synthesis and evaluation of novel oxoisoindoline derivatives as acetylcholinesterase inhibitors

ORIGINAL PAPER

Synthesis and evaluation of novel oxoisoindoline derivativesas acetylcholinesterase inhibitors

Ayeh Rayatzadeh • Mina Saeedi • Mohammad Mahdavi •

Zahra Rezaei • Reyhaneh Sabourian • Mohammad Hossein Mosslemin •

Tahmineh Akbarzadeh • Alireza Foroumadi • Abbas Shafiee

Received: 28 June 2014 / Accepted: 15 October 2014

� Springer-Verlag Wien 2014

Abstract An efficient synthetic route for the synthesis of

novel oxoisoindoline derivatives was described through

Ugi reaction of 2-formylbenzoic acids, amines, and iso-

cyanides. All compounds were evaluated for their

acetylcholinesterase inhibitory activity and among them, 2

of 12 tested compounds showed good activity as compared

to other derivatives.

Keywords Oxoisoindoline � Ugi reaction �Acetylcholinesterase � 2-Formylbenzoic acid

Introduction

Oxoisoindolines (isoindolinones) are one of the most

important heterocyclic scaffolds ubiquitous in natural

products such as aristoyagonine, nuevamine, lennoxamine,

and chilenine (Fig. 1) [1]. Although their exclusive struc-

tural aspects have absorbed the attention of organic

chemists, their biological activities are not widely reported.

At this juncture, cytotoxicity of aristoyagonine against

various tumoral cell lines has been well established [2].

In contrast to naturally occurring oxoisoindolines, syn-

thetic analogs exhibited precious biological and medicinal

properties. For example, some derivatives represented

ADAMTS (A disintegrin and metalloproteinase with

thrombospondin motifs) inhibitory activity as an osteoar-

thritis agent [3] and various reports indicated anti-cancer

[4], anti-HIV [5], prolyl oligopeptidase (POP) inhibitory

[6], anti-pain [7], cardioselective [8], and anti-Alzheimer

[9] activities.

Alzheimer’s disease (AD) is the most common neuro-

degenerative disorder, known as origin of dementia among

older people [10]. Cholinergic system disorders accelerated

aggregation of b-amyloid peptides and the dyshomeostasis

of biometals are the most common factors causing AD [11–

15]. Considering the fact that most of the AD symptoms

arise from acetylcholine-producing system dysfunction

[16], recent research has focused on inhibition of acetyl-

cholinesterase (AChE), the enzyme which hydrolyzes

acetylcholine as one of the most important neurotransmitter.

Since incidence statistics of AD is growing up and it has

imposed considerable economic costs on many countries,

this disease has attracted the attention of medical chemist

community [17]. In spite of the fact that various efficient

cholinesterase inhibitor drugs such as donepezil [18], riv-

astigmine [19], and galanthamine [20] have been

developed, there is still significant demand for drug dis-

covery leading to efficient anti-Alzheimer’s agents.

In continuation of our efforts to develop new acetyl-

cholinesterase agents [21–24], herein, we focused on the

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00706-014-1334-2) contains supplementarymaterial, which is available to authorized users.

A. Rayatzadeh � M. H. Mosslemin

Department of Chemistry, Yazd Branch, Islamic Azad

University, Yazd, Iran

e-mail: [email protected]

M. Mahdavi � A. Foroumadi � A. Shafiee (&)

Department of Medicinal Chemistry, Faculty of Pharmacy and

Pharmaceutical Sciences Research Center, Tehran University of

Medical Sciences, 14176 Tehran, Iran

e-mail: [email protected]

Z. Rezaei � T. Akbarzadeh

Department of Medicinal Chemistry, Faculty of Pharmacy,

Tehran University of Medical Sciences, 14176 Tehran, Iran

M. Saeedi � R. Sabourian � T. Akbarzadeh

Persian Medicine and Pharmacy Research Center, Tehran

University of Medical Sciences, Tehran, Iran

123

Monatsh Chem

DOI 10.1007/s00706-014-1334-2

http://dx.doi.org/10.1007/s00706-014-1334-2

synthesis of novel oxoisoindoline derivatives and evaluated

the title compounds for their acetylcholinesterase inhibi-

tory activity (Scheme 1).

