Study on synthesis and inhibitory activities of NR2B ca 2 flux of isoindoline derivatives and study on chemical constituents of marsdenia tenacissima and calotropis gigantea

学校代码：10251
学 号：010110066

.

博士学位论文
题 目：

异吲哚啉衍生物的合成及其抑制 NR2B Ca2+
流量研究和通关藤和牛角瓜的化学成分研究

专 业：
研究方向：

有机化学
天然药物化学

姓 名：

范文康 (PHAM VAN KHANG)

导 师：

胡立宏 教授

定稿时间： 2014 年 01 月 15 日


分类号： O62

密级：

U D C：

华 东 理 工 大 学
学 位 论 文
异吲哚啉衍生物的合成及其抑制 NR2B Ca2+流量
研究和通关藤和牛角瓜的化学成分研究
范文康 (PHAM VAN KHANG)
指导教师姓名：

胡立宏 教授, 华东理工大学药学院
马磊 副教授, 华东理工大学药学院

申请学位级别：

博士

专

论文定稿日期： 2014-01-15

业 名 称：

论文答辩日期：

学位授予单位：

有机化学
2014-04-28

华东理工大学

学位授予日期：

答辩委员会主席：

沈旭

教授

评

李剑

教授

柳红

教授

刘璇

教授

沈旭

教授

段文虎

教授

阅

人：


作者说明
我郑重声明: 本人悟守学术道德, 崇尚严谨学风。所呈交的学位论文, 是本人在导师的指
导下, 独立进行研究工作所取得的结果。除文中明确注明和引用的内容外, 本论文不包
含任何他人已经发表或撰写过的内容。论文为本人亲自撰写, 并对所写内容负责。
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华东理工大学博士学位论文

第I页

Study on synthesis and inhibitory activities of NR2B Ca2+-flux of isoindoline
derivatives, and study on chemical constituents of Marsdenia tenacissima
and Calotropis gigantea
Abstract
The whole thesis consists of four parts: (1) Study on synthesis of isoindolin-1-imine
derivatives via one-pot reaction and their inhibitory activities of NR2B Ca2+-flux; (2) Study

on synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives via one-pot reaction;
(3) Study on C21 steroidal glycosides from the Stems of Marsdenia tenacissima; (4) Study on
the isolation, structural determination and cytotoxicities of cardenolides from the bark of
Calotropis gigantea.
Isoindolin-1-imine derivatives are an important class of isoindole scaffold, which exhibit
typical pharmacological activities as NR2B-selective NMDA (N-Methyl-D-aspartate) receptor
antagonists, and the thrombin receptor (PAR-1) inhibitors. In an effort to explore new method
for synthesis of isoindolin-1-imine derivatives, a novel one-pot method for the synthesis of
isoindolin-1-imine derivatives has been developed via a simple three-component condensation
of 2-cyanobenzaldhyde, ammonium acetate, and 4-hyroxycoumarin derivatives or 1,3dicarbonyl compounds, or 4-hydroxyquinolin-2(1H)-one in ethanol under reflux condition for
20-60 min with excellent yields. Moreover, a new, simple, efficient procedure for the
preparation of 3-(2-substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one analogs
is decribed in this thesis via a three-component condensation of 2-cyanobenzaldehyde,
primary amine, and 4-hydroxycoumarin derivatives in dry dichloromethane without catalyst at
room temperature. The condensation reactions are proceeded smoothly and quickly to afford
products in excellent yields. The inhibitory activities of NR2B Ca2+-flux of synthesized
isoindolin-1-imine analogs have been evaluated via the fluorescence measurement of free
concentrations of intracellular calcium of L(tk-) cells expressing NR1a/NR2B receptors. The
results showed that all tested isoindolin-1-imine derivatives exhibited potent inhibitory
activity of Ca2+flux in cells.
An efficient one-pot procedure for the synthesis of 2-substituted-3-(2oxoalkyl)isoindolin-1-one analogs has been developed from phthalaldehydic acid, primary
amine, and ketone in aqueous solution under reflux condition in the presence of VB1. Various
substrates can be applied to this procedure with operational simplicity, good yields, short
reaction time, and environmental friendly conditions.
Marsdenia tenacissima (Roxb.) Wight et Arn., is known as a famous traditional Chinese
medicine, which is widely used in the treatment of cancer, and other diseases. From ethanolic
extract of stem of Marsdenia tenacissima, 20 compounds have been isolated, including 3 new
compounds and 17 known ones, and their structures have been elucidated via NMR
spectroscopic identification and LC-MS analysis.
Calotropis species are known as a source of biological active substances, in particular it

第 II 页

华东理工大学博士学位论文

is one of a good source of cardenolides. From the 90% ethanolic extract of the bark of
Calotropis gigantea (C. gigantea), three new cardenolides and eleven known ones have been
isolated, and their structures have been elucidated via NMR spectroscopic identification and
LC-MS analysis. The inhibitory activities of all isolated compounds have been evaluated
against non-small cell lung carcinoma (A549) and human cervix epithelial adenocarcinoma
cell line (HeLa), and several cardenolides exhibit strong potent cytotoxicities.
Keywords: Isoindolin-1-imine;
gigantea; cardenolides.

