Synthesis and evaluation of novel rutaecarpine derivatives and related alkaloids derivatives as selective acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2009.12.044

A series of novel rutaecarpine derivatives and related alkaloid derivatives 3-aminoalkanamido-substituted rutaecarpine 4a-f and 7,8-dehydrorutaecarpine 5a-c, and 6-aminoalkanamido-substituted 3-[2-(3-Indolyl)ethyl]-4(3a)-quinazolinones 8a-c, were synthesized and subjected to pharmacological evaluation as acetylcholinesterase (AChE) inhibitors. The synthetic compounds exhibited strong inhibitory activity for AChE and high selectivity for AChE over BuChE. The structure-activity relationships were discussed and their binding conformation and simultaneous interactions mode were further clarified by kinetic characterization and the molecular docking studies.

Full text of DOI:10.1016/j.ejmech.2009.12.044

European Journal of Medicinal Chemistry 45 (2010) 1415–1423
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of novel rutaecarpine derivatives and related alkaloids
derivatives as selective acetylcholinesterase inhibitors
,
Bin Wang a b, Yi-Chi Mai a, Yue Li a, Jin-Qiang Hou a, Shi-Liang Huang a, Tian-Miao Ou a,
,
a,
*
Jia-Heng Tan a, Lin-Kun An a, Ding Li a, Lian-Quan Gu a , Zhi-Shu Huang
*
a School of Pharmaceutical Sciences, Sun Yat-sen University, Waihuan East Road 132, Guangzhou 510006, People’s Republic of China
b Department of Chemistry and Life Science, Xiangnan University, Chenzhou, Hunan 423000, People’s Republic of 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 novel rutaecarpine derivatives and related alkaloid derivatives 3-aminoalkanamido-
Received 7 October 2009
Received in revised form
9 December 2009
Accepted 18 December 2009
Available online 4 January 2010
substituted rutaecarpine 4a–f and 7,8-dehydrorutaecarpine 5a–c, and 6-aminoalkanamido-substituted
3-[2-(3-Indolyl)ethyl]-4(3a)-quinazolinones 8a–c, were synthesized and subjected to pharmacological
evaluation as acetylcholinesterase (AChE) inhibitors. The synthetic compounds exhibited strong inhibi-
tory activity for AChE and high selectivity for AChE over BuChE. The structure–activity relationships were
discussed and their binding conformation and simultaneous interactions mode were further clarified by
kinetic characterization and the molecular docking studies.
Keywords:
Ó 2009 Elsevier Masson SAS. All rights reserved.
Rutaecarpine derivatives
Acetylcholinesterase inhibitors
Synthesis
Selectivity
1. Introduction
Several compounds have been previously synthesized and deter-
mined as reversible and selective AChE inhibitors (AChEI) for
Alzheimer’s disease (AD) is an age-related chronic, neurode-
generative disorder characterized by progressive memory loss and
other cognitive impairments, which are thought to be related to the
degeneration of cholinergic neurons in the cerebral cortex and
subcortical structures [1]. Although many factors have been
implicated in AD, its etiology and pathogenesis remain unclear. The
cholinergic hypothesis, which postulates that at least some of the
cognitive decline experienced by AD patients results from a defi-
ciency in acetylcholine (ACh) and thus in cholinergic neurotrans-
mission, represents one of the most useful approaches involved in
the design of new agents for the treatment of AD [2]. This strategy is
based on the development of drugs with an acetylcholinesterase
(AChE) inhibition profile in order to rectify the deficiency of cere-
bral acetylcholine. Meanwhile, reversibility of inhibitors is an
important aspect in the design of AChE inhibitors in AD. Actually
reversible inhibitors are useful in AD, and irreversible inhibitors
would become nerve agents like organophosphorus type inhibitors.
symptomatic treatment of AD, such as donepezil, galantamine and
rivastigmine.
Cholinesterases constitute a family of enzymes that catalyze the
hydrolysis of the neurotransmitter acetylcholine into choline and
acetic acid. There are two types of cholinesterases including AChE
(EC 3.1.1.7) and BuChE (EC 3.1.1.8) in various tissues of body [3]. The
main function of AChE is terminating the impulse transmission at
cholinergic synapses rapid hydrolysis by AChE into ACh. Recent
studies have identified that AChE could also play a key role in
accelerating senile amyloid
b
-peptide (Ab) plaques deposition [4]. It
is likely that AChE interacted with (A
b
) and promoted amyloid fibril
formation through a pool of amino acids located in the proximity of
peripheral anionic site (PAS) [5].
However, the functions of BuChE are still not clear. There is some
evidence to suggest that BuChE activity may be involved in the
pathogenesis of Alzheimer’s disease [6]. This has led to the
hypothesis that the use of nonselective cholinesterase inhibitors
inhibit both BuChE and AChE, which may be more beneficial to
patients with Alzheimer’s disease than the use of selective
cholinesterase inhibitors that inhibit AChE alone [7]. However,
inhibition of BuChE, highly abundant outside the brain, may
contribute to the peripheral side effects of ChE inhibitors [8]. Dose-
response curves for tremor (central effect) and salivation (periph-
eral effect) showed that more selective AChE inhibitor donepezil
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE,
butyrylcholinesterases; Ab, amyloid b-peptide; PAS, peripheral anionic site; DHED,
dehydroevodiamine; SAR, structure–activity relationship.
* Corresponding author. Tel.: þ86 20 39949056; fax: þ86 20 39943056.
E-mail addresses: cesglq@mail.sysu.edu.cn (L.-Q. Gu), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.044

1416
B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
possessed a more favorable therapeutic index than tacrine, which
could inhibit both of AChE and BuChE inhibitor (Fig. 1) [9]. In
clinical treatment of AD, tacrine shows hepatotoxicity, significant
drug–drug interactions, and serious side effects, compared with
donepezil, rivastigmine, and galantamine [10]. In addition, it was
recently reported that BuChE activity was decreased in synapses of
AD patients in vivo [11]. All these findings indicated that drug
development would be better targeted to increasing AChE inhibi-
tion and high selectivity for AChE over BuChE.
The design strategy for AChE inhibitors is based on the study of
crystallographic structure of AChE. X-ray crystallography of the
AChE/inhibitor complexes indicated that AChE has a catalytic site at
the bottom of deep narrow gorge and a peripheral anionic site (PAS)
at the entrance. Simultaneous binding with the active site and PAS
has been suggest to be important in designing powerful and
selective AChE inhibitors [12,13].
compound 12. Elimination of CF3H was accomplished by treating 12
with alcoholic KOH to afford 1.
The intermediate 6-amino-3-[2-(3-indolyl)ethyl]-4(3a)-quina-
zolinon 6 was prepared as illustrated in Scheme 3. Treatment of
tryptamine with nitro isatoic anhydride in EtOH under reflux gave
13. Catalytic reduction of 13 with hydrogen in the presence of 10%
Pd/C gave the compound 14. The compound 14 was reacted with
refluxing triethyl orthoformate to afford 6.
The acylation of 1 with the appropriate acid halide gave the
haloalkanamide 2a–b. Compound 2a was treated with DDQ in
dioxane to give the haloalkanamide 7,8-dehydrorutaecarpine 3.
However, dehydrogenation of compound 2b with DDQ in dioxane
was failed, therefore the chain elongation was not performed for
compounds 5 (5d–f). Finally, the target compounds 4a–f and 5a–c
were obtained by aminolysis of the compounds 2a–b and 3 under
reflux by the treatment with the appropriate secondary amines. In
addition, 6-amino-alkanamido-substituted 3-[2-(3-indolyl)ethyl]-
(3a)-quinazolinones derivatives 8a–c were also synthesized
following similar procedures.
