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

Study on synthesis and inhibitory activities of NR2B Ca2+-flux of isoindoline derivatives, and study on chemical constituents of Marsdenia tenacissima and Calotropis gigantea of 2-cyan

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Study on synthesis and inhibitory activities of NR2B Ca2+-flux of isoindoline

derivatives, and study on chemical constituents of Marsdenia tenacissima

and Calotropis gigantea

of 2-cyanobenzaldhyde, ammonium acetate, and 4-hyroxycoumarin derivatives or

1,3-dicarbonyl 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

2-substituted-3-(2-oxoalkyl)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

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; isoindolin-1-one; Marsdenia tenacissima; Calotropis

gigantea; cardenolides

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，

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

Table of contents

Chapter 1 Study on synthesis of isoindolin-1-imine derivatives via one-pot reaction and their

inhibitory activities of NR2B Ca 2+ -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

Chapter 3 Study on C 21 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 ofCalotropis 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

Appendix 2 94

Appendix 3 104

Publications 106

致谢 107

Chapter 1 Study on synthesis of isoindolin-1-imine

derivatives via one-pot reaction and their inhibitory activities

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-1-imine 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

N-heterocyclic compounds [12]

Up to date, several methods for the formation of isoindolin-1-imine structures have been

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 imines via a three-component reaction of 2-cyanobenzaldehyde, primary amine, and alcohol

isoindolin-1-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 4-hydroxycoumarin for the synthesis of isoindolin-1-imine analogues, we considered it worthwhile to investigate its multi-component condensations with 2-cyanobenzaldehyde and

an amine

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 hydroxycoumarin derivatives as reaction substrates to attempt whether isoindolin-1-imine structure can be constructed

4-Initially, we screened different solvents for condensation reaction of cyanobenzaldehyde and 4-hydroxycoumarin with ammonium acetate or benzyl amine Notably, when ethanol was selected as a reaction solvent, these reactions yielded new

2-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-4-one (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-chromen-4-one 4a in excellent yield (95%) Structure of 4a was determined by using NMR data and

LC-MS

Ethanol Reflux

NH 4 OAc

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

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,

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

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

1.3-1.5, 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) 4- Hydroxycoumarin 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

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

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

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)

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

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),

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 1 H and 13 C 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 imine 4 or 10 via intramolecular cyclization

isoindolin-1-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-(2-

substituted-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-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-2H-chromen-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

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 11 a

24 11y n-Butyl 3c 90c

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 2H-chromen-2-one) 3b or 4-hydroxy-2H-benzo[h]chromen-2-one 3c under room temperature

4-hydroxy-6-methyl-in dry dichloromethane for 5 m4-hydroxy-6-methyl-inutes for synthesis of the series of iso4-hydroxy-6-methyl-indol4-hydroxy-6-methyl-in-1-im4-hydroxy-6-methyl-ines 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,N-dimethylaminoethyl (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 3-methylbutan-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

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]

The inhibitory activities of isoindolin-1-imines 4 and 11 were evaluated against NR2B 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

Ca2+-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 from97.2±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

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 Ca 2+ -flux by isoindolin-1-imine analogs

CHO CN +

OH

O O +

HN NH

O

Ethanol Reflux

1' 2'

3' 4' 4a' 5' 6'

7 '

8' 8a'

2+

-flux

IC50 (nM) Positive

Entry R1 R2 Reagent 3 Product 4 NR2B Ca

3d

OH O

1.3.3.2.2 Effect of products 11

The isoindolin-1-imine products 11 were prepared by one-pot procedure via three-component

condensation of 2-cyanobenzaldehyde, primary amines, and 4-hydrocoumarin derivatives in

dry DCM The inhibitory evaluation results of isoindolin-1-imine analogs 11 against NR2B

calcium ion-flux are summarized in Table 1.9

Table 1.9 Inhibition of NR2B Ca 2+ -flux by products 11

As shown in Table 1.9, all the tested compounds 11 are potent NR2B calcium ion-flux

inhibitors with IC50 values range from 84.2±7.5 to 2500.7±68.7 nM, but lower than that of ifenprodil (IC50 = 75.3±7.3 nM)

In the case of compounds were substituted by various substituent at 2-position of

isoindolin-1-imine core (as shown in entries 1-16 of Table 1.9), all benzyl substituent compounds (11a-f) showed better inhibitory effects than compounds containing alkyl group

