70.4	168.7-170.2
V₄	-CH₂CH₂-		4-F	77.1	154.8-156.3

Table 1 Structures and physical properties of target compounds I-V

Table 2 Spectral data of target compounds I-V

No.	¹H NMR,MS,Elemental analysis
I₁	¹H NMR (DMSO-d₆): 1.08-1.17 (m,3H),1.48 (q,2H,J = 6.6 Hz),1.67 (d,2H,J = 10.0 Hz),1.85 (t,2H,J = 10.8 Hz),2.75 (d,2H,J = 11.4 Hz),3.41 (s,2H),3.53 (t,2H,J = 7.1 Hz),6.27 (s,2H),7.22-7.32 (m,5H),7.37 (s,2H); ESI-MS (m/z): 393 [M+H]⁺; Calcd. (%): C 70.39; H 6.16; N 7.14; Found (%): C 70.42; H 6.18; N 7.12
I₂	¹H NMR (CDCl₃): 1.24-1.32 (m,3H),1.58 (q,2H,J = 7.5 Hz),1.75 (d,2H,J = 10.8 Hz),2.00 (t,2H,J = 11.0 Hz),2.89 (d,2H,J = 11.5 Hz),3.57 (s,2H),3.64 (t,2H,J = 7.4 Hz),6.15 (s,2H),6.99-7.03 (m,1H),7.07-7.11 (m,1H),7.19 (s,2H),7.21-7.23 (m,1H),7.37 (t,1H,J = 7.1 Hz); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 67.31; H 5.65; N 6.83; Found (%): C 67.29; H 5.64; N 6.8
I₃	¹H NMR (CDCl₃): 1.27-1.34 (m,3H),1.57-1.62 (m,2H),1.74 (d,2H,J = 9.4 Hz),1.93 (t,2H,J = 10.7 Hz),2.84 (d,2H,J = 11.3 Hz),3.46 (s,2H),3.65 (t,2H,J = 7.2 Hz),6.15 (s,2H),6.89-6.94 (m,1H),7.03-7.07 (m,2H),7.19 (s,2H),7.21-7.27 (m,1H); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 67.31; H 5.65 ; N 6.83; Found (%): C 67.33; H 5.66; N 6.81
I₄	¹H NMR (DMSO-d₆): 1.08-1.20 (m,3H),1.48 (q,2H,J = 6.7 Hz),1.67 (d,2H,J = 10.2 Hz),1.84 (t,2H,J = 11.0 Hz),2.74 (d,2H,J = 11.4 Hz),3.39 (s,2H),3.53 (t,2H,J = 7.2 Hz),6.27 (s,2H),7.09-7.14 (m,2H),7.28-7.31 (m,2H),7.37 (s,2H); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 67.31; H 5.65 ; N 6.83; Found (%): C 67.35; H 5.63; N 6.85
II₁	¹H NMR (CDCl₃): 1.26-1.36 (m,3H),1.57 (q,2H,J = 7.5 Hz),1.73 (d,2H,J = 9.2 Hz),1.93 (t,2H,J = 10.6 Hz),2.87 (d,2H,J = 11.2 Hz),3.48 (s,2H),3.60 (t,2H,J = 7.5 Hz),4.23 (s,2H),6.04 (s,2H),6.83 (s,1H),7.21 (s,1H),7.22-7.26 (m,1H),7.29-7.30 (m,4H); ESI-MS (m/z): 379 [M+H]⁺; Calcd. (%): C 72.99; H 6.92; N 7.40; Found (%): C 73.01; H 6.94; N 7.38
II₂	¹H NMR (CDCl₃): 1.29-1.34 (m,3H),1.57 (q,2H,J = 5.4 Hz),1.74 (d,2H,J = 8.8 Hz),1.99 (t,2H,J = 10.2 Hz),2.88 (d,2H,J = 10.7 Hz),3.56 (s,2H),3.59 (t,2H,J = 7.2 Hz),4.22 (s,2H),6.03 (s,2H),6.82 (s,1H),7.00 (t,1H,J = 9.2 Hz),7.08 (t,1H,J = 7.3 Hz),7.20-7.22 (m,2H),7.35 (t,1H,J = 7.1 Hz); ESI-MS (m/z): 397 [M+H]⁺; Calcd. (%): C 69.68; H 6.36; N 7.07; Found (%): C 69.70; H 6.35; N 7.09