isoindolin-1-one; Marsdenia tenacissima; Calotropis


华东理工大学博士学位论文

第 III 页

异吲哚啉衍生物的合成及其抑制 NR2B Ca2+流量研究和
通关藤和牛角瓜的化学成分研究
摘要
本论文内容包括四个部分：（1）异吲哚啉-1-亚胺衍生物的多组分合成及其抑制
NR2B Ca2+流量抑制研究；（2）异吲哚啉-1-酮衍生物的多组分合成方法学研究；（3）通
关藤茎中 C21 甾体皂苷的化学成分研究；（4）牛角瓜化学成分及其细胞毒活性研究。
异吲哚啉-1-亚胺衍似物是一类非常重要的异吲哚类化合物，它们具有多种生物活
性，如拮抗 N-甲基-D-天冬氨酸（NMDA）受体, 抑制凝血酶受体(PAR-1)和抗增值等作

用。为了开发异吲哚啉-1-亚胺衍似物的新合成方法，通过探索研究，我们发现了以邻
醛基氰基取代的苯环与相应的亲核试剂发生缩合反应，可高效, 简便, 绿色构建异吲哚
啉-1-亚胺衍生物类骨架。首先探索了 2-氰基苯甲醛, 乙酸铵，与 4-香豆素, 或 1,3-二羰
基化合物, 或 2,4-二羟基喹啉等亲核试剂构建异吲哚啉-1-亚胺骨架的多组分合成反应的
最佳反应条件和底物适用性。其次，发现以烷基伯胺替代乙酸铵，该反应在二氯甲烷,
室温条件下以高产率得到 2-取代-3-烷氧基异吲哚啉-1-亚胺-香豆素类衍生物。最后，对
所合成的 2-（4-羟基香豆素取代）异吲哚啉-1-亚胺衍似物进行了抑制 NR2B Ca2+流量
活性评价。结果显示大多数 2-（4-羟基香豆素取代）异吲哚啉-1-亚胺衍似物是对细胞
钙外流的都具有强的抑制作用。
此外，我们还开发了一种简单，有效的三组分法反应，三组分苯醛酸, 伯胺, 酮，
在 VB1 催化下，在水中回流以高产率构建 2-取代-3-(2-氧代烷基) 异吲哚啉-1-酮衍生物
类似物。该反应底物适应性广, 操作简单, 产率高, 反应时间短, 绿色环保。
通关藤是一种常用的抗肿瘤中药，广泛生长在中国的西南部以及热带地区。我们
从通关藤的茎中分离鉴定了 20 个化合物，其中包括 3 个新化合物。
牛角瓜是一种萝藦科植物，文献报道其主要化学成分是强心苷类。我们从牛角瓜
皮的 90%乙醇提取物中分离得到 3 个新的强心苷，11 个已知强心苷。 化合物的结构通
过 NMR 及 LC-MS 得到鉴定。部分化合物显示强的细胞毒活性。

关键词 异吲哚林-1-亚胺； 异吲哚林；通关藤；牛角瓜；强心苷.


第 IV 页

华东理工大学博士学位论文

Table of contents
Chapter 1. Study on synthesis of isoindolin-1-imine derivatives via one-pot reaction and their
inhibitory activities of NR2B Ca2+-flux........................................................................................... 1
1.1 Introduction ...................................................................................................................... 1
1.2 Design for synthesis of isoindolin-1-imine derivatives ...................................................... 3

1.3 Experiment ....................................................................................................................... 3
1.3.1 Synthesis of 3-substituted isoindolin-1-imine derivatives ............................................. 3
1.3.1.1 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)................ 3
1.3.1.2 The optimization of reaction conditions .................................................................. 4
1.3.1.3 The scope and limitations of reaction substrates ..................................................... 5
1.3.1.4 The structural determination ................................................................................... 9
1.3.1.5 The plausible mechanism for synthesis of isoindolin-1-imine ............................... 10
1.3.2 Synthesis of 2,3-disubstituted isoindolin-1-imine derivatives ....................................... 10
1.3.2.1 The optimization of reaction conditions ................................................................ 11
1.3.2.2 The scopes and limitations of reaction substrates .................................................. 12
1.3.2.3 The plausible mechanism for synthesis of isoindolin-1-imines 11.............................. 13
1.3.3 Activity of isoindolin-1-imine derivatives .................................................................. 14
1.3.3.1 Introduction.......................................................................................................... 14
1.3.3.2 Effect of isoindolin-1-imine derivatives as NR2B Ca2+ flux inhibitor ................... 14
1.3.3.2.1 Effect of products 4 .................................................................................. 14
1.3.3.2.2 Effect of products 11 ................................................................................. 17
1.3.4 Preparation section .................................................................................................... 18
1.3.4.1 Preparation of isoindolin-1-imines 4, 8, and 10..................................................... 18
1.3.4.1.1 Preparation of products 4 .......................................................................... 18
1.3.4.1.2 Preparation of products 8 .......................................................................... 22
1.3.4.1.3 Preparation of products 10 ........................................................................ 22
1.3.4.2 Preparation of products 11 ...................................................................................... 23
1.3.4.3 Preparation of Calcium flux functional assay ........................................................... 28
1.4 Conclusions .................................................................................................................... 29
Chapter 2. Study on synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-one derivatives via
one-pot reaction.............................................................................................................................. 30
2.1 Introduction .................................................................................................................... 30
2.2 Results and Discussion ................................................................................................... 32
2.2.1 The optimization of reaction catalyst ......................................................................... 32
2.2.2 The optimization of reaction solvent .......................................................................... 33