(R1-) such as n-butyl (11h), i-butyl (11i) and cyclohexyl (11l ) These results suggested that

the substitution of benzyl by alkyl group at 2-position had no obvious contribution to activities

However, compound 11m containing ethanol-2-yl (IC50 = 352.8±29.5 nM, entry 12) and

than compounds containing alkyl group They also exhibited nearly equivalent activity of

p-chlorobenzyl compound 11b (IC50 = 345.4±28.5 nM, entry 2), but revealed about 3 to 4-fold

lower potential activity than that of benzyl 11a (IC50 = 106.3±11.2 nM, entry 1) and

compounds 11e-f with methoxyl group at the phenyl ring (IC50 = 84.2±7.5, 89.1±11.5 nM, respectively)

Comparing the IC50 values of compounds containing various substituent group at the phenyl ring at 2-position of isoindolin-1-imine core, showed that p-halogenobenzyl

derivatives (11b-d, 11s) displayed reduced effects compared to benzyl group (11a, 11r) in

their inhibition activity p-Methoxyl substituent derivatives (11e, 11t) had better activity than that of p-halogen substituent (11b, 11s) and benzyl group (11a, 11r, 11x) Otherwise, p-

chloro- 11b (IC50 = 345.4±28.5, entry 2) exhibited better inhibitory activity than p-bromo- 11c

(IC50 = 642.2±23.5 nM, entry 3) and p-floro- 11d (IC50 = 501.2±34.2 nM, entry 4) substituent compounds

Compounds 11r with 3b (entry 17) and 11x with 3c (entry 23) at position 3 of 1-imine core had weaker inhibitory potency as compared to 11a with 3a (entry 1) and a clear order of the introduction of 3 at position 3 for this activity was observed as the following:

isoindolin-3a > 3b > 3c (11a > 11r > 11x)

Finally, all above isoindolin-1-imine derivatives revealed inhibitory activities of Ca2+flux in cells expressing the NR2B subunit (NR2B Ca2+-flux) Among them, compounds 11e,

11f, 11t exhibited stronger potent inhibitory activities Thus, the substitution at 2-postion on

isoindolin-1-imine core by substituted benzyl structures was potential for further synthesis of derivatives as Ca2+-flux inhibitors

4-resulting mixture was stirred under reflux untill precipitate was afforded After completion of the reaction, solid was filtered off, washed with dichloromethane and acetone, respectively, to

give the corresponding product 4a-o (89-95%)

2-hydroxy-3-(3-iminoisoindolin-1-yl)-4H-chromen-4-one (4a)

White solid, yield 95%, mp 185-186oC; 1H NMR (400 MHz, DMSO-d 6) δ 10.38 (s, 1H),

9.30 (s, 1H), 8.79 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.80 (dd, J = 7.7, 1.7 Hz, 1H), 7.61 (m, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.42-7.33 (m, 2H), 7.12 (td, J = 7.7, 1.0 Hz, 1H), 7.08 (dd, J =

8.2, 1.0 Hz, 1H), 6.51 (s, 1H); 13C NMR (101 MHz, DMSO-d 6) δ 173.6, 162.9, 162.8, 154.3, 151.0, 133.4, 130.9, 130.8, 129.1, 127.6, 125.1, 123.3, 122.6, 122.4, 116.1, 89.5, 60.2; MS

(ESI): m/z 293.35 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C17H13N2O3: 293.2802;

Found: 293.2801

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (4b)

White solid, yield 95%, mp 188-189oC; 1H NMR (400 MHz, DMSO-d 6) δ 10.39 (s, 1H),

9.32 (s, 1H), 8.81 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.64-7.57 (m, 2H), 7.51 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.18 (dd, J = 8.2, 2.3 Hz, 1H), 7.00-6.94 (d, J = 8.2 Hz, 1H), 6.50 (s,

6.89 (s, 1H), 6.46 (s, 1H), 2.32 (s, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 173.8, 163.1, 162.5, 152.4, 151.0, 133.4, 131.5, 131.3, 129.1, 127.6, 125.0, 123.1, 122.9, 122.6, 115.9, 89.6, 60.2,

21.1; MS (ESI): m/z 307.57 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C18H15N2O3:

