Discovery of novel 2,6-disubstituted pyridazinone derivatives as acetylcholinesterase inhibitors

Abstract

2,6-Disubstituted pyridazinone 4 was identified by HTS as a novel acetylcholinesterase (AChE) inhibitor. Under SAR development, compound 17e stood out as displaying high AChE inhibitory activity and AChE/butyrylcholinesterase (BuChE) selectivity in vitro. Docking studies revealed that 17e might interact with the catalytic active site (CAS) and the peripheral anionic site (PAS) simultaneously. Based on this novel binding information, 6-ortho-tolylamino and N-ethyl-N-isopropylacetamide substituted piperidine were disclosed as new PAS and CAS binders.

Full text

Original article
Discovery of novel 2,6-disubstituted pyridazinone derivatives
as acetylcholinesterase inhibitors
Weiqiang Xing
a
,
1
,YanFu
b
,
1
, Zhangxing Shi
a
, Dong Lu
a
, Haiyan Zhang
b
,
*
, Youhong Hu
a
,
*
a
State Key Laboratory of Drug Research, Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Shanghai 201203, China
b
State Key Laboratory of Drug Research, Department of Neuropharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Shanghai 201203, China
article info
Article history:
Received 3 November 2012
Received in revised form
24 January 2013
Accepted 27 January 2013
Available online 8 February 2013
Keywords:
Pyridazinone
Acetylcholinesterase
Alzheimer’s disease
Docking study
abstract
2,6-Disubstituted pyridazinone 4was identified by HTS as a novel acetylcholinesterase (AChE) inhibitor.
Under SAR development, compound 17 e stood out as displaying high AChE inhibitory activity and AChE/
butyrylcholinesterase (BuChE) selectivity in vitro. Docking studies revealed that 17e might interact with
the catalytic active site (CAS) and the peripheral anionic site (PAS) simultaneously. Based on this novel
binding information, 6-ortho-tolylamino and N-ethyl-N-isopropylacetamide substituted piperidine were
disclosed as new PAS and CAS binders.
Ó2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Alzheimer’s disease (AD) is a complex neurodegenerative dis-
order of the central nervous system. It is estimated that nearly 36
million people worldwide are now suffering from AD, and the
figure would be increased to about 66 million by 2030 if no
breakthroughs were made. Acetylcholinesterase (AChE), a serine
protease, is responsible for acetylcholine hydrolysis and plays a
fundamental role in impulse transmission by terminating the
action of the neurotransmitter acetylcholine at the cholinergic
synapses and neuromuscular junction [1]. Among the various
approaches for treating AD, inhibition of AChE is still prevailing in
treating or alleviating the symptoms of AD. Tacrine (1,Fig. 1), a
nonselective AChE/butyrylcholinesterase (BuChE) inhibitor, was
the first drug approved by FDA in 1993. Other selective inhibitors,
such as donepezil (2,Fig.1), also reached the market sequentially. In
recent years, novel AChE inhibitors are continuingly discovered
from natural resources or by synthetic approaches, bearing such as
berberine [2e6], coumarin [7,8], benzofuran [9],
b
-carboline [10],
quinoline [11e13], benzophenone [14,15], triazin [16], ferulic acid
[17] and naphthyridine [18] frameworks as the primary pharma-
cophoric scaffolds.
The most interesting structural character of AChE protein is the
presence of a deep narrow gorge, at the bottom of which a triad lies
in the catalytic anionic site (CAS). Besides, the peripheral anionic
site (PAS) lies at the entrance of the gorge as a regulatorysite. Based
on these findings, Pang et al. first reported a tacrine dimer 3(Fig. 1)
as a bivalent AChE inhibitor which interacts with CAS and PAS
simultaneously [19]. This compound displayed much higher AChE
inhibitory activity and specificity over BuChE than the parent
compound 1. Through this strategy, the second generation AChE
inhibitors are developed by utilizing one or two known scaffolds of
AChE inhibitor and most of them possess elevated potency and
improved AChE/BuChE selectivity profile [20e28].
Through an in vitro HTS campaign with diverse compounds
libraries inhouse, pyridazinone derivative 4(Fig. 1) stood out as a
potential AChE inhibitor with an IC
50
of 1.66
m
M. To our best
knowledge, only a few compounds bearing this scaffold were
reported with a liner pyridazine-donepezil hybrid 5(Fig. 1) in the
literature [29e31], which behaved as a dual site binding inhibitor.
By optimizing the 3,6-substitutions on the pyridazine ring, high
AChE affinity of the compound was achieved. However, this type of
molecules displayed low AChE/BuChE selectivity, which might lead
*Corresponding authors.
E-mail addresses: hzhang@mail.shcnc.ac.cn (H. Zhang), yhhu@mail.shcnc.ac.cn
(Y. Hu).
1
Authors equally contributed to this work.
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
0223-5234/$ esee front matter Ó2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.056
European Journal of Medicinal Chemistry 63 (2013) 95e103

to undesirable peripheral side effects. As the compound 4bears
different substituents on pyridazinone ring from 5a/5b and does
not share any structural similarity with donepezil, we realized that
the SAR of 4might not parallel with that of 5a/5b or donepezil, thus
leading this de novo pyridazinone molecule to be worth of opti-
mization for finding diverse AChE inhibitors with high potency and
AChE/BuChE selectivity.