2.2.3 The scope and limitations of reaction substrates ......................................................... 34
2.3 The plausible mechanism ................................................................................................ 35
2.4 Conclusions .................................................................................................................... 35
2.5 Experiment section ......................................................................................................... 36


华东理工大学博士学位论文

第V页

Chapter 3. Study on C21 steroidal glycosides from the stems of Marsdenia tenacissima ........... 38
3.1 Introduction .................................................................................................................... 38
3.1.1 Chemical compositions of M. tenacissima ................................................................. 38
3.1.2 Biological activities of of M. tenacissima................................................................... 43
3.1.2.1 Biological activities of the total extract of M. tenacissima......................................... 43
3.1.2.2 Biological activities of M. tenacissima steroids .................................................... 45
3.2 Results and discussion .................................................................................................... 46
3.2.1 New isolated compounds ........................................................................................... 47
3.2.1.1 Compound 10 ....................................................................................................... 47
3.2.1.2 Compound 13 ....................................................................................................... 48
3.2.1.3 Compound 14 ....................................................................................................... 50
3.2.2 Known compounds .................................................................................................... 52
3.3 Conclusions .................................................................................................................... 58
3.4 Experimental section....................................................................................................... 58
3.4.1 General experimental procedures ............................................................................... 58
3.4.2 Extraction and isolation ............................................................................................. 58
3.4.3 Hydrolysis of compounds ............................................................................................. 59
Chapter 4. Study on the isolation, structural determination, and cytotoxicities of cardenolides
from the bark of Calotropis gigantea (Linb.) ................................................................................ 60
4.1 Introduction ..................................................................................................................... 60

4.2 Chemical components ..................................................................................................... 61
4.3 Pharmacological activities of extracts and isolated components ...................................... 63
4.4 Aims of study.................................................................................................................. 66
4.5 Results and discussion .................................................................................................... 67
4.5.1 Structural determination of isolated compounds.............................................................. 67
4.5.1.1 New compounds .................................................................................................... 68
4.5.1.1.1 Compound 1 ............................................................................................. 68
4.5.1.1.2 Compounds 11 and 12 ................................................................................ 69
4.5.1.2 Known compounds............................................................................................... 70
4.5.2 Inhibitory activity against cancer cell lines................................................................. 73
4.6 Conclusions .................................................................................................................... 74
4.7 Experimental .................................................................................................................. 74
4.7.1 General experimental procedures ............................................................................... 74
4.7.2 Plant material ............................................................................................................ 75
4.7.3 Extraction and isolation ............................................................................................. 75
4.7.4 Hydrolysis of compounds 11 and 12 .............................................................................. 76
4.7.5 Cytotoxicity assays .................................................................................................... 76
Chapter 5. Summary ..................................................................................................................... 77
References....................................................................................................................................... 79
Appendix 1...................................................................................................................................... 91


第 VI 页

华东理工大学博士学位论文

Appendix 2...................................................................................................................................... 94
Appendix 3.................................................................................................................................... 104
Publications .................................................................................................................................. 106
致谢 ............................................................................................................................................... 107

华东理工大学博士学位论文

第1页

Chapter 1. Study on synthesis of isoindolin-1-imine
derivatives via one-pot reaction and their inhibitory activities
of NR2B Ca2+-flux
1.1 Introduction
Isoindole derivatives are one of the most important classes of N-heterocyclic biological active
compounds [1-7]. They have been received considerable attention from synthetic pharmacists
and chemists due to their potent therapeutic and pharmacological activities [1, 3-7]. Isoindolin-1imine series have exhibited typical pharmacological activities, such as NR2B-selective
NMDA (N-Methyl-D-aspartate) receptor antagonists [4], the thrombin receptor (PAR-1)
inhibitors [5-6], and antiproliferative effect [7].

Fig. 1.1 Several ioindolin-1-imine analogues [4]

Since the first multicomponent reaction (MCR) was accomplished in 1850 by Strecker[8],
MCRs show especial significance to the organic synthesis due to their operational simplicity,
high productivity, short reaction time, and high yield without isolating the intermediates from
simple and popular starting materials [9-12]. Up to date, many well-known MCRs such as
Alkynes trimerisation, Kabachnik–Fields reaction, Biginelli reaction, Asinger reaction,
Mannich reaction, Passerini reaction, and Ugi reaction have been developed [12-14]. The
important aspects of MCRs is widely recognized and applied as a powerful tool for the
general synthesis of important biologically active compounds, particularly the synthesis of Nheterocyclic compounds [12].
Up to date, several methods for the formation of isoindolin-1-imine structures have been


第2页

华东理工大学博士学位论文

reported, but most of methods include some kind of drawbacks such like multi-step strategies,
required tough reaction conditions, prolonged reaction time, low yields, and so on [4-6, 9-11].

