Design, synthesis and evaluation of isaindigotone derivatives as dual inhibitors for acetylcholinesterase and amyloid beta aggregation

Article abstract of DOI:10.1016/j.bmc.2012.02.061

A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual inhibitors of cholinesterases (ChEs) and self-induced β-amyloid (Aβ) aggregation. The synthetic compounds had IC₅₀ values at micro or nano molar range for cholinesterase inhibition, and some compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much better than the isaindigotone derivatives previously reported by our group. Most of these compounds showed higher self-induced Aβ aggregation inhibitory activity than a reference compound curcumin. The structure-activity relationship studies revealed that the derivatives with higher inhibition activity on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibition for both AChE and self-induced Aβ aggregation was further studied using CD, EM, molecular docking and kinetics.

Full text of DOI:10.1016/j.bmc.2012.02.061

Bioorganic & Medicinal Chemistry 20 (2012) 2527–2534
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of isaindigotone derivatives as dual
inhibitors for acetylcholinesterase and amyloid beta aggregation
⇑
Jin-Wu Yan, Yan-Ping Li, Wen-Jie Ye, Shuo-Bin Chen, Jin-Qiang Hou, Jia-Heng Tan , Tian-Miao Ou,
Ding Li, Lian-Quan Gu, Zhi-Shu Huang
⇑
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isaindigotone derivatives and analogues were designed, synthesized and evaluated as dual
inhibitors of cholinesterases (ChEs) and self-induced b-amyloid (Ab) aggregation. The synthetic com-
pounds had IC50 values at micro or nano molar range for cholinesterase inhibition, and some compounds
exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE, which were much
better than the isaindigotone derivatives previously reported by our group. Most of these compounds
showed higher self-induced Ab aggregation inhibitory activity than a reference compound curcumin.
The structure–activity relationship studies revealed that the derivatives with higher inhibition activity
on AChE also showed higher selectivity for AChE over BuChE. Compound 6c exhibiting excellent inhibi-
tion for both AChE and self-induced Ab aggregation was further studied using CD, EM, molecular docking
and kinetics.
Received 2 January 2012
Revised 25 February 2012
Accepted 27 February 2012
Available online 7 March 2012
Keywords:
Isaindigotone derivatives
AChE inhibition
Ab aggregation inhibition
Anti-Alzheimer agents
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
a key role in accelerating senile amyloid b-peptide (Ab) plaques
deposition.9 AChE has a catalytic site (CAS) at the bottom of deep
Alzheimer’s disease (AD) is a progressive neurodegenerative
brain disorder which is affecting millions of elder people, and the
number of patients is expected to reach 70 million by 2050.1–3
Although many factors have been implicated in AD, its etiology is
not completely clear. There are diverse pathologic factors responsi-
ble for the AD, such as low levels of acetylcholine, b-amyloid (Ab)
deposits and Tau-protein aggregation.4,5 Current treatment of AD
with AChE (Acetylcholinesterase) inhibitors (tacrine, donepezil,
narrow gorge and a peripheral anionic site (PAS) at the entrance.10
The simultaneous binding to both sites has been suggested to be
important in designing powerful and selective AChE inhibitors.
On the other hand, the amyloid cascade hypothesis implies that
the progressive deposition of Ab is fundamental to the develop-
ment of neurodegenerative pathology. The cell toxicity associated
with Ab fibril aggregation provides an explanation for the neuronal
cell loss found in AD patients. Inhibition of Ab fibril aggregation
has been proved to be a promising approach for the treatment of
AD.11–13 Meanwhile, due to the multi-pathogenesis of AD, an
attractive strategy is to develop novel anti-Alzheimer agents with
multiple functions. In recent years, many series of promising com-
pounds have been designed and developed to inhibit both AChE
and Ab aggregation.14–16
Isaindigotone (a, Fig. 1) is a naturally occurring alkaloid which
is commonly used in traditional Chinese medicine.17 The structure
of isaindigotone is consisted of a deoxyvasicinone moiety conju-
gated with a substituted benzylidene moiety. The structure of
deoxyvasicinone (b, Fig. 1) is similar to that of tacrine (c, Fig. 1).
