Searching for the Multi-Target-Directed Ligands against Alzheimer's disease: Discovery of quinoxaline-based hybrid compounds with AChE, H 3R and BACE 1 inhibitory activities

Article abstract of DOI:10.1016/j.bmc.2011.09.061

A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were designed by lending the core structural elements required for H₃R antagonists and hybridizing BACE 1 inhibitor 1 with AChE inhibitor BYYT

Full text of DOI:10.1016/j.bmc.2011.09.061

Bioorganic & Medicinal Chemistry 19 (2011) 7158–7167
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Searching for the Multi-Target-Directed Ligands against Alzheimer’s
disease: Discovery of quinoxaline-based hybrid compounds with AChE,
H3R and BACE 1 inhibitory activities
Wenhai Huang a,b, Li Tang a, Ying Shi c, Shufang Huang a, Lei Xu d, Rong Sheng a, Peng Wu a, Jia Li d,
Naiming Zhou c, Yongzhou Hu a,
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zijingang Campus, Zhejiang University, Hangzhou 310058, China
b Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310058, China
c Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China
d The National Center for Drug Screening, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were
designed by lending the core structural elements required for H3R antagonists and hybridizing BACE 1
inhibitor 1 with AChE inhibitor BYYT-25. A virtual database consisting of quinoxaline derivatives was first
screened on a pharmacophore model of BACE 1 inhibitors, and then filtered by a molecular docking model of
AChE. Seventeen quinoxaline derivatives with high score values were picked out, synthesized and evalu-
ated for their biological activities. Compound 11a, the most effective MTDL, showed the potent activity
to H3R/AChE/BACE 1 (H3R antagonism, IC50 = 280.0 98.0 nM; H3R inverse agonism, IC50 = 189.3
95.7 nM; AChE, IC50 = 483 5 nM; BACE 1, 46.64 2.55% inhibitory rate at 20 lM) and high selectivity over
H1R/H2R/H4R. Furthermore, the protein binding patterns between 11a and AChE/BACE 1 showed that it
makes several essential interactions with the enzymes.
Received 3 August 2011
Revised 27 September 2011
Accepted 28 September 2011
Available online 5 October 2011
Keywords:
Alzheimer’s disease
BACE 1
AChE
H3R
Ó 2011 Elsevier Ltd. All rights reserved.
Multi-Target-Directed Ligands
1. Introduction
Amyloid-b (Ab), formed by the continuously proteolytic pro-
cessing of b-amyloid precursor protein (APP) by b and
c-secre-
Alzheimer’s disease (AD), the most common form of dementia, is
a complex neurodegenerative disorder.1,2 The fact that complexity
and multiple etiologies of AD make single-target strategy difficult
to shed desirable therapeutic effect makes the choice of Multi-
Target-Directed Ligand (MTDL) to be a potentially more effective
strategy.3 MTDL, whose goal is to enhance efficacy and improve
safety, is rationally designed to hit multiple targets for a particular
disease to improve pharmacological profiles.4–6
tase,9,10 plays a central role in the pathogenesis of AD. Recent
evidence indicated certain links between Ab and AChE.11 On one
hand, the presence of Ab increases AchE activity, and on the other
hand, AChE could form a complex with Ab, which changes the con-
formation of Ab and then promotes the aggregation Ab. Thus,
simultaneously inhibition of AChE and b-secretase (BACE 1) not
only reduces Ab generation and hydrolysis of ACh but also weakens
the interaction between ACh and Ab.
Until now, most drugs approved for AD treatment are AChE
inhibitors, which improve the ACh level in the brain by decreasing
the hydrolysis of ACh. In contrast, histamine H3 receptor
antagonists can also increase ACh via a distinct mechanism. Since
activated presynaptic histamine H3 receptor decreases the release
of ACh from cholinergic neurons and H3 receptor antagonists in-
creases ACh release, the approach of integrating AChE inhibitors
and H3R antagonists, both contribute to the same result of increas-
ing synaptic levels of ACh although through different mechanisms,
may form synergistic effect in the treatment of AD.7,8
Previously, we reported the establishment of a pharmacophore
model for BACE 1 inhibitor, Hypo 1, which was validated by Enrich-
ment and ROC method.12 We also reported the potent dual-site
binding AChE inhibitor BYYT-25 (IC50 = 0.05 lM) with the interac-
tion between pyrrolidine and center active site.13
Considering the above, we focused on Multi-Target-Directed
Ligands which have effects on AChE, H3R and BACE 1 in this study.
According to reported studies, H3R antagonists may contain a basic
center, a ring core and a linker between the two centers.14 Based on
previous work, 2-amino-3,4-dihydroquinazoline of BACE 1 inhibi-
tor 1 which has an IC50 around 0.011 lM was selected as the core
ring moiety of H3R antagonist, and benzyl pyrrolidine fragment of
BYYT-25 was chosen to function as the basic center of H3R to
⇑
Corresponding author. Tel./fax: +86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.061

W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
7159
Figure 1. Structure of the parent compounds, compound (1) and BBYT-25, and the common structure for compounds included in the virtual database.
design the Multi-Target-Directed Ligands with new scaffold
(Fig. 1). A virtual database consisting of new scaffold compounds
was built and screened on pharmacophore model of BACE 1 inhib-
itor which was built in previous work,12 and then filtered by a
molecular docking model of AChE inhibitor. According to the
screening result, 17 quinoxaline derivatives were picked out, syn-
thesized, and subsequently evaluated for their AChE/BACE 1 inhib-
itory activity and H3R antagonistic action.
operating curve (ROC) method.12 The EF of test set consisting of
1200 and 18 compounds screened by the molecular docking model
at 2%, 5% and 10% were 67.6, 18.3 and 8.7, respectively. In addition,
the ROC curve of the test set screening by the molecular docking
model (Fig. 2) and an AUC of 0.91 meant that a selected randomly
active compound has a higher score than a randomly selected inac-
tive compound, 9.1 times out of 10. The results of the EF and ROC
metric suggested that the molecular docking model would be a
valuable and reliable tool in identifying compounds with AChE
inhibitory activity.