Scheme 1.1 Synthesis of isoindolin-1-imine derivatives 1-4 [4]

Recently, our research group reported new procedures for 2,3-disubstituted isoindolin-1imines via a three-component reaction of 2-cyanobenzaldehyde, primary amine, and alcohol
in the presence of acetic acid [15] (5, Scheme 1.2) or 3-methyl-1H-pyrazol-5(4H)-one [16] (6,
Scheme 1.3) with good yields and wide scope. Dueing to the reactive activity of the adjacent
formyl and cyano groups of 2-cyanobenzaldehyde, cascade condesation reactions can be
carried out with different nucleophilic reagents via Knoevenagel condensation and Michael
addition. For further development, we turned our attention towards other substrates

Scheme 1.2 Synthesis of products 5 [15]

Scheme 1.3 Synthesis of products 6 [16]

4-Hydroxycoumarin scaffolds are abundant in natural products [17], and offen exhibit
remarkable pharmacological properties [18-29]. In addition, due to the active methylene group,
4-hydroxycoumarin is served as starting scaffold for the synthesis of many different structures
with various functional groups [20-28]. In an effort to exploit the potential of 4hydroxycoumarin for the synthesis of isoindolin-1-imine analogues, we considered it
worthwhile to investigate its multi-component condensations with 2-cyanobenzaldehyde and
an amine.


华东理工大学博士学位论文

第3页

Fig. 1.2 The biologically active 4-hydroxycoumarin derivatives

1.2 Design for synthesis of isoindolin-1-imine derivatives
According to the summary of the existing literatures, the formation of isoindolin-1-imine
skeleton most needed benzonitrile as a substrate fragment, and the reaction of a halogen
substituent compound combined with a primary amine to give the target product. Otherwise,
benzaldehyde could react with a primary amine to give unstable imines, and could be formed
isoindolin-1-imine structure by cyclization. In our previous work, isoindolin-1-imine
skeletons can be afforded via condensation from 2-cyanobenzaldehyde, primary amine, and
alcohol [15] or 3-methyl-1H-pyrazol-5(4H)-one [16] in high yields. Based on the previous results,
we choose 2-cyanobenzaldehyde, ammonium acetate or primary amine, and 4hydroxycoumarin derivatives as reaction substrates to attempt whether isoindolin-1-imine
structure can be constructed.
Initially, we screened different solvents for condensation reaction of 2cyanobenzaldehyde and 4-hydroxycoumarin with ammonium acetate or benzyl amine.
Notably, when ethanol was selected as a reaction solvent, these reactions yielded new
structures as 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a, Scheme 1.4)
under reflux condition, and 3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4one (11a, Scheme 1.9) without catalyst under room temperature. Thus, we decided to study
the optimization of reaction conditions, the scope for further systematic study and accurately
identify their structures.

1.3 Experiment
1.3.1 Synthesis of 3-substituted isoindolin-1-imine derivatives
1.3.1.1 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)
Based on our previous results [15-16], we continued to explore the three-component
condensation reaction of 2-cyanobenzaldehyde 1a (2 mmol), ammonium acetate 2 (2 mmol),
4-hydroxycoumarin (4-hydroxy-2H-chromen-2-one) 3a (2 mmol) in dry ethanol under reflux
for 20 minutes (Scheme 1.4). The condensation reaction was carried out smoothly, quickly,
and a white precipitate was afforded. When the reaction completed, the white solid was
filtered and washed with acetone to give 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen4-one 4a in excellent yield (95%). Structure of 4a was determined by using NMR data and
LC-MS.

华东理工大学博士学位论文

第4页
CHO

O
+

NH4OAc

OH

Ethanol

+

CN
1a

O

O
Reflux

O

3aOH

2

HN

4a

NH

Scheme 1.4 Synthesis of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)

1.3.1.2 The optimization of reaction conditions
Initially, the influence of different solvents on this condensation was investigated. The results
summarized in Table 1.1 showed that the target product 4a was afforded in low to moderate
yields in DCM (entries 1, 2), toluen (entry 3), DCE (entry 4), CH3CN (entry 5), THF (entry 6),
and H2O (entries 11-13), and good yield in MeOH (entry 10). Particularly, product 4a was
obtained in excellent yield in EtOH under reflux condition (entry 8), but the product 4a was
only given in about 30% yield under room temperature (entry 7). Thus, dry ethanol was
selected as an ideal solution for this three-component condensation. To further investigate the
influence of reaction time in yield, the multi-component reaction was carried out in EtOH
under reflux condition with different times. When the reaction was proceeded for 120 minutes,
the product was obtained in 96% yield (entry 9). Consequently, the optimized reaction
condition was obtained in ethanol solution under reflux for 20 minutes without any catalyst,
and this condition was chosen to further synthesis of isoindolin-1-imine derivatives.
Table 1.1 Solvent screening for the synthesis of 4a

Entry
1
2
3
4

5
6
7
8
9
10
11
12
13

Solvent
DCM
DCM
Toluene
DCE
CH3CN
THF
EtOH
EtOH
EtOH
MeOH
H2O
H2O
H2O

Time (min)
20
120
20
20

20
20
20
20
120
20
20
20
120

Yield 4ab(%)
33
46
30
35
47
32
30d
95
96
88
15d
60
65

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), ammonium acetate (2 mmol),
4-hydroxycoumarin 3a (2 mmol), solvent (4 mL), reflux. bIsolated yields. d Room temperature.