II₃	¹H NMR (DMSO-d₆): 1.12-1.17 (m,3H),1.50 (q,2H,J = 5.4 Hz),1.68 (d,2H,J = 8.5 Hz),1.87 (t,2H,J = 9.4 Hz),2.75 (d,2H,J = 10.6 Hz),3.43 (s,2H),3.49 (t,2H,J = 7.1 Hz),4.31 (s,2H),6.12 (s,2H),7.03-7.12 (m,5H),7.32-7.37 (m,1H); ESI-MS (m/z): 397 [M+H]⁺; Calcd. (%): C 69.68; H 6.36; N 7.07; Found (%): C 69.66; H 6.38; N 7.08
II₄	¹H NMR (CDCl₃): 1.26-1.32 (m,3H),1.57 (q,2H,J = 5.0 Hz),1.72-1.74 (m,2H),1.92 (t,2H,J = 10.6 Hz),2.83 (d,2H,J = 10.9 Hz),3.43 (s,2H),3.60 (t,2H,J = 7.4 Hz),4.23 (s,2H),6.04 (s,2H),6.83 (s,1H),6.96-7.00 (m,2H),7.21 (s,1H),7.24-7.27 (m,2H); ESI-MS (m/z): 397 [M+H]⁺; Calcd. (%): C 69.68; H 6.36; N 7.07; Found (%): C 69.65; H 6.37; N 7.05
III₁	¹H NMR (CDCl₃): 1.34-1.42 (m,3H),1.46 (d,3H,J = 6.7 Hz),1.59-1.64 (m,2H),1.72-1.75 (m,1H),1.86-1.89 (m,1H),1.97-2.04 (m,2H),2.94 (t,2H,J = 9.1 Hz),3.21-3.28 (m,1H),3.55 (s,2H),3.97-4.05 (m,1H),4.46 (q,1H,J = 6.7 Hz),6.09 (s,2H),6.87 (s,1H),7.26 (s,1H),7.29-7.32 (m,1H),7.34-7.37 (m,4H); ESI-MS (m/z): 393 [M+H]⁺; Calcd. (%): C 73.44; H 7.19; N 7.14; Found (%): C 73.47; H 7.20; N 7.12
III₂	¹H NMR (CDCl₃): 1.36-1.42 (m,3H),1.46 (d,3H,J = 6.7 Hz),1.58-1.63 (m,2H),1.73-1.76 (m,1H),1.87-1.89 (m,1H),2.05-2.10 (m,2H),2.95 (m,2H),3.20-3.27 (m,1H),3.63 (s,2H),3.98-4.05 (m,1H),4.46 (q,1H,J = 6.6 Hz),6.10 (s,2H),6.87 (s,1H),7.07 (t,1H,J = 9.1 Hz),7.16 (t,1H,J = 7.4 Hz),7.26-7.35 (m,2H),7.43 (t,1H,J = 7.1 Hz); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 70.22; H 6.63; N 6.82; Found (%): C 70.25; H 6.61; N 6.84
III₃	¹H NMR (CDCl₃): 1.34-1.42 (m,3H),1.46 (d,3H,J = 6.8 Hz),1.59-1.64 (m,2H),1.72-1.75 (m,1H),1.87-1.89 (m,1H),1.99-2.04 (m,2H),2.92 (t,2H,J = 8.9 Hz),3.21-3.28 (m,1H),3.54 (s,2H),3.97-4.05 (m,1H),4.47 (q,1H,J = 6.7 Hz),6.10 (s,2H),6.88 (s,1H),6.96-7.01 (m,1H),7.10-7.15 (m,2H),7.26 (s,1H),7.30-7.34 (m,1H); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 70.22; H 6.63; N 6.82; Found (%): C 70.25; H 6.64; N 6.81
III₄	¹H NMR (CDCl₃): 1.32-1.40 (m,3H),1.46 (d,3H,J = 6.7 Hz),1.59-1.64 (m,2H),1.73-1.75 (m,1H),1.86-1.88 (m,1H),1.95-2.00 (m,2H),2.90 (t,2H,J = 8.2 Hz),3.21-3.28 (m,1H),3.50 (s,2H),3.98-4.05 (m,1H),4.47 (q,1H,J = 6.7 Hz),6.10 (s,2H),6.88 (s,1H),7.02-7.06 (m,2H),7.26 (s,1H),7.31-7.34 (m,2H); ESI-MS (m/z): 411 [M+H]⁺; Calcd. (%): C 70.22; H 6.63; N 6.82; Found (%): C 70.18; H 6.65; N 6.83
IV₁	¹H NMR (DMSO-d₆): 1.18-1.23 (m,2H),1.26-1.33 (m,1H),1.40 (s,6H),1.50-1.56 (m,2H),1.70 (d,2H,J = 11.2 Hz),1.89 (t,2H,J = 10.4 Hz),2.77 (d,2H,J = 11.2 Hz),3.35 (t,2H,J = 7.8 Hz),3.42 (s,2H),6.12 (s,2H),7.05 (s,1H),7.21-7.25 (m,2H),7.27-7.33 (m,4H); ESI-MS (m/z): 407 [M+H]⁺; Calcd. (%): C 73.86; H 7.44; N 6.89; Found (%): C 73.83; H 7.45; N 6.90