Results and discussion

Chemistry

Recently, we reported novel oxoindolinylidene methyl

pyridinium halides as acetylcholinesterase inhibitors pos-

sessing very good activities (IC50 = 0.44 – 677 nM)

(Fig. 2) [24]. It was demonstrated that some derivatives

were more potent than the reference drug, donepezil

(IC50 = 14 nM). In this study, focusing on the efficacy of

indolinones for the inhibition of AChE, we prepared novel

oxoisoindolines and evaluated their AChE inhibitory

effect.

Isocyanide-based multicomponent reactions (IMCRs)

[25] have absorbed medicinal chemists’ attention due to

their ability to generate versatile functional groups in the

corresponding products. Recently, using bifunctional

starting materials such as 2-formylbenzoic acids in IMCRs

have led to the formation of interesting cyclic compounds

[6, 26–30].

Herein, to obtain novel oxoisoindoline possessing ace-

tylcholinesterase inhibitory activity 4, we considered Ugi

reaction of 2-formylbenzoic acids 1, amines 2, and isocy-

anides 3 as depicted in Scheme 1. Trusting on our

experience related to isocyanide-based multicomponent

reactions [31–33], we found that conducting the reaction in

MeOH at 50 �C in the absence of any catalysts or additives

led to the formation of compound 4. As shown in Table 1,

various oxoisoindoline derivatives 4 were synthesized in

good to excellent yields. For this purpose, we specially

paid attention to amines or 2-formylbenzoic acid deriva-

tives similar to the structure of the drugs, donepezil and

rivastigmine. It should be noted that the reaction exhibited

good tolerance toward different 2-formylbenzoic acids,

amines, and isocyanides and all products 4 were obtained

in satisfactory yields.

The reaction sequence has been depicted in Scheme 2. It

was initiated by the reaction of 2-formylbenzoic acid 1 and

amine 2 to lose water and formation of imine 5. The imine

5 was activated (6) through proton exchange with the

carboxyl group and prone to be attacked by isocyanide 3 to

form 7. The latter intermediate 7 underwent the second

nucleophilic addition of carboxylate anion to nitrilium ion

to obtain 8. Finally, Mumm rearrangement with acyl group

transfer led to the formation of compound 4.

All products 4a–4l were characterized using spectral

data. The combination of all information from IR, NMR

spectroscopy, and mass spectrometry fragmentation pattern

analysis confirmed the given structure.

Scheme 1

Fig. 2 Indolinone-based acetyl-

cholinesterase inhibitors

Fig. 1 Naturally occurring

oxoisoindoline derivatives

A. Rayatzadeh et al.

123

Biological activity

All compounds 4a–4l were evaluated as AChE inhibitors

using modified colorimetric Ellman’s method [34] and

compared with rivastigmine as the reference drug

(Table 1). The IC50 of most compounds was higher than

100 lM and showed no significant inhibitory activity.

Among the synthesized compounds, 2-(1-benzylpiperidin-

4-yl)-3-oxo-N-(2,4,4-trimethylpentan-2-yl)isoindoline-1-

carboxamide (4a) and N-(tert-butyl)-2-[2-(diethylamino)-

ethyl]-3-oxoisoindoline-1-carboxamide (4k) showed rela-

tive good activity with IC50 values of 41.04 and

83.05 lM, respectively. Clearly, insertion of methoxy

groups onto oxoisoindoline ring did not improve AChE

inhibitory activity. However, presence of benzyl piperi-

dine group in compound 4a led to the notable increase

of AChE inhibitory activity in comparison to 4k. Alto-

gether, the compounds described in this study were not

as effective as reported oxoindolinylidene methyl pyrid-

inium halides [24].

Conclusion

In conclusion, we have successfully developed a practical

procedure for the preparation of novel oxoisoindoline

derivatives starting from 2-formylbenzoic acids, amines,

and isocyanides. All compounds were evaluated for their

inhibitory activity against acetylcholinesterase using a

modified Ellman’s method and among them, 4a and 4k

showed relatively good activity. However, their IC50

values were not in the low lM range as described

recently for oxoindolinylidene methyl pyridinium halides

[24]. It seems that pyridinium plays a main role in AChE

inhibition.