2. Materials and methods
2.1. Data preparation
3.2. Build in-house database and virtual screening
The virtual database consisting of new scaffold compound was
built using the Build 3D database protocol within the Pipeline Pilot
software. Various stereoisomers protocol was used to expend the
database, which contains 222291 compounds. Followed Lipinski
filtration (AlogP was changed as P2, with the remaining parame-
ters were set as default) led to a reduced set of 118232 compounds.
All selected compounds were then optimized by using the Discov-
ery Studio 2.1 software package.
Eighteen AChE inhibitors as the positives for the molecular
docking model study were collected from literatures,13,15 and
1200 negatives were retrieved from Available Chemicals Directory
(ACD) database using the Random Percent Filter protocol within
PipelinePilot. The test set contains 18 positives and 1200 negatives.
All compounds were optimized by Discovery Studio 2.1 software
package.
Discovery Studio 2.1/Ligand Pharmacophore Mapping protocol
was used to screen the designed database using Hypo 1 as the tem-
plate. Thousand two seventy three compounds with high Fitvalues
(bigger than 3.00, the maximum of the Fitvalue is 5.00) were
picked out and screened by the molecular docking model of AChE
inhibitor. Finally, 17 compounds were selected based on LigScore1
values and synthesized.
2.2. The pharmacophore model of BACE 1 inhibitor
The pharmacophore model of BACE 1 inhibitor (Hypo 1) was
built in a previous work.12
2.3. The molecular docking model of AChE inhibitor
Discovery Studio 2.1/Ligandfit was employed to construct the
molecular docking model of AChE inhibitor by using the crystal
structure of E2020/AChE complex (PDB ID: 1EVE). First, docking
and subsequent scoring were performed using default parameters
except for the smart Minimization Algorithm process, followed by
the best conformation selection based on score values calculated
using scoring functions. The parameters for docking and scoring
were then changed until the best conformation of docked E2020
was similar with that of E2020 in the crystal structure.
3. Results and discussion
3.1. Molecular docking model validation
The best score function LigScore1 was used to evaluate the re-
sult for the docking study. The validation of the molecular docking
model was ascertained by the Enrichment Factor (EF) and receiver
Figure 2. The receiver operating curve of the molecular docking model (Blue curve,
test set ROC; green curve, ROC for the random classification of the compounds).

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3.3. Chemistry
3.4.1. H3R antagonistic and inverse agonistic action
As shown in Table 1, six compounds showed equivalent or more
potent H3R antagonistic activities in comparison with that of thio-
peramide. In addition, three compounds (11a,b,p) displayed potent
H3R inverse agonistic activity which may be beneficial for the
treatment of AD. Compound 11j exhibited the most potent H3R
antagonistic action with an IC50 value of 52.4 23.6 nmol/L, and
11d showed the most effective inverse agonistic activity with an
IC50 value of 170.6 98.7 nmol/L. Compound 11a with a piperidine
ring at the end of benzyl exhibited more potent H3R antagonistic
activity over that with an inserted piperidine moiety between
quinoxaline and benzyl fragments (11c). Similarly, compounds
11n and 11i, containing inserted piperidine moiety, showed no
H3R antagonistic activity.
The synthetic route for 11a–f is illustrated in Scheme 1. Reaction
of methyl 4-(bromomethyl)benzoate with piperidine or pyrrolidine
in refluxing CH2Cl2 gave 2, followed by hydrolysis to yield substi-
tuted benzoic acid 3. Condensation of 3 or 4 with 2,3-diaminoqui-
noxaline in the presence of EDC/HOBt resulted in compounds
11a–c. Furthermore, compounds 11a,b were salted using HCl satu-
rated ethylacetate to give target compounds 11d,e. In addition,
further reduction of amide group of compound 11a afforded com-
pound 11f.
On the other hand, reaction of 2-chloroacetyl chloride with 5
gave 6 in good yields. Treatment of 7 with 1,2-dibromoethane or
1,3-dibromopropane afforded compounds 8a–d. Then reaction of
6, 8 with 2,3-diaminoquinoxaline in the presence of NaH in DMF
gave the target compounds 11g–m (Scheme 2).
3.4.2. Selectivity for H1R, H2R, H4R
Condensation of 3-aminoquinoxaline-2-carboxylic acid with 5
in the presence of EDC/HOBt afforded target compounds 11n–p.
In addition, 11o could be further transformed into 11q by treat-
ment with aluminum lithium hydride. Reaction of 2,3-diaminoqui-
noxaline with 4-(piperidin-1-ylmethyl)benzaldehyde 10 in the
presence of EDC gave compound 11r (Scheme 3).
It has been revealed that each of the yet identified four subtypes
of histamine GPCRs (namely H1R, H2R, H3R and H4R) bears distinct
biological effects, which makes selectivity an issue of considerable
importance in designing H3R antagonist. Thus, six compounds with
potent H3R antagonistic activities were selected to evaluate their
biological activities for H1R, H2R and H4R, using thioperamide as
the reference standard. The results are showed in Figs. 3–5 (H: His-
tamine; T: Thioperamide; F: Forslin; FH: Forslin and Histamine).
3.4. Biological activity and SAR
As shown in Figs. 3 and 4, cAMP accumulation induced by 1 lM
histamine in HEK-293 cells stably expressing H1R and H2R did not
been significantly influenced by tested compounds at a dose of
H3R antagonistic action and H3R inverse agonist action of syn-
thesized compounds were tested by using CRE-luciferace tran-
scription assay with thioperamide as the reference standard.
AChE inhibitory activities were evaluated according to a modified
Ellman method with donepezil as the reference standard and BACE
1 inhibitory activities were assayed using fluorescence resonance
energy transfer (FRET) assay with OM99-2 as the reference stan-
dard. The results are summarized in Table 1.