307.1023; Found: 307.1025

2-hydroxy-3-(3-iminoisoindolin-1-yl)-6-methoxy-4H-chromen-4-one (4d)

White solid, yield 92%, mp 179-180oC; 1H NMR (400 MHz, DMSO-d 6) δ 10.36 (s, 1H),

9.27 (s, 1H), 8.78 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.61 (td, J = 7.7, 1.1 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.34 (dd, J = 7.6, 1.1 Hz, 1H), 7.27 (d, J = 3.0 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 6.97 (dd, J = 8.8, 3.0 Hz, 1H), 6.46 (s, 1H), 3.75 (s, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 173.8, 162.9, 162.6, 155.8, 154.3, 151.0, 133.3, 130.8, 129.1, 127.6, 124.3, 123.3, 122.6,

(dd, J = 8.3, 2.2 Hz, 1H), 6.79 (s, 1H), 6.40 (s, 1H), 3.93 (t, J = 6.7 Hz, 2H), 3.76 (s, 3H), 2.31 (s, 3H), 1.78 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 173.7, 162.9 (2C), 154.3, 152.4, 148.3, 145.6, 131.4, 131.0, 125.0, 123.0, 120.5, 115.9, 106.6, 104.7,

59.8, 56.3, 22.4, 10.8; MS (ESI): m/z 431.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for

C25H23N2O5:431.1502; Found: 431.1501

1.3.4.1.2 Preparation of products 8

To a stirred dry ethanol solution of 2-cyanobenzaldehyde (1, 2 mmol), and 1,3-dicarbonyl compounds (7, 2 mmol) was added ammonium acetate (2, 2 mmol) The resulting mixture was

stirred under reflux for 60 minutes After completion of the reaction, solvent was removed in

vacuo, and crude product was purified by silica gel column chromatography with gradient eluent system DCM/MeOH (50/1-20/1) to give product 8 in yields of 83-90%

6-hydroxy-5-(3-iminoisoindolin-1-yl)-2,2-dimethyl-4H-1,3-dioxin-4-one (8a)

Yellow solid, yield 90%, mp 180-181oC; 1H NMR (400 MHz, DMSO-d 6) δ 10.24 (s, 1H),

9.23 (s, 1H), 8.69 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 7.7 Hz, 1H), 5.98 (s, 1H), 1.51 (s, 6H); 13C NMR (101 MHz, DMSO-d6) δ 175.0, 164.6, 162.2, 151.4, 133.4, 128.7, 127.6, 123.3, 122.7, 100.4, 67.8, 61.6, 26.4 (2C); MS

(ESI): m/z 275.41 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C14H15N2O4: 275.1014;

59.7, 51.0, 31.7, 29.2 (2C); MS (ESI): m/z 271.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C16H19N2O2:271.1411; Found: 271.1410

1.3.4.1.3 Preparation of products 10

To a stirred dry ethanol solution of 2-cyanobenzaldehyde (1, 1 mmol), and

4-hydroxyquinolin-2(1H)-one (9, 1 mmol) was added ammonium acetate (2, 1 mmol) The

resulting mixture was stirred under reflux for 20 minutes After completion of the reaction,

solvent was removed in vacuo, and crude product was purified by silica gel column

chromatography with gradient eluent system ethyl acetate/MeOH (20/1-5/1) to give product

10 in yields of 84-85%

2-hydroxy-3-(3-iminoisoindolin-1-yl) quinolin-4(1H)-one (10a)

White solid, yield 85%, mp > 280oC; 1H NMR (400 MHz, MeOD) δ 8.05 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 6.8 Hz, 1H), 7.68 (t, J = 7.3 Hz, 1H), 7.55 (t, J = 7.5 Hz , 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.12 (t, J = 6.8 Hz, 1H), 6.70 (s,

(2H), 9.03 (s, 1H), 8.11 (d, J = 7.4 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.64 (s, 1H), 7.58-7.53 (m, 2H), 7.46 (t, J = 7.4 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 7.8 Hz, 1H), 6.58 (s, 1H), 2.25 (s, 3H); 13C NMR (101 MHz, DMSO-

70.5, 60.2, 56.2, 22.4, 10.8; MS (ESI): m/z 380.43 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C21H22N3O4:380.1518; Found: 380.1514