Rutaecarpine (Ru) is a major quinazolinocarboline alkaloid
isolated from Evodia rutaecarpaas shown in Fig. 1. It has been
reported to possess a wide spectrum of pharmacological activities,
such as vasodilation, antithrombosis, and anti-inflammation
[14–16]. It has also been recently reported that the rutaecarpine-
type alkaloid dehydroevodiamine (DHED) shows strong anti-
3. Results and discussion
amnesic activity in vivo with an IC50 value of 6.3 mM, combined with
a moderate AChE inhibition in vitro [17,18]. Based on the structural
information of AChE and AChE inhibitors, we designed the AChE
inhibitors that can interact simultaneously with both the catalytic
and peripheral sites (PAS) of AChE. The general structure designed
includes two components separated by a spacer group with a suit-
able length, with the aromatic ring binding with the PAS of AChE
and with the side chain terminal amino group interacting with the
catalytic site of AChE. Our introduction of a side chain with terminal
amino group in the rutaecarpine could greatly improve AChE
inhibitory activity and AChE/BuChE selectivity based on our initial
docking study.
The inhibitory activity of synthetic derivatives and the original
lead compound (Ru) was evaluated against AChE and BuChE using
tacrine as positive control. The IC50 values and selectivity index for
the inhibition of AChE and BuChE are summarized in Table 1. All
compounds were tested in at least five concentrations covering the
range producing less than 20% and greater than 80% inhibition, but
limited to 100 mM. Concentration inhibition curves were obtained
and their inhibitory concentration (IC50) calculated by nonlinear
regression. For comparison, tacrine was tested under the same
assay conditions, and gave IC50 values of 222.7 nM and 29.98 nM for
the inhibition of AChE and BuChE, respectively.
Based on our above strategy, a series of new 3-aminoalkanamido-
substituted rutaecarpine derivatives were designed and synthesized,
and their inhibitory activities for AChE and BuChE were tested
according to the modified Ellman method [19]. The structure–
activity relationships (SARs) were then discussed.
These results showed that all of synthetic derivatives gave higher
inhibitory activity against AChE and selectivity for AChE over BuChE
comparing with the original lead compound (Ru). Compound 5c was
the most potent for AChE inhibition, which presented an IC50 value
of 10.1 nM. It was indicated that the introduction of the amino-
alkanamido-substituted group could significantly increase the
inhibitory activity of derivatives. These side chains might be
protonated at physiological pH, thus they could occupy the anionic
2. Chemistry
binding site of quaternary amino group via cation–p interaction, and
further promote the simultaneous binding interaction of rutae-
carpine moieties with PAS of AChE.
The preparation of a series of rutaecarpine derivatives 4a–f,
5a–c and 8a–c was accomplished using the general methods out-
lined in Scheme 1. The key intermediates 3-amino-rutaecarpine 1
After comparing inhibitory activity of synthetic derivatives with
different rings (4a–c, 5a–c and 8a–c), we found that the
compounds with aromatic C ring (5a–c) showed a better activity,
which are approximately 5–11-fold stronger than those without
aromatic C ring (4a–c) (Table 1). Derivatives with opened C ring
(8a–c) led to a huge (more than 130-fold compared with 5a–c)
decrease in activity, indicating the aromatic planar moiety of
compounds could be in favor for interacting to the enzyme-binding
site, while the flexibility of molecules might be a disadvantageous
factor for binding affinity of compounds with enzyme.
and 6-amino-3-[2-(3-indolyl)ethyl]-4(3a)-quinazolinon
prepared according to the Bergman procedure [20], as shown in
Schemes 2 and 3.
The intermediate 3-amino-rutaecarpine 1 was synthesized as
outlined in Scheme 2. First, the nitro isatoic anhydride was
combined with tryptamine in pyridine containing trifluoroacetic
anhydride to give 10. Then cyclization of 10 provided 11 under
reflux in acidic conditions (HCl–AcOH) for 30 min. Treatment of the
11 with hydrogen in the presence of 10% Pd/C provided the
6 were
According to the screening data, the structure for terminal
groups of side chain also had effects on their inhibitory activities
(Table 1). The results were the same as those reported for oxoi-
soaporphine derivatives by our group [21,22]. The higher inhibitory
potency was found to be associated with piperidine at the end of
side chain (4c, 4f, 5c, and 8c). Diethylamine derivatives (4a, 4d, 5a,
and 8a) showed less inhibitory activity, which were approximately
3–6-fold lower than corresponding piperidine derivatives. These
results indicated that the semi-rigid side chain could be in favor of
their entering into the corresponding binding pocket of the
9
8
7
10
11
NH2
6
O
O
13
N
N
N
N
H
5
12
H
N
N
4
14
H3C
N
1
3
2
Tacrine
Ru
DHED
Fig. 1. Structures of donepezil, tacrine, dehydroevodiamine (DHED), rutaecarpine (Ru).

B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
1417
C
E
D
O
O
O
N
N
B
N
i
ii
N
H
N
N
H
H
N
N
N
O
O
A
R
Cl
n
N
H
n
N
H
NH2
1
2a n = 1; 2b n = 2
4a-c
4d-f
n =1;
n = 2
iii
E
C
D
O
N
B
O
N
iv
N
N
H
H
N
N
O
O
A
R
Cl
N
N
H
H
5a-c
3
E
3
4
D
O
O
N
O
N
N
B
vi
v
2
N
H
N
H
N
H
N
N
N
1
5
O
O
A
R
Cl
8
N
H
N
6
NH2
7
H
6
7
8a-c
Scheme 1. Synthesis of 3-aminoalkanamido-substituted rutaecarpine and 7,8-dehydro-rutaecarpine, 6-aminoalkanamido-substituted 3-[2-(3-Indolyl)ethyl]-4(3a)-quinazolinones
derivatives. Reagents and conditions: (i) ClCO(CH2)nCl, CH2Cl2, reflux; (ii) HNR2, EtOH, KI, reflux; (iii) DDQ, Dioxane, reflux; (iv) HNR2, EtOH, KI, reflux; (v) ClCOCH2Cl, CH2Cl2, reflux;
(vi) HNR2, EtOH, KI, reflux. (‘R-’ are shown in Table 1).
enzyme. In addition, the elongation of side chain (4d–f) signifi-
cantly increased AChE and BuChE inhibition.
reversible inhibition. Furthermore, the reversibility of the inhibi-
tion process was supported by dialysis of incubation mixture. After
dialysis, AChE activity was almost completely recovered (Fig. 2).
These results indicate that the inhibition is reversible.
The nature of AChE inhibition caused by the most potent
compound 5c was investigated by the graphical analysis of steady-
state inhibition data (Fig. 3). Reciprocal plots (Lineweaver–Burk
plots) describing 5c inhibition showed both increasing slopes and
increasing intercepts with higher inhibitor concentration. The
pattern indicated the mixed-type inhibition which was similar to
that of tacrine. The result revealed that compound 5c was able to
bind both of the active site and PAS of AChE, which was also in
agreement with the results of molecular modeling studies.
Most of the derivatives were found to be selective for AChE over
BuChE. Particularly, Compound 5c gave the highest inhibitory
activity and also the highest selectivity index (539-fold). The
derivatives with piperidine at the end of side chain gave more
significant selectivity than those with diethylamine or pyrrolidine.
Moreover, the selectivity of the derivatives was related with the
different backbone and the length of the side chain. The derivatives
of the backbone with aromatic C ring (5a–c) showed better selec-
tivity than non-aromatic C ring (4a–c), while the derivatives of
backbone with opened C ring (8a–c) were less selective.