1
l
M.
The inhibition of cAMP accumulation induced by 1
mine (FH) could be partly decreased by co-incubation with 1
l
M Hista-
l
M
11a and 11d or thioperamide, respectively (Fig. 5). Further evalua-
tion results are summarized in Table 2.
As shown above, all tested compounds, except 11a and 11d
with the weak function to H4R, have no effect to H1R, H2R or
Scheme 1. Reagents and conditions: (a) amine, Et3N, acetontrile, reflux; (b) NaOH, MeOH/H2O, rt; (c) 2,3-diaminoquinoxaline, HOBt, EDCꢀHCl rt; (d) HCl/AcOEt, rt; (e) AlLiH4,
THF, 35 °C.

W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
7161
Scheme 2. Reagents and conditions: (a) ClCH2COCl, CH2Cl2, rt; (b) BrCH2(CH2)nBr, NaOH/H2O, reflux; (c) 2,3-diaminoquinoxaline, NaH,DMF, rt.
Scheme 3. Reagents and conditions: (a) 5a–c, HOBt, EDCꢀHCl. rt; (b) AlLiH4, THF, 35 °C; (c) 3-diaminoquinoxaline, EDCꢀHCl, acetontrile rt.
H4R, which indicated that the selectivity of quinoxaline derivatives
over H1R, H2R, and H4R is higher than that of thioperamide.
the NHCH2 linkage to N@CH (11r) decreases activity. This result
indicated that the nitrogen atom in the linkage may form a hydro-
gen bond interaction with the enzyme. Compounds (11a,o and 11j)
with piperidine ring at the end of benzyl are more effective than
those including pyrrolidine, diethylamine and N-methylpiperazine
moiety (11e, p, q and 11l).
3.4.3. AChE inhibitory activity
The results of AChE inhibitory activity of synthesized com-
pounds showed that most quinoxaline derivatives exhibited mod-
erate to potent AChE inhibitory activities (Table 1). Compound 11d
displayed the most potent AChE inhibitory activity, with an IC50 va-
3.4.4. BACE 1 inhibitory activity
lue of 0.313 0.211
clusion can be drawn as follows:
l
M. Based on these results, some general con-
According to the results of BACE 1 inhibitory activities summa-
rized in Table 1, all synthesized compounds displayed moderate to
weak activity. The most effective compound 11a and its hydrochlo-
The variation of linker between quinoxaline and benzylamine
exhibited obvious effect on AChE inhibitory activity, compounds
(11a,d and 11e) containing NHCO linkage are more potent than that
of containing CONH (11o,p), NHCH2CONH (11g,h) or NHCH2CH2O (
11j–l). In addition, reduction of amide to amine (11a vs 11f) has no
significantly influence for AChE inhibitory activity, while changing
ride (11d) showed almost 50% inhibitory rates at 20 lM.
3.4.5. Molecular docking study
Based on results of biological evaluation, compound 11a
showed the most potent activity to H3R/AChE/BACE 1 and high

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Table 1
The biological activity of quinoxaline derivatives
L
N
N
R1
NH2
Compd.
L
R1
H3R antagonistic action at
M (%) and (IC50, nM)a,b
H3R inverse agonistic
action IC50 (nM)
AChE inhibitory
BACE 1 inhibitory
activity at 20 l
g/mla (%)
a
a
1
l
activity IC50
(lM)
Thioperamide
Donepezil
OM99-2
(517.5 66.5)
NT
NT
NAc
NT
NT
NTd
5.0 1.5 nM
NT
NT
NT
0.25 0.03 lM
11a
11d
11e
Piperidine
74.4 2.7 (280.0 98.0)
65.3 5.4 (511.0 96.0)
0.8 1.3
189.3 95.7
170.6 98.7
NA
0.483 0.005
0.313 0.211
NA
46.64 2.55
44.79 9.44
10.59 1.39
O
O
H
N
PiperidineꢀHCl
PyrrolidineꢀHCl
H
N
11f
Piperidine
0.2 2.4
NA
0.476 0.210
1.06 0.23
11g
11h
Piperidine
Pyrrolidine
15.0 2.1
16.9 4.8
NA
NA
1.155 0.125
1.173 0.437
0.79 1.27
36.05 8.13
H
N
N
H
11j
11k
11l
Piperidine
Diethylamine
N-Methylpiperazine
82.3 8.1 (52.4 23.6)
65.5 3.1 (182.0 52.4)
7.4 1.2
NA
NA
NA
1.599 0.478
3.866 2.756
NA
NA
NA
NA
H
N
O
H
N
11m
O
Piperidine
40.5 1.4
NA
1.929 0.544
6.56 2.32
11o
11p
Piperidine
pyrrolidine
34.6 11.9
72.8 1.6 (260.0 77.0)
NA
0.892 0.132
1.498 0.058
NA
4.58 5.05
O
H
378.5 157.5
N
H
11q
11r
Piperidine
Piperidine
78.2 4.8 (129.7 66.3)
0.5 2.2
NA
NA
NA
NA
NA
N
N
0.748 0.014
O
H
N
N
N
11c
0.6 0.2
NA
NA
8.96 2.35
N
N
NH2
O
H
N
N
N
11n
11i
1.8 2.3
NA
NA
NA
NA
NH2
O
N
H
N
N
N
H
4.5 1.4
1.329 0.486
30.80 9.49
N
NH2
a
b
c
Data are means standard deviation of duplicate independent experiments.
IC50 value only determined for compounds attaining over 50% H3 antagonism at 1 lM and put in brackets.
NA, no activity.