To further demonstration the scope of this condensation reaction, we continued to screen
the significant effects of reagent 2 to this three-component reaction on the yields (Table 1.2).

When reagent 2 was ammonium salts of strong acids, such as HCl and H2SO4 (Table 1.2, entries 5,
6), only a trace amount of compound 4a was observed in reaction solution. When NH3,


华东理工大学博士学位论文

第5页

NH3/AcOH (1/1, eq/eq) or NH4OAc was used as reagent 2 under similar conditions (Table 1.2,
entries 1-4, 7-11), compound 4a was obtained in moderate to excellent isolated yields (40-95%).
When ammonia solution or ammonia/AcOH solution was used, the yield of this condensation
reached 78% (Table 1.2, entries 10, 11), whereas ammonium acetate 2 was the most effective
reagent in term of yields of product 4a (Table 1.2, entries 1-4). When the amount of ammonium
acetate increased from 1.0 (eq.) to 2.0 (eq.), no significant impact had on the overall yields of
product 4a. Therefore, ammonium acetate was chosen as the ideal reagent 2 for this condensation.
Table 1.2 The effect of reagent 2 for the synthesis of 4a

Entry
1
2
3
4
5
6
7
8
9
10
11

Reagent 2
NH4OAc
NH4OAc
NH4OAc
NH4OAc
NH4Cl
(NH4)2SO4
NH3
NH3/AcOH (1/1, eq/eq)
NH3/AcOH (1/1, eq/eq)
NH3/AcOH (1/1, eq/eq)
NH3/AcOH (1/1, eq/eq)

Ratio (eq.)
1.0
1.2
1.5
2.0
1.0
1.0
1.0
1.0
1.2
1.5
2.0

Yield 4ab(%)
95
95
94

96
Trace
Trace
40
75
77
78
78

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), 4-hydroxycoumarin 3a (2 mmol),
solvent (4 mL), reflux, 20 minutes. bIsolated yields.

1.3.1.3 The scope and limitations of reaction substrates
To demonstrate the scope and limitations of this reaction, some series of isoindolin-1-imine
derivatives 4, 8, and 10 were synthesized from different starting materials 1 and 3 (Table 1.31.5, Scheme 1.5-1.7).

Scheme 1.5 Synthesis of 3-substituted isoindolin-1-imine derivatives 4

Firstly, a series of 2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one derivatives 4
were prepared via condensations of 2-cyanobenzaldehyde derivatives 1, ammonium acetate 2
and 4-hydroxycoumarin derivatives 3 in dry ethanol in excellent yields (Table 1.3). 4Hydroxycoumarin 3 carrying different substituent groups on the aromatic ring had no
significant impact on the overall yields of the corresponding products 4 (entries 1-8). To
further investigate the effects of substituted 2-cyanobenzaldehydes for this condensation, the
reactions of 2-cyanobenzaldehydes bearing either electron-donating groups such as methoxy


华东理工大学博士学位论文

第6页

or n-propoxy group (entries 9-14) or electron-withdrawing groups such as nitro group with
ammonium acetate 2 and 4-hydroxycoumarin derivatives 3 were also investigated. The results
showed that the reaction of 2-cyano-4-propoxy-5-methoxybenzaldehyde (entries 9-14) gave
the corresponding products 4 in excellent yields, but 2-cyano-4-nitrobenzaldehyde (entry 15)
or 2-cyano-5-nitrobenzaldehyde (entry 16) did not give the corresponding product under
similar reaction conditions.
Table 1.3 Synthesis of isoindole-1-imine analogs products 4

Reagent 1
Entry
1

1

2

R

R

H

H

Reagent 3

Product 4

Yieldb (%)

4a

95

4b

95

4c

94

4d

92

4e

90

4f

91

4g

90

3a
2

H

H

3b
3

H

H

3c
4

H

H

3d
5

H

H

3e
6

H

H

3f
7

H

H

3g


华东理工大学博士学位论文

第7页

Reagent 1
Entry
8

1

2

R

R

H

H

Reagent 3

Product 4

Yieldb (%)

4h

89

4i

93

4k

94

4l

92

4m

91

4n

93

4o

92

4p

Trace

3h
9

Methoxy

n-Propoxy

3a
10

Methoxy

n-Propoxy

3b
11

Methoxy

n-Propoxy

3d
12

Methoxy

n-Propoxy

3e
13

Methoxy

n-Propoxy

3g
14

Methoxy

n-Propoxy

3h
15

H

Nitro

3a


华东理工大学博士学位论文

第8页
Reagent 1
Entry
16

1

Reagent 3

2

R

R

Nitro

H

Product 4

Yieldb (%)

4q

Trace

3a
Conditions: 1 (2 mmol), ammonium acetate 2 (2 mmol), 3 (2 mmol), solvent (4 mL), reflux, 20 minutes.
b
Isolated yields.