Experimental

All the chemicals were purchased from Merck to Sigma-

Aldrich and used without further purification. Melting point

was taken on a Kofler hot stage apparatus. 1H and 13C NMR

spectra were recorded on a Bruker FT-300 MHz, using TMS

as an internal standard. The IR spectra were obtained on a

Nicolet Magna FTIR 550 spectrophotometer (in KBr). Mass

spectra were determined on an Agilent Technology (HP)

mass spectrometer operating at an ionization potential of

70 eV. The elemental analysis was performed with an Ele-

mentar Analysensystem GmbH VarioEL CHNS mode.

General procedure for the synthesis of oxoisoindoline

derivatives 4

A solution of 2-formylbenzoic acid derivative 1 (1 mmol),

amine 2 (1 mmol), and isocyanide 3 (1.2 mmol) in 6 cm3

methanol was heated at 50 �C for 12 h. Upon completion

of the reaction, monitored by TLC, the reaction mixture

was cooled to room temperature and a small amount of

water was added. The precipitate was filtered off and

recrystallized from ethanol to obtain pure product 4.

2-(1-Benzylpiperidin-4-yl)-3-oxo-N-(2,4,4-trimethylpen-

tan-2-yl)isoindoline-1-carboxamide (4a, C29H39N3O2)

White crystals; m.p.: 172–175 �C; 1H NMR (300 MHz,

DMSO-d6): d = 8.18 (s, 1H, NH), 7.64 (d, J = 7.4 Hz,

1H, H4), 7.57 (t, J = 7.4 Hz, 1H, H6), 7.49–7.44 (m, 2H,

H5, H7), 7.33–7.21 (m, 5H, Ph), 5.26 (s, 1H, H1), 3.85–3.82

(m, 1H, CH), 3.45 (s, 2H, CH2), 2.84–2.83 (m, 2H,

2 9 Heq), 2.03-1.57 (m, 8H, 4 9 CH2), 1.36 (s, 3H, CH3),

1.31 (s, 3H, CH3), 1.00 [s, 9H, C(CH3)3] ppm; 13C NMR

(75 MHz, DMSO-d6): d = 167.9, 166.6, 142.4, 138.7,

132.4, 131.4, 128.5, 128.4, 128.1, 126.8, 122.6, 121.8,

62.3, 61.8, 54.8, 52.6, 50.9, 50.8, 31.3, 31.2, 29.4, 28.7,

28.4 ppm; IR (KBr): v = 3,294, 3,062, 2,947, 2,886, 1,690,

Scheme 2

Synthesis and evaluation of novel oxoisoindoline derivatives

123

1,659 cm-1; MS (70 eV): m/z (%) = 461.30 (M?, 51), 172

(51), 82 (100).

2-(1-Benzylpiperidin-4-yl)-4,5-dimethoxy-3-oxo-N-(2,4,4-

trimethylpentan-2-yl)isoindoline-1-carboxamide

(4b, C31H43N3O4)

Off-white crystals; m.p.: 130–132 �C; 1H NMR (300 MHz,

DMSO-d6): d = 8.10 (s, 1H, NH), 7.30–7.21 (m, 6H, Ph,

H7), 7.08 (d, J = 8.1 Hz, 1H, H6), 5.10 (s, 1H, H1), 3.85 (s,

3H, OCH3), 3.84–3.82 (m, 1H, CH), 3.79 (s, 3H, OCH3),

3.45 (s, 2H, CH2), 2.84–2.82 (m, 2H, 2 9 Heq), 2.02–1.57

(m, 8H, 4 9 CH2), 1.31 (s, 3H, CH3), 1.29 (s, 3H, CH3),

0.99 [s, 9H, C(CH3)3] ppm; 13C NMR (75 MHz, DMSO-

d6): d = 167.0, 166.2, 152.2, 145.8, 138.8, 135.4, 128.5,

128.1, 126.9, 124.3, 117.0, 116.5, 61.9, 61.7, 61.1, 56.4,

54.7, 52.6, 50.9, 50.8, 31.4, 31.3, 29.4, 28.7, 28.4 ppm; IR

(KBr): v = 3,297, 2,935, 1,693, 1,622 cm-1.