IV₂	¹H NMR (DMSO-d₆): 1.13-1.22 (m,2H),1.23-1.28 (m,1H),1.39 (s,6H),1.48-1.54 (m,2H),1.70 (d,2H,J = 11.6 Hz),1.94 (t,2H,J = 10.9 Hz),2.78 (d,2H,J = 11.2 Hz),3.33 (t,2H,J = 7.7 Hz),3.48 (s,2H),6.10 (s,2H),7.03 (s,1H),7.12-7.18 (m,2H),7.24 (s,1H),7.27-7.32 (m,1H),7.39 (t,1H,J = 7.4 Hz); ESI-MS (m/z): 425 [M+H]⁺; Calcd. (%): C 70.73; H 6.89; N 6.60; Found (%): C 70.70; H 6.90; N 6.61
IV₃	¹H NMR (DMSO-d₆): 1.15-1.24 (m,2H),1.25-1.31 (m,1H),1.40 (s,6H),1.50-1.55 (m,2H),1.71 (d,2H,J = 10.8 Hz),1.91 (t,2H,J = 11.2 Hz),2.77 (d,2H,J = 11.5 Hz),3.35 (t,2H,J = 8.2 Hz),3.45 (s,2H),6.11 (s,2H),7.03-7.14 (m,4H),7.25 (s,1H),7.32-7.38 (m,1H); ESI-MS (m/z): 425 [M+H]⁺; Calcd. (%): C 70.73; H 6.89; N 6.60; Found (%): C 70.75; H 6.88; N 6.62
IV₄	¹H NMR (DMSO-d₆): 1.13-1.22 (m,2H),1.25-1.29 (m,1H),1.41 (s,6H),1.50-1.55 (m,2H),1.70 (d,2H,J = 11.2 Hz),1.89 (t,2H,J = 10.7 Hz),2.76 (d,2H,J = 11.2 Hz),3.35 (t,2H,J = 7.7 Hz),3.40 (s,2H),6.12 (s,2H),7.04 (s,1H),7.12 (t,2H,J = 8.9 Hz),7.25 (s,1H),7.29-7.33 (m,2H); ESI-MS (m/z): 425 [M+H]⁺; Calcd. (%): C 70.73; H 6.89; N 6.60; Found (%): C 70.69; H 6.91; N 6.58
V₁	¹H NMR (CDCl₃): 1.24-1.33 (m,5H),1.49-1.57 (m,4H),1.72 (d,2H,J = 9.6 Hz),1.96 (t,2H,J = 10.8 Hz),2.87 (d,2H,J = 11.4 Hz),3.19 (t,2H,J = 7.8 Hz),3.49 (s,2H),6.03 (s,2H),6.42 (s,1H),7.22-7.25 (m,2H),7.28-7.32 (m,4H); ESI-MS (m/z): 405 [M+H]⁺; Calcd. (%): C 74.23; H 6.98; N 6.93; Found (%): C 74.76; H 6.96; N 6.94
V₂	¹H NMR (CDCl₃): 1.23-1.31 (m,5H),1.48-1.55 (m,4H),1.72 (d,2H,J = 9.2 Hz),2.02 (t,2H,J = 10.3 Hz),2.88 (d,2H,J = 11.2 Hz),3.18 (t,2H,J = 7.9 Hz),3.56 (s,2H),6.02 (s,2H),6.41 (s,1H),6.99-7.03 (m,1H),7.07-7.11 (m,1H),7.19-7.25 (m,2H),7.35-7.39 (m,1H); ESI-MS (m/z): 423 [M+H]⁺; Calcd. (%): C 71.07; H 6.44; N 6.63; Found (%): C 71.04; H 6.45; N 6.65
V₃	¹H NMR (CDCl₃): 1.24-1.32 (m,5H),1.49-1.56 (m,4H),1.71 (d,2H,J = 10.2 Hz),1.95 (t,2H,J = 10.7 Hz),2.83 (d,2H,J = 11.2 Hz),3.19 (t,2H,J = 7.7 Hz),3.45 (s,2H),6.02 (s,2H),6.41 (s,1H),6.89-6.93 (m,1H),7.03-7.07 (m,2H),7.21-7.27 (m,2H); ESI-MS (m/z): 423 [M+H]⁺; Calcd. (%): C 71.07; H 6.44; N 6.63; Found (%): C 71.10 ; H 6.42; N 6.62
V₄	¹H NMR (CDCl₃): 1.24-1.32 (m,5H),1.49-1.56 (m,4H),1.72 (d,2H,J = 10.2 Hz),1.94 (t,2H,J = 10.7 Hz),2.84 (d,2H,J = 11.4 Hz),3.18 (t,2H,J = 7.9 Hz),3.44 (s,2H),6.02 (s,2H),6.42 (s,1H),6.95-7.01 (m,2H),7.24 (s,1H),7.25-7.28 (m,2H); ESI-MS (m/z): 423 [M+H]⁺; Calcd. (%): C 71.07; H 6.44; N 6.63; Found (%): C 71.09; H 6.46; N 6.61