1.3.4.2 Preparation of products 11

General procedure the synthesis of 11: To a stirred dichloromethan solution of

2-cyanobenzaldehyde 1 (2 mmol) and 4-hydroxycoumarin derivatives 3 (2 mmol) was added primary amines 2 (2mmol) The resulting mixture was stirred at room temperature After the

reaction was completed, solid was filtered off, washed with dichloromethane and acetone,

respectively, recrystallized to give the corresponding product 11a-11x (90-98%)

3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11a)

White solid, yield 98%, mp 243–244oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.74 (s, 1H),

9.22 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 7.6, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.55 (d, J

= 7.3 Hz, 1H), 7.27–7.45 (m, 7H), 7.17 (t, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.62 (s, 1H), 4.99 (d, J = 15.5 Hz, 1H), 4.47 (d, J = 15.5 Hz, 1H); 13C NMR (101 MHz, DMSO-d 6)

(t, J = 7.5, 1.0 Hz, 1H), 7.35-7.45 (m, 6H) , 7.16 (t, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.96 (d, J = 15.7 Hz, 1H), 4.51 (d, J = 15.7 Hz, 1H); 13C NMR (100 MHz,

δ 175.0, 161.9, 161.1, 161.0, 154.6, 149.0, 133.6, 131.7, 131.1, 130.5 (3J CF = 7.0 Hz) (2C), 128.8, 128.0, 125.5, 123.2, 122.8, 122.7, 122.5, 116.3, 115.9 (2J CF = 21.3 Hz) (2C), 87.8, 63.9,

44.9; MS (ESI): m/z 401.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C24H18FN2O3:

401.4123; Found: 401.4112

2-hydroxy-3-(3-imino-2-(4-methoxybenzyl) isoindolin-1-yl)-4H-chromen-4-one (11e)

White solid, yield 95%, mp 244-245oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.72 (s, 1H),

9.20 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.54 (t, J = 7.3 Hz, 1H), 7.38-7.46 (m, 3H), 7.35(d, J = 7.4 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.92 (d, J = 15.5 Hz, 1H ), 4.37 (d, J = 15.5 Hz, 1H),

3.72 (s, 3H); 13C NMR (100 MHz, DMSO-d 6) δ 174.9, 161.9, 160.7, 159.3, 154.6, 148.9, 133.5, 131.7, 131.1, 130.0 (2C), 129.9, 128.8, 128.0, 127.4, 125.6, 122.9, 122.6, 116.3, 114.5

(2C), 87.9, 63.7, 55.5, 44.9; MS (ESI): m/z 413.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C25H21N2O4:413.4212; Found: 413.4208

3-(2-(3,4-dimethoxyphenethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11f)

White solid, yield 95%, mp 237-238oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.48 (s, 1H),

8.95 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.3 Hz ,1H), 7.53 (t, J = 7.3 Hz, 1H), 7.36-7.47 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.09 (dd, J = 8.0, 1.0 Hz, 1H), 6.82 (d, J = 7.3 Hz, 1H), 6.79 (s, 1H), 6.71 (d, J = 7.3 Hz, 1H), 6.66 (s, 1H), 3.99 (m, 1H ),

3.68 (s, 3H), 3.67 (s, 3H), 3.43 (m, 1H), 2.99(m, 1H), 2.87 (m, 1H); 13C NMR (100 MHz,

DMSO-d 6) δ 175.0, 161.9, 160.4, 158.4, 154.6, 149.1, 147.9, 133.3, 131.1, 130.8, 128.8, 127.9, 125.6, 123.4, 123.0, 122.8, 122.5, 121.0, 116.3, 112.6, 112.1, 87.8, 63.5, 55.9, 55.5,

43.8, 32.7; MS (ESI): m/z 457.44 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C27H24N2O5:457.1707; Found: 457.1692

2-hydroxy-3-(3-imino-2-propylisoindolin-1-yl)-4H-chromen-4-one (11g)

White solid, yield 97%, mp 247-248 oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.62 (s, 1H),

9.06 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.6, Hz, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.53 (m, 3H), 7.19 (d, J = 7.6 Hz, 1H), 7.10 (dd, J = 8.0, 1.0 Hz, 1H), 6.65 (s, 1H), 3.69 (m,