The AChE inhibition caused by the most potent compound 5c
was investigated for its time-independence and dialysis after
incubation (Fig. 2). For compound 5c the AChE activity was moni-
tored during incubation with 5c. It was found that the inhibition
was not time-dependent, which exhibits a typical pattern of
To explore the possible binding conformation and interaction
mode of the compound with TcAChE (PDB code: 1EVE), a molecular
modeling study was performed using the docking program
AUTODOCK 4.0 package with PyMOL program as shown in Fig. 4
O
O
N
O
O2N
O2N
i
O
ii
iii
O
N
H
F3C
N
NO2
N
H
CF3
N
H
O
10
9
O
O
O
N
N
N
v
iv
N
N
N
H
H
H
NH
NH
N
F3C
F3C
NH2
NH2
NO2
1
12
11
Scheme 2. Synthesis of 3-amino-rutaecarpine 1 according to the Bergman procedure [19]. Reagents and conditions: (i) (CF3CO)2O, pyridine, 25 ꢀC, 30 min; (ii) tryptamine, 115 ꢀC,
3 h; (iii) HCI, AcOH, reflux, 1 h; (iv) 10% Pd/C, H2, MeOH, 4 h; (v) KOH, H2O, EtOH, reflux, 30 min.

1418
B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
Table 1
O
In vitro inhibition IC50 and selectivity index of derivatives 4a–f, 5a–c, and 8a–c on
AChE and BuChE.
HN
O2N
O
i
N
H
O
ii
Compound
R
n
AChE inhibitiona, BuChE
Selectivity
H2N
N
H
O
nM
inhibitionb, nM Indexc
Ru
4a
>100,000
>100,000
–
NO2
9
13
–N(CH2CH3)2
1
1
372.3 ꢁ 0.9
131.2 ꢁ 0.8
17,620 ꢁ 150
47.4
O
iii
N
HN
4b
696.1 ꢁ 1.1
5.3
N
O
N
H
N
H
N
H2N
NH2
4c
4d
4e
1
2
2
111.4 ꢁ 0.9
80.20 ꢁ 0.93
29.24 ꢁ 0.44
33,020 ꢁ 90
2848 ꢁ 6
297.5
35.5
28.9
N
NH2
6
14
–N(CH2CH3)2
N
Scheme 3. Synthesis of 6-amino-3-[2-(3-indolyl)ethyl]-4(3a)-quinazolinon 6 accord-
ing to the Bergman procedure [19]. Reagents and conditions: (i) tryptamine, EtOH,
reflux, 1 h; (ii) 10% Pd/C, H2, MeOH, 3 h; (iii) triethyl orthoformate, reflux, 6 h.
844.5 ꢁ 1.8
[23,24]. The most probable conformation of the ligand 5c was
chosen based on the docked energy value (Fig. 5). The docking
result demonstrated that the aromatic A ring in 5c could stack
against PAS residue Phe331 with the ring-to-ring distance being
4.1 Å, as shown in Fig. 3A. The conformation of the side chain made
good fit with the shape of the gorge, in the bottom of the gorge, the
4f
2
1
21.40 ꢁ 0.05
57.09 ꢁ 0.68
2112 ꢁ 2
98.7
N
5a
–N(CH2CH3)2
11,360 ꢁ 64
198.9
N
5b
1
23.56 ꢁ 0.13
428.2 ꢁ 0.5
18.2
charged nitrogen of piperidine made a cation–
p interaction with
the Trp84 and the distance was 4.0 Å. The simultaneous interac-
tions of 5c in the peripheral pocket and catalytic triad side of
TcAChE explained the higher inhibitory potency of AChE, however,
different interaction mode were found in 5c in complex with
N
5c
8a
1
1
10.07 ꢁ 0.32
7676 ꢁ 44
5429 ꢁ 38
539.1
5.4
–N(CH2CH3)2
N
41,620 ꢁ 140
HuBuChE (PDB code: 1POI), E ring in 5c could make
p–p stacking
8b
8c
1
1
4044 ꢁ 94
25,860 ꢁ 370
6.4
with the Trp82 in catalytic triade side and the distance was 4.5 Å.
Besides this, no other obvious interactions were observed between
5c and HuBuChE, (Fig. 5). As a result, the compound gave weaker
inhibitory activity for BuChE and exhibited higher selectivity for
AChE/BuChE. The interaction mode of other derivatives with AChE
and BuChE was similar to 5c. The calculated binding free energy of
derivatives in molecular docking was found to correlate well with
inhibition, as shown in Table 2.
N
1520 ꢁ 24
222.7 ꢁ 2.3
12,950 ꢁ 220
29.98 ꢁ 0.27
8.5
0.1
Tacrine
a
50% inhibitory concentration (means ꢁ SEM of at least four independent
experiments) of AChE from electric eel.
b
50% inhibitory concentration (means ꢁ SEM of at least four independent
experiments) of BuChE from equine serum.
c
Selectivity Index for AChE ¼ IC50 (BuChE)/IC50 (AChE).
4. Conclusion
3-Aminoalkanamido-substituted rutaecarpine (4a–f) and
7,8-dehydro-rutaecarpine (5a–c), as well as 6-aminoalkanamido-
substituted 3-[2-(3-indolyl)ethyl]-4(3a)-quinazolinones (8a–c)
derivatives were synthesized and subjected to pharmacological
evaluation. The results showed that the synthetic compounds
possessed high AChE inhibitory potency. The 3-aminoalkanamido-
substituted 7,8-dehydrorutaecarpine derivatives (5a–c) showed
higher inhibitory effects on AChE. The compounds with piperidine at
the end of side chain possessed higher inhibitory activity. In addition,
the elongation of side chain significantly increased AChE and BuChE
inhibition. Moreover, our synthetic compounds also showed high
selectivity for AChE over BuChE. The molecular docking, reversibility
and kinetic analysis revealed that the simultaneous interactions in
the central pocket, gorge, and peripheral pocket of TcAChE may
explain the high reversible inhibitory potency of AChE. Finally, our
results indicate that these new compounds represent useful
templates for the development of new anti-AD agents.
spectrometer at 400.132 MHz and 100.614 MHz, respectively; MS
spectra were recorded on a Shimadzu LCMS-2010A instrument
with an ESI or ACPI mass selective detector. Melting points (mp)
were determined using an SRS-OptiMelt automated melting point
instrument without correction. Elemental analysis was carried out
on an Elementar Vario EL CHNS Elemental Analyzer.
6-Nitro-1H-benzo[d][1,3]oxazine-2,4-dione
according to a literature procedure [25].
9 was prepared
5.1.1. 3-Amino-rutaecarpine (1)
A mixture of 3-amino-13b-(trifluoromethyl)-13b,l4-dihydror-
utaecarpine 12 (3.2 g) was added to a hot well-stirred solution of
KOH (4.0 g) in ethanol (50 mL) and water (15 mL). The reflux was
continued for 30 min, whereupon the mixture was cooled, the
precipitate was filtered off and was further purified by recrystalli-
zation from hot methanol to give the compound 1 as yellow brown
solid in 35% yield; m.p. 270–272 ꢀC; 1H NMR (400 MHz, DMSO-d6):
5. Experimental section
d
11.72 (s, 1H), 7.61 (d, 1H, J ¼ 7.9), 7.45 (dd, 2H, J ¼ 8.3, 4.2), 7.29
(s, 1H), 7.22 (t, 1H, J ¼ 7.5), 7.08 (dd, 2H, J ¼ 17.5, 9.1), 5.68 (s, 2H),
4.43 (t, 2H, J ¼ 6.6), 3.13 (t, 2H, J ¼ 6.6); HRMS (ESI): calcd for
(M þ H)þ (C18H14N4O) requires m/z 303.1246, found 303.1237. Anal.
Calcd for C18H14N4O: C, 71.51; H, 4.67; N, 18.53. Found: C, 71.37; H,
4.72; N, 18.33.
5.1. Chemistry
1H and 13C NMR spectra were recorded using TMS as the internal
standard in DMSO-d6 or CDCl3 with a Bruker BioSpin GmbH

B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
1419
110
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
90
70
50
Control
5c (5nM)
30
0
15
30
45
60
75
0
30
60
90
120
150
Time(min)
A
Time (min)
B
Fig. 2. Reversible inhibition of AChE by 5c. (A) Time-independence of AChE inhibition; (B) The recovery of AChE activity after dialysis.