Table 2 Spectral data of target compounds I-V

2 乙酰胆碱酯酶抑制活性

体外乙酰胆碱酯酶抑制活性实验表明,制备的20个化合物对乙酰胆碱酯酶 (AChE) 均有一定的抑制作用 (表 3),IC₅₀值在微摩尔级。其中化合物I₁ (IC₅₀ = 0.086 µmol·L^-1) 和I₂(IC₅₀ = 0.080 µmol·L^-1) 对AChE的抑制活性较好,与对照药 (donepezil,IC₅₀ = 0.094 µmol·L^-1) 相当; 化合物III₄和IV₁～IV₄活性弱 (IC₅₀值均大于2.0 µmol·L^-1); 其他化合物对AChE的抑制活性接近 (IC₅₀值在0.1～0.9 µmol·L^-1之间)。

Table 3 Inhibitory activities on AChE of target compounds I-V

Compd.	IC₅₀/µmol·L^-1	Compd.	IC₅₀/µmol·L^-1
I₁	0.086	III₃	0.889
I₂	0.080	III₄	>2.0
I₃	0.269	IV₁	>2.0
I₄	0.167	IV₂	>2.0
II₁	0.502	IV₃	>2.0
II₂	0.483	IV₄	>2.0
II₃	0.486	V₁	0.202
II₄	0.658	V₂	0.154
III₁	0.601	V₃	0.376
III₂	0.239	V₄	0.706
Donepezil	0.094