7.37-1H), 3.26 (m, 7.37-1H), 1.96 (m, 2H), 0.85 (m, 3H); 13C NMR (100 MHz, DMSO-d 6) δ 174.9, 161.9, 160.7, 154.5, 148.9, 133.3, 131.1, 129.0, 127.9, 125.5, 123.4, 123.0, 122.8, 122.5,

116.3, 88.3, 63.5, 43.9, 20.4, 19.8; MS (ESI): m/z 333.45 [M-H]-; HRMS (ESI) [M-H]- Calcd

for C20H17N2O3:333.1367; Found: 333.1364

3-(2-butyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11h)

White solid, yield 95%, mp 230-231oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.59 (s, 1H),

9.02 (s, 1H), 8.13 (d, J = 7.7 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.57-7.64 (m, 2H), 7.50 (t, J = 7.3, 1H), 7.36-7.45 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1.0, 1H), 6.64 (s,

1H), 3.72 (m, 1H), 3.26 (m, 1H), 1.64 (m, 1H), 1.27 (m, 1H), 0.82 (m, 3H); 13C NMR (100 MHz, DMSO) δ 174.9, 161.9, 160.6, 154.5, 148.8, 133.3, 131.1, 129.1, 127.9, 125.5, 123.3,

123.0, 122.7, 122.5, 116.3, 88.2, 63.5, 42.2, 29.5, 19.9, 14.0; MS (ESI): m/z 349.5 [M+H]+; HRMS (ESI) Calcd for C21H21N2O3 [M+H]+:349.1547; Found: 349.1543

2-hydroxy-3-(3-imino-2-isobutylisoindolin-1-yl)-4H-chromen-4-one (11i)

White solid, yield 94%, mp 255-256 oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.55 (s, 1H),

8.98 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.55– 7.52 (m, 2H), 7.35–7.45 (m, 2H), 7.18 (t, J = 7.5 Hz, 1H), 7.08 (dd, J = 8.0, 1.0 Hz, 1H), 6.61

(s, 1H), 3.53 (m, 1H), 3.07 (m, 1H), 2.17 (m, 1H), 0.87 (m, 6H); 13C NMR (100 MHz

DMSO-d 6) δ 174.7, 161.8, 161.1, 154.5, 148.9, 133.4, 131.1, 128.9, 127.9, 125.6, 123.4, 123.0, 122.8,

122.5, 116.3, 88.0, 63.8, 49.2, 27.2, 20.4, 19.8; MS (ESI): m/z 347.40 [M-H]-; HRMS (ESI) [M-H]- Calcd for C21H19N2O3:347.1545; Found: 347.1543

3-(2-(cyclopropylmethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11k)

White solid, yield 97%, mp 251-252oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.57 (s, 1H),

9.02 (s, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.53 (t, J = 7.3 Hz, 1H), 7.36-7.45 (m, 2H), 6.75 (s, 1H), 3.74 (m, 1H), 2.99 (m, 1H), 1.22 (m, 1H),

0.42-0.58 (m, 3H), 0.20-0.29 (m, 1H); 13C NMR (100 MHz, DMSO-d 6) δ 174.8, 161.8, 160.3, 154.5, 149.0, 133.4, 131.1, 129.0, 127.9, 125.5, 123.4 123.0, 122.8, 122.5, 116.2, 88.0, 63.7,

46.6, 9.8, 4.1, 3.4; MS (ESI): m/z 347.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for

C21H19N2O3:347.1342; Found: 347.1340

3-(2-cyclohexyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-one (11l)

White solid, yield 91%, mp 252-253oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.45 (s, 1H),

8.88 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.95 (dd, J = 7.6, 1.0 Hz, 1H), 7.57 (t, J = 7.3 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (dd, J = 7.3, 1.0 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.06 (dd, J = 8.0, 1.0 Hz, 1H), 6.66 (s, 1H), 3.84 (m, 1H), 1.74-2.00 (m, 4H),

1.40-1.62 (m, 3H), 1.19-1.36 (m, 2H), 0.86 (m, 1H); 13C NMR (100 MHz, DMSO-d 6) δ 173.4, 161.7, 160.3, 154.4, 149.1, 133.1, 130.9, 129.2, 127.7, 125.6, 123.5, 122.6, 122.5, 122.0,