NT, not tested.
d
selectivity over H1R/H2R/H4R. In an attempt to understand the
molecular interaction between 11a and AChE/BACE 1, a molecular
docking study was performed using the Discovery Studio 2.1/
CDOCKER protocol. The crystal structure of OM99-2/BACE 1 com-
plex (PDB ID: 2ZHR) and E2020/AChE complex (PDB ID:1EVE) were
used as the templates. As shown in Fig. 6a, hydrogen bond interac-
tion was obse0rved between amide group and Tyr121 with a dis-
4. Conclusions
Based on SAR analysis of H3R antagonists, the strategy of hybrid-
izing AChE inhibitor BYYT-25 with BACE 1 inhibitor 1 was utilized to
design quinoxaline-based hybrid compounds as Multi-Target-Di-
rected Ligands in this study. The designed database of new scaffold
was built and screened against a previous built and validated BACE
1 inhibitor pharmacophore model and filtered using an AChE molec-
ular docking model. Seventeen compounds with high score values
were selected, synthesized and evaluated for their biological activi-
ties. Most compounds showed moderate to potent inhibitory/antag-
onistic activities to the AChE, BACE 1 and H3R. Compound 11a
showed the potent activity to H3R/AChE/BACE 1 and high selectivity
to H1R/H2R/H4R. In addition, further receptor-binding studies of 11a
and AChE/BACE 1 showed that it can make several essential interac-
tions to the enzymes. These result suggested that the development
tance of 2.14 ÅA. The charged nitrogen made a cation- interaction
p
with Trp84, benzene and quinoxaline ring formed p–p stacking
with Tyr334 and Trp279, respectively. Besides, the result of molec-
ular docking between 11a and BACE 1 showed that the amino
group formed hydrogen bond with catalytic aspartate Asp32, with
0
a distance of 2.45 ÅA (Fig. 6b). In addition, hydrogen bonds were also
presented between nitrogen atom and Thr72 with a distance of
0
2.41 and 2.03 0ÅA, and between amide group and Thr72 with a dis-
tance of 2.36 ÅA.

W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
7163
Figure 3. Selectivity of compounds over H1R in a CRE-luciferace transcription assay.
Figure 4. Selectivity of compounds over H2R in a CRE-luciferace transcription assay
of quinoxaline derivatives may shed instructive light for the treat-
ment of AD in the near future.
a previous work.12 Molecular model studies were performed using
the Discovery Studio 2.1.
5.2. Chemistry
5. Experimental
5.1. General
Compounds 2,14 4,16 5a–b,17 7,13 9,18 and 1019 were synthesized
according to the known methods.
Melting points were recorded on a Buchi apparatus and uncor-
rected, IR spectra were recorded on a Bruker VECTOR 22 FTIR spec-
trophotometer. 1H NMR spectra were recorded on a Bruker Avance
III 500 M instrument (chemical shifts are expressed as d values
relative to TMS as internal standard). Mass spectra (MS) were re-
corded on an Esquire-LC-00075 spectrometer. The database of
quinoxaline derivatives was built by Pipeline Pilot software. The
pharmacophore model (Hypo 1) of BACE 1 inhibitors was built in
5.2.1. General procedure for the preparation of compounds 3
Methyl benzoate 2 (7.6 mmol) was dissolved in a mixture of
40 mL methanol and 10 mL water. NaOH (0.33 g, 8.5 mmol) was
added, and the mixture was refluxed for 7 h. After methanol evap-
oration, the remaining aqueous layer pH was adjusted to 7. The
solid precipitate was filtered and washed with water (2 ꢁ 10 mL)
to obtain white solid 3.

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Figure 5. Selectivity of compounds over H4R in a CRE-luciferace transcription assay.
Table 2
The selectivity of compounds 11a and 11d for H4R
Compd.
11a
11d
Thioperamide
Function to H4R IC50
>10
lM
>10
l
M
667 114.0 nM
5.2.1.1. 4-(Piperidin-1-ylmethyl)benzoic acid (3a). White solid
(50%); mp: 234–236 °C. 1H NMR (d, DMSO-d6): 12.73 (s, 1H, –
OH), 7.88 (d, 2H, J = 8.0 Hz, ArH), 7.40 (d, 2H, J = 8.0 Hz, ArH),
3.49 (s, 2H, ArCH2N–), 2.28–2.35 (m, 4H, piperidine), 1.47–1.51
(m, 4H, piperidine), 1.37–1.40 (m, 2H, piperidine).
5.2.1.2. 4-(Pyrrolidin-1-ylmethyl)benzoic acid (3b). White solid
(51%); mp: 227–229 °C. 1H NMR (d, DMSO-d6): 12.75 (s, 1H, –OH),
7.89 (d, 2H, J = 7.5 Hz, ArH), 7.40 (d, 2H, J = 7.5 Hz, ArH), 3.51 (s, 2H,
ArCH2N–), 2.43–2.49 (m, 4H, pyrroline), 1.73–1.78 (m, 4H,
pyrroline).
5.2.2. N-(3-aminoquinoxalin-2-yl)-4-(piperidin-1-ylmethyl)
benzamide (11a)
Compound 3a (219 mg, 1 mmol), EDCꢀHCl (316 mg, 1.65 mmol)
and HOBt (223 mg, 1.65 mmol) were dissolved in a solution of
3.5 mL CH2Cl2 and 1.5 mL DMF, and the mixture was stirred for
1 h at 0 °C. 2,3-Diaminoquinoxaline (320 mg, 2 mmol) was added
to the mixture, and then the mixture was refluxed for 12 h. The so-
lid precipitate was filtered and washed with CH2Cl2 (2 ꢁ 5 mL) to
obtain 11a. Yellow solid (78%); mp: >250 °C. IR (KBr): 3450,
2932, 2779, 1718, 1633, 1393, 1327 cmꢂ1 1H NMR (d, DMSO-d6):
;
7.83 (d, 2H, J = 7.0 Hz, ArH), 7.75–7.78 (m, 2H, ArH), 7.46 (d, 2H,
J = 7.0 Hz, ArH), 7.12–7.13 (m, 2H, ArH), 4.76 (s, 2H, –NH2), 2.88–
3.05 (m, 4H, piperidine), 2.73 (s, 2H, –NCH2), 1.71–1.75 (m, 4H,
piperidine), 1.40–1.60 (m, 2H, piperidine); MS (ESI): m/z = 362
[M+H]+.