In recent years, some deoxyvasicinone derivatives have been found
to be ChE inhibitors with a wide range of ChE inhibition activ-
ity.18,19 Our research group has designed and synthesized a series
of isaindigotone derivatives as cholinesterases inhibitors with mul-
tiple binding modes,20 targeting both cholinesterases. However,
their inhibition activities have been found to be lower than that
of tacrine with only moderate inhibition selectivity for AChE over
BuChE. In addition, the capability of isaindigotone derivatives to
rivastigmine and galantamine) and an NMDA (N-methyl D-aspar-
tate) antagonist memantine could only improve symptoms but
not address AD’s etiology, therefore, much effort has been made
to develop more effective drug for the treatment of AD.6,7
Among the diverse pathologic factors, ACh and Ab both play sig-
nificant roles in the disease. The observation of a deficiency in cho-
linergic neurotransmission in AD has led to the statement of
cholinergic hypothesis. Two types of ChE enzymes have been found
in the central nervous system, including acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE), and current treatment
of AD mainly focuses on the inhibition of AChE activity in order
to rectify the deficiency of cerebral acetylcholine.8 AChE plays a
pivotal role in central and peripheral nervous systems, and its main
function is to terminate the impulse transmission at cholinergic
synapses. Recent studies have identified that AChE could also play
⇑
Corresponding authors. Tel./fax: +86 20 39943056.
E-mail addresses: tanjiah@mail.sysu.edu.cn (J.-H. Tan), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.061

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J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
O
O
NH2
N
O
N
N
OH
N
N
O
a
b
c
O
O
R
HN
N
N
H
N
HN
N
N
H
n O
m
NH
n
Cl
Cl
d
e
Figure 1. Chemical structures of isaindigotone (a), deoxyvasicinone (b), tacrine (c), and 6-chlorotacrine hybrids (d, e).
inhibit Ab aggregation has not been investigated and the structural
diversity of these compounds was not enough for the study of the
structure–activity relationship of the interaction. Herein, we ratio-
nally designed and synthesized two new series of isaindigotone
derivatives, and their inhibitory activities for cholinesterases
(ChEs) and self-induced b-amyloid (Ab) aggregation were investi-
gated. It has been reported that the substitution of a chlorine atom
at position 6 of tacrine and related compounds strongly increased
the binding affinity of tacrine with AChE (d and e, Fig. 1).21,22 Thus,
we attached a chlorine atom at the similar position of our deoxyva-
sicinone moiety, in order to figure out whether the chlorine atom
on the deoxyvasicinone moiety has the similar effect as the chlo-
rine atom on tacrine in AChE inhibition. We also replaced the ether
linkage of our previous compounds with N-phenylalkanamide or
N-alkylbenzamide bond in order to increase their hydrogen bond-
ing interaction. Our previous studies have shown that expanding
the aliphatic ring size from five to six would resulted in decreased
planarity of the aromatic core,23 therefore, in the present study we
varied the size of the aliphatic ring to investigate the effect of the
planarity and linearity of the chromophore on their interactions
with ChEs and Ab. Molecular docking studies were carried out to
study the binding mode and selectivity of these compounds for
AChE, and the structure–activity relationships of these compounds
were further investigated and discussed.
NOESY experiments of compounds (3a–3d) showed the correlation
between H-1 and H-2/H-20, thus the double bond was assigned as
the E configuration.
2.2. Inhibition studies of AChE and BuChE
The inhibitory activity of our synthetic compounds against
AChE (from electric eel) and BuChE (from equine serum) was evalu-
ated using the method of Ellman et al.,26 with tacrine as a positive
control. Their IC50 values and selectivity index for AChE over BuChE
were summarized as shown in Table 1. Most of our compounds
showed selective inhibition of AChE over BuChE, with IC50 values
at micro or nano molar range. Obviously, the series 6 compounds
with five-member aliphatic ring in the skeleton showed preferable
inhibition and selectivity for AChE, suggesting that the more planar
and linear chromophore was favored for occupying the PAS of
AChE. The size of the aliphatic ring could impact the planarity
and linearity of the skeleton, which may also adjust the orientation
and rotation of the benzylidene moiety. We found that the com-
pounds with various terminal amine groups in their side chains
showed slightly different activity, and compound 6e with piperidyl
group exhibited optimal activity.
Comparing to the series 6 with the N-phenylalkanamides side
chains, most of the series 7 compounds with the N-alkylbenza-
mides moiety exhibited decreased AChE inhibitory activity, except
compound 7a. These results indicated that electron-withdrawing
effect of the N-alkylbenzamide bond may reduce the electronic
2. Results and discussion
2.1. Chemistry
density of the chromophore, and consequently, diminish the
p–p
stacking interaction between the chromophore and the PAS of
AChE. On the other hand, some of series 7 compounds showed bet-
ter BuChE inhibitory activity than series 6 compounds, such as
compounds 7c and 7g. For BuChE, the peripheral site is surrounded
with aliphatic amino acids instead of large aromatic residues for
AChE, thus, the BuChE inhibition was weakly influenced by the
reduction of electronic density of the chromophore.