5.1.2. 6-Amino-3-[2-(3-indolyl)ethyl]-4(3a)-quinazolinon (6)
A mixture of N-(2-(1H-indol-3-yl)ethyl)-2,5-diaminobenzamide
14 (3.2 g) was refluxed with triethyl orthoformate (45 mL) for 6 h,
whereupon the clear solution was concentrated to a syrup. The
mixture was carefully loaded on a silica gel column and chroma-
tographed using CH2Cl2/MeOH (10:1) as eluent to give the
compound 6 as pink solid in 31% yield; m.p. 206–207 ꢀC; 1H NMR
green solid in 77% yield; m.p. 281–283 ꢀC; 1H NMR (400 MHz,
DMSO-d6):
d
11.86 (s, 1H), 10.67 (s, 1H), 8.49 (d, 1H, J ¼ 2.1), 7.98
(dd, 1H, J ¼ 8.8, 2.3), 7.67 (dd, 2H, J ¼ 16.1, 8.4), 7.48 (d, 1H, J ¼ 8.3),
7.26 (t, 1H, J ¼ 7.7), 7.09 (t, 1H, J ¼ 7.5), 4.46 (t, 2H, J ¼ 6.8), 4.32 (s,
2H), 3.18 (t, 2H, J ¼ 6.8); HRMS (ESI): calcd for (M þ H)þ
(C20H15N4O2Cl) requires m/z 379.0962, found 379.0941. Anal. Calcd
for C20H15N4O2Cl: C, 63.41; H, 3.99; N, 14.79. Found: C, 63.28; H,
3.98; N, 14.50.
(400 MHz, DMSO-d6):
d 11.61 (s, 1H), 9.70 (s, 1H), 7.60 (s, 1H), 7.51
(s, 2H), 7.41 (d, 1H, J ¼ 8.6), 7.26 (s, 1H), 7.24 (s, 1H), 7.05 (s, 1H), 6.85
(t, 1H, J ¼ 7.3), 5.69 (s, 2H), 3.35 (s, 2H), 2.30 (s, 2H); HRMS (ESI):
calcd for (M þ H)þ (C18H16N4O) requires m/z 305.1402, found
305.1394. Anal. Calcd for C18H16N4O: C, 71.04; H, 5.30; N, 18.41.
Found: C, 70.09; H, 5.32; N, 18.46.
5.1.4. 3-(2-Chloro-propionamino)-rutaecarpine (2b)
The compound 1 was treated with 3-chloropropanoyl chloride
according to general acylation procedure to afford 2b as green solid
in 70% yield; m.p. 275–277 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d
11.84 (s, 1H), 10.44 (s, 1H), 8.51 (d, 1H, J ¼ 2.3), 8.00 (dd, 1H, J ¼ 8.8,
5.1.2.1. General acylation procedure. A solution of the appropriate
acid halide (4.8 mmol) in CH2Cl2 (5 mL) was added during to a well-
stirred mixture of the amino-substituted compounds 1 or 6,
(4 mmol) and K2CO3 (0.8 g) in CH2Cl2 (75 mL) at room temperature.
After completed addition the solution was finally refluxed for 4–6 h.
After cooling to 0–5 ꢀC, the precipitate formed was filtered off and
was further purified by flash column chromatography with petro-
leum ether/EtOAc (10:1) elution to give the compounds 2a–b or 7.
2.4), 7.66 (dd, 2H, J ¼ 14.1, 8.4), 7.49 (d, 1H, J ¼ 8.2), 7.26 (t, 1H,
J ¼ 7.5), 7.09 (t, 1H, J ¼ 7.4), 4.46 (t, 2H, J ¼ 6.8), 3.93 (t, 2H, J ¼ 6.2),
3.17 (t, 2H, J ¼ 6.8), 2.89 (t, 2H, J ¼ 6.2); HRMS (ESI): calcd for
(M þ H)þ (C21H17N4O2Cl) requires m/z 393.1118, found 393.1097.
Anal. Calcd for C21H17N4O2Cl: C, 64.21; H, 4.36; N, 14.26. Found: C,
64.04; H, 4.33; N, 13.98.
5.1.5. 6-(2-Chloro-acetamino)-3-[2-(3-indolyl)ethyl]-4(3a)-
quinazolinon (7)
5.1.3. 3-(2-Chloro-acetamino)-rutaecarpine (2a)
The compound 6 was treated with chloroacetyl chloride
The compound
1
was treated with chloroacetyl chloride
according to general acylation procedure to afford 7 as white-pink
according to general acylation procedure to afford 2a as yellow-
solid in 67% yield; m.p. 270–272 ꢀC; 1H NMR (400 MHz, DMSO-d6):
4
3
4
3
36
A
B
2
2
36
32
1
1
32
28
0
0
-1
28
-2
-1
1
2
3
-2
1
2
3
24
20
16
12
8
24
20
16
12
8
Control
5.0 nM
10.0 nM
15.0 nM
Control
2.5 µM
5.0 µM
7.5 µM
4
4
0
0
-4
0
4
8
12
16
20
24
28
32
-4
0
4
8
12
16
20
24
28
32
1/[S] 1/mM
1/[S] 1/mM
Fig. 3. Lineweaver–Burk plots of the inhibition kinetics of AChE (A) and BuChE (B) by compound 5c. Inserts were the enlarged plots.

1420
B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
Fig. 4. Docking models of compound–enzyme complex. Representations of compound 5c interacting with residues in the binding site of TcAChE (left) and HuBuChE (right). The
compounds are rendered in blue stick models, and the residues are rendered in golden sticks. Pictures are created with PyMOL [24].
d
10.84 (s,1H),10.72 (s,1H), 8.53 (s,1H), 8.00 (d,1H, J ¼ 1.9), 7.96–7.86
5.1.6.1. General aminolysis procedure. To
a
stirred refluxing
(m, 1H), 7.65–7.54 (m, 2H), 7.33 (d,1H, J ¼ 8.1), 7.11 (s, 1H), 7.06 (t, 1H,
J ¼ 7.5), 6.94 (d, 1H, J ¼ 14.8), 4.31 (d, 2H, J ¼ 1.0), 4.23 (t, 4H, J ¼ 7.2),
3.12 (t, 2H, J ¼ 7.1); HRMS (ESI): calcd for (M þ H)þ (C20H17N4O2Cl)
requires m/z 381.1118, found 381.1094. Anal. Calcd for C20H17N4O2Cl:
C, 63.08; H, 4.50; N, 14.71. Found: C, 63.28; H, 4.52; N, 14.75.
suspension of the haloalkanamido-substituted compounds 2a–b, 3
or 7 (1.5 mmol) and NaI (0.15 g) in EtOH (40 mL) was added
dropwise appropriate secondary amine (1.0 mL) in EtOH (10 mL).
The mixture was stirred at reflux for 3 h, cooled to 0 ꢀC, filtered, and
washed with ether and water, then evaporated under vacuum. The
crude solid was purified by chromatography with petroleum ether/
EtOAc (20:1) elution or crystallization from EtOH/DMF (5:1 v/v) to
afford 4a–f, 5a–c and 8a–c.