5.2.3. 4-Carboxamide-N-(3-aminoquinoxalin-2-yl)-1-benzyl
piperidine (11c)
Using the previous procedure and starting from 1-benzylpiperi-
dine-4-carboxylic acid 4 (219 mg, 1 mmol) and 2,3-diaminoquinox-
aline (320 mg, 2 mmol), 11c was obtained as yellow solid (79%);
Figure 6. (a) The binding pattern of 11a into AChE; (b) The binding pattern of 11a
into BACE 1.
mp: 122–125 °C. IR (KBr):3449, 2931, 2851, 1636, 1389 cmꢂ1 1H
;

W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
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NMR (d, CDCl3): 8.15–8.22 (m, 2H, ArH), 7.71–7.74 (m, 2H, ArH),
5.2.8. General procedure for the preparation of compounds
11f,q
7.32–7.37 (m, 4H, ArH), 7.27–7.29 (m, 1H, ArH), 3.62 (s, 2H, –
NCH2), 3.18–3.24 (m, 1H, piperidine), 3.08–3.11 (m, 2H, piperidine),
2.15–2.30 (m, 6H, piperidine); MS (ESI): m/z = 362 [M+H]+.
To a solution of 11a or 11o (0.14 mmol) in 5 mL THF, AlLiH4
(10.5 mg) was added. After the mixture was stirred for 3 h at
50 °C, the reaction was stopped by addition of 5 mL water. The
THF was evaporated, and the remaining aqueous layer was ex-
tracted with AcOEt (2 ꢁ 15 mL). The combined AcOEt layers were
dried over Na2SO4. The solvent was removed under vacuum to give
crude product, which was purified by silica gel column chromatog-
raphy (PE:AcOEt:TEA = 2:1:0.1) to afford target compounds 11f,q.
5.2.4. 2-Carboxamide-3-amino-N-(1-benzylpiperidin-4-yl)quino
xaline (11n)
Using the previous procedure and starting from 3-aminoqui-
noxaline-2-carboxylic acid 9 (189 mg, 1 mmol) and 4-(piperidin-
1-ylmethyl)benzenamine (380 mg, 2 mmol), 11n was obtained as
yellow solid (73%); mp: >250 °C. IR (KBr):3452, 2933, 2857, 1635,
1557, 1375 cmꢂ1
;
1H NMR (d, CDCl3): 8.06 (d, 1H, J = 8.5 Hz, ArH),
5.2.8.1. 2,3-Diamine-N2-(4-(piperidin-1-ylmethyl)benzyl)quin-
oxaline (11f). Yellow solid (34%); mp: >250 °C. IR (KBr):3456,
7.87 (d, 1H, J = 8.5 Hz, ArH), 7.65–7.69 (m, 2H, ArH), 7.46 (t, 1H,
J = 7.0 Hz, ArH), 7.34–7.37 (m, 2H, ArH), 6.99 (t, 1H, J = 7.0 Hz,
ArH), 6.92 (t, 1H, J = 7.0 Hz, ArH), 6.24 (s, 1H, –CONH), 3.87 (s,
2H, –NCH2Ph), 2.75–2.78 (m, 2H, piperidine), 2.56–2.59 (m, 1H,
piperidine), 2.46–2.49 (m, 2H, piperidine), 2.00–2.06 (m, 4H, piper-
idine); MS (ESI): m/z = 362 [M+H]+.
2929, 2856, 1636, 1474, 1382 cmꢂ1 1H NMR (d, CDCl3): 8.41 (d,
;
1H, J = 8.0 Hz, ArH), 8.15–8.17 (m, 2H, ArH), 7.79–7.81 (m, 2H,
ArH), 7.64 (d, 2H, J = 8.0 Hz, ArH), 3.65 (m, 1H, –NHCH2), 3.55 (s,
2H, –NCH2Ph), 3.07–3.10 (m, 2H, –NHCH2), 2.40–2.49 (m, 4H,
piperidine), 1.55–1.62 (m, 4H, piperidine), 1.43–1.47 (m, 2H, piper-
idine); MS (ESI): m/z = 348 [M+H]+.
5.2.5. 2-Carboxamide-3-amino-N-(4-(piperidin-1-ylmethyl)
phenyl)quinoxaline (11o)
5.2.8.2. 2-Amine-3-((4-(piperidin-1-ylmethyl)phenylamino)met
Using the previous procedure and starting from 3-aminoqui-
noxaline-2-carboxylic acid 9 (189 mg, 1 mmol) and 4-(pyrrolidin-
1-ylmethyl)benzenamine (380 mg, 2 mmol), 11o was obtained as
yellow solid (83%); mp: >250 °C. IR (KBr):3450, 2931, 2639, 1635,
hyl)quinoxalin (11q). Yellow solid (26%); mp: >250 °C. IR
(KBr):3451, 2932, 1635, 1513, 1371 cmꢂ1 1H NMR (d CDCl3):
;
7.35 (d, 2H, J = 8.5 Hz, ArH), 7.14 (d, 2H, J = 8.0 Hz, ArH), 6.99 (t,
1H, J = 7.0 Hz, ArH), 6.93 (t, 1H, J = 7.0 Hz, ArH), 6.62–6.64 (m, 4H,
ArH), 6.54–6.56 (m, 2H, –NH2), 3.70–3.74 (m, 1H, –NHCH2), 3.42–
3.44 (m, 4H, –NHCH2, –NCH2), 2.34–2.42 (m, 4H, piperidine),
1.55–1.61 (m, 4H, piperidine), 1.41–1.44 (m, 2H, piperidine); MS
(ESI): m/z = 348 [M+H]+.