7.3 Hz, 1H), 4.05 (m, 1H), 3.60 (m, 1H), 3.24 (m, 1H), 3,09 (m, 1H); 13C NMR (100 MHz,

DMSO-d 6) δ 175.2, 162.2, 160.6, 154.6, 148.9, 136.5, 133.3, 131.3, 129.1, 127.9, 127.4, 125.7, 123.6, 123.0, 122.8, 122.7, 122.5, 121.4, 118.8, 118.7, 116.4, 111.8, 110.8, 88.4, 63.5,

43.4, 23.3; MS (ESI): m/z 436.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C27H22N3O3:436.1612; Found: 436.1610

3-(2-benzyl-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one (11q)

White solid, yield 98%, mp 248–249oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.73 (s, 1H),

9.21 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.54 (m, 1H), 7.33–7.45 (m, 5H), 7.30(d, J = 7.6 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.01 (dd, J = 8.0, 1.0 Hz, 1H), 6.60 (s, 1H), 4.98 (d, J = 15.5 Hz, 1H), 4.46 (d, J = 15.5 Hz, 1H), 2.35 (s, 3H); 13C NMR

(101 MHz, DMSO-d 6) δ 175.0, 162.0, 161.0, 152.6, 149.0, 135.5, 133.6, 131.0, 129.1 (2C), 128.8, 128.2 (2C), 128.1, 128.0, 125.4, 123.2, 123.1, 122.7, 122.5, 116.1, 87.8, 64.0, 45.5,

20.9; MS (ESI): m/z 397.45 [M+H]+; HRMS (ESI) Calcd for C25H21N2O3 [M+H]+:397.1527; Found: 397.1520

3-(2-(4-chlorobenzyl)-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one

(11r)

White solid, yield 97%, mp 250-251oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.77 (s, 1H),

9.25 (s, 1H), 8.16 (d, J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.54 (t, J = 7.5

Hz, 1H), 7.33-7.44 (m, 5H), 7.22 (d, J = 7.3 Hz, 1H), 7.01 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.95 (dd, J = 15.5 Hz, 1H), 4.48 (d, J = 15.5 Hz, 1H), 2.25-2.35 (3H); 13C NMR (101

MHz, DMSO-d 6) δ 175.1, 161.3, 161.1, 152.6, 149.1, 134.5, 133.6, 132.8, 131.8, 131.4, 130.1 (2C), 129.0 (2C), 128.7, 128.0, 125.4, 123.2, 122.7, 122.4, 116.1, 87.8, 64.1, 45.0, 20.9; MS

(ESI): m/z 431.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C25H20ClN2O3:431.1117; Found: 431.1110

2-hydroxy-3-(3-imino-2-(4-methoxybenzyl)isoindolin-1-yl)-6-methyl-4H-chromen-4-one

(11s)

White solid, yield 96%, mp 251-252oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.72 (s, 1H),

9.22 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.62 (t, J = 7.3 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.32-7.41 (m, 3H), 7.23 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.0, 1.0 Hz, 1H), 6.59 (s, 1H), 4.92 (dd, J = 15.7 Hz, 1H), 4.48 (d, J = 15.7 Hz, 1H), 3.71 (s, 3H), 2.25-2.36 (3H); 13C NMR

(101 MHz, DMSO-d 6) δ 175.0, 162.1, 160.3, 159.3, 152.6, 149.0, 135.5, 133.6, 131.8, 131.4, 130.1 (2C), 128.8, 128.0, 127.4, 125.4, 123.2, 122.6, 122.5, 116.1, 114.5 (2C), 87.9, 63.7, 55.5,

44.9, 20.9; MS (ESI): m/z 427.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C26H22N2O4:427.1621; Found: 427.1612

3-(2-(3,4-dimethoxyphenethyl)-3-iminoisoindolin-1-yl)-2-hydroxy-6-methyl-4H-chromen-4-one (11t)

White solid, yield 97%, mp 227-228oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.49 (s, 1H),

8.69 (s, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.78(s, 1H), 7.64 (t, J = 7.3 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.36 (m, 3H), 7.23 (t, J = 7.8 Hz, 1H), 7.03 (dd, J = 8.0, 1.0 Hz, 1H), 6.81(d, J = 7.9 Hz, 1H), 6.78 (s, 1H), 6.72 (d, J = 7.9 Hz, 1H), 3.99 (m, 1H), 3.57-3.67 (6H), 3.48 (m, 1H), 2.98