2. Results and discussion
2.1. Chemistry
The general synthetic route to build the focused AChE-targeted
inhibitor library is illustrated in Scheme 1. Compound 9could be
obtained by a sequential process involving alkylation of corre-
sponding pyridazinone (6)[32e34] and followed by reduction of
the nitro group under Fe/HOAc in refluxing EtOH. The final products
were obtained by 9condensing the substituted piperidines in the
presence of triphosgene.
2.2. SAR study and docking study
Structural inspections of the compounds in our HTS library
revealed that the terminal 4-diethyl amide piperidine in 4may be
essential for activity. Firstly, we commenced with our work by
changing the substituents on the pyridazinone ring (Table 1).
Replacing the phenyl in 4by a single hydrogen (10) caused a slight
loss in activity, which indicated that the phenyl might function as a
hydrophobic pharmacophore. As the AChE binding pocket is a
narrow hydrophobic gorge, a small hydrophobic methyl group was
added to the phenyl (11aec) for modulating the binding affinity.
Compound 11c,ameta-methyl substituted pattern, displayed a
slight enhancement in activity with an IC
50
of 0.746
m
M. Besides,
other meta-halo substituted analogs (11d ee) remained biological
activity at enzymatic level. When the conjugated system in 4was
extended by replacing the phenyl with naphthyl (12aeb), or by
adding a cyano group on meta position (12c), the potency of com-
pounds was evidently enhanced. The most promising compound
12b displayed a potent activity with an IC
50
of 0.188
m
M, which was
about 9 folds more potent than the no-conjugation extended
compound 4.
In order to get a comprehensive understanding of the SAR,
docking study was performed. Crystal complex (code: 1EVE) of
donepezil and AChE protein was downloaded from Protein Data
Bank, in which the positively charged piperidine nitrogen in
donepezil achieves a caution-
p
interaction with Phe330 and the
terminal benzyl ring involves in
p
e
p
stacking with aromatic Trp84.
Docking results showed that 12b may function as a bi-functional
AChE inhibitor with novel binding conformation, which the naph-
thyl occupies in the PAS pocket and the 4-diethyl amide piperidine
is located at the CAS near the bottom of the gorge. The carbonyl
oxygen on pyridazinone ring possibly takes part in hydrogen
bonding interaction with Phe288 and the 2-benzyl onpyridazinone
involves in
p
e
p
stacking with Tyr334 (Fig. 2). No direct hydrogen
bond interaction was observed in the urea motif of 12b, however,
just as donepezil complex, a water bridged hydrogen bonding
might occur in biological circumstances.
Fig. 1. Representative AChE inhibitors.
Scheme 1. Synthesis of analogues
a
.
Table 1
AChE inhibitory activity of 6-aryl substituted pyridazinones.
No. R IC
50
(
m
M) No. R IC
50
(
m
M)
41.66 11d 0.623
10 H 4.90 11e 0.648
11a 2.61 12a 0.462
11b 2.01 12b 0.188
11c 0.746 12c 0.220
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e10396

As shown in Fig. 3, docking results revealed that the naphthyl for
PAS binder in 12b not only acts as a hydrophobic group involving in
the van der Waals interactions with the protein, but also functions
as a
p
e
p
interaction element, which mainly works by the extended
aromatic ring B. Considering the hydrophobic effects induced by
the phenyl A in 12b and the methyl in 11c , a novel 6-aniline pyr-
idazinone scaffold was designed, in which the ring B is remained as
a
p
e
p
interaction motif and ring A is broken by a single nitrogen
linker. In this new scaffold,
p
e
p
stacking efficiency of ring B may
also be elevated due to the increased rotation freedom.
Based on above analyses, 6-substituted aniline pyridazinones
were synthesized. As shown in Table 2, when the naphthyl
(hydrophobic-
p
motif) in 12b was replaced by a single aniline
(nitrogen-
p
motif, 13), the AChE inhibitory activity of 13 decreased
drastically (about 17 folds). However, the activity of 14 was restored
when the aniline was changed to more hydrophobic naphthalen-1-
amine, which indicated that the hydrophobic substitutions on the
aniline ring played a great part in binding affinity. Subsequently,
other analogs with different substituted anilines were synthesized
(15aef). The activity of compounds was decreased in the presence
of para substitutions (15c,f). The ortho substitutions significantly
increased the binding affinity probably due to a similar hydro-
phobic effect induced by ring A in 12b. In addition, a prevailing
‘Magic Methyl’effect emerged in these pyridazinone compounds
[35], a single 2-methyl aniline derivative (15a) showed the prom-
inent inhibitory potency with an IC
50
of 0.049
m
M. It was about 34
and 4-fold more potent than 4and 12b, respectively. Further
enlarging the steric hindrance to dimethyl, ethyl or isopropyl (16ae
c) decreased the activity.
Docking studies showed that 15a displays a similar binding
mode as 12b (Fig. 4A). Overlapping compounds 15a,4and 12b
(Fig. 4B) revealed that changing the phenyl in 4with naphthyl (12b)
or adding a conjugated group (12c) made it possible for
p
e
p
stacking with Trp279, which led to an increase in activity. In the
case of 15a , the 2-methyl aniline does act as a bi-functional group:
the aromatic phenyl stacks with Trp279 while the terminal methyl
fit within the PAS hydrophobic pocket perfectly.