Chloro-isaindigotone derivatives were facilely prepared accord-
ing to the synthetic route as shown in Scheme 1. Most of the inter-
mediates were prepared following the procedures reported in our
previous work.23,24 The synthesis began with commercially avail-
able 2-amino- 4-chlorobenzoic acid, which was dissolved in tolu-
ene and heated with pyrrolidin-2-one or piperidin-2-one in the
presence of POCl3. The intermediates (2a, 2b) were reacted with
4-nitrobenzaldehyde through Claisen–Schmidt condensation to
provide the nitro intermediates (3a, 3b) at high yield.25 The reduc-
tion of the nitro intermediates with sodium sulfide gave the amino
intermediates (4a, 4b), which were subsequently treated with acyl
chloride to give the chloro intermediates (5a, 5b). Their further
amination with various alkylamines produced the desired com-
pounds 6a–6h. The carboxyl intermediates (3c, 3d) were obtained
through the Claisen–Schmidt condensation of the intermediates
(2a, 2b) with 4-formylbenzoic acid. The condensation of the car-
boxyl intermediates (3c, 3d) with various linear aliphatic amines
catalyzed with BOP in DMF, gave the desired compounds 7a–7h.
2.3. Molecular modeling and kinetic studies
In order to study the binding mode and selectivity of our com-
pounds with two cholinesterases, molecular modeling was carried
out using the docking program AUTODOCK 4.0 package,27 and the
results were shown in Figure 2 with MOE 2008.10 software
package.
Compound 6c, with strong inhibitory activity and high selectiv-
ity to AChE, exhibited multiple binding modes with AChE, which
were in agreement with those of our previous isaindigotone
derivatives. The deoxyvasicinone moiety adopted an appropriate

J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
2529
O
O
N
COOH
NH2
i
ii
m
N
N
1
m
R2
CHO
Cl
Cl
Cl
N
2'
2a
2b
R2
1
m = 1;
m = 2
2
3a
3b
3c
3d
m = 1, R2 = NO2;
m = 2, R2 = NO2;
m = 1, R2 = COOH;
m = 2, R2 = COOH
O
N
N
O
N
N
iii
iv
m
m
Cl
Cl
NH2
NO2
3a m = 1; 3b m = 2
4a m = 1; 4b m = 2
O
O
N
N
v
m
m
Cl
N
Cl
N
O
O
R1
2
N
N
H
Cl
H
5a m = 1; 5b m = 2
6a
6b
6c
6d
6e
6f
m = 1, R1 = Pyridyl-
m = 2, R1 = Pyridyl-
m = 1, R1 = Pyrrolidino-
m = 1, R1 = Diethylamino-
m = 1, R1 = Piperidino-
m = 2, R1 = Pyrrolidino-
m = 2, R1 = Diethylamino-
m = 2, R1 = Piperidino-
6g
6h
O
N
O
N
vi
m
m
Cl
N
Cl
N
H
N
R1
n
COOH
O
7a m = 1, n = 2, R1 = Pyrrolidino-
3c m = 1; 3d m = 2
7b m = 1, n = 2, R1 = Dimethylamino-
7c m = 1, n = 3, R1 = Pyrrolidino-
7d m = 1, n = 3, R1 = Dimethylamino-
7e m = 2, n = 2, R1 = Pyrrolidino-
7f m = 2, n = 2, R1 = Dimethylamino-
7g m = 2, n = 3, R1 = Pyrrolidino-
7h m = 2, n = 3, R1 = Dimethylamino-
Scheme 1. Synthetic route for isaindigotone derivatives. Reagents and conditions: (i) toluene, POCl3, 2-pyrrolidinone or piperidin-2-one, reflux; (ii) AcOH, AcONa, reflux; (iii)
NaOH, Na2Sꢁ9H2O, EtOH, reflux; (iv) CH2Cl2, 3-chloropropionyl chloride, reflux; (v) R1H, KI, EtOH, reflux; (vi) NH2(CH2)nR1, BOP, triethylamine, DMF, 70 °C.
orientation for its binding to PAS, via the
p
–
p
stacking interaction
Meanwhile, the calculated binding free energy of compound 6c
with AChE is much lower than that with HuBuChE (ꢀ12.74 and
ꢀ10.48 Kcal/mol, respectively), thus, compound 6c exhibited excel-
lent selectivity for the inhibition of AChE over BuChE.
We also performed the docking studies of compound 7c with
AChE (shown in Supplementary data). From the docking result,
with Trp279, and the ring-to-ring distance was 3.8 Å. But this pre-
ferred binding mode was different with that of 6-chlorotacrine,
which interacted with CAS, and no significant interaction between
chlorine atom of the deoxyvasicinone moiety with the residues of
PAS was found. The binding affinity can be attributed to the
p–p
stacking interaction between the benzylidene moiety and Phe331
with a distance of 3.8 Å. The conformation of the side chain could
fit well with the shape of the gorge. The positively-charged nitrogen
only the cation–p interaction between positively-charged nitrogen
of pyrrolidine and Trp84 was observed, and no stacking interaction
between the chromophore and the PAS of AChE was found. This
result supported that the electron-withdrawing effect of the N-
of pyrrolidine made a cation–p interaction (4.5 Å) with Trp84, and
the hydrogen on the nitrogen of pyrrolidine was hydrogen-bonded
with Glu199 with a distance of 2.9 Å. These results may rationalize
the potent inhibition of compound 6c for AChE. On the other hand,
compound 6c could also occupy the large catalytic cavity of Hu-
BuChE, but without strong interactions except a hydrogen bond be-
tween Glu197 and the hydrogen on the nitrogen of pyrrolidine.
alkylbenzamide bond may reduce the p–p stacking interaction
between the chromophore and the PAS of AChE.