(m, 1H), 2.86 (m, 1H), 2.24-2.37 (m, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 175.1, 162.1, 160.4, 152.6, 149.1, 149.0, 147.9, 133.3, 131.8, 131.4, 130.8, 128.9, 127.8, 125.4, 123.0,

3.68 (m, 1H), 3.23 (m, 1H), 2.24-2.36 (3H), 1.63 (m, 2H), 1.27 (m, 2H), 0.84 (m, 3H); 13C

NMR (101 MHz, DMSO-d 6) δ 174.9, 161.9, 160.5, 152.6, 149.0, 133.2, 131.2, 131.7, 131.3, 129.0, 127.8, 125.4, 123.1, 122.9, 122.5, 116.1, 87.9, 63.7, 42.2, 29.4, 20.9, 19.9, 14.0; MS

(ESI): m/z 363.45 [M+H]+; HRMS (ESI): m/z [M+H]+ Calcd for C22H23N2O3: 363.1623; Found: 363.1614

2-hydroxy-3-(2-(2-hydroxyethyl)-3-iminoisoindolin-1-yl)-6-methyl-4H-chromen-4-one (11v)

White solid, yield 91%, mp 233-234oC; 1H NMR (400 MHz, DMSO-d 6) δ 9.50 (s, 1H),

8.91 (s, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.72 (s, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.53 (d, J = 7.5

7.53-(101 MHz, DMSO-d 6) δ 175.5, 161.7, 161.1, 150.8, 148.9, 135.5, 134.9, 133.6, 131.1, 129.1 (2C), 128.9, 128.2 (2C), 128.1, 127.8, 126.6, 123.6, 123.3, 122.8, 122.6, 122.1, 122.0, 121.7,

118.4, 117.8, 88.2, 64.0, 45.6; MS (ESI): m/z 433.45 [M+H]+; HRMS (ESI) Calcd for

C28H21N2O3 [M+H]+:433.1527; Found: 433.1520

3-(2-butyl-3-iminoisoindolin-1-yl)-2-hydroxy-4H-benzo[h]chromen-4-one (11x)

White solid, yield 90%, mp 237–238oC; 1H NMR (400 MHz, DMSO-d 6): δ 9.58 (s, 1H),

9.00 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 7.7 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.93 (dd, J = 7.6 Hz, 1H), 7.50-7.69 (m, 5H), 7.41 (m, 1H), 6.67 (s, 1H), 3.74 (m, 1H), 3.30 (m,

1H), 1.66 (m, 2H), 1.28 (m, 2H), 0.83 (m, 3H); 13C NMR (101 MHz, DMSO-d 6) δ 175.4, 161.6, 160.7, 150.7, 148.8, 134.9, 133.3, 129.1, 128.2, 127.8, 126.6, 123.6, 123.0, 122.5,

122.1, 122.0, 121.1, 118.3, 117.7, 88.3, 63.5, 42.3, 29.5, 19.9, 14.0; MS (ESI): m/z 399.40

[M+H]+; HRMS (ESI) Calcd for C25H23N2O3 [M+H]+:399.1607; Found: 399.1601

1.3.4.3 Preparation of Calcium flux functional assay

NR1a/NR2B receptor transfected L(tk-) cells were plated in 96-well format at 3 x 104cells/well and grown for 1 day in normal growth medium (Dulbeccos MEM with Na pyruvate,

4500 mg glucose, pen/strep, glutamine, 10% FCS and 0.5 mg/mL geneticin) Receptor expression was induced by the addition of 10 nM dexamethasone in the presence of 500 µM ketamine for 15–24 h Solutions of NR2B antagonists were prepared in DMSO and serially diluted with DMSO to yield 10 solutions differing by 3-fold in concentration 96-well drug plate was prepared by diluting the DMSO solution 250-fold into assay buffer (Hanks balanced salt solution (HBSS) Mg2+ free containing 20 mM HEPES, 2 mM CaCl2, 0.1% BSA, and 250

µM probenecid After induction, the cells were washed twice with assay buffer and loaded with 4 µM of the calciumfluorescence indicator fluo-3 AM in assay buffer containing pluronic F-127 and 100 µM ketamine at 37 oC for 1 h The cells were then washed five times with assay buffer leaving 40 µL of buffer in each well Fluorescence intensity was measured in a fluorometric imaging plate reader (FLIPR), using an excitation of 488 nm, and emission at