5.1.6. 3-(2-Chloro-acetamino)-7,8-dehydrorutaecarpine (3)
A solution of DDQ (0.54 g) in dioxane (10 mL) was added to a hot
solution of 2a (0.76 g) in dioxane (50 mL). After a reflux period 4 h
the precipitate formed was filtered and treated with a solution of
KOH (1.5 g) in water (25 mL). This procedure was repeated until all
DDQ-2H had been removed. The solid obtained was purified by
recrystallization from EtOH/DMF (5:1 v/v) to give the compound 3
as brown solid in 26% yield; m.p. 299–301 ꢀC; 1H NMR (400 MHz,
5.1.7. 3-(2-Diethylamino-acetamino)-rutaecarpine (4a)
The compound 2a was treated with excess diethylamine
according to general acylation procedure to afford 4a, after column
chromatography with petroleum ether/EtOAc (20:1) elution, as
gray solid in 52% yield; m.p. 271–272 ꢀC; 1H NMR (400 MHz, DMSO-
DMSO-d6):
d
12.68 (s, 1H), 10.69 (s, 1H), 8.71 (s, 1H), 8.61
d6):
d
11.83 (s, 1H), 10.02 (s, 1H), 8.54 (d, 1H, J ¼ 2.4), 8.06 (dd, 1H,
(d, 1H, J ¼ 7.4), 8.16 (d, J ¼ 7.9, 1H), 8.07 (d, 1H, J ¼ 8.8), 7.84 (dd, 2H,
J ¼ 14.0, 8.3), 7.69 (d, 1H, J ¼ 8.2), 7.49 (t, 1H, J ¼ 7.5), 7.29 (t, 1H,
J ¼ 7.4), 4.34 (s, 2H); HRMS (ESI): calcd for (M þ H)þ (C20H13N4O2Cl)
requires m/z 377.0805, found 377.0763. Anal. Calcd for
C20H13N4O2Cl: C, 63.75; H, 3.48; N, 14.87. Found: C, 63.71; H, 3.64;
N, 14.75.
J ¼ 8.8, 2.4), 7.65 (dd, 2H, J ¼ 8.4, 3.1), 7.49 (d, 1H, J ¼ 8.3), 7.26 (t, 1H,
J ¼ 7.6), 7.09 (t, 1H, J ¼ 7.4), 4.46 (t, 2H, J ¼ 6.8), 3.19 (dd, 4H, J ¼ 15.4,
8.6), 2.63 (q, 4H, J ¼ 7.1), 1.04 (t, 6H, J ¼ 7.1); HRMS (ESI): calcd for
(M þ H)þ (C24H25N5O2) requires m/z 416.2087, found 416.2037.
Anal. Calcd for C24H25N5O2$H2O: C, 66.49; H, 6.28; N, 16.16. Found:
C, 66.28; H, 6.24; N, 16.16.
Fig. 5. Clustering analysis of docking conformation of 5c with AChE (A) and BuChE(B), the red bar are the most probable conformations with the lowest docked energy.

B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
1421
Table 2
The calculated free energies of derivatives in molecular docking.
5.1.12. 3-(2-N-Piperidyl-propionamino)-rutaecarpine (4f)
The compound 2b was treated with excess piperidine according
to general acylation procedure to afford 4f, after column chroma-
tography with petroleum ether/EtOAc (20:1) elution, as gray solid
in 22% yield; m.p. 260–262 ꢀC; 1H NMR (400 MHz, DMSO-d6):
Compound
Calculated free energy
(kcal/mol)/AChE
Calculated free energy
(kcal/mol)/BuChE
4c
4f
5a
5b
5c
8c
ꢂ10.56
ꢂ11.06
ꢂ10.89
ꢂ11.08
ꢂ11.28
ꢂ10.20
ꢂ4.78
ꢂ6.32
ꢂ4.91
ꢂ5.65
ꢂ5.74
ꢂ5.38
d
11.84 (s, 1H), 10.59 (s, 1H), 8.50 (d, 1H, J ¼ 2.2), 7.98 (dd, 1H, J ¼ 8.8,
2.4), 7.65 (dd, 2H, J ¼ 8.2, 6.1), 7.48 (d,1H, J ¼ 8.3), 7.26 (t,1H, J ¼ 7.6),
7.09 (t, 1H, J ¼ 7.5), 4.45 (t, 2H, J ¼ 6.8), 3.17 (t, 2H, J ¼ 6.8), 2.63
(d, 2H, J ¼ 6.5), 2.53 (d, 2H, J ¼ 6.7), 2.49–2.29 (m, 4H), 1.57–1.46
(m, 4H), 1.40 (d, 2H, J ¼ 4.8); HRMS (ESI): calcd for (M þ H)þ
(C26H27N5O2) requires m/z 442.2243, found 442.2212. Anal. Calcd
for C26H27N5O2$H2O: C, 67.95; H, 6.36; N, 15.24. Found: C, 67.68; H,
6.16; N, 15.22.
5.1.8. 3-(2-N-Pyrrolyl-acetamino)-rutaecarpine (4b)
The compound 2a was treated with excess pyrrolidine according
to general acylation procedure to afford 4b, after column chroma-
tography with petroleum ether/EtOAc (20:1) elution, as beige solid
in 40% yield; m.p. 269–271 ꢀC; 1H NMR (400 MHz, DMSO-d6):
5.1.13. 3-(2-Diethylamino-acetamino)-7,8-dehydrorutaecarpine (5a)
The compound
3 was treated with excess diethylamine
d
11.84 (s, 1H), 10.10 (s, 1H), 8.55 (d, 1H, J ¼ 2.4), 8.06 (dd, 1H, J ¼ 8.8,
according to general acylation procedure to afford 5a, after
2.5), 7.65 (d, 2H, J ¼ 8.7), 7.48 (d, 1H, J ¼ 8.3), 7.26 (t, 1H, J ¼ 7.5), 7.09
(t, 1H, J ¼ 7.4), 4.46 (t, 2H, J ¼ 6.8), 3.30 (s, 2H), 3.18 (t, 2H, J ¼ 6.8),
2.69–2.53 (m, 4H), 1.76 (dt, 4H, J ¼ 6.2, 3.1); HRMS (ESI): calcd for
(M þ H)þ (C24H23N5O2) requires m/z 414.1930, found 414.1867. Anal.
Calcd for C24H23N5O2$H2O: C, 66.81; H, 5.84; N, 16.23. Found: C,
66.78; H, 5.51; N, 16.28.
crystallization from EtOH/DMF (5:1 v/v), as brown solid in 27%
yield; m.p. 297–299 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 12.71
(s, 1H), 10.12 (s, 1H), 8.77 (d, 1H, J ¼ 10.8), 8.62 (d, 1H, J ¼ 7.4), 8.18
(d, 2H, J ¼ 8.1), 7.85 (t, 2H, J ¼ 10.8), 7.69 (d, 1H, J ¼ 8.1), 7.50 (t, 1H,
J ¼ 7.4), 7.30 (t, 1H, J ¼ 7.3), 3.26 (s, 2H), 2.68 (t, 4H, J ¼ 18.0), 1.06
(t, 6H, J ¼ 6.9); HRMS (ESI): calcd for (M þ H)þ (C24H23N5O2)
requires m/z 414.1930, found 414.1859. Anal. Calcd for
C25H23N5O2$2H2O: C, 65.06; H, 5.90; N, 15.17. Found: C, 65.03; H,
5.91; N, 15.10.
5.1.9. 3-(2-N-Piperidyl-acetamino)-rutaecarpine (4c)
The compound 2a was treated with excess piperidine
according to general acylation procedure to afford 4c, after
column chromatography with petroleum ether/EtOAc (20:1)
elution, as beige solid in 67% yield; m.p. 267–269 ꢀC; 1H NMR
5.1.14. 3-(2-N-Pyrrolyl-acetamino)-7,8-dehydrorutaecarpine (5b)
The compound 3 was treated with excess pyrrolidine according
to general acylation procedure to afford 5b, after crystallization
from EtOH/DMF (5:1 v/v), as brown solid in 30% yield; m.p.