Table 1 Synthesis of oxoisoindoline derivatives 4

Entry X R1 R2 Product 4 Yield /%a IC50 /µM

1 H 1,1,3,3-Tetramethylbutyl 4a 82 41.04

2 OMe 1,1,3,3-Tetramethylbutyl 4b 88 > 100

3 H Cyclohexyl 4c 83 > 100

4 OMe Cyclohexyl 4d 80 > 100

5 H tert-Butyl 4e 90 > 100

6 OMe tert -Butyl 4f 75 > 100

7 H 1,1,3,3-Tetramethylbutyl 4g 73 > 100

8 OMe 1,1,3,3-Tetramethylbutyl 4h 76 > 100

9 H Cyclohexyl 4i 70 > 100

10 OMe Cyclohexyl 4j 70 > 100

11 H tert-Butyl 4k 75 83.05

12 OMe tert-Butyl 4l 78 > 100

Rivastigmine 11.07

a Yield of isolated product


123

2-(1-Benzylpiperidin-4-yl)-N-cyclohexyl-3-oxoisoindoline-

1-carboxamide (4c, C27H33N3O2)

Yellow crystals; m.p.: 118–20 �C; 1H NMR (300 MHz,

DMSO-d6): d = 8.57 (d, J = 7.6 Hz, 1H, NH), 7.65 (d,

J = 7.3 Hz, 1H, H4), 7.57 (t, J = 7.3 Hz, 1H, H6), 7.50–

7.42 (m, 2H, H5, H7), 7.34–7.23 (m, 5H, Ph), 5.23 (s, 1H,

H1), 3.90–3.88 (m, 1H, CH), 3.52–3.50 (m, 1H, CH), 3.45

(s, 2H, CH2), 2.84 (m, 2H, 2 9 Heq), 2.04–1.98 (m, 2H,

CH2), 1.85–1.70 (m, 8H, 4 9 CH2), 1.57–1.14 (m, 6H,

3 9 CH2) ppm; 13C NMR (75 MHz, DMSO-d6):

d = 168.0, 167.0, 142.3, 138.6, 132.2, 131.7, 128.7,

128.5, 128.2, 126.9, 122.7, 121.8, 61.9, 61.6, 52.6, 52.4,

50.8, 48.0, 32.1, 29.5, 29.3, 25.2, 24.4 ppm; IR (KBr):

v = 3,263, 3,084, 2,925, 2,846, 1,696, 1,653 cm-1.

2-(1-Benzylpiperidin-4-yl)-N-cyclohexyl-4,5-dimethoxy-3-

oxoisoindoline-1-carboxamide (4d, C29H37N3O4)


DMSO-d6): d = 8.46 (d, J = 7.8 Hz, 1H, NH), 7.33–7.20

(m, 6H, Ph, H7), 7.06 (d, J = 8.2 Hz, 1H, H6), 5.06 (s, 1H,

H1), 3.90–3.87 (m, 1H, CH), 3.85 (s, 3H, OCH3), 3.78 (s,

3H, OCH3), 3.51–3.49 (m, 1H, CH), 3.44 (s, 2H, CH2),

2.83 (m, 2H, 2 9 Heq), 2.01–1.94 (m, 2H, CH2), 1.71–1.68

(m, 8H, 4 9 CH2), 1.56–1.14 (m, 6H, 3 9 CH2) ppm; 13C

NMR (75 MHz, DMSO-d6): d = 167.4, 166.2, 152.3,

145.8, 138.6, 135.3, 128.7, 128.2, 126.9, 124.2, 116.9,

116.7, 61.9, 61.7, 60.5, 56.4, 52.7, 52.5, 50.7, 47.9, 32.1,

29.4, 29.2, 25.2, 24.4 ppm; IR (KBr): v = 3,297, 3,062,

2,928, 2,852, 2,828, 1,696, 1,659 cm-1.