In continuing to demonstrate the scope and limitations of this reaction, the condensation
of 2-cyanobenzaldehyde 1, ammonium acetate 2, and 1,3-dicarbonyl compounds 7 were
carried out (Scheme 1.6). In this case, the condensation could also be accomplished quickly
and smoothly to give formation of products 8 with lower yields and longer reaction time as
described in Table 1.4.

Scheme 1.6 Synthesis of compounds 8
Table 1.4 Synthesis of products 8

Product 8
8a

8b

Reagent 7

Yieldb (%)
90

83

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), ammonium acetate 2 (2 mmol), 4 (2 mmol), solvent (4 mL),
reflux, 60 minutes. b Isolated yields.

On the other hand, this three-component condensation was also extended to other
nucleophilic reagents such as 4-hydroxyquinolin-2(1H)-one derivative, which exhibited useful
biological benefits [33-37]. In previous reports, we showed that 4-hydroxyquinolin-2(1H)-one
was one of the common starting substances in the synthetic medicinal chemistry [34-35]. Thus,
the three-component condensation reactions of 2-cyanobenzaldehyde 1, ammonium acetate 2,
and 4-hydroxyquinolin-2(1H)-one derivatives 9 were also explored (Scheme 1.7).
Interestingly, the condensation of 4-hydroxyquinolin-2(1H)-one derivatives 9 with 1 and 2
gave the corresponding 10 quickly and smoothly in high yields (entries 1-3, Table 1.5).


华东理工大学博士学位论文

第9页

However, 2-cyano-4-nitrobenzaldehyde (entry 4, Table 1.5) only gave a trace amount of
product 10 under similar reaction conditions.

Scheme 1.7 Synthesis of compounds 10
Table 1.5 Synthesis of products 10

Reagent 1
Entry
1

1

2

R

R

H

H

Reagent 9

Product 10

Yieldb (%)

10a

85

10b

85

10c

84

10d

Trace

9a

2

H

H

9b
3

Methoxy

n-Propoxy

9c
4

H

Nitro

9a
Conditions: 2-cyanobenzaldehyde 1 (1 mmol), ammonium acetate 2 (1 mmol), 9 (1 mmol), solvent (4 mL),
reflux, 20 minutes. b Isolated yields.

1.3.1.4 The structural determination
The structures of isoindolin-1-imine derivatives 4a-4o, 8a-8b, and 10a-10c were determined by
NMR spectral data and LC-MS data, as illustrated by the representative example 4b (Figure
1.3). In its 1H NMR spectrum in DMSO-d6, a signal of H-3 proton occurred as a singlet at δ =
6.50 that showed HMBC correlation with C-1 (δ =163.0), C-3 (δ = 89.5), C-2’ (δ = 162.8),

第 10 页

华东理工大学博士学位论文

and C-4’ (δ = 173.8), respectively. H-2 proton (s, δ = 10.39) also had correlation with C-1 (δ =
163.0) and C-3 (δ = 60.2). H-5’ proton showed important HMBC correlations with C-4’
(Figure 1.4).

Fig. 1.3 Important 1H and 13C NMR assignments for compound 4b

Fig. 1.4 Key HMBC correlations for compound 4b

1.3.1.5 The plausible mechanism for synthesis of isoindolin-1-imine
Thus, a plausible mechanism can reasonably be proposed for the series of products 4 and 10
(Scheme 1.8) [15-16]. In the initial step, 2-cyanobenzaldehyde 1 reacted with ammonia by
decomposition of ammonium acetate under reflux condition to give adduct 5. Then the adduct
5 gave unstable imine intermediate 6 by protonation and dehydration under acidic condition.
Subsequent nucleophilic addition of 3 to 6 afforded 7, which afforded the title isoindolin-1imine 4 or 10 via intramolecular cyclization.

Scheme1.8 Possible mechanism for the formation of target products

1.3.2 Synthesis of 2,3-disubstituted isoindolin-1-imine derivatives
As part of a continuing effort in our laboratory toward the synthesis of isoindolin-1-imine
derivatives, we herein describe a novel one-pot procedure for the synthesis of 3-(2substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one derivatives 11 via cascade
three-component condensation. This reaction was carried out by condensation of 2-


华东理工大学博士学位论文

第 11 页

cyanobenzaldehyde 1, primary amine 2 (NH4OAc replaced by RNH2), and 4-hydrocoumarin
derivatives 3 with catalyst-free under room temperature in dry dichloromethane with excellent
yields, fast reactive time, and wide scope (Scheme 1.9).