(400 MHz, DMSO-d6):
d 11.85 (s, 1H), 10.07 (s, 1H), 8.53 (d, 1H,
J ¼ 2.4), 8.05 (dd, 1H, J ¼ 8.8, 2.4), 7.66 (dd, 2H, J ¼ 8.0, 5.7), 7.48
(d, 1H, J ¼ 8.2), 7.26 (t, 1H, J ¼ 7.5), 7.09 (t, 1H, J ¼ 7.5), 4.46 (t, 2H,
J ¼ 6.8), 3.34 (s, 2H), 3.18 (t, 4H, J ¼ 6.8), 3.13 (s, 2H), 1.67–1.48 (m,
4H), 1.42 (d, 2H, J ¼ 4.2); HRMS (ESI): calcd for (M þ H)þ
(C25H25N5O2) requires m/z 428.2087, found 428.2052. Anal. Calcd
for C25H25N5O2$H2O: C, 67.40; H, 6.11; N, 15.72. Found: C, 67.33;
H, 5.98; N, 15.50.
261–263 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 12.70 (s, 1H), 10.20 (s,
1H), 8.78 (s, 1H), 8.62 (d, 1H, J ¼ 7.4), 8.22–8.10 (m, 2H), 7.83 (d, 2H,
J ¼ 6.6), 7.69 (d, 1H, J ¼ 8.0), 7.50 (t, 1H, J ¼ 7.2), 7.30 (t, 1H, J ¼ 7.2),
3.34 (s, 2H), 2.75–2.57 (m, 4H),1.89–1.68 (m, 4H); HRMS (ESI): calcd
for (M þ H)þ (C24H21N5O2) requires m/z 412.1774, found 412.1765.
Anal. Calcd for C24H21N5O2$2H2O: C, 64.42; H, 5.63; N,15.65. Found:
C, 64.53; H, 5.64; N, 15.73.
5.1.10. 3-(2-Diethylamino-propionamino)-rutaecarpine (4d)
The compound 2b was treated with excess diethylamine
according to general acylation procedure to afford 4d, after column
chromatography with petroleum ether/EtOAc (20:1) elution, as
white-pink solid in 37% yield; m.p. 315–317 ꢀC; 1H NMR (400 MHz,
5.1.15. 3-(2-N-Piperidyl-acetamino)-7,8-dehydrorutaecarpine (5c)
The compound 3 was treated with excess piperidine according
to general acylation procedure to afford 5b, after crystallization
from EtOH/DMF (5:1 v/v), as brown solid in 21% yield; m.p. 281–
DMSO-d6):
d
11.84 (s,1H),10.55 (s,1H), 8.50 (d,1H, J ¼ 2.2), 8.00 (dd,
283 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 12.71 (s, 1H), 10.16 (s, 1H),
1H, J ¼ 8.8, 1.9), 7.64 (dd, 2H, J ¼ 8.1, 5.6), 7.48 (d, 1H, J ¼ 8.2), 7.26 (t,
1H, J ¼ 7.6), 7.09 (t, 1H, J ¼ 7.5), 4.45 (t, 2H, J ¼ 6.7), 3.17 (t, 2,H
J ¼ 6.7), 2.77 (t, 2H, J ¼ 6.9), 2.51 (dd, 4H, J ¼ 10.6, 3.6), 2.47 (d, 2H,
J ¼ 7.2), 0.99 (t, 6H, J ¼ 7.1); HRMS (ESI): calcd for (M þ H)þ
(C25H27N5O2) requires m/z 430.2243, found 430.2210. Anal. Calcd
for C25H27N5O2$H2O: C, 67.09; H, 6.53; N, 15.65. Found: C, 67.00; H,
6.44; N, 15.38.
8.77 (s, 2H), 8.62 (d, 1H, J ¼ 7.4), 8.27–8.08 (m, 2H), 7.84 (d, 2H,
J ¼ 6.3), 7.68 (d, 1H, J ¼ 8.1), 7.50 (t, 1H, J ¼ 7.4), 7.30 (t, 1H, J ¼ 7.2),
3.16 (s, 2H), 2.49–2.42 (m, 2H), 1.51 (d, 6H, J ¼ 70.1); HRMS (ESI):
calcd for (M þ H)þ (C25H23N5O2) requires m/z 426.1930, found
426.1894. Anal. Calcd for C25H23N5O2$2H2O: C, 65.06; H, 5.90; N,
15.17. Found: C, 65.02; H, 5.93; N, 15.18.
5.1.16. 6-(2-Diethylamino-acetamino)-3-[2-(3-indolyl)ethyl]-
5.1.11. 3-(2-N-Pyrrolyl-propionamino)-rutaecarpine (4e)
4(3a)-quinazolinon (8a)
The compound 2b was treated with excess pyrrolidine accord-
ing to general acylation procedure to afford 4e, after column
chromatography with petroleum ether/EtOAc (20:1) elution, as
beige solid in 28% yield; m.p. 255–256 ꢀC; 1H NMR (400 MHz,
The compound 7 was treated with excess diethylamine
according to general acylation procedure to afford 8a, after
column chromatography with petroleum ether/EtOAc (20:1)
elution, as white solid in 67% yield; m.p. 183–185 ꢀC; 1H NMR
DMSO-d6):
d
11.84 (s, 1H),10.50 (s, 1H), 8.52 (d, 1H, J ¼ 2.2), 7.99 (dd,
(400 MHz, DMSO-d6): d 10.89 (s, 1H), 10.06 (s, 1H), 8.60 (d, 1H,
1H, J ¼ 8.8, 2.3), 7.64 (dd, 2H, J ¼ 8.3, 4.9), 7.48 (d,1H, J ¼ 8.3), 7.26 (t,
1H, J ¼ 7.6), 7.09 (t, 1H, J ¼ 7.5), 4.46 (t, 2H, J ¼ 6.8), 3.17 (t, 2H,
J ¼ 6.8), 2.76 (t, 2H, J ¼ 7.0), 2.55 (t, 2H, J ¼ 7.1), 2.51–2.44 (m, 4H),
1.77–1.62 (m, 4H); HRMS (ESI): calcd for (M þ H)þ (C25H25N5O2)
requires m/z 428.2087, found 428.2061. Anal. Calcd for
C25H25N5O2$H2O: C, 67.40; H, 6.11; N, 15.72. Found: C, 67.21; H,
5.96; N, 15.65.
J ¼ 1.9), 8.03 (d, 1H, J ¼ 2.1), 7.99 (s, 1H), 7.59 (t, 2H, J ¼ 9.4), 7.35 (d,
1H, J ¼ 8.1), 7.12 (d, 1H, J ¼ 2.2), 7.07 (t, 1H, J ¼ 7.5), 6.96 (t, 1H,
J ¼ 7.4), 4.25 (t, 2H, J ¼ 7.1), 3.22 (s, 2H), 3.13 (t, 2H, J ¼ 7.1), 2.62 (q,
4H, J ¼ 7.1), 1.04 (t, 6H, J ¼ 7.1); HRMS (ESI): calcd for (M þ H)þ
(C24H27N5O2) requires m/z 418.2243, found 418.2231. Anal. Calcd
for C24H27N5O2: C, 69.04; H, 6.52; N, 16.77. Found: C, 68.86; H,
6.35; N, 16.50.

1422
B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
5.1.17. 6-(2-N-Pyrrolyl-acetamino)-3-[2-(3-Indolyl)ethyl]-4(3a)-
quinazolinon (8b)
The compound 7 was treated with excess pyrrolidine according
to general acylation procedure to afford 8b, after column chroma-
tography with petroleum ether/EtOAc (20:1) elution, as white-pink
solid in 40% yield; m.p. 188–190 ꢀC; 1H NMR (400 MHz, DMSO-d6):
NMR (400 MHz, DMSO-d6): d 11.11 (s, 1H), 7.95 (s, 1H), 7.57 (d, 1H,
J ¼ 7.8), 7.50 (d, 1H, J ¼ 8.2), 7.20 (m, 2H), 7.09 (m, 2H), 6.79(s, 1H),
6.71(m, 1H), 6.63(d,1H, J ¼ 8.4), 2.89(s, 2H), 2.84 (m, 2H); ESI-MS m/
z: 373 [M þ H]þ.