2-(1-Benzylpiperidin-4-yl)-N-(tert-butyl)-3-oxoisoindoline-

1-carboxamide (4e, C25H31N3O2)


DMSO-d6): d = 8.70 (s, 1H, NH), 7.81 (d, J = 7.3 Hz,

1H, H4), 7.66–7.43 (m, 3H, H5, H6, H7), 7.33–7.20 (m, 5H,

Ph), 5.37 (s, 1H, H1), 3.90–3.89 (m, 1H, CH), 3.46 (s, 2H,

CH2), 2.83–2.79 (m, 2H, 2 9 Heq), 2.07–1.61 (m, 6H,

3 9 CH2), 1.29 [s, 9H, C(CH3)3] ppm; 13C NMR (75 MHz,

DMSO-d6): d = 168.0, 167.2, 142.7, 138.6, 132.3, 131.5,

128.7, 128.4, 128.2, 126.8, 122.6, 121.8, 62.4, 61.9, 61.8,

51.6, 50.6, 29.1, 28.2 ppm; IR (KBr): v = 3,418, 2,944,

2,807, 1,690, 1,655 cm-1.

2-(1-Benzylpiperidin-4-yl)-N-(tert-butyl)-4,5-dimethoxy-3-

oxoisoindoline-1-carboxamide (4f, C27H35N3O4)


DMSO-d6): d = 8.29 (s, 1H, NH), 7.34–7.21 (m, 6H, Ph,

H7), 7.06 (d, J = 8.2 Hz, 1H, H6), 5.08 (s, 1H, H1), 3.85 (s,

3H, OCH3), 3.84–3.82 (m, 1H, CH), 3.79 (s, 3H, OCH3),

3.48 (d, J = 13.2 Hz, 1H, CH2a), 3.43 (d, J = 13.2 Hz,

1H, CH2b), 2.86–2.82 (m, 2H, 2 9 Heq), 2.02–1.95 (m, 2H,

CH2), 1.73–1.66 (m, 4H, 2 9 CH2), 1.27 s, 9H, C(CH3)3]

ppm; 13C NMR (75 MHz, DMSO-d6): d = 167.5, 166.2,

152.2, 145.8, 138.6, 135.5, 128.7, 128.2, 126.9, 124.2,

116.8, 116.6, 61.9, 61.6, 60.6, 56.4, 52.7, 52.4, 50.6, 50.5,

29.5, 29.1, 28.2 ppm; IR (KBr): v = 3,284, 3,056, 2,959,

2,937, 1,696, 1,655 cm-1; MS (70 eV): m/z (%) = 465.26

(M?, 68), 293 (20), 172 (100), 91 (96).

2-[2-(Diethylamino)ethyl]-3-oxo-N-(2,4,4-trimethylpentan-

2-yl)isoindoline-1-carboxamide (4g, C23H37N3O2)


DMSO-d6): d = 8.24 (s, 1H, NH), 7.66 (d, J = 7.2 Hz,

1H, H4), 7.62–7.53 (m, 2H, H6, H7), 7.48 (t, J = 7.2 Hz,

1H, H5), 5.32 (s, 1H, H1), 3.93–3.84 (m, 1H, CH2a), 2.98–

2.90 (m, 1H, CH2b), 2.62–2.39 (m, 6H, 3 9 CH2), 1.94 (d,

J = 14.6 Hz, 1H, CH2a’), 1.54 (d, J = 14.6 Hz, 1H,

CH2b’), 1.35 (s, 3H, CH3), 1.28 (s, 3H, CH3), 0.97 [s,

9H, C(CH3)3], 0.86 (t, J = 7.1 Hz, 6H, 2 9 CH3) ppm;13C NMR (75 MHz, DMSO-d6): d = 167.8, 165.7, 141.9,

132.0, 131.4, 128.4, 122.7, 122.3, 63.9, 54.7, 50.4, 50.1,

46.6, 31.3, 31.2, 29.2, 28.9, 11.8 ppm; IR (KBr):

v = 3,318, 3,047, 2,965, 2,867, 2,807, 1,696, 1,665 cm-1.