After the modification of the active compounds for PAS binding
site, we optimized the CAS binding motifs to synthesize compounds
17a ef(Table 3). Cyclic analog 17a with the terminal morpholine
motif lost the activity totally. Among the acyclic amide series (17b e
f), the inhibitory activity of the compounds strongly paralleled with
the hydrophobic effect of the substitutions. Among them, the N-
ethyl-N-isopropyl amide derivative 17e showed slightly more
potent than 15a . This phenomenon is consistent with the hydro-
phobic environment of the CAS binding pocket.
2.3. Selectivity profile for AChE/BuChE
Moreover, the most potent and representative compounds were
selected for evaluation of AChE/BuChE selectivity profile. As shown
in Table 4, compared with the reported 3-amino-6-aryl pyridazine
series [29,30], 2,6-disubstituted pyridazin-3-one series displayed
about 200-fold or higher AChE/BuChE selectivity. For this scaffold,
AChE inhibitory activity of the compounds could be enhanced by
modifying PAS binding motif without affecting the BuChE affinity
by comparing 4with 12b,or13 with 15 a. In the catalytic binding
pocket, increasing the hydrophobic groups of the amide (17d ee)
lowered the IC
50
s for AChE accompanying with slight increasing
for the binding affinities of BuChE, However, the selectivity profiles
were still maintained.
Fig. 2. Model of compound 12a bound to AChE. The naphthyl and 4-substituted
piperidine interact with PAS and CAS respectively.
Fig. 3. Designing strategy for aniline series.
Table 2
AChE inhibitory activity of 6-aniline substituted pyridazinones.
No. Ar IC
50
(
m
M) No. Ar IC
50
(
m
M)
13 3.28 15d 0.119
14 0.126 15e 0.135
15a 0.049 15f 4.84
15b 1.42 16a 0.607
15c 19.8 16b 0.188
16c 1.50
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e103 97

3. Conclusion
In this report, a series of novel 2,6-disubstituted pyridazinone
derivatives for AChE inhibition was optimized. Under the SAR
development, high AChE affinity of the compounds was achieved
by optimizing different substituents on the pyridazinone ring,
without sacrificing the AChE/BuChE selectivity profile.
Docking study revealed that these pyridazinones might behave
as dual binding site inhibitors with novel binding conformation.
Utilizing this information, a delicate SAR study was performed at
PAS binding site and 6-ortho-tolylamino substitution was opti-
mized for involving in both
p
e
p
stacking and “Magic Methyl”
hydrophobic interaction. Moreover, rather than the prevailing N-
benzyl piperidine reported in donepezil, a novel hydrophobic 4-
substituted piperidine motif for catalytic pocket binder was dis-
closed, which also provided a new platform for designing AChE
inhibitor. Further biological evaluations of these pyridazinone ser-
ies are ongoing.
4. Experimental section
4.1. General information
Reagents were purified prior to use unless otherwise stated.
Column chromatography was carried out on silica gel (200e
300 mesh).
1
H NMR and
13
C NMR spectral data were recorded in
DMSO-d
6
,CD
3
OD or acetone-d
6
on Varian Mercury 500, 400 or 300
NMR spectrometer and Chemical shifts (
d
) were reported in parts
per million (ppm), and the signals were described as brs (broad
singlet), d (doublet), dd (doublet of doublet), m (multiple), q
(quarter), s (singlet), and t (triplet). Coupling constants (Jvalues)
were given in Hz. Low-resolution mass spectra (ESI) was obtained
using Agilent HPLC-MS (1260-6120B) and high-resolution mass
spectra (ESI) were obtained using Waters Q-Tof Ultima apparatus.
4.2. General procedures
To a solution of pyridazinone derivative [32e34] (0.5 mmol) in
DMF (10 mL) was added 1-(chloromethyl) 3-nitrobenzene
(0.52 mmol) and Cs
2
CO
3
(0.55 mmol), the resulting reaction mix-
ture was stirred at 40e50

C until no starting materials was
detected by TLC (about 3 h). The solvent was removed under
reduced pressure and the residue was dissolved in EtOAc (30 mL),
washed with brine (3 10 mL). The organic layer was dried over
anhydrous Na
2
SO
4
and concentrated in vacuo. The crude product
was dissolved in 95% ethanol (50 mL) containing 10 mmol acetic
acid. Iron powder (2 mmol) was added and the resulting mixture
Fig. 4. (A) Model of compound 15 a bound to AChE. The ortho-tolylamino and
4-substituted piperidine interact with PAS and CAS, respectively. (B) Overlap of 4
(magenta), 12b (yellow), and 15a (green). The naphthyl in 12b and ortho-methyl amine
in 15a function as
p
e
p
stacking element and involve in PAS van der Waals interaction.
Table 3
SAR study at catalytic binding site.
No. R IC
50
(
m
M)
17a NA
17b 7.60
17c 0.473
17d 0.046
15a 0.050
17e 0.028
17f 0.116
Table 4
Selectivity profile of representative compounds for AChE versus BuChE.