530 nm Five seconds after starting the recording of fluorescence intensity, 50 µL of agonist solution (40 µM glutamate/glycine, the final concentration 10 µM) was added and after 1 min, when fluorescence signal was stable, 50 µL of NR2B antagonists and control solutions from

the drug plate were added, and then the fluorescence intensity recorded for an other 30 min The IC50 values were determined by non-linear least squares fitting of the end fluorescence values [46-47]

1.4 Conclusions

In conclusion, a novel one-pot method for the synthesis of isoindolin-1-imine derivatives (4, 8, and 10) was devoloped This method is a simple three-component condensation of 2-

cyanobenzaldhyde, ammonium acetate, and 4-hyroxycoumarin derivatives or 1,3-dicarbonyl

compounds or 4-hydroxyquinolin-2(1H)-one in ethanol under reflux for 20-60 minutes with

excellent yields In addition, we have also developed a new protocol, simple, efficient

procedure for the preparation of

3-(2-substituted-3-iminoisoindolin-1-yl)-2-hydroxy-4H-chromen-4-ones 11 via three-component condensation reaction of 2-cyanobenzaldehyde,

primary amine, and 4-hydroxycoumarin derivatives in dry dichloromethane with catalyst-free condition at room temperature These methodologies offer significant efficient for the synthesis of isoindolin-1-imine derivatives with regard to operational simplicity, excellent yields, fast reactive time, without catalyst, and little environmental impact, and are good valid

contribution to the synthesis of N-heterocyclic compounds

The inhibitory activities of the synthesized isoindolin-1-imine analogs against NR2B

Ca2+ flux were investigated. The evalution results showed that all tested isoindolin-1-imine derivatives exhibited acceptable inhibitory effects against Ca2+flux in cells expressing the NR2B subunit (NR2B Ca2+-flux) Noteably, compounds 4l, 11e, 11f, and 11t exhibited

stronger potent inhibitory activities

Chapter 2 Study on synthesis of oxoalkyl)isoindolin-1-one derivatives via one-pot reaction

2-substituted-3-(2-2.1 Introduction

Isoindolin-1-ones are one of the most important classes of N-heterocyclic biological active

substances [50-54].They have been attracted considerable attention from synthetic pharmacists and chemists due to their potent therapeutic and pharmacological activities [50-56] A series of isoindolin-1-one bridged to 4-(1,2-benzisothiazol-3-yl)-1-piperazinyl moiety were revealed

acceptable dopamine D2, serotonin 5-HT1a, and 5-HT2 receptor antagonistic actions in vitro,

and could antagonize the apomorphine-induced climbing ability in mice [51] In addition, isoindolin-1-ones also exhibited as inhibitory agents of the MDM2-p53 interaction [53-54], enzymatic and potent cellular inhibitory of KDR kinase activities [55] Otherwise, a series of 2-piperazino-isoindolin-1-one derivatives as nonpeptide urotensin-II (U-II) receptor antagonists

in vitro, in vivo were reported [56] (Fig 2.1)

Nonpeptide Urotensin-II Receptor Antagonists Sedative and anxiolytic drug

N O O

H 3 CO OH OCH 3

1

MDM2-p53 IC 50 = 17.9 uM

N O

O N N Ph H

O

O

3 JNJ-28318706

N O N N Cl O

N O O

2 (S)-Pazinaclone

Fig 2.1 Structure of isoindolin-1-one activity analogs

Due to the potential of isoindolin-1-one derivatives, several methods have been developed and published for the synthesis of 2-substituted-3-(2-oxoalkyl)isoindolin-1-ones [57-59]

In 1999, Nitya G K et al carried out the synthesis of isoindolin-1-ones by a mixture of

2-iodobenzamide or its N-substituted derivatives and a selected acetylenic carbinol in the

presence of bis(triphenylphosphine)palladium(II) dichloride and copper(I) iodide as catalyst at 80oC for 24 h (Method A) to give target products in good yields Otherwise, when the palladium and copper (I) iodide as co-catalyst, synthetic reactions were carried out at room temperature only to give disubstituted alkynol mediate products The target products isoindolin-1-ones could be obtain by cyclation of disubstituted alkynols in the presence of sodium ethoxide in ethanol under reflux condition (Method B) (Scheme 2.1) [57]