2-[2-(Diethylamino)ethyl]-4,5-dimethoxy-3-oxo-N-(2,4,4-

trimethylpentan-2-yl)isoindoline-1-carboxamide

(4h, C25H41N3O4)


DMSO-d6): d = 8.14 (s, 1H, NH), 7.24 (d, J = 8.2 Hz,

1H, H7), 7.16 (d, J = 8.2 Hz, 1H, H6), 5.15 (s, 1H, H1),

3.86 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 2.92–2.83 (m, 1H,

CH2a), 2.60–2.42 (m, 7H, 3 9 CH2, CH2b), 1.92 (d,

J = 14.6 Hz, 1H, CH2a’), 1.53 (d, J = 14.6 Hz, 1H,

CH2b’), 1.34 (s, 3H, CH3), 1.28 (s, 3H, CH3), 0.97 [s,


145.8, 134.8, 124.0, 117.4, 116.4, 62.9, 61.7, 56.4, 54.7,

50.2, 46.7, 31.3, 31.2, 29.2, 28.9, 11.9 ppm; IR (KBr):

v = 3,323, 2,971, 2,945, 2,813, 1,699, 1,662 cm-1.

N-Cyclohexyl-2-[2-(diethylamino)ethyl]-3-oxoisoindoline-

1-carboxamide (4i, C21H31N3O2)

Pale yellow crystals; m.p.: 113–115 �C; 1H NMR

(300 MHz, DMSO-d6): d = 8.58 (d, J = 7.8 Hz, 1H,

NH), 7.66 (d, J = 7.4 Hz, 1H, H4), 7.62–7.47 (m, 3H,

H5, H6, H7), 5.36 (s, 1H, H1), 3.92–3.84 (m, 1H, CH), 3.55–

3.53 (m, 1H, CH2a), 3.03–2.93 (m, 1H, CH2b), 2.64–2.34

(m, 6H, 3 9 CH2), 1.77–1.12 (m, 10H, cyclohexyl), 0.91

(t, J = 7.0 Hz, 6H, 2 9 CH3) ppm; 13C NMR (75 MHz,

DMSO-d6): d = 167.7, 165.8, 141.8, 131.8, 131.5, 128.5,

122.7, 122.2, 63.2, 50.6, 48.0, 46.5, 40.3, 32.3, 25.1, 24.4,

11.7 ppm; IR (KBr): v = 3,297, 2,971, 2,934, 2,855, 2,801,

1,705, 1,656 cm-1.

N-Cyclohexyl-2-[2-(diethylamino)ethyl]-4,5-dimethoxy-3-

oxoisoindoline-1-carboxamide (4j, C23H35N3O4)


DMSO-d6): d = 8.47 (d, J = 7.8 Hz, 1H, NH), 7.24 (d,


123

J = 8.2 Hz, 1H, H7), 7.16 (d, J = 8.2 Hz, 1H, H6), 5.19 (s,

1H, H1), 3.86 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.56–3.54

(m, 1H, CH), 3.33–3.32 (m, 1H, CH2a), 2.96–2.87 (m, 1H,

CH2b), 2.62–2.36 (m, 6H, 3 9 CH2), 1.70–1.18 (m, 10H,

cyclohexyl), 0.91 (t, J = 7.1 Hz, 6H, 2 9 CH3) ppm; 13C

NMR (75 MHz, DMSO-d6): d = 166.2, 166.0, 152.3,

145.8, 134.8, 123.8, 117.3, 116.5, 62.6, 61.6, 56.3, 50.5,

47.9, 46.6, 40.3, 32.3, 25.1, 24.4, 11.7 ppm; IR

(KBr):v = 3,281, 2,968, 2,937, 2,855, 1,693, 1,647 cm-1.