No. IC
50
(
m
M) Ratio of IC
50
(BuChE/AChE)
AChE BuChE
41.66 >40 >24
12b 0.188 >40 >212
13 3.28 >40 >12
15a 0.049 >40 >816
17c 0.473 >40 >84
17d 0.046 10.0 217
17e 0.028 5.50 196
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e10398

was stirred for 5 h. After cooled to room temperature, the reaction
mixture was filtered through celite and the filter cake was washed
with 95% ethanol (3 15 mL). The combined ethanol layers were
evaporated in vacuo and the residue was re-dissolved in ethyl
acetate (30 mL). The organic layer was washed with brine
(3 10 mL) and 2 M NaOH (10 mL) sequentially. The organic layer
was dried over anhydrous Na
2
SO
4,
evaporated in vacuo to afford the
crude 2-aminobenzyl-6-substituted-pyridazin-3(2H)-ones, which
were used without further purification.
To a stirred solution of 2-aminobenzyl-6-substituted-pyridazin-
3(2H)-one and triphosgene (1 mmol) in dry dichloromethane
(5 mL) was added triethylamine (2 mmol) under nitrogen atmos-
phere. A solution of the corresponding piperidine (1 mmol) in
dichloromethane (5 mL) was added 5e10 min later and the mixture
was stirred at room temperature overnight. The reaction mixture
was diluted with dichloromethane (15 mL) and washed with water
(3 20 mL). The organic phases were separated, dried over anhy-
drous Na
2
SO
4
and concentrated in vacuo. The residue was purified
by column chromatography to afford the products.
4.2.1. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-phenylpyridazin-1(6H)-yl)
methyl)phenyl)piperidine-1,4-dicarboxamide (4)
Yield: 81%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.03e7.87 (m, 4H),
7.61e7.38 (m, 5H), 7.19 (t, J¼7.9 Hz, 1H), 7.04 (d, J¼7.7 Hz, 1H), 6.99
(d, J¼9.9 Hz,1H), 5.32 (s, 2H), 4.30e4.15 (m, 2H), 3.47e3.37 (m, 2H),
3.32 (d, J¼6.8 Hz, 2H), 2.99e2.74 (m, 3H), 1.68 (s, 4H), 1.18 (t,
J¼7.0 Hz, 3H), 1.03 (t, J¼6.9 Hz, 3H);
13
C NMR (125 MHz,MeOD-d
4
)
d
174.67,160.25, 156.20, 145.43, 139.91, 136.53, 134.31, 131.02, 129.41,
129.22, 128.51, 128.35, 125.74, 122.58, 120.41, 120.20, 55.33 (eCH
2
e
Phe), 43.31 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.84 (eCON(CH
2
CH
3
)
2
),
40.29 (eCON(CH
2
CH
3
)
2
), 38.32 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.39
(eNHCON(CH
2
CH
2
)
2
CHCOe), 13.85 (eCON(CH
2
CH
3
)
2
), 11.90 (e
CON(CH
2
CH
3
)
2
); HRMS(ESI): m/z[M þH]
þ
488; HRMS(ESI) calcd
for C
28
H
33
N
5
O
3
Na [M þNa]
þ
510.2481, found 510.2492.
4.2.2. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxopyridazin-1(6H)-yl)methyl)
phenyl)piperidine-1,4-dicarboxamide (10)
Yield: 58%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.06 (s, 1H), 7.82
(dd, J¼3.6, 1.4 Hz, 1H), 7.53 (d, J¼8.2 Hz,1H), 7.47 (s,1H), 7.33(dd,
J¼9.5, 3.8 Hz, 1H), 7.16 (t, J¼7.9 Hz, 1H), 6.94 (d, J¼7.7 Hz, 1H),
6.87 (dd, J¼9.5, 1.6 Hz, 1H), 5.21 (s, 2H), 4.29e4.15 (m, 2H), 3.43
(q, J¼7.1 Hz, 2H), 3.32 (q, J¼7.0 Hz, 2H), 2.98e2.77 (m, 3H), 1.73e
1.59 (m, 4H), 1.18 (t, J¼7.1 Hz, 3H), 1.03 (t, J¼7.0 Hz, 3H);
13
C
NMR (125 MHz, MeOD-d
4
)
d
174.73, 161.04, 156.17, 139.92, 137.38,
136.56,132.36, 129.08, 128.25, 122.32, 120.22,120.04, 54.88 (eCH
2
e
Phe), 43.29 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.81 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.28 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.39 (eNHCON(CH
2
CH
2
)
2
CHCOe), 13.73 (eCON(CH
2
CH
3
)
2
), 11.78
(eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z412 [M þH]
þ
; HRMS(ESI) calcd
for C
22
H
30
N
5
O
3
[M þH]
þ
412.2349, found 412.2331.
4.2.3. Diethyl-N
1
-(3-((6-oxo-3-(o-tolyl)pyridazin-1(6H)-yl)methyl)
phenyl)piperidine-1,4-dicarboxamide (11a)
Yield: 69%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.01 (s, 1H), 7.53 (t,
J¼8.3 Hz, 3H), 7.41 (d, J¼7.1 Hz, 1H), 7.35e7.23 (m, 3H), 7.17 (t,
J¼7.7 Hz, 1H), 7.03e6.92 (m, 2H), 5.27 (s, 2H), 4.31e4.16 (m, 2H),
3.42 (q, J¼7.0 Hz, 2H), 3.34e3.26 (m, 2H), 2.87 (ddd, J¼21.2, 14.9,
7.1 Hz, 3H), 2.32 (s, 3H), 1.75e1.61 (m, 4H),1.17 (t, J¼7.1 Hz, 3H),1.02
(t, J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.72, 159.99,
156.17, 147.71, 139.97, 136.68, 135.94, 134.90, 134.25, 130.60, 128.91,
128.80, 128.71,128.29, 125.72, 122.49,120.40, 120.06, 55.02 (eCH
2
e
Phe), 43.30 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.29 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.39
(eNHCON(CH
2
CH
2
)
2
CHCOe), 19.20 (eNHePheCH
3
), 13.73 (e
CON(CH
2
CH
3
)
2
), 11.79 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
502; HRMS(ESI) calcd for C
29
H
35
N
5
O
3
Na [M þNa]
þ
524.2638, found
524.2657.