Table 3 Inhibitory activities on AChE of target compounds I-V

由表 3数据可知,B处取代基 (R¹,R²) (图 2) 对活性的影响: IV系列双甲基取代 (CH₃,CH₃) 活性最弱,III系列单甲基取代 (H,CH₃)、II系列未被取代 (H,H) 活性较弱,V系列三元螺环 (-(CH₂)₂-) 取代较强,I系列羰基 (=O) 取代的活性最强,例如表 1化合物的IC₅₀值: IV₁ > III₁ > II₁ > V₁ > I₁、IV₂ > II₂ > III₂ > V₂ > I₂、IV₃ > III₃ > II₃ > V₃ > I₃; D处(图 2) 苯环上R³取代基对活性的影响: 2-氟取代较未被取代化合物活性增强 (IC₅₀值: I₁ > I₂、II₁ > II₂、III₁ > III₂、V₁ > V₂); 除II系列化合物外,3-或4-氟取代化合物较未被取代化合物活性减弱 (IC₅₀值: I₃ > I₄ > I₁、III₄ > III₃ > III₁、V₄ > V₃ > V₁); D处 (图 2) 苯环氟取代 (R³=F) 的位置对活性影响: 2-氟取代较3-或4-氟取代化合物活性增强 (IC₅₀值: I₃ > I₄ > I₂、II₄ > II₃ > II₂、III₄ > III₃ > III₂、V₄ > V₃ > V₂)。 3 动物避暗实验测试

为了进一步研究化合物对动物学习记忆功能的影响,选择对乙酰胆碱酯酶抑制活性较好的I系列化合物 (I₁～I₄) 进行了小鼠避暗实验测试,结果如图 3所示。结果表明与模型组相比,除I₃ 5 mg·kg^-1组外,其余各组均可显著延长进入暗室的潜伏期,且与阳性药多奈哌齐无明显差异; 同时在减少进入暗室的错误次数方面,除I₁～I₃ (5 mg·kg^-1) 组外,其余各组与模型组均有显著性差异,且与阳性药多奈哌齐无明显差异。可见,在避暗实验中I₁～I₄均具有改善由东莨菪碱引起的小鼠记忆障碍的作用。

Figure 3 The effects of compounds I₁-I₄ on memory impairment induced by scopolamine in step-through test. A: Latency; B: Error number. ^*P < 0.05 vs model group,^**P < 0.01 vs model group,^***P < 0.001 vs model group

实验部分

乙酰胆碱酯酶 (AChE)、多奈哌齐、碘化硫代乙酰胆碱 (ATCH)、5,5'-二硫代双 (2-硝基苯甲酸) (DTNB) 和东莨菪碱购于Sigma公司; 小鼠避暗实验记录仪为大正制药株式会社产品 (型号PA M5,日本); 实验用小鼠购于中国医学科学院实验动物研究所; NMR使用Bruker Avance 400型核磁共振仪测定 (TMS为内标,DMSO-d₆、CDCl₃、D₂O为溶剂); ESI- MS采用Agilent-1100-LCQ-Advantage质谱仪; 薄层色谱硅胶GF254、硅胶H为青岛海洋化工有限公司生产; 其他试剂均为市售AR或CP级。 1 化学合成 1.1 4-[2-(5,7-二氧代-^[1,3]二氧杂环戊烯并[4,5-f]异吲哚-6-基)乙基]哌啶-1-羧酸叔丁酯 (3) 的合成

将1 (130 g,0.68 mol) 加入到800 mL DMSO中,60 ℃加热溶解,加入溶有KOH (58.5 g,1.05 mol) 的乙醇溶液250 mL,再加入溶有2 (292.5 g,0.78 mol) 的DMSO溶液400 mL,60 ℃反应5 h。加入1.25 L乙酸乙酯和750 mL水萃取,无水硫酸钠干燥,蒸除溶剂,柱色谱分离得180 g化合物3,收率65.8%; ¹H NMR (DMSO-d₆): 0.90～1.00 (m,2H),1.36 (s,10H),1.46 (q,2H,J = 7.0 Hz),1.66 (d,2H,J = 11.9 Hz),2.62 (br s,2H),3.51 (t,2H,J = 7.0 Hz),3.88 (d,2H,J = 11.5 Hz),6.24 (s,2H),7.32 (s,2H); MS (ESI): m/z 425 [M+Na]⁺。 1.2 4-[2-(7-氧代-5H-^[1,3]二氧杂环戊烯并[4,5-f]异吲哚-6-基)乙基]哌啶-1-羧酸叔丁酯 (4) 的合成