N-(tert-Butyl)-2-[2-(diethylamino)ethyl]-3-oxoisoindoline-

1-carboxamide (4k, C19H29N3O2)


DMSO-d6): d = 8.40 (s, 1H, NH), 7.66 (d, J = 7.0 Hz,

1H, H4), 7.16 (m, 2H, H6, H7), 7.48 (t, J = 7.0 Hz, 1H,

H5), 5.37 (s, 1H, H1), 3.91–3.83 (m, 1H, CH2a), 3.04–2.95

(m, 1H, CH2b), 2.63–2.38 (m, 6H, 3 9 CH2), 1.29 [s, 9H,

C(CH3)3], 0.91 (t, J = 7.0 Hz, 6H, 2 9 CH3) ppm; 13C

NMR (75 MHz, DMSO-d6): d = 167.8, 166.0, 142.0,

131.9, 131.4, 128.3, 122.7, 122.1, 63.8, 50.7, 50.6, 46.6,

40.3, 28.3, 11.7 ppm; IR (KBr): v = 3,430, 3,071, 2,971,

2,952, 2,867, 1,699, 1,656 cm-1; MS (70 eV): m/z

(%) = 331.23 (M?, 85), 160 (28), 99 (27), 86 (100).

N-(tert-Butyl)-2-[2-(diethylamino)ethyl]-4,5-dimethoxy-3-

oxoisoindoline-1-carboxamide (4l, C21H33N3O4)

Off-white crystals; m.p.: 140–142 �C; 1H NMR (300 MHz,

DMSO-d6): d = 8.30 (s, 1H, NH), 7.24 (d, J = 8.3 Hz,

1H, H7), 7.16 (d, J = 8.3 Hz, 1H, H6), 5.18 (s, 1H, H1),

3.86 (s, 3H, OCH3), 3.80–3.76 (m, 4H, OCH3, CH2a), 2.96–

2.87 (m, 1H, CH2b), 2.63–2.37 (m, 6H, 3 9 CH2), 1.28 [s,


145.8, 135.0, 123.9, 117.2, 116.5, 62.8, 61.6, 56.3, 50.7,

50.4, 46.6, 40.3, 28.3, 11.8 ppm; IR (KBr): v = 3,315,

2,974, 2,931, 2,834, 2,807, 1,699, 1,675 cm-1.

In vitro AChE assay

Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V–S,

lyophilized powder, from electric eel, 1,000 unit), acetyl-

thiocholine iodide (ATCI), and 5,5-dithiobis-(2-

nitrobenzoic acid) (DTNB) were purchased from Sigma-

Aldrich. Potassium dihydrogen phosphate, dipotassium

hydrogen phosphate, potassium hydroxide, and sodium

hydrogen carbonate were obtained from Fluka. The solutions

of the title compounds were prepared in a mixture of 5 cm3

DMSO and 5 cm3 methanol and diluted in 0.1 M KH2PO4/

K2HPO4 buffer (pH = 8.0) to obtain final assay concentra-

tions. All experiments were achieved at 25 �C. Six different

concentrations were tested for each compound in triplicate to

obtain the range of 20–80 % inhibition for AChE.

To measure in vitro AChE activity, modified Ellman

method was performed [34] using a 96-well plate reader

(BioTek ELx808). Each well contained 50 mm3 potas-

sium phosphate buffer (KH2PO4/K2HPO4, 0.1 M,

pH = 8), 25 mm3 sample dissolved in 50 % methanol

and 50 % DMSO and 25 mm3 enzyme (final concentra-

tion 0.22 U/cm3 in buffer). They were preincubated for

15 min at room temperature, then 125 mm3 DTNB

(3 mM in buffer) was added. Characterization of the

hydrolysis of ATCI catalyzed by AChE was performed

spectrometrically at 405 nm followed by the addition of

substrate (ATCI 3 mM in water). The change in absor-

bance was measured at 405 nm after 15 min. The IC50

values were determined graphically from inhibition curves

(log inhibitor concentration vs. percent of inhibition). A

control experiment was performed under the same con-

ditions without inhibitor and the blank contained buffer,

water, DTNB, and substrate.

Acknowledgments We gratefully acknowledge the partial financial

support from Tehran University of Medical Sciences Research

Council and Iran National Science Foundation (INSF).

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synthesis and evaluation of novel oxoisoindoline derivatives as acetylcholinesterase inhibitors

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