Scheme 1.9 Synthesis of products 11

1.3.2.1 The optimization of reaction conditions
Due to our previously results [15-16], we continued to explore a three-component condensation
reaction of 2-cyanobenzaldehyde 1 (2 mmol), benzylamine 2a (2 mmol), 4-hydroxy-2Hchromen-2-one 3a (2 mmol) (Scheme 1.9). It noted that the reaction processed smoothly in
dry dichloromethane at room temperature for 5 minutes and obtained a new structure white
solid product 11a, its structure was determined by NMR and LC-MS data.
To investigate the influence of solvents, the condensation reaction was carried out in
different solvents. The results were summarized in Table 1.6.
Table 1.6 Solvent screening for the synthesis of 11a

Entry
1
2
3
4
5
6
7
8
9
10
11
12

13

Solvent
DCM
DCM
Benzene
DCE
CH3CN
THF
EtOH
EtOH
EtOH
MeOH
H2O
H2O
H2O

Time (min)
5
30
5
5
5
5
5
180
180
5
5
180

180

Yield 11ab (%)
98
98
50
85
50
55
65
70
90c
60
10
30
80c


华东理工大学博士学位论文

第 12 页
a

Conditions: 2-cyanobenzaldehyde 1 (2 mmol), benzyl amine 2a (2 mmol), 4-hydroxycoumarin 3a (2
mmol), solvent (5 mL), room temperature.
b
Isolated yields. c Conditions: Reflux.

The results showed that the product 11a was afforded with excellent yield in DCM for 5
and 30 min (entries 1, 2). The multi-component reaction was also carried out in Benzene

(entry 3), DCE (entry 4), CH3CN (entry 5), and THF (entry 6), MeOH (entry 10) at room
temperature to yield 11a in low to good yields. Then, this reaction was also carried out in
ethanol and in water for 5 and 180 minutes under room temperature and reflux condition. The
yields of 11a in ethanol were 65%, 70%, and 90% (entries 7-9), and in water were 10%, 30%,
and 80% (entries 11-13), respectively. Thus, the optimized reaction condition was in dry
dichloromethane under room temperature with catalyst-free for 5 minutes. This condition was
chosen for further synthesis of isoindolin-1-imine derivatives.
1.3.2.2 The scopes and limitations of reaction substrates
Table 1.7 Synthesis of isoindole-1-imine analogs products 11a

Entry

Product 11

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

17
18
19
20
21
22
23

11a
11b
11c
11d
11e
11f
11g
11h
11i
11k
11l
11m
11n
11o
11p
11q
11r
11s
11t
11u
11v
11w

11x

R1
Benzyl
4-ClC6H4CH2
4-BrC6H4CH2
4-FC6H4CH2
4-MeOC6H4CH2
3,4-(MeO)2C6H3(CH2)2
n-Propyl
n-Butyl
i-Butyl
Cyclopropylmethyl
Cyclohexyl
Ethanol-2-yl
Propan-1-ol-3-yl
3-Methylbutan-1-ol-2-yl
N,N-Dimethylaminoethyl
2-(1H-Indol-3-yl)ethyl
Benzyl
4-ClC6H4CH2
4-MeOC6H4CH2
3,4-(MeO)2C6H3(CH2)2
n-Butyl
Ethanol-2-yl
Benzyl

Reagent 3

Yieldb (%)

3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3b
3b
3b
3b
3b
3b
3c

98
98
97
95
95

93
97
95
94
97
91
94
92
92
94
93
98
97
96
97
95
91
92c


华东理工大学博士学位论文
24

11y

第 13 页

n-Butyl

90c

3c

a

Conditions: 2-cyanobenzaldehyde 1a (2 mmol), amine 2 (2 mmol), 3 (2 mmol), solvent (5 mL), room
temperature, 5 minutes. b Isolated yields.
c
Conditions: 2-cyanobenzaldehyde 1a (1 mmol), amine 2 (1 mmol), 3c (1 mmol), solvent (5 mL), room
temperature for 30 minutes.

To further demonstrate the scope and limitations of this procedure, we prepared the
condensation reaction of 2-cyanobenzaldehyde 1, alkyl amine 2, and 4-hydroxycoumarin (or
4-hydroxy-2H-chromen-2-one) 3a or 4-hydroxy-6-methylcoumarin (or 4-hydroxy-6-methyl2H-chromen-2-one) 3b or 4-hydroxy-2H-benzo[h]chromen-2-one 3c under room temperature
in dry dichloromethane for 5 minutes for synthesis of the series of isoindolin-1-imines 11. The
structure of compound 11 was determined by using NMR and LC-MS data analysis. The
results were summarized in Table 1.7. The reaction scope and limitations were explored
according to amine and 4-hydroxycoumarin derivatives.
As shown in the Table 1.7, we showed that the electronic effect from the substrate had no
significant impact on the overall yields of the products 11. For example, arylalkylamines
carrying either electron-withdrawing or electron-donating substituents (entries 2-6, 16; entries
17-20, 23) reacted quickly to afford the desired products in excellent yields. Alkyl amines
such as n-propyl (entry 7), n-butyl (entries 8, 21, 24), i-butyl (entry 9), N,Ndimethylaminoethyl (entry 15), cyclopropylmethyl (entry 10), cyclohexyl (entry 11), and
hydroxyalkyl amines as ethanol-2-yl (entries 12, 22), propan-1-ol-3-yl (entry 13) or 3methylbutan-1-ol-2-yl (entry 14) could also react quickly and smoothly to give formation of
products 4 in excellent yields.
1.3.2.3 The plausible mechanism for synthesis of isoindolin-1-imines 11
A plausible mechanism can reasonably be proposed for the series of products 11 (Scheme 1.10)
[15-16]
. In the initial step, 2-cyanobenzaldehyde 1 reacted with amine to give adduct 5. Then the
adduct 5 gave unstable imine intermediate 6 by protonation and dehydration. Subsequent

nucleophilic addition of 3 to 6 afforded 7, which afforded the title isoindolin-1-imine 11 via
intramolecular cyclization.
OH
O