1525, 1415, 1373 cmꢂ1 1H NMR (d, CDCl3): 10.10 (s, 1H, –CONH),
;
7.94 (d, 1H, J = 8.0 Hz, ArH), 7.66–7.73 (m, 4H, ArH), 7.48 (t, 1H,
J = 6.0 Hz, ArH), 7.39 (d, 2H, J = 8.5 Hz, ArH), 3.53 (s, 2H, –NCH2Ph),
2.40–2.47 (m, 4H, piperidine), 1.58–1.63 (m, 4H, piperidine), 1.43–
1.47 (m, 2H, piperidine); MS (ESI): m/z = 362 [M+H]+.
5.2.9. General procedure for the preparation of compounds
11g–i
5.2.6. 2-Carboxamide-3-amino-N-(4-(pyrrolidin-1-ylmethyl)
phenyl)quinoxaline (11p)
To a solution of 5 (1 mmol) in 3 mL CH2Cl2, 2-chloroacetyl chlo-
ride (0.08 mL, 1.1 mmol) was added. After the mixture was stirred
for 0.5 h, the solid was filtered and purified by silica gel column
chromatography (PE:AcOEt:TEA = 3:1:0.1) to afford 6. NaH
(29 mg, 1.24 mmol) was added to 2,3-diaminoquinoxaline
(125 mg, 0.79 mmol)20 in 3 mL DMF. After the mixture was stirred
for 0.5 h, 6 (238 mg, 0.79 mmol) was added, and the mixture was
stirred for 0.5 h at 35 °C. Water (10 mL) was added to the mixture,
and the crude product was extracted with AcOEt (2 ꢁ 15 mL), the
organic layer was washed with water (2 ꢁ 20 mL) and dried over
Na2SO4. The solvent was removed under vacuum to give crude
product, which was purified by silica gel column chromatography
(PE:AcOEt:TEA = 3:1:0.1) to afford target compounds 11g–i.
Using the previous procedure and starting from 3-aminoqui-
noxaline-2-carboxylic acid 9 (189 mg, 1 mmol) and 1-benzylpi-
peridin-4-amine (380 mg, 2 mmol), 11p was obtained as yellow
solid (79%); mp: >250 °C. IR (KBr):3451, 2932, 2854, 1637, 1533,
1412, 1373 cmꢂ1 1H NMR (d, CDCl3): 10.16 (s, 1H, –CONH), 7.96
;
(d, 1H, J = 7.0 Hz, ArH), 7.78 (d, 1H, J = 7.0 Hz, ArH), 7.67–7.71 (m,
2H, ArH), 7.50 (d, 2H, J = 8.0 Hz, ArH), 6.98–7.03 (m, 1H, ArH),
6.93 (t, 1H, J = 7.0 Hz, ArH), 3.88 (s, 2H, –NCH2Ph), 2.75–2.84 (m,
4H, pyrrolidine), 1.91–1.97 (m, 4H, pyrrolidine); MS (ESI): m/
z = 348 [M+H]+.
5.2.7. General procedure for the preparation of compounds
11d,e
11a or 11b (0.1 mmol) was dissolved in 2 mL HCl saturated
ethyl acetate, stirred at room temperature for 3 h. The precipitate
was filtered to obtain compound 11d or 11e.
5.2.9.1. 2-(3-Aminoquinoxalin-2-ylamino)-N-(4-(piperidin-1-yl
methyl)phenyl)acetamide (11g). Yellow oil (11%); IR (KBr):
3457, 2929, 1637, 1381, 1118 cmꢂ1 1H NMR (d, DMSO-d6): 10.17
;
5.2.7.1.
N-(3-aminoquinoxalin-2-yl)-4-(piperidin-1-ylmethyl)
(s, 1H, –CONH), 7.59 (d, 2H, J = 8.0 Hz, ArH), 7.39 (d, 2H,
J = 7.5 Hz, ArH), 7.24 (d, 2H, J = 8.0 Hz, ArH), 7.20 (t, 2H,
J = 7.5 Hz, ArH), 6.80 (s, 2H, –NH2), 4.50 (m, 1H, –NHCH2), 4.30
(d, 2H, J = 5.5 Hz, –NCH2CO), 3.43 (s, 2H, –NCH2Ph), 2.33 (m, 4H,
piperidine), 1.49–1.52 (m, 4H, piperidine), 1.40–1.42 (m, 2H, piper-
idine); MS (ESI): m/z = 391 [M+H]+.
benzamide hydrochloride (11d). Yellow solid (91%); mp:
>250 °C. IR (KBr): 3454, 2775, 2632, 1718, 1633, 1473, 1393 cm
ꢂ1
;
1H NMR (d, DMSO-d6): 10.75 (s, 1H, –CONH), 8.12–8.14 (m,
2H, ArH), 7.91 (d, 2H, J = 7.0 Hz, ArH), 7.76–7.78 (m, 2H, ArH),
7.48 (d, 2H, J = 7.0 Hz, ArH), 4.37 (s, 2H, –NH2), 2.85–2.95 (m, 4H,
piperidine), 2.71 (s, 2H, –NCH2), 1.70–1.80 (m, 4H, piperidine),
1.32–1.40 (m, 2H, piperidine); MS (ESI): m/z = 398 [M+H]+.
5.2.9.2. 2-(3-Aminoquinoxalin-2-ylamino)-N-(4-(pyrrolidin-1-
ylmethyl)phenyl)acetamide (11h). Yellow oil (12%); IR (KBr):
5.2.7.2. N-(3-Aminoquinoxalin-2-yl)-4-(pyrrolidin-1-ylmethyl)
benzamide hydrochloride (11e). Yellow solid (84%); mp:
3455, 2931, 2855, 1636, 1572, 1494, 1381 cmꢂ1 1H NMR1H NMR
;
(500 MHz, CD3OD) d: 7.67–7.69 (m, 1H, ArH), 7.61–7.63 (m, 1H,
ArH), 7.54–7.58 (m, 2H, ArH), 7.45–7.49 (m, 2H, ArH), 7.26–7.31
(m, 2H, ArH), 4.78 (s, 2H, –NH2), 4.21–4.23 (m, 2H, –NHCH2),
3.67 (s, 2H, –NCH2Ph), 3.45–3.55 (m, 4H, pyrrolidine), 2.12–2.20
(m, 4H, pyrrolidine); MS (ESI): m/z = 377 [M+H]+.