5.1.22. N-(2-(1H-Indol-3-yl)ethyl)-2-amino-5-nitrobenzamide (13)
d
10.86 (s, 1H), 10.11 (s, 1H), 8.59 (d, 1H, J ¼ 2.4), 8.01 (d, 1H, J ¼ 2.5),
A mixture of tryptamine (3.2 g) and 6-nitro-1H-benzo[-
7.99 (s, 1H), 7.59 (dd, 2H, J ¼ 11.2, 8.4), 7.34 (d, 1H, J ¼ 8.1), 7.12 (d,
1H, J ¼ 2.2), 7.06 (dd, 1H, J ¼ 11.1, 4.0), 6.99–6.94 (m, 1H), 4.24 (t, 2H,
J ¼ 7.2), 3.31 (s, 2H), 3.13 (t, 2H, J ¼ 7.2), 2.62 (t, 4H, J ¼ 5.2),1.81–1.74
(m, 4H); HRMS (ESI): calcd for (M þ H)þ (C24H25N5O2) requires m/z
416.2087, found 416.2058. Anal. Calcd for C24H25N5O2: C, 69.38; H,
6.06; N, 16.86. Found: C, 69.19; H, 6.07; N, 16.87.
d][1,3]oxazine-2,4-dione 9 (5.2 g) was refluxed in ethanol (20 mL)
for 1 h. After cooling the crystals formed were collected and dried to
afford a light yellow solid (67%). m.p. 181–182 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
d
10.83(s,1H), 8.85 (s,1H), 8.49(d,1H, J ¼ 2.6),
8.02 (dd, 1H, J ¼ 9.2, 2.6), 7.79 (s, 2H), 7.59(d, 1H, J ¼ 7.8), 7.34(d, 1H,
J ¼ 8.1), 7.18(d, 1H, J ¼ 2.1), 7.07(t, 1H, J ¼ 7.8), 6.98(t, 1H, J ¼ 7.4),
6.80(d, 1H, J ¼ 9.2), 3.51(dd, 2H, J ¼ 13.5, 7.0), 2.96(t, 2H, J ¼ 7.4);
ESI-MS m/z: 325 [M þ H]þ.
5.1.18. 6-(2-N-Piperidyl-acetamino)-3-[2-(3-Indolyl)ethyl]-4(3a)-
quinazolinon (8c)
The compound 7 was treated with excess piperidine according
to general acylation procedure to afford 8b, after column chroma-
tography with petroleum ether/EtOAc (20:1) elution, as yellow-
white solid in 14% yield; m.p. 161–163 ꢀC; 1H NMR (400 MHz,
5.1.23. N-(2-(1H-Indol-3-yl)ethyl)-2,5-diaminobenzamide (14)
N-(2-(1H-Indol-3-yl)ethyl)-2-amino-5-nitrobenzamide 13 (2.9 g)
ina solution ofMeOH (300 mL) at 40–45 lb/inch2 ofH2 using 10%Pd/C
(1.0 g) as catalyst for 8 h. The catalyst was filtered off, and the filtrate
was evaporated under reduced pressure to give an gray solid (87%).
DMSO-d6):
d
10.86 (s, 1H), 10.05 (s, 1H), 8.58 (d, 1H, J ¼ 2.4), 8.01
(d, 1H, J ¼ 2.5), 7.99 (d, 1H, J ¼ 2.5), 7.59 (t, 2H, J ¼ 8.7), 7.34 (d, 1H,
J ¼ 8.1), 7.12 (d, 1H, J ¼ 2.2), 7.10–7.03 (m, 1H), 6.99–6.93 (m, 1H),
4.25 (t, 2H, J ¼ 7.2), 3.30 (s, 2H), 3.15–3.10 (m, 4H),1.61–1.55 (m, 4H),
1.42 (d, 2H, J ¼ 4.8), 1.20 (d, J ¼ 24.7, 2H); HRMS (ESI): calcd for
(M þ H)þ (C25H27N5O2) requires m/z 430.2243, found 430.2214.
Anal. Calcd for C25H27N5O2: C, 69.91; H, 6.34; N, 16.31. Found: C,
70.05; H, 6.58; N, 16.49.
m.p.178–179 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 10.80 (s,1H), 8.19 (t,
1H, J ¼ 5.6), 7.58 (d,1H, J ¼ 7.8), 7.34 (d,1H, J ¼ 8.0), 7.17 (d,1H, J ¼ 2.1),
7.07 (m,1H), 6.99(t,1H, J ¼ 7.0), 6.71(d,1H, J ¼ 2.4), 6.55(dd,1H, J ¼ 8.5,
2.5), 6.49(d, 1H, J ¼ 8.5), 5.39(s, 2H), 4.33(s, 2H), 3.46(d, 2H, J ¼ 8.1),
2.84 (m, 2H); ESI-MS m/z: 373 [M þ H]þ.
5.2. Biological activity and molecular modeling
5.1.19. 3-(2-(1H-Indol-3-yl)ethyl)-6-nitro-2-(trifluoromethyl)-
quinazolin-4(3H)-one (10)
5.2.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyl-
cholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-dithiobis-
(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), butylthiocholine
chloride (BTC), acetylthiocholine chloride (ATC), and tarcine hydro-
chloride were purchased from SigmaeAldrich.
In vitro AChE assay: All the assays were under 0.1 M KH2PO4/
K2HPO4 buffer, pH 8.0, using a Shimadzu 2450 Spectrophotometer.
Enzyme solutions were prepared to give 2.0 units/mL in 2 mL
aliquots. The assay medium contained phosphate buffer, pH 8.0
To a solution of the 6-Nitro-1H-benzo[d][1,3]oxazine-2,4-dione
9 (5.2 g) in dried pyridine (100 mL) a solution of trifluoroacetic
anhydride (3.7 mL) in dried pyridine (4 mL)was dropwise added at
room temperature and the stirring was continued for an additional
30 min. After completed addition the solution was finally refluxed
for 30 min, whereupon tryptamine (3.2 g) was added and the reflux
then continued for 3 h. After cooling the reaction mixture was
poured into cold water (500 mL), and the precipitate formed was
filtered off, washed with water. The solid obtained was treated with
hot methanol to yield yellow crystals (82%). m.p. 244–246 ꢀC; 1H
(1 mL), 50 ml of 0.01 M DTNB, 10 mL of enzyme, and 50 mL of 0.01 M
substrate (Acetylthiocholine chloride). The substrate was added to
the assay medium containing enzyme, buffer, and DTNB with
inhibitor after 15 min of incubation time. The activity was deter-
mined by measuring the increase in absorbance at 412 nm at 1 min
intervals at 37 ꢀC. Calculations were performed according to the
method of the equation in Ellman [19] et al. In vitro BuChE assay use
the similar method described above.
NMR (400 MHz, DMSO-d6):
d 10.97 (s, 1H), 9.12 (s, 1H), 8.56 (d, 1H,
J ¼ 2.7), 8.26 (dd, 1H, J ¼ 9.0, 2.7), 7.59 (dd, 1H, J ¼ 11.4, 8.1), 7.27
(t, 1H, J ¼ 7.1), 7.10 (m, 2H), 3.00 (dd, 2H J ¼ 15.7, 3.1), 2.84 (m, 2H);
ESI-MS m/z: 401 [M ꢂ H]ꢂ.