The nature of AChE inhibition of compound 6c was further
investigated using graphical analysis of steady-state inhibition
data.28 As shown in Figure 3, a mixed type of inhibition was con-
firmed, which indicated that compound 6c could bind to both

2530
J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
Table 1
Inhibition IC50 values and selectivity index of isaindigotone derivatives on AChE and BuChE
Compound
R1
n
m
IC50 (nM)
Selectivityc for AChE
AChEa
49
BuChEb
6a
6b
—
—
1
2
7
1700 490
3380 270
35
4.12
N
N
N
N
N
820 350
6c
6f
—
—
1
2
41 14
>1000
3820 260
6450 680
93
—
6d
6g
—
—
1
2
60
1
6440 900
2340 410
107
—
>1000
6e
6h
—
—
1
2
23
31
3
1
2880 340
5990 110
130
190
7a
7c
7e
7g
2
3
2
3
1
1
2
2
45
7
11060 830
610 120
6750 2300
500 80
250
0.42
4.5
0.4
1440 730
1510 290
1210 140
7b
7d
2
3
1
1
160
1420 540
0
>20000
5180 260
>125
3.6
N
7f
7h
2
3
2
2
360 70
2050 280
>40000
6640 730
>111
3.2
Tacrine
96
2
14
1
0.15
a
b
c
Inhibitor concentration (mean _ SEM of three experiments) required for 50% inactivation of AChE.
Inhibitor concentration (mean _ SEM of three experiments) required for 50% inactivation of BuChE.
Selectivity for AChE: IC50 (BuChE)/IC50 (AChE).
PAS and CAS of AChE. This result was in agreement with the results
of molecular modeling studies.
incubated with compound 6c. This EM experimental result showed
that compound 6c could inhibit the Ab1–40 fibrils formation, which
was in agreement with the results from ThT and CD studies.
2.4. Inhibition of self-mediated Ab1–40 aggregation
3. Conclusion
To investigate the activity of our compounds to inhibit the self-
mediated Ab1–40 aggregation, the Thioflavin T (ThT) fluorescence
assay was performed.29 As shown in Table 2, most of our com-
pounds showed better inhibitory activity of Ab1–40 aggregation at
In summary, two new series of chloro-substituted isaindigotone
derivatives and analogues were synthesized as dual inhibitors for
AChE and amyloid beta aggregation. Some of these compounds
were found to have improved inhibitory activity and selectivity
for AChE compared with the isaindigotone derivatives developed
previously in our group. The structure–activity relationship studies
revealed that the compounds containing the five-member aliphatic
ring had higher inhibition activity on AChE and much higher selec-
tivity for AChE over BuChE, compared to the compounds with the
six-member aliphatic ring. Most compounds showed higher self-
induced Ab aggregation inhibitory activity than a reference com-
pound curcumin. Further investigation of compound 6c through
CD and EM experiments confirmed that compound 6c could reduce
or inhibit b-sheet aggregation and fibril formation. Our present re-
sults indicated that isaindigotone derivatives could become lead
compounds for further development for new anti-Alzheimer
agents.
10 lM compared to the reference compound curcumin. Interest-
ingly, compound 6c with excellent inhibitory activity and high
selectivity to AChE, also gave the optimal inhibition of Ab1–40
aggregation (62.31%) at 10 lM, which indicated that our com-
pounds were dual inhibitors for AChE and Ab aggregation.
2.5. Effect of compound 6c on Ab b-sheet formation
It has been reported that Ab comprises a conformational mix-
ture of
a-helix, b-sheet, and random coil in the aqueous solution,
and forms intramolecular b-sheet structure in the fibrillation
through a conformational change.30 CD spectroscopy was em-
ployed to study the effect of compound 6c on the structural change
of Ab1–40. As shown in Figure 4, there was no aggregation for Ab1–40
(black and red line) before incubation. After 3 days incubation, the
CD spectrum of Ab1–40 alone (blue line) was found to have a nega-
tive band around 217 nm and positive band around 197 nm, which
4. Experimental section
4.1. Chemistry
were assigned to
a-helix structure and b-sheet structure, respec-
tively. Notably, the addition of compound 6c (green line) led to sig-
nificant decrease of the band around 197 nm, with slight change to
the intensity of the band around 217 nm. These results revealed
that compound 6c could reduce the b-sheet structure formation,
1H and 13C NMR spectra were recorded using TMS as the inter-
nal standard in CDCl3 or DMSO-d6 with a Bruker BioSpin GmbH
spectrometer at 400 MHz and 100 MHz, respectively. High resolu-
tion mass spectra (HRMS) were recorded on Shimadzu LCMS-IT-
TOF. Flash column chromatography was performed with silica gel
(200–300 mesh) purchased from Qingdao Haiyang Chemical Co.