OH
NH 2 R, 2
N

1
O

O
7

R

NHR

5

-H2 O

N

6

O

O

NH

O
N

NR

11

O

O

N

OH

N
R

NH

Scheme 1.10 Possible mechanism for the formation of compounds 11

3


第 14 页

华东理工大学博士学位论文

1.3.3 Activity of isoindolin-1-imine derivatives
1.3.3.1 Introduction
N-methyl-D-aspartate receptors (NMDARs) are well known as ligandgated cation-selective
channels that are highly expressed in the central nervous system crucial to brain functions
such as circuit development, learning, and memory [38-42]. NMDARs containing different NR2
(A, B, C or D) subunits have different pharmacological and kinetic properties [40-42].
NMDARs are unusual ligandgated ion channels because their activation requires the relief of
Mg2+ block by membrane depolarization and the concomitant binding of two agonists: glycine
and L-glutamate. The opening of NMDARs might lead to an influx of cations, including Ca2+,
which initiates the signal transduction cascade [38-42].
In order to development of NR2B subtype selective NMDA antagonist drugs, many
NMDA receptor antagonists such as ifenprodil, besonprodil CI-104, Ro-25-6981, traxoprodil,
and carbamates, 4-substituted-3-phenylquinolin-2(1H)-ones, have been reported [4-6, 42-49].
Particularly, ioindolin-1-imines exhibited strong inhibition activities of NR2B Ca2+-flux [4].
1.3.3.2 Effect of isoindolin-1-imine derivatives as NR2B Ca2+ flux inhibitor
The inhibitory activities of isoindolin-1-imines 4 and 11 were evaluated against NR2B Ca2+flux as compared to ifenprodil as a positive control. This assay is depending on the
fluorescence measurement of free concentrations of intracellular calcium of L(tk-) cells
expressing NR1a/NR2B receptor by fluorometric imaging plate reader (FLIPR) method. From
obtained results, several structure activities relationships were discussed as follow.
1.3.3.2.1 Effect of products 4
The inhibitory effects of NR2B Ca2+-flux by compounds 4 are summarized in the Table 1.8.
According to the assay results, we showed that all the tested compounds 4a-4n exhibited
inhibitory activities against NR2B calcium ion-flux with IC50 values range from 97.2±10.1 to
1925.3±42.5 nM, but lower than that of ifenprodil (IC50 = 75.3±7.3 nM).
Compounds 4a-h with R1=R2=H (entries 1-8) displayed less potent activities compared
to compounds 4i-n (entries 9-13) with R1=methoxyl and R2=n-propoxyl. These results
suggested that the introduction of the 5- and 6-alkoxyl group into the aromatic ring was
beneficial to inhibitory activity.

Compound 4d (IC50 = 250.3±20.1 nM) and 4l (IC50 = 97.2±10.1 nM) with methoxyl
substituent on aromatic ring of coumarin moiety showed better inhibitory activities than
methyl, tert-butyl, and iso-propyl substituent compounds. Otherwise, the presence of methyl
at position 6’ (4b, entry 2) or 7’ (4c, entry 3) of the aromatic ring of coumarin moiety had no
obvious contribution to activities.
Replacing the methyl group by tert-butyl (4e, entry 5), iso-propyl (4f, entry 6) did not
lead to significant effect on the inhibitory activities. All alkyl substituent groups on coumarin
moiety derivatives had one to two-fold potent activities reduction compared to unsubstituted
compounds (4a, entry 1; 4i, entry 10). These activity results showed that the presence of the


华东理工大学博士学位论文

第 15 页

alkyl group at aromatic ring of coumarin is critical.
Among the compounds above, compound 4l exhibited the most effective inhibition
activity, and could be a hit compound for further SAR study as a potential NR2B calcium ion
flux antagonists.
Table 1.8 Inhibition of NR2B Ca2+-flux by isoindolin-1-imine analogs
OH
1

R

CHO
+

R2

NH4OAc

CN
1

Entry
Positive
control
1

Ethanol

+
O

O

H

R2

Reflux

R3
7'

8'

Reagent 3

H

R1

1'
8a'

O

2'

4a'

6'

OH 4
3

5

3a

6

3'

4'

5'

HN

O

3

2

R1

R3

4

7a 7

2
1

NH

Product 4

NR2B Ca2+-flux
IC50 (nM)

Ifenprodil

75.3±7.3

4a

432.5±21.3

4b

812.6±23.5

4c

823.1±25.8

4d

250.3±20.1

4e

710.6±25.5

4f

807.9±28.5

3a
2

H

H

3b
3

H

H

3c
4

H

H

3d
5

H

H

3e
6

H

H

3f

R2