5.1.20. 3-Nitro-13b-(trifluoromethyl)-13b,l4-dihydrorutaecarpine (11)
A
mixture of 3-(2-(1H-indol-3-yl)ethyl)-6-nitro-2-(trifluoro-
To determine the time-dependence of AChE inhibition, AChE
was incubated in the presence of 5c at 25 ꢀC, and the residual
activity was determined at various time intervals (from 15 to
75 min). The enzyme (0.6 unit) was incubated with 5 nM of 5c in
methyl)quinazolin-4(3H)-one 10 (8.0 g), acetic acid (40 mL) and
hydrochloric acid (6 mL) was heated under reflux for 1 h. After the
reaction mixture was cooled, it was diluted with water (50 mL) and
the precipitate formed was filtered off, washed with water. The
solid obtained was dried under vacuum to give yellow-green
powder (8). m.p. 266–269 ꢀC; 1H NMR (400 MHz, DMSO-d6):
phosphate buffer of a total volume of 500 mL. After 30 min incu-
bation at 25 ꢀC, the mixture was chilled in an ice bath. The samples
were dialyzed in phosphate buffer at 4 ꢀC, and the residual activity
was recorded after various dialysis time. The residual enzyme
activity was determined before and after dialysis. A control exper-
iment was performed under identical conditions without inhibitor.
The AChE inhibitory activity was assayed using the method of
Ellman.
d
10.97 (s, 1H), 9.12 (s, 1H), 8.56 (d, 1H, J ¼ 2.7), 8.26 (dd, 1H, J ¼ 9.0,
2.7), 7.59 (dd, 1H, J ¼ 11.4, 8.1), 7.27 (m, 1H), 7.10 (m, 2H), 2.98(m,
2H), 2.84 (m, 2H); ESI-MS m/z: 401 [M ꢂ H]ꢂ.
5.1.21. 3-Amino-13b-(trifluoromethyl)-13b,l4-dihydro-
rutaecarpine (12)
Kinetic characterization of AChE and BuChE was performed
using a reported method. Six different concentrations of substrate
were mixed in the 1 mL 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0),
containing 50 mL of DTNB, 10 mL AChE, and 50 mL substrate. Test
compound was added into the assay solution and pre-incubated
with the enzyme at 37 ꢀC for 15 min, followed by the addition of
3-Nitro-13b-(trifluoromethyl)-13b,l4-dihydrorutaecarpine 11
(4.0 g) was hydrogenated in a solution of MeOH (300 mL) at
40–45 lb/inch2 of H2 using 10% Pd/C (1.2 g) as catalyst for 8 h. The
catalyst was filtered off, and the filtrate was evaporated under
reduced pressure to give an brown solid (80%). m.p. 177–179 ꢀC; 1H

B. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 1415–1423
1423
substrate. Kinetic characterization of the hydrolysis of ATC cata-
Acknowledgment
lyzed by AChE was done spectrometrically at 412 nm. A parallel
control with no inhibitor in the mixture, allowed adjusting activi-
ties to be measured at various times. Kinetic characterization of
BuChE assay use the similar method described above.
We thank the National Nature Science Foundation of China
(20772159, 90813011, U0832005), the NSFC/RGC joint Research
Scheme (30731160006) for financial support of this study.
5.2.2. Molecular modeling
References
The crystal structure of acetylcholinesterase complexed with
E2020 (code ID:1EVE) and the Human Butyrylcholinesterase
complexed with Echothiophate (code ID: 1POI) were obtained in
the Protein Data Bank after eliminating the inhibitor (E2020) and
water molecules. The 3D Structure of 5c was built and performed
geometry optimization by molecular mechanics. Further prepa-
ration of substrates included addition of Gasteiger charges,
removal of hydrogen atoms and addition of their atomic charges to
skeleton atoms, and finally, assignment of proper atomic types.
Autotors was then used to define the rotatable bonds in the
ligands.
Docking studies were carried out using the AUTODOCK 4.0
program. 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.
[1] P. Davies, A.J.F. Maloney, Lancet 2 (1976) 1403.
[2] R.T. Bartus, R.L. Dean III, B. Beer, A.S. Lippa, Science 217 (1982) 408–414.
[3] M.J. assoulie´, F. Bacou, E. Barnard, A. Chatonnet, B.P. Doctor, D.M. Quinn (Eds.),
Cholinesterases: Structure, Function, Mechanism, Genetics and Cell Biology,
American Chemical Society, Washington, 1991.
[4] J. Hardy, D.J. Selkoe, Science 297 (2002) 353–356.
[5] G.V. De Ferrari, M.A. Canales, I. Shin, L.M. Weiner, I. Silman, N.C. Inestrosa,
Biochemistry 40 (2001) 10447–10457.
[6] M.M. Mesulam, M. Asuncion Moran, Ann. Neurol. 22 (1987) 223–228.
[7] M. Weinstock, CNS Drugs 12 (1999) 307–323.
[8] G. Pacheco, R. Palacios-Esquivel, D.E. Moss, J. Pharmacol. Exp. Ther. 274 (1995)
767–770.
[9] D.R. Liston, J.A. Nielsen, A. Villalobos, D. Chapin, S.B. Jones, S.T. Hubbard,
A. Shalaby, A. Ramirez, D. Nason, W.F. White, Eur. J. Pharmacol. 486 (2004) 9–17.
[10] D.E. Kuhl, R.A. Koeppe, S.E. Snyder, S. Minoshima, K.A. Frey, M.R. Kilbourn, Ann.
Neurol. 59 (2006) 13–20.
[11] E.H. Rydberg, B. Brumshtein, H.M. Greenblatt, D.M. Wong, D. Shaya,
L.D. Williams, et al., J. Med. Chem. 49 (2006) 5491–5500.
[12] N. Akula, L. Lecanu, J. Greeson, V. Papadopoulos, Bioorg. Med. Chem. Lett. 16
(2006) 6277–6280.
AUTOGRID performed a precalculated 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. The centre of the grid box was placed at the bottom of the
active site gorge (AChE [2.781 64.383 67.971]; BuChE [112.0 20.0
40.0]). The dimensions of the active site box were set at 50
ꢃ 46 ꢃ 46 Å.
[13] G. Kryger, I. Silman, J.L. Sussman, Structure 7 (1999) 297–307.
[14] T.H. Tsai, T.F. Lee, C.F. Chen, L.C.H. Wang, Pharmacol. Biochem. Behav. 50 (1995)
293–298.
[15] J.R. Sheu, Cardiovasc. Drug Rev. 17 (1999) 237–298.
[16] T.C. Moon, M. Murakami, I. Kudo, K.H. Son, H.P. Kim, S.S. Kang, H.W. Chang,
Inflamm. Res. 48 (1999) 621–625.
[17] C.H. Park, S.H. Kim, W. Choi, Y.L. Lee, J.S. Kim, S.S. Kang, Y.H. Suh, Planta Med.
62 (1996) 405–409.
[18] M. Decker, Eur. J. Med. Chem. 40 (2005) 305–313.
[19] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, Biochem. Phar-
macol. 7 (1961) 88–95.
Flexible ligand docking was performed for the compounds.
Docking calculations were carried out using the Lamarckian genetic
algorithm (LGA) and all parameters were the same for each dock-
ing. We used initially a population of random individuals (pop-
ulation size: 150), a maximum number of 2,500,000 energy
evaluations, a maximum number of generations of 27,000, At the
end of a docking procedure (100 docking runs), the resulting
positions were clustered according to a root mean square criterion
of 0.5 Å.
[20] J. Bergman, S. Bergman, J. Org. Chem. 50 (1985) 1246–1255.
[21] H. Tang, Y.-B. Wei, C. Zhang, F.-X. Ning, W. Qiao, S.-L. Huang, L. Ma, Z.-S. Huang,
L.-Q. Gu, Eur. J. Med. Chem. 44 (2009) 2523–2532.
[22] H. Tang, F.-X. Ning, Y.-B. Wei, S.-L. Huang, Z.-S. Huang, A.S.-C. Chan, L.-Q. Gu,
Bioorg. Med. Chem. Lett. 17 (2007) 3765–3768.
[23] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, J. Comput. Chem. 19 (1998) 1639–1662.
[24] W.L. DeLano, The PyMOL Molecular Graphics System. DeLano Scientific. San
Carlos, CA.
[25] J. Das, M.S. Kumar, D. Subrahmanyam, T.V.R.S. Sastry, et al., Bioorg. Med. Chem.
14 (2006) 8032–8042.