Ltd. The purity of synthesized compounds was confirmed to be
higher than 95% through analytical HPLC performed with a dual
pump Shimadzu LC-20AB system equipped with an Ultimate XB-
without significant effect on the content of a-helix structure.
2.6. Effect of compound 6c on abundance of Ab fibrils
Finally, electron microscopy (EM) was employed to monitor and
clarify the effect of compound 6c on Ab aggregation. As shown in
Figure 5, the control sample of Ab1–40 alone was mostly aggregated
into mature and bulky amyloid fibrils after 4 days of incubation. In
contrast, only a few short fibrils were found for the Ab1–40 sample
C18 column (4.6 ꢂ 250 mm, 5
lm) and eluted with methanol/
water (40:60 to 60:40) containing 0.1% TFA at a flow rate of
0.5 mL/min. All chemicals were purchased from commercial

J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
2531
Figure 2. Docking models of compound 6c with TcAChE (A) and HuBuChE (B) generated with MOE.
sources unless otherwise specified. All the solvents were of analyt-
ical reagent grade and were used without further purification.
J = 8.5, 2.0 Hz, 1H), 4.20 (t, J = 8.0 Hz, 2H), 3.18 (t, J = 8.0 Hz, 2H),
2.35–2.25 (m,2H). LC-MS m/z: 221 [M+H]+.
4.2. Synthesis of intermediate 2a–2b
4.2.2. 3-Chloro-8,9-dihydro-6H-pyrido[2,1-b]quinazolin-11(7H)-
one (2b)
4.2.1. 6-Chloro-2,3-dihydropyrrolo[2,1-b]quinazolin-9(1H)-one
(2a)
Following the method for preparation of 2a, 2-pyrrolidinone
was replaced with piperidin-2-one, and a white solid 2b was ob-
tained (2.98 g, 64.4%): 1H NMR (400 MHz, CDCl3): d 8.18 (d,
J = 8.6 Hz, 1H), 7.59 (d, J = 1.9 Hz, 1H), 7.37 (dd, J = 8.6, 2.0 Hz,
1H), 4.06 (t, J = 6.2 Hz, 2H), 2.99 (t, J = 6.7 Hz, 2H), 2.06–1.91 (m,
4H). LC-MS m/z: 235 [M+H]+.
To a solution of 2-pyrrolidinone (6.01 g, 60 mmol) and 2-amino-
4-chlorobenzoic acid 1 (3.36 g, 20 mmol) in 50 mL anhydrous tolu-
ene, phosphorus oxychloride (11 mL) was added dropwise at room
temperature. The mixture was then stirred under reflux for 5 h.
After concentrating to give a slurry, the residue was poured onto
ice, and then aqueous NaOH was added to make the solution basic.
The mixture was extracted with three 100 mL portions of EtOAc.
The combine organic phase was washed with 80 mL water, dried
over magnesium sulfate, and concentrated under reduced pres-
sure. The crude product was purified by using flash column chro-
matography with EtOAc/petroleum ether (1:4) elution to afford a
white solid compound 2a (2.91 g, 63.0%): 1H NMR (400 MHz,
CDCl3): d 8.19 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.39 (dd,
4.3. General procedures for the preparation of compounds (3a–
3d)
A mixture of 2a/2b (10 mmol), different aromatic aldehyde
(11 mmol) and AcOH (30 mL), sodium acetate (8 mmol) was
heated under reflux for 10–24 h. After cooling to room tempera-
ture, the mixture was filtered and washed with water, CH2Cl2,
and EtOH, to give desired compounds.

2532
J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
Figure 4. CD spectroscopy of Ab1–40 alone and with compound 6c incubated for 0 or
3 days.
7.74 (s, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.5 Hz, 1H), 4.04
(t, J = 6.0 Hz, 2H), 2.91 (t, J = 5.4 Hz, 2H), 2.01–1.93 (m, 2H). LC-
MS m/z: 367 [M+H]+.
Figure 3. Lineweaver-Burk plot for the inhibition of AChE by compound 6c.