>250 °C. IR (KBr):3451, 2933, 2857, 1636, 1383, 1109 cmꢂ1 1H
;
NMR (d, CD3OD): 7.94 (d, 2H, J = 8.0 Hz, ArH), 7.81 (d, 2H,
J = 9.0 Hz, ArH), 7.60–7.64 (m, 2H, ArH), 7.49–7.51 (m, 2H, ArH),
3.45 (s, 2H, –NCH2Ph), 2.95 (m, 4H, pyrrolidine), 1.94 (m, 4H, pyr-
rolidine); MS (ESI): m/z = 385 [M+H]+.

7166
W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
5.2.9.3. 2-(3-Aminoquinoxalin-2-ylamino)-N-(1-benzylpiperi-
din-4-yl)acetamide (11i). Yellow oil (10%); IR (KBr): 3457,
were dissolved in 3 mL acetontrile, and the mixture was refluxed
for 3 h. After cooled to room temperature, 10 mL water was added
to the mixture, and extracted with ethyl acetate (2 ꢁ 20 mL). The or-
ganic layer was dried over Na2SO4. The solvent was evaporated. The
residue was purified by silica gel column chromatography (PE:A-
cOEt:TEA = 4:1:0.1) to afford 11r. Yellow oil (35%); IR (KBr): 3456,
2933, 2857, 1637, 1375, 1120 cmꢂ1 1H NMR (d, CDCl3): 7.37–
;
7.39 (m, 2H, ArH), 7.28–7.34 (m, 5H, ArH), 7.20–7.22 (m, 2H,
ArH), 3.59 (s, 2H, –NCH2Ph), 2.51–2.53 (m, 5H, piperidine, –
NHCH2CONH), 2.00–2.03 (m, 6H, piperidine); MS (ESI): m/z = 391
[M+H]+.
2927, 2858, 1637, 1469, 1384, 611 cmꢂ1 1H NMR (d, CDCl3): 8.35
;
(d, 1H, J = 8.0 Hz, ArH), 8.10–8.12 (m, 2H, ArH), 7.74–7.75 (m, 2H,
ArH), 7.58 (d, 2H, J = 8.0 Hz, ArH), 6.61 (s, 1H, –NCH–), 3.56 (s, 2H,
–NCH2Ph), 2.31–2.40 (m, 4H, piperidine), 1.50–1.53 (m, 4H, piperi-
dine), 1.37–1.42 (m, 2H, piperidine); MS (ESI): m/z = 346 [M+H]+
5.2.10. General procedure for the preparation of compounds
11j–m
To a solution of 7 (5 mmol) in 5 mL 1,2-dibromoethane or 1,3-
dibromopropane, 40% NaOH solution 5 mL and tetrabutyl ammo-
nium bromide (0.05 g) were added. After the mixture was refluxed
for 4 h, the organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (2 ꢁ 15 mL). The combined organic layer
was washed with brine, dried over Na2SO4. The solvent was re-
moved under vacuum to give crude product, which was purified
by silica gel column chromatography (PE:AcOEt:TEA = 10:1:0.1)
to afford compound 8 .
5.3. In vitro BACE 1 inhibit activity screening
All synthesized compounds were tested for their BACE 1 inhib-
itor activities, using fluorescence resonance energy transfer (FRET)
assay, which uses purified insect-expressed BACE 1 and a specific
substrate. An excitation wavelength of 355 nm and an emission
wavelength of 460 nm were used to monitor the hydrolysis of sub-
Compounds 11j–m were obtained by the procedure of 11g–i
using compound 8 as the starting material instead of compound 6.
strate. Compounds with inhibitory rates at 20
were tested for IC50 values.
lg/mL above 50%
5.2.10.1. 2,3-Diamine-N2-(2-(4-(piperidin-1-ylmethyl)phenoxy
)ethyl)quinoxaline (11j). Yellow oil (18%); IR (KBr):3445, 2857,
5.4. In vitro AChE inhibitory activity screening
1628, 1511, 1459, 1404, 1307 cmꢂ1
;
1H NMR (d, CD3OD): 7.53–
AChE activities were measured by the spectrophotometric
method with slight modification with rat cortex homogenate was
used as the resource of AChE. The brain homogenate was preincu-
bated for 5 min with tetraisopropyl pyrophosphoramide (iso-
OMPA) (0.04 mmol/L), a selective inhibitor of BuChE. For assay of
AChE activity, a reaction mixture of 200 ml containing acetylthi-
ocholine iodide 0.3 mmol/L, sodium phosphate buffer (0.1 mmol/
L, pH 7.4) 100 mL, homogenate or serum 20 mL and different con-
centrations of test compounds 20 mL were incubated at 37 °C for
15 min. The reaction was terminated by adding 50 mL 3% sodium
lauryl sulfate, then, 50 mL 0.2% 5,5-dithio-bis(2-nitrobenzoic acid)
was added to produce the yellow anion of 5-thio-2-nitrobenzoic
acid. The rate of color production was measured spectrophotomet-
rically at 440 nm. Assays were performed with at least seven con-
centrations of compounds and IC50 (drug concentration that
inhibits 50% AChE activity) was calculated according to the inhibi-
tion curve. Donepezil was applied as the positive control. All sam-
ples were assayed in duplicate.