将3(92 g,0.23 mol) 溶于250 mL CH₃OH和250 mL THF,搅拌,在0～10 ℃加入NaBH₄ (10 g,0.26 mol),保温反应20～30 min,加入100 mL水,减压浓缩至干,加入500 mL乙酸乙酯和300 mL水萃取,无水硫酸钠干燥,蒸除溶剂至少量,加入300 mL石油醚,析出固体,过滤,烘干得64.7 g产物,收率70%。

将制备所得产物 (55 g,0.136 mol)、385 mL CH₂Cl₂、Et₃N (43 mL,0.31 mol) 和4-二甲氨基吡啶 (1.8 g,0.015 mol) 加入500 mL反应瓶中,搅拌,加入乙酸酐 (31 mL,0.33 mol),室温反应1 h,加入200 mL水,萃取,无水硫酸钠干燥,蒸除溶剂,剩余物溶于400 mL乙酸乙酯,加入17 g5%钯炭 (50% 水),于60 ℃常压氢化7～8 h,过滤,蒸除溶剂至少量,滴加石油醚,冷却析出固体,过滤,得33 g化合物4,收率: 62.5%; ¹H NMR (DMSO-d₆): 0.94～1.04 (m,2H),1.39 (s,10H),1.47～1.54 (m,2H),1.69 (d,2H,J = 11.6 Hz),2.64 (br s,2H),3.50 (t,2H,J = 7.0 Hz),3.90 (d,2H,J = 11.3 Hz),4.32 (s,2H),6.12 (s,2H),7.08 (s,1H),7.11 (s,1H); MS (ESI): m/z 411 [M+Na]⁺。 1.3 4-[2-(5-甲基-7-氧代-5H-^[1,3]二氧杂环戊烯并[4,5-f]异吲哚-6-基)乙基]哌啶-1-羧酸叔丁酯 (5) 的合成

将4(30 g,0.077 mol) 溶于300 mL THF,氮气保护,在-15～-10 ℃滴加二异丙基氨基锂 (2 mol·L^-1) 的正庚烷溶液 (78 mL,0.16 mol) (30 min滴完),保温反应30 min,再滴加CH₃I (4.8 mL,0.077 mol),室温反应2 h,加入600 mL& lt; span style='font-family:宋体;color:black'>乙酸乙酯和300 mL水萃取,无水硫酸钠干燥,蒸除溶剂,柱色谱分离得14.5 g化合物5,收率: 46.7%; ¹H NMR (CDCl₃): 1.07～1.21 (m,2H),1.41 (d,3H,J = 6.7 Hz),1.45 (s,10H),1.55 (q,2H,J = 7.4 Hz),1.66～1.69 (m,1H),1.80～1.83 (m,1H),2.64～2.71 (m,2H),3.17～3.24 (m,1H),3.92～4.00 (m,1H),4.06～4.09 (m,2H),4.41 (q,1H,J = 6.7 Hz),6.05 (s,2H),6.82 (s,1H),7.20 (s,1H); MS (ESI): m/z 403 [M+H]⁺。 1.4 4-[2-(7,7-二甲基-5-氧代-^[1,3]二氧杂环戊烯并[4,5-f]异吲哚-6-基)乙基]哌啶-1-羧酸叔丁酯 (6) 的合成

以5为原料,按照1.3的方法进一步合成化合物6,收率: 54.2%; MS (ESI): m/z 417 [M+H]⁺。 1.5 4-[2-(5-氧代螺环[^[1,3]二氧杂环戊烯并[4,5-f]异吲哚-7,1'-环丙烷]-6-基)乙基]哌啶-1-羧酸叔丁酯 (7) 的合成

将3 (75 g,0.19 mol) 溶于500 mL THF中,氮气保护,在0～10 ℃缓慢滴加400 mL CH₃MgI的乙醚溶液 (1.2 mol·L^-1),反应1 h,加入少量水至无气泡产生,滴加浓盐酸,调pH至酸性,加入500 mL乙酸乙酯和300 mL水萃取,无水硫酸钠干燥,蒸除溶剂至少量,滴加石油醚,析出固体,过滤,烘干得47.6 g产物,收率: 63.8%。