4.3.1. (E)-6-chloro-3-(4-nitrobenzylidene)-2,3-dihydropyrrolo
[2,1-b]quinazolin-9(1H)-one (3a)
Intermediate 2a was treated with 4-nitrobenzaldehyde accord-
ing to general procedure to give the desired product 3a as pale yel-
low solid (2.37 g, 67.1%). 1H NMR (400 MHz, DMSO-d6): d 8.30 (d,
J = 8.6 Hz, 2H), 8.15 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.6 Hz, 2H),
7.83 (s, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.55 (dd, J = 8.2, 1.7 Hz, 1H),
4.21 (t, J = 7.0 Hz, 2H), 3.38–3.32 (m, 2H).. LC-MS m/z: 354 [M+H]+.
4.4. Synthesis of intermediate 4a–4b
4.4.1. (E)-3-(4-Aminobenzylidene)-6-chloro-2,3-dihydropyrrolo
[2,1-b]quinazolin-9(1H)-one (4a)
To a stirred suspension of 3a (3.5 g,10 mmol) in ethanol (50 mL)
was added a solution of Na2S.9H2O (12 g, 50 mmol) and NaOH (8 g,
200 mmol) in water (100 mL). The mixture was heated at 90 °C for
8 h. The ethanol was removed under vacuo. After cooling to room
temperature, the precipitate was collected through filtration,
repeatedly washed with water and ethanol, and dried, to afford
the product 4a (2.05 g, 64.3%). 1H NMR (400 MHz, DMSO-d6): d
8.07 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.57 (t, J = 2.3 Hz,
1H), 7.42 (dd, J = 8.5, 2.0 Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.65 (d,
J = 8.5 Hz, 2H), 5.79 (s, 2H), 4.14 (t, J = 6.0 Hz, 2H), 3.21–3.11 (m,
2H). LC-MS m/z: 324 [M+H]+.
4.3.2. (E)-3-Chloro-6-(4-nitrobenzylidene)-8,9-dihydro-6H-
pyrido[2,1-b]quinazolin-11(7H)-one (3b)
Intermediate 2b was treated with 4-nitrobenzaldehyde accord-
ing to general procedure to give the desired product 3b as pale yel-
low solid (2.35 g, 63.9%). 1H NMR (400 MHz, CDCl3): d 8.32–8.27
(m, 3H), 8.22 (d, J = 8.5 Hz, 1H), 7.74 (d, J = 1.8 Hz, 1H), 7.62 (d,
J = 8.5 Hz, 2H), 7.42 (dd, J = 8.6, 2.0 Hz, 1H), 4.18 (t, J = 8.0 Hz,
2H), 2.99–2.90 (m, 2H), 2.12–2.03 (m, 2H). LC-MS m/z: 368 [M+H]+.
4.3.3. (E)-4-((6-Chloro-9-oxo-1,2-dihydropyrrolo[2,1-b]
quinazolin-3(9H)-ylidene)methyl) benzoic acid (3c)
4.4.2. (E)-6-(4-Aminobenzylidene)-3-chloro-8,9-dihydro-6H-
pyrido[2,1-b]quinazolin-11(7H)-one (4b)
Intermediate 2a was treated with 4-formylbenzoic acid accord-
ing to general procedure to give the desired product 3c as pale yel-
low solid (2.31 g, 65.6%). 1H NMR (400 MHz, DMSO-d6): d 13.14 (s,
1H), 8.13 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.83–7.73 (m,
4H), 7.53 (dd, J = 8.5, 1.8 Hz, 1H), 4.19 (t, J = 7.0 Hz, 2H), 3.38–
3.29 (m, 3H). LC-MS m/z: 353 [M+H]+.
Following the method for preparation of 4a, a red solid 4b
(2.23 g, 66.1%) was obtained. 1H NMR (400 MHz, CDCl3): d 8.22–
8.10 (m, 2H), 7.69 (d, J = 1.7 Hz, 1H), 7.41–7.30 (m, 3H), 6.71 (d,
J = 8.4 Hz, 2H), 4.14 (t, J = 8.0 Hz, 2H), 3.91 (s, 2H), 2.95 (t,
J = 6.3 Hz, 2H), 2.11–1.95 (m, 2H). LC-MS m/z: 338 [M+H]+.