4.2.4. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(p-tolyl)pyridazin-1(6H)-yl)
methyl)phenyl)piperidine-1,4-dicarboxamide(11 b )
Yield: 61%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.02 (s, 1H), 7.90 (d,
J¼9.7 Hz, 1H), 7.78 (d, J¼8.3 Hz, 2H), 7.53 (d, J¼10.8 Hz, 2H), 7.27
(d, J¼8.0 Hz, 2H), 7.17 (t, J¼7.8 Hz, 1H), 7.02 (d, J¼7.4 Hz, 1H), 6.95
(d, J¼9.7 Hz, 1H), 5.29 (s, 2H), 4.30e4.15 (m, 2H), 3.42 (q, J¼7.2 Hz,
2H), 3.31 (q, J¼7.0 Hz, 2H), 2.96e2.73 (m, 3H), 2.35 (s, 3H),1.71e1.58
(m, 4H), 1.17 (t, J¼7.1 Hz, 3H), 1.02 (t, J¼7.0 Hz, 3H);
13
C NMR
(126 MHz, MeOD-d
4
)
d
174.72, 160.21, 156.21, 145.44, 139.94, 139.53,
136.66, 131.53, 130.93,129.26, 129.11, 128.29, 125.62, 122.50, 120.31,
120.13, 55.18 (eCH
2
ePhe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe),
41.79 (eCON(CH
2
CH
3
)
2
), 40.24 (eCON(CH
2
CH
3
)
2
), 38.28 (eNHCON
(CH
2
CH
2
)
2
CHCOe), 28.37 (eNHCON(CH
2
CH
2
)
2
CHCOe), 19.80 (e
NHePheCH
3
), 13.71 (eCON(CH
2
CH
3
)
2
), 11.77 (eCON(CH
2
CH
3
)
2
);
LCMS(ESI) m/z[M þH]
þ
502; HRMS(ESI) calcd for C
29
H
35
N
5
O
3
Na
[M þNa]
þ
524.2638, found 524.2622.
4.2.5. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(m-tolyl)pyridazin-1(6H)-yl)
methyl)phenyl)piperidine-1,4-dicarboxamide (11 c )
Yield: 73%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.08 (s, 1H), 7.88 (d,
J¼9.8 Hz, 1H), 7.72 (s, 1H), 7.65 (d, J¼7.8 Hz, 1H), 7.61 (s, 1H), 7.51
(d, J¼8.8 Hz, 1H), 7.32 (t, J¼7.7 Hz,1H), 7.22 (d, J¼7.6 Hz, 1H), 7.17
(t, J¼7.9 Hz,1H), 7.01 (d, J¼7.5 Hz,1H), 6.94 (d, J¼9.7 Hz, 1H), 5.29
(s, 2H), 4.28e4.15 (m, 2H), 3.47e3.28 (m, 4H), 3.00e2.71 (m, 3H),
2.37 (s, 3H), 1.75e1.55 (m, 4H), 1.22e1.10 (t, J¼7.0 Hz, 3H), 1.01 (t,
J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.66, 160.15,
156.08, 145.40, 140.02, 138.35, 136.63, 134.19, 131.01, 129.91, 129.23,
128.40,128.33, 126.24,122.88, 122.43,120.22, 120.00, 55.24 (eCH
2
e
Phe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.24 (eCON(CH
2
CH
3
)
2
), 38.25 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.39 (eNHCON(CH
2
CH
2
)
2
CHCOe), 20.13 (eNHePheCH
3
e), 13.79
(eCON(CH
2
CH
3
)
2
), 11.86 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z
[M þH]
þ
502; HRMS(ESI) calcd for C
29
H
35
N
5
O
3
Na [M þNa]
þ
524.2638, found 524.2637.
4.2.6. N
1
-(3-((3-(3-Bromophenyl)-6-oxopyridazin-1(6H)-yl)
methyl)phenyl)-N
4
,N
4
-diethylpiperidine-1,4-dicarboxamide (11d)
Yield: 86%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.08 (s, 1H), 8.03e
7.93 (m, 2H), 7.90 (d, J¼8.0 Hz, 1H), 7.60 (s, 2H), 7.51 (d, J¼8.1 Hz,
1H), 7.42 (t, J¼7.7 Hz, 1H), 7.18 (t, J¼7.7 Hz, 1H), 7.00 (t, J¼9.3 Hz,
2H), 5.31 (s, 2H), 4.28e4.15 (m, 2H), 3.42 (d, J¼7.0 Hz, 2H), 3.31 (d,
J¼7.2 Hz, 2H), 3.01e2.76 (m, 3H), 1.67 (s, 4H), 1.17 (t, J¼6.8 Hz, 3H),
1.02 (t, J¼6.9 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.72,
160.14, 156.16, 143.77, 139.99, 136.47, 132.03,130.76, 130.25, 129.50,
128.54, 128.33, 124.49, 122.54, 120.30, 120.12, 55.28 (eCH
2
ePhe),
43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
), 40.25
(eCON(CH
2
CH
3
)
2
), 38.27 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.38 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 13.72 (eCON(CH
2
CH
3
)
2
), 11.78 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
566; HRMS(ESI) calcd
for C
28
H
32
N
5
O
3
NaBr [M þNa]
þ
588.1586, found 588.1569.