7.55 (m, 1H, ArH), 7.38–7.42 (m, 3H, ArH), 7.22–7.25 (m, 2H,
ArH), 7.09 (d, 2H, J = 9.0 Hz, ArH), 4.33 (t, 2H, J = 5.5 Hz, -OCH2CH2),
4.09 (s, 2H, –NCH2), 3.98 (t, 2H, J = 5.5 Hz, –OCH2CH2), 3.02–3.06
(m, 4H, piperidine), 1.76–1.81 (m, 4H, piperidine), 1.62 (m, 2H,
piperidine); MS (ESI): m/z = 378 [M+H]+.
5.2.10.2.
2,3-Diamine-N2-(2-(4-((diethylamino)methyl)phen-
oxy)ethyl)quinoxaline (11k). Yellow oil (12%). IR (KBr):3447,
2929, 2861, 1731, 1636, 1512, 1463, 1381 cmꢂ1; 1H NMR (d, CD3OD):
7.69–7.74 (m, 2H, ArH), 7.52–7.55 (m, 2H, ArH), 7.41 (d, 2H,
J = 8.5 Hz, ArH), 6.94 (d, 2H, J = 8.5 Hz, ArH), 5.92 (m, 1H, –
NHCH2CH2O), 5.37 (s, 2H, –NH2), 4.08 (d, 2H, J = 6.5 Hz, –NHCH2),
3.95 (s, 2H, –NCH2Ph), 2.94 (q, 4H, J = 7.5 Hz, –N(CH2CH3)2), 2.06
(d, 2H, J = 6.5 Hz, –NHCH2CH2O), 0.90 (t, 6H, J = 7.5 Hz,
–
N(CH2CH3)2); MS (ESI): m/z = 366 [M+H]+.
5.2.10.3.
2,3-Diamine-N2-(2-(4-((4-methylpiperazin-1-yl)
methyl)phenoxy)ethyl)quinoxaline (11l). Yellow solid (15%);
mp: 114–117 °C. IR (KBr):3447, 2977, 2734, 1639, 1570, 1464,
5.5. In vitro H1R, H2R, H3R, H4R antagonistic action screening
1398 cmꢂ1 1H NMR (d, CD3OD): 7.67 (d, 1H, J = 7.0 Hz, ArH), 7.54
;
(d, 1H, J = 7.0 Hz, ArH), 7.37–7.40 (m, 4H, ArH), 7.12 (d, 2H,
J = 8.0 Hz, ArH), 5.06 (s, 2H, –NH2), 4.42 (t, 2H, J = 5.5 Hz, –
NHCH2CH2O), 4.09 (t, 2H, J = 5.5 Hz, –NHCH2CH2O), 3.75 (s, 2H, –
NCH2Ph), 3.10–3.16 (m, 4H, piperazine), 2.81–2.87 (m, 4H, pipera-
zine), 2.77 (s, 3H, –NCH3); MS (ESI): m/z = 393 [M+H]+.
5.5.1. Cell culture and generation of stable cell lines
HEK-293 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum,
100 U/mL penicillin, and 100 lg/mL streptomycin in a humidified
atmosphere of 95% air and 5% CO2 at 37 °C. For transfection, H1R,
H2R, H3R or H4R cDNA plasmid constructs were transfected or
co-transfected into HEK-293 cells using Lipofectamine 2000 (Invit-
rogen) according to the manufacturer’s instructions. Stable trans-
5.2.10.4. 2,3-Diamine-N2-(3-(4-(piperidin-1-ylmethyl)phenoxy)
propyl)quinoxaline (11m). Yellow solid (8%); mp: 117–121 °C.
IR (KBr):3452, 2930, 1636, 1486, 1414, 1372 cmꢂ1 1H NMR (d,
;
CD3OD): 7.84–7.87 (m, 1H, ArH), 7.75–7.77 (m, 1H, ArH), 7.62–
7.63 (m, 1H, ArH), 7.51–7.53 (m, 1H, ArH), 7.42 (d, 2H, J = 8.5 Hz,
ArH), 7.09 (d, 2H, J = 8.5 Hz, ArH), 4.76 (s, 1H, –NHCH2CH2CH2O),
4.29 (t, 2H, J = 5.5 Hz, –NHCH2CH2CH2O), 3.91 (m, 4H, –NCH2Ph, –
NHCH2CH2CH2O), 2.82–2.90 (m, 4H, piperidine), 2.32–2.36 (m,
2H, –NHCH2CH2CH2O), 1.81–1.84 (m, 4H, piperidine), 1.65–1.70
(m, 2H, piperidine); MS (ESI): m/z = 392 [M+H]+.
fectants were selected in the presence of 800 lg/mL G418.
5.5.2. cAMP accumulation
Stable HEK-293 cells co-transfected with H3R and pCRE-Luc
were seeded in a 48-well plate overnight and were grown to 90–
95% confluence. Next, the cells were stimulated with 10
lM for-
skolin or 10 M forskolin plus different concentrations of hista-
l
mine or compound in serum-free DMEM, and the cells were
incubated for 5 h at 37 °C. Luciferase activity was detected using
a firefly luciferase kit (Promega, Madison, WI). When required,
cells were treated overnight with or without PTX (100 ng/mL) in
serum-free DMEM before the experiment.
5.2.11. 2,3-Diamine-N2-(4-(piperidin-1-ylmethyl)benzylidene)
quinoxaline (11r)
2,3-Diaminoquinoxaline (40 mg, 0.25 mmol), 4-(piperidin-1-
ylmethyl)benzaldehyde 10(20 mg, 0.1 mmol) and 100 mg EDCꢀHCl

W. Huang et al. / Bioorg. Med. Chem. 19 (2011) 7158–7167
7167
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Acknowledgment
The authors thank the Zhejiang Provincial Natural Foundation
of China (No. R2110297) for financial support.
11. Magdolna, P.; János, K. Neurochem. Int. 2008, 53, 103.
12. Huang, W. H.; Yu, H. P.; Sheng, R.; Li, J.; Hu, Y. Z. Bioorg. Med. Chem. 2008, 16,
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