4.5. Synthesis of intermediate (5a–5b)
4.3.4. (E)-4-((3-Chloro-11-oxo-7,8,9,11-tetrahydro-6H-pyrido
[2,1-b]quinazolin-6-ylidene) methyl) benzoic acid (3d)
4.5.1. (E)-3-Chloro-N-(4-((6-chloro-9-oxo-1,2-
Intermediate 2b was treated with 4-formylbenzoic acid accord-
ing to general procedure to give the desired product 3d as yellow
solid (2.45 g, 66.8%). 1H NMR (400 MHz, DMSO-d6): d 13.02 (s,
1H), 8.20 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 8.01 (d, J = 8.1 Hz, 2H),
dihydropyrrolo[2,1-b]quinazolin-3(9H)-ylidene)
methyl)phenyl)propanamide (5a)
To a stirred suspension of 4a (0.96 g, 3 mmol) in CH2Cl2 (20 mL),
3-chloropropanoyl chloride (1 mL, 12 mmol) was added. The
Table 2
Inhibition of self-induced Ab1–40 aggregation by isaindigotone derivatives with curcumin as a reference
Compound
Inhibition ratio (%)
Compound
Inhibition ratio (%)
Compound
Inhibition ratio (%)
Compound
Inhibition ratio (%)
6a
6b
6c
6d
49/96 4.45
32.96 2.55
62.31 0.28
39.00 1.42
48.76 0.83
6e
6f
6g
6h
49.80 4.86
53.46 2.03
54.76 3.97
52.94 3.70
7a
7b
7c
7d
57.39 2.13
36.84 6.76
36.25 2.65
56.46 1.60
7e
7f
7g
7h
53.60 3.57
54.47 3.44
53.28 1.78
49.75 3.60
Curcumin
The ThT method was used, the mean SD of three independent experiments and the measurements were carried out in presence of 10 lM compounds.

J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
2533
Figure 5. EM images of Ab1–40 (100 lM) in the absence (left) and presence (right) of 10 lM compound 6c, after 4 days of incubation.
mixture was then heated under reflux for 6 h. After cooling to room
temperature, the precipitate was separated from solvent through
filtration and washed with ethanol to afford a yellow solid 5a
(1.13 g, 90.9%). 1H NMR (400 MHz, DMSO-d6): d 10.40 (s, 1H),
8.12 (d, J = 8.5 Hz, 1H), 7.79–7.69 (m, 4H), 7.64 (d, J = 8.4 Hz, 2H),
7.50 (d, J = 7.0 Hz, 1H), 4.18 (t, J = 8.0 Hz, 2H), 3.90 (t, J = 6.0 Hz,
2H), 3.27 (t, J = 6.0 Hz, 2H), 2.88 (t, J = 6.0 Hz, 2H). LC-MS m/z:
414 [M+H]+.
DMF(10 ml) was heated at 70 °C for 12–20 h. After cooling to room
temperature, the mixture was filtered. The filtrate was washed
with water and methanol, to give desired products 7a–7h.
4.8. Biological assay
4.8.1. In vitro AChE and BuChE inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acet-
ylthiocholine chloride (ATC), butyrylthiocholine chloride (BTC)
were purchased from Sigma–Aldrich. Isaindigotone derivatives
were dissolved in DMSO and then diluted in 0.1 M KH2PO4/
K2HPO4 buffer (pH 8.0) to provide a final concentration range.
All the assays were carried out in 0.1 M KH2PO4/K2HPO4 buffer,
pH 8.0, using a Shimadzu 2450 Spectrophotometer. Enzyme solu-
tions were prepared at the concentration of 2.0 unit/mL. The assay
medium contained phosphate buffer (pH 8.0), 0.01 M DTNB, AChE,
and 0.01 M substrate (ATC). The substrate was added to the med-
ium with inhibitor after incubation for 15 min. The activity was
determined by measuring the increase in absorbance at 412 nm
in 1 min interval at 37 °C. The data were calculated based on the
method of Ellman et al. In vitro BuChE assays were carried out
using a similar method as described above.26
4.5.2. (E)-4-Chloro-N-(4-((3-chloro-11-oxo-7,8,9,11-tetrahydro-
6H-pyrido[2,1-b]quinazolin-6-
ylidene)methyl)phenyl)butanamide (5b)
Following the method for preparation of 5a, a yellow solid 5b
(1.07 g, 83.3%) was obtained. 1H NMR (400 MHz, DMSO-d6): d
10.34 (s, 1H), 8.16–8.07 (m, 2H), 7.79–7.69 (m, 3H), 7.55 (d,
J = 8.4 Hz, 2H), 7.49 (dd, J = 8.6, 1.3 Hz, 1H), 4.05 (t, J = 6.0 Hz,
2H), 3.90 (t, J = 6.2 Hz, 2H), 2.92 (t, J = 5.8 Hz, 2H), 2.87 (t,
J = 6.2 Hz, 2H), 2.03–1.90 (m, 2H). LC-MS m/z: 428 [M+H]+.
4.6. General procedure for the preparation of compounds (6a–
6h)
To a stirred suspension of 5a/5b (0.25 g, 0.5 mmol) and KI
(0.05 g) in EtOH (7 mL), different amine (2.5 mmol) was added
dropwise. The mixture was stirred under reflux for 3–6 h. After cool-
ing to 0 °C, the precipitate was collected through filtration and
washed with water and ethanol, to give desired products 6a–6h.