4.2.7. N
1
-(3-((3-(3-Chlorophenyl)-6-oxopyridazin-1(6H)-yl)
methyl)phenyl)-N
4
,N
4
-diethylpiperidine-1,4-dicarboxamide (11e)
Yield: 92%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.06 (s, 1H), 7.96 (d,
J¼9.8 Hz, 2H), 7.85 (d, J¼7.1 Hz, 1H), 7.63 (s, 1H), 7.55e7.43 (m, 3H),
7.18 (t, J¼7.8 Hz, 1H), 7.02 (t, J¼8.1 Hz, 2H), 5.32 (s, 2H), 4.30e4.19
(m, 2H), 3.50e3.25 (m, 4H), 3.01e2.75 (m, 3H), 1.78e1.60 (m, 4H),
1.18 (dd, J¼8.3, 5.8 Hz, 3H), 1.03 (t, J¼7.0 Hz, 3H);
13
C NMR
(125 MHz, MeOD-d
4
)
d
174.72, 160.16, 156.18, 143.90, 139.98, 136.47,
136.29, 134.54, 130.79, 130.03, 129.50, 129.05, 128.32, 125.61,
124.07, 122.55, 120.33, 120.13, 55.28 (eCH
2
ePhe), 43.27 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 41.79 (eCON(CH
2
CH
3
)
2
), 40.24 (e
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e103 99

CON(CH
2
CH
3
)
2
), 38.27 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.37 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 13.71 (eCON(CH
2
CH
3
)
2
), 11.77 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
522; HRMS(ESI) calcd
for C
28
H
32
N
5
O
3
NaCl [M þNa]
þ
544.2091, found 544.2084.
4.2.8. N
4
,N
4
-Diethyl-N
1
-(3-((3-(naphthalen-2-yl)-6-oxopyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (12a)
Yield: 88%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.39 (s, 1H), 8.25e
7.70 (m, 6H), 7.68 (s, 1H), 7.53 (d, J¼6.3 Hz, 3H), 7.19 (t, J¼7.8 Hz,
1H), 7.06 (d, J¼7.6 Hz, 1H), 6.98 (d, J¼9.8 Hz, 1H), 5.33 (s, 2H),
4.30e4.15 (m, 2H), 3.44e3.29 (m, 4H), 2.98e2.73 (m, 3H), 1.68 (m,
4H), 1.23e1.09 (t, J¼7.0 Hz, 3H), 1.02 (t, J¼7.0 Hz, 3H);
13
C NMR
(125 MHz, MeOD-d
4
)
d
174.69, 160.16, 156.18, 145.07, 140.00, 136.63,
133.66, 133.14, 131.52, 130.89,129.24, 128.33, 128.22, 128.20,127.23,
126.61, 126.21,125.33, 122.82, 122.57, 120.33, 120.09, 55.24 (eCH
2
e
Phe), 43.27 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.78 (eCON(CH
2
CH
3
)
2
),
40.23 (eCON(CH
2
CH
3
)
2
), 38.25 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.36 (eNHCON(CH
2
CH
2
)
2
CHCOe), 13.70 (eCON(CH
2
CH
3
)
2
), 11.77
(eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
538; HRMS(ESI) calcd
for C
32
H
35
N
5
O
3
Na [M þNa]
þ
560.2638, found 560.2643.
4.2.9. N
4
,N
4
-Diethyl-N
1
-(3-((3-(naphthalen-1-yl)-6-oxopyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (12b)
Yield: 76%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.19e7.87 (m, 4H),
7.7 2e7.46 (m, 7H), 7.22 (t, J¼7.8 Hz, 1H), 7.03 (q, J¼8.7 Hz, 2H), 5.34
(s, 2H), 4.30e4.16 (m, 2H), 3.47e3.27 (m, 4H), 3.15e2.25 (m, 3H),
1.80e1.50(m, 4H), 1.23e1.12 (t, J¼7.0 Hz, 3H), 1.03 (t, J¼7.0 Hz, 3H);
13
C NMR (100 MHz, MeOD-d
4
)
d
174.74, 160.15, 156.21,
147.00,140.08, 136.73,134.90, 133.93,132.83,130.49,129.56,129.22,
128.46, 128.20,127.13, 126.62, 125.93,124.87,124.70, 122.86, 120.70,
120.26, 55.04 (eCH
2
ePhe), 43.35 (eNHCON(CH
2
CH
2
)
2
CHCOe),
41.88 (eCON(CH
2
CH
3
)
2
), 40.34 (eCON(CH
2
CH
3
)
2
), 38.36 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 28.46 (eNHCON(CH
2
CH
2
)
2
CHCOe),
13.86 (eCON(CH
2
CH
3
)
2
), 11.91 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z
[M þH]
þ
538; HRMS(ESI) calcd for C
32
H
35
N
5
O
3
Na [M þNa]
þ
560.2638, found 560.2631.