将210 mL (1 mol·L^-1) 二乙基锌的己烷溶液和210 mL CH₂Cl₂加入到反应瓶中,氮气保护,在0～ 10 ℃滴加含有 (16.2 mL,0.22 mol) CF₃COOH的二氯甲烷溶液150 mL,反应20 min,滴加含有CH₂I₂ (16.8 mL,0.21 mol) 的二氯甲烷溶液200 mL,反应20 min,再滴加溶有42 g(0.11 mol) 以上新制备产物的二氯甲烷溶液200 mL,升温至30 ℃反应3～4 h,加入350 mL水,以1 mol·L^-1盐酸调至中性,萃取,无水硫酸钠干燥,蒸除溶剂得化合物7。¹H NMR (CDCl₃): 1.08～1.19 (m,2H),1.28 (dd,2H,J = 6.2,7.4 Hz),1.45 (s,9H),1.48～1.57 (m,5H),1.72 (d,2H,J = 12.7 Hz),2.69 (t,2H,J = 11.6 Hz),3.20 (t,2H,J = 7.6 Hz),4.07 (d,2H,J = 13.1 Hz),6.03 (s,2H),6.43 (s,1H),7.23 (s,1H); MS (ESI): m/z 437 [M+Na]⁺。 1.6 中间体8～12的合成通法

向反应瓶中加入3～7 (0.03 mol) 和240 mL含10% 氯化氢的乙酸乙酯溶液,室温反应2 h,过滤,洗涤,烘干得化合物8～12。

8: ¹H NMR (D₂O): 1.40～1.64 (m,5H),2.02 (d,2H,J = 13.4 Hz),2.96～3.03 (m,2H),3.45～3.50 (m,4H),6.12 (s,2H),6.79 (s,2H)。

9: ¹H NMR (D₂O): 1.38～1.49 (m,2H),1.59～1.61 (m,3H),1.98 (d,2H,J = 13.5 Hz),2.91～2.98 (m,2H),3.42 (d,2H,J = 12.8 Hz),3.49 (t,2H,J = 7.0 Hz),4.13 (s,2H),5.97 (s,2H),6.72 (s,1H),6.76 (s,1H); MS (ESI): m/z 289 [M-Cl]⁺。

10: ¹H NMR (D₂O): 1.24 (d,3H,J = 7.0 Hz),1.44～1.58 (m,5H),1.98 (m,2H),2.96 (m,2H),3.21～3.25 (m,1H),3.44 (d,2H,J = 12.4 Hz),3.64～3.72 (m,1H),4.34 (q,1H,J = 6.6 Hz),5.94 (s,2H),6.72 (s,1H),6.79 (s,1H); MS (ESI): m/z 303 [M+H]⁺。

11: ¹H NMR (D₂O): 1.27 (s,6H),1.45 (m,2H),1.56 (q,2H,J = 6.9 Hz),1.69 (br s,1H),2.00 (d,2H,J = 13.9 Hz),3.00 (t,2H,J = 12.6 Hz),3.37 (t,2H,J = 7.6 Hz),3.45 (d,2H,J = 12.3 Hz),5.96 (s,2H),6.79 (s,1H),6.86 (s,1H); MS (ESI): m/z 317 [M-Cl]⁺。

12: ¹H NMR (D₂O): 1.06 (t,2H,J = 6.7 Hz),1.32～1.46 (m,6H),1.60 (m,1H),1.91 (d,2H,J = 13.5 Hz),2.91～3.03 (m,4H),3.39 (d,2H,J = 12.8 Hz),5.90 (s,2H),6.18 (s,1H),6.68 (s,1H); MS (ESI): m/z 315 [M-Cl]⁺。 1.7 化合物I～V的合成通法 (以I₁～I₄为例<)