4.8.2. Molecular modeling studies
The crystal structure of the torpedo acetylcholinesterase com-
plexed with tacrine (code ID: 1ACJ) and the human butyrylcholin-
esterase complexed with echothiophate (code ID: 1POI) were
obtained from the Protein Data Bank after removing the inhibitor
and water molecules. The 3D structure of compound 6c was built,
and its geometry optimization was performed with molecular
mechanics. Further preparation of substrates included addition of
Gasteiger charges, removal of hydrogen atoms and addition of their
atomic charges to skeleton atoms, and finally, assignment of prop-
er atomic types. Autotors was then used to define the rotatable
bonds in the ligand.
Docking studies were carried out using the AUTODOCK 4.0 pro-
gram. By using ADT, polar hydrogen atoms were added to amino
acid residues and Gasteiger charges were assigned to all atoms of
the enzyme. The resulting enzyme structure was used as an input
for the AUTOGRID program. AUTOGRID performed a pre-calculated
atomic affinity grid maps for each atom type in the ligand plus an
electrostatics map and a separate desolvation map present in the
substrate molecule. All maps were calculated with 0.375 Å spacing
between grid points. Flexible ligand docking was performed for the
compound. Docking calculations were carried out using the
4.6.1. (E)-N-(4-((6-Chloro-9-oxo-1,2-dihydropyrrolo[2,1-
b]quinazolin-3(9H)-ylidene)methyl) phenyl)-3-(pyrrolidin-1-
yl)propanamide (6c)
Intermediate 5a was reacted with pyrrolidine following the
general procedure to give the desired product 6c as an orange solid
with a yield of 66.8%. Mp 237–240 °C. 1H NMR (400 MHz, CDCl3): d
11.62 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.79 (s, 1H), 7.73 (d, J = 1.6 Hz,
1H), 7.59 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.37 (dd, J = 8.5,
1.7 Hz, 1H), 4.29 (t, J = 7.3 Hz, 2H), 3.29 (t, J = 6.0 Hz, 2H), 2.88 (t,
J = 8.0 Hz, 2H), 2.77–2.65 (m, 4H), 2.57 (t, J = 6.0 Hz, 2H), 1.99–
1.86 (m, 4H). 13C NMR (100 MHz, CDCl3): d 171.03, 160.67,
150.99, 150.89, 140.32, 139.89, 131.17, 130.88, 130.53, 129.42,
127.75, 126.70, 126.44, 119.66, 119.29, 53.10, 51.26, 44.10, 34.65,
25.47, 23.75. Purity: 98.8% by HPLC. LC-MS m/z: 449 [M+H]+. HRMS
(ESI): calcd for (M+H)+ (C25H25N4O2Cl) 449.1739, found 449.1731.
4.7. General procedure for preparation of compounds (7a–7h)
A mixture of 3c/3d (1 mmol), BOP (0.66 g, 1.5 mmol), triethyl-
amine (0.28 ml, 2 mmol), different amine (2 mmol) and

2534
J.-W. Yan et al. / Bioorg. Med. Chem. 20 (2012) 2527–2534
Lamarckian genetic algorithm (LGA), and all parameters were the
same for each docking. The results were shown with Molecular
Operating Environment (MOE) program (Chemical Computing
Group, Montreal, Canada).
the International S&T Cooperation Program of China (No.
2010DFA34630).
Supplementary data
Supplementary data (the preparation of the compounds, 1H
NMR, 13C NMR, HPLC and HRMS spectra of target compounds
(6a–6h, 7a–7h), NOSEY spectra of compounds (3a–3d), and dock-
ing result of compound 7c with AChE) associated with this article
can be found, in the online version, at doi:10.1016/j.bmc.
2012.02.061.
4.8.3. Kinetic studies of AChE inhibition
Kinetic characterization of AChE was performed using a re-
ported method.28 Compound 6c was added into the assay solution
and pre-incubated with the enzyme at 37 °C for 15 min, followed
by the addition of ATC. The assay solution (200
compound 6c (20, 40, 80 nM), DTNB (0.5 mM), 10
l
L) containing
lL AChE and
ATC (0.03, 0.1, 0.2, 0.3, 0.4 mM) dissolved in 0.1 M KH2PO4/
K2HPO4 buffer (pH 8.0). Kinetic characterization for the hydrolysis
of ATC catalyzed by AChE was carried out spectrometrically at
412 nm. The parallel control experiment was carried out without
compound 6c in the mixture.
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l
l
l
NaOH buffer, pH 8.0) was added. Fluorescence was measured at
450 nm (kex) and 485 nm (kem). Each compound was examined in
triplicate. The fluorescence intensities were recorded, and the per-
centage of inhibition on aggregation was calculated with the fol-
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Ab1–40 peptide (Anaspec Inc) was dissolved in 0.1 M phosphate
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lM, respectively. After 4 days incubation, aliquots
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lL samples were placed on carbon-coated copper/rhodium
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Acknowledgements
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This work was supported by National Natural Science
Foundation of China (No. 90813011, 21172272, 81001400),