4.2.10. N
1
-(3-((3-(3-Cyanophenyl)-6-oxopyridazin-1(6H)-yl)
methyl)phenyl)-N
4
,N
4
-diethyl piperidine-1,4-dicarboxamide (12c)
Yield: 79%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.31 (s, 1H), 8.23 (d,
J¼7.8Hz, 1H), 8.05 (s, 1H), 8.01 (s,1H), 7.82 (d, J¼7.6 Hz, 1H), 7.70e
7.65 (m, 2H), 7.48 (d, J¼7.9 Hz, 1H), 7.19 (t, J¼7.8 Hz, 1H), 7.05 (d,
J¼7.7Hz, 1H), 7.00 (d, J¼9.8 Hz, 1H), 4.30e4.17 (m, 2H), 3.43e3.25
(m, 4H), 3.00e2.80 (m, 3H),1.70e1.60 (m, 4H), 1.12 (t, J¼7.0 Hz, 3H),
1.03(t, J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.71,160.04,
156.10, 143.14, 140.06, 136.39, 135.65, 132.42, 130.56, 130.07, 129.64,
129.27, 128.36, 122.58, 120.29, 120.01, 117.87, 112.71, 55.26 (eCH
2
e
Phe), 43.29 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.27 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.39
(eNHCON(CH
2
CH
2
)
2
CHCOe), 13.74 (eCON(CH
2
CH
3
)
2
), 11.80 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
513; HRMS(ESI) calcd for
C
29
H
32
N
6
O
3
Na [M þNa]
þ
535.2434, found 535.2439.
4.2.11. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(phenylamino)pyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (13 )
Yield: 64%:
1
H NMR (300 MHz, CD
3
OD-d
4
)
d
7.47 (d, J¼8.2 Hz,
2H), 7.41 (s, 1H), 7.33 (d, J¼8.7 Hz, 1H), 7.25e7.07 (m, 4H), 7.08 (d,
J¼7.6 Hz,1H), 6.92 (t, J¼7.8 Hz, 2H), 5.20 (s, 2H), 4.30e4.15 (m, 2H),
3.50e3.30 (m, 4H), 3.05e2.72 (m, 3H), 1.80e1.64 (m, 4H), 1.23 (t,
J¼7.0 Hz, 3H),1.10 (t, J¼7.1 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.74, 158.48, 156.24, 145.41, 140.36, 139.85, 137.09, 130.15, 128.22,
128.17, 122.72, 121.03,120.72, 120.05, 117.66, 101.92, 54.02 (eCH
2
e
Phe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.79 (eCON(CH
2
CH
3
)
2
),
40.24 (eCON(CH
2
CH
3
)
2
), 38.30 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.38
(eNHCON(CH
2
CH
2
)
2
CHCOe), 13.69 (eCON(CH
2
CH
3
)
2
), 11.75 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
503; HRMS(ESI) calcd
for C
28
H
34
N
6
O
3
Na [M þNa]
þ
525.2590, found 525.2618.
4.2.12. N
4
,N
4
-Diethyl-N
1
-(3-((3-(naphthalen-1-ylamino)-6-
oxopyridazin-1(6H)-yl)methyl)phenyl) piperidine-1,4-
dicarboxamide (14)
Yield: 81%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.23 (d, J¼8.8 Hz,
1H), 8.16 (s, 1H), 8.03 (s,1H), 7.99e7.91 (m, 1H), 7.88 (d, J¼4.7 Hz,
1H), 7.63e7.38 (m, 7H), 7.18 (t, J¼7.8 Hz,1H), 6.99 (d, J¼7.8 Hz, 1H),
6.87 (d, J¼9.8 Hz, 1H), 5.10 (s, 2H), 4.30e4.15 (m, 2H), 3.48e3.39
(m, 2H), 3.39e3.25 (m, 2H), 3.00e2.18 (m, 3H), 1.79e1.62 (m, 4H),
1.19 (t, J¼6.8 Hz, 3H), 1.04 (t, J¼7.1 Hz, 3H);
13
C NMR (125 MHz,
MeOD-d
4
)
d
174.84, 158.86, 156.34, 146.75, 139.88, 137.14, 135.47,
134.45, 130.31, 128.29, 128.10, 128.03, 127.27, 125.56, 125.40,125.33,
123.40, 122.83, 121.69, 120.85, 120.16, 117.56, 54.11 (eCH
2
ePhe),
43.39 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.91 (eCON(CH
2
CH
3
)
2
), 40.36
(eCON(CH
2
CH
3
)
2
), 38.41 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.51 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 13.82 (eCON(CH
2
CH
3
)
2
), 11.89 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
553; HRMS(ESI) calcd
for C
32
H
36
N
6
O
3
Na [M þNa]
þ
575.2747, found 575.2739.