将新制备的8 (5 g,0.015 mol)、K₂CO₃ (1.4 g,0.01 mol)、100 mL乙腈和相应的苄氯 (0.02 mol) 加入到反应瓶中,加热至50 ℃反应3～4 h,加入200 mL乙酸乙酯和100 mL水萃取,无水硫酸钠干燥,蒸除溶剂,柱色谱分离得化合物I₁～I₄。分别以9、10、11、12为原料,类似方法合成II₁～II₄、III₁～III₄、IV₁～IV₄、V₁～V₄,化合物波谱数据见表 2。 2 乙酰胆碱酯酶活性测定

采用改良的Ellman法^[21]在体外考察化合物I～V对乙酰胆碱酯酶的抑制作用,同时用空白组、多奈哌齐为阳性对照组进行实验。配制磷酸缓冲液PBS的pH为7.2,以碘化硫代乙酰胆碱 (ATCH) 为底物 (浓度为0.01 mol·L^-1),以5,5'-二硫代双 (2-硝基苯甲酸) [5,5'-dithiobis-(2-nitrobenzoic acid),DTNB] 为显色剂 (浓度为0.01 mol·L^-1),乙酰胆碱酯酶 (AChE) 为0.006 U (PBS缓冲液配制),待测样品用DMSO溶解。

分别取40 µL PBS缓冲液、10 µL AChE、20 µL显色剂DTNB、10 µL各浓度的待测化合物于96孔板中,37 ℃孵育10 min,然后再加入20 µL底物ATCH,充分混匀。37 ℃保温15 min后,加入30 µL 1% 十二烷基磺酸钠 (SDS) 终止反应,使用全功能检测仪在412 nm处读取每孔吸光度 (A)。空白组用10 µL PBS缓冲液分别代替底物和待测化合物,标准组用10 µL PBS缓冲液代替待测样品,每个化合物在不同浓度均实验3次,然后以抑制率I[I = 100×(A_测 - A_空) / (A_标 - A_空)%] 对抑制剂浓度C作图,根据抑制曲线求得各种化合物的IC₅₀值。 3 动物避暗实验

实验装置为一条件反射箱,分为明暗两部分,两室中有一小孔洞使小鼠可自由通行,箱底有铜栅,其中暗室底部的铜栅通以36 V电压。实验时将小鼠放入明室并背向暗室,因小鼠有喜暗及钻洞的习性,所以在实验训练期间会多次钻入暗室 (此行为即为错误反应)。当小鼠一走入暗室四足接触铜栅时就会受到电击,它会马上设法退出暗室。第一次训练时,记录小鼠在2 min内进入暗室的次数及第一次进入暗箱的时间 (即为潜伏期)。24 h后再进行记忆检测,观察5分钟内小鼠首次进入暗箱的时间 (即潜伏期latency) 和小鼠进入暗箱的次数 (即错误次数error number)。

雄性昆明种小鼠 (20 ± 2 g),饲养于明暗交替 (12 h/12 h) 清洁级动物房中,自由进食饮水,在动物房中适应环境一周后开始进行实验。采用东莨菪碱所致学习记忆障碍模型,多奈哌齐 (donepezil) 为阳性对照药。将小鼠随机分成对照组 (control)、东莨菪碱模型组 (model)、I₁ (5 mg·kg^-1组、10 mg·kg^-1组)、I₂ (5 mg·kg^-1组、10 mg·kg^-1组)、I₃ (5 mg·kg^-1组、10 mg·kg^-1组)、I₄(5 m g·kg^-1组、10 mg·kg^-1组) 和多奈哌齐组 (donepezil,5 mg·kg^-1)。每组16只小鼠,其中对照组、模型组每天早上固定时间灌胃给予蒸馏水,I₁～I₄组分别按体重给予相应剂量的药物,共给药4天。第3天模型组、I₁～I₄组分别在训练前20 min给予东莨菪碱 (1 mg·kg^-1) 造成记忆障碍模型,然后进行避暗实验训练,24 h后 (即给药第4天) 进行正式测试,分别记录各组各只小鼠潜伏期和错误次数。所有数值均采用均数和标准误差来表示 (Mean ± SEM), 分析方法采用t检验。

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药学学报 2015, Vol. 50 Issue (2): 191-198	PDF