4.2.13. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(o-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (15a)
Yield: 80%:
1
H NMR (300 MHz, DMSO-d
6
)
d
8.51 (s, 1H), 7.93 (s,
1H), 7.60 (d, J¼9.0 Hz, 1H), 7.38 (m, 2H), 7.31e7.05 (m, 3H), 6.96e
6.79 (m, 3H), 4.20e4.12 (m 2H), 3.40e3.24 (m, 4H), 2.94e2.69 (m,
3H), 2.16 (s, 3H), 1.67e1.44 (m, 4H), 1.13 (t, J¼7.0 Hz, 3H), 0.99 (t,
J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.73, 158.67,
156.21, 146.39, 139.77, 137.96, 137.05, 130.17, 130.02, 129.93, 128.15,
127.87, 125.84, 123.29, 122.65, 121.85, 120.75, 120.04, 53.87 (eCH
2
e
Phe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.30 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.40
(eNHCON(CH
2
CH
2
)
2
CHCOe), 16.77 (eNHePheCH
3
), 13.71 (e
CON(CH
2
CH
3
)
2
), 11.77 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
517; HRMS(ESI) calcd for C
29
H
36
N
6
O
3
Na [M þNa]
þ
539.2747, found
539.2742.
4.2.14. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(m-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (15b)
Yield: 78%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.15 (s, 1H), 8.01 (s,
1H), 7.56 (s, 2H), 7.40 (s,1H), 7.34 (d, J¼7.9 Hz,1H), 7.26e7.10 (m,3H),
7.07 (d, J¼7.5 Hz, 1H), 6.83 (d, J¼9.8 Hz,1H), 6.74 (d, J¼6.8 Hz,1H),
5.15 (s, 2H), 4.30e4.20 (m, 2H), 3.44 (t, J¼7.0 Hz, 2H), 3.34 (q,
J¼7.0Hz, 2H), 3.00e2.92 (m, 3H), 2.28 (s, 3H),1.75e1.64(m, 4H), 1.20
(d, J¼7.0 Hz, 3H), 1.05 (t, J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-
d
4
)
d
174.66, 158.38, 156.14, 145.39, 140.29, 139.92, 138.00,
137.23, 129.97, 128.30, 128.19, 128.14, 122.79, 121.82, 120.80, 120.09,
118.22, 114.84, 53.96 (eCH
2
ePhe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.81 (eCON(CH
2
CH
3
)
2
), 40.25 (eCON(CH
2
CH
3
)
2
), 38.26
(eNHCON(CH
2
CH
2
)
2
CHCOe), 28.41 (eNHCON(CH
2
CH
2
)
2
CH COe),
20.41 (eNHePheCH
3
), 13.80 (eCON(CH
2
CH
3
)
2
), 11.87 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
517; HRMS(ESI) calcd for
C
29
H
36
N
6
O
3
Na [M þNa]
þ
593.2747, found 593.2744.
4.2.15. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-(p-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide (15c)
Yield: 82%:
1
H NMR (300 MHz, DMSO-d
6
)
d
8.88 (s, 1H), 8.49 (s,
1H), 7.45 (s, 1H), 7.40e7.32 (m, 3H), 7.20e7.13 (m, 2H), 7.02 (d,
J¼8.5 Hz, 2H), 6.88e6.78 (m, 2H), 4.15e4.05 (m, 2H), 3.30e3.19 (m,
4H), 2.90e2.70 (m, 3H), 2.19 (s, 3H), 1.65e1.40 (m, 4H), 1.11 (t,
J¼7.1 Hz, 3H), 0.97 (t, J¼7.0 Hz, 3H);
13
C NMR (100 MHz, MeOD-d
4
)
d
174.79,158.51, 156.32, 145.63,139.90, 137.90, 137.17,130.55,130.06,
128.74, 128.28, 128.22, 122.79, 120.78, 120.11, 117.87, 54.12 (eCH
2
e
Phe), 43.36 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.86 (eCON(CH
2
CH
3
)
2
),
40.31(eCON(CH
2
CH
3
)
2
), 38.36 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.46
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e103100

(eNHCON(CH
2
CH
2
)
2
CHCOe), 19.40 (eNHePheCH
3
), 13.77 (e
CON(CH
2
CH
3
)
2
), 11.83 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
517; HRMS(ESI) calcd for C
29
H
36
N
6
O
3
Na [M þNa]
þ
593.2747, found
593.2745.
4.2.16. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-((2-(trifluoromethyl)phenyl)
amino)pyridazin-1(6H)-yl)methyl)phenyl)piperidine-1,4-
dicarboxamide (15d)
Yield: 75%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.06 (s, 1H), 7.91 (d,
J¼8.4 Hz, 1H), 7.64 (d, J¼7.9 Hz, 1H), 7.61e7.53 (m, 2H), 7.50 (d,
J¼8.1 Hz, 1H), 7.39 (d, J¼9.8 Hz, 2H), 7.25e7.10 (m, 2H), 6.97 (d,
J¼7.6 Hz, 1H), 6.88 (d, J¼9.8 Hz, 1H), 5.07 (s, 2H), 4.30e4.15 (m,
2H), 3.50e3.40 (m, 2H), 3.40e3.25 (m, 2H), 2.90e2.77 (m, 3H),
1.78e1.63 (m, 4H), 1.23e1.14 (t, J¼7.0 Hz, 3H), 1.04 (t, J¼7.0 Hz,
3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.68, 158.74, 156.12, 145.73,
139.89,137.65,136.99, 132.35,130.67,128.23, 127.90, 125.92, 125.88,
125.10, 123.35, 122.67, 121.71, 121.48, 120.76, 120.03, 53.98 (eCH
2
e
Phe), 43.30 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.82 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.29 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.44 (eNHCON(CH
2
CH
2
)
2
CHCOe), 13.80 (eCON(CH
2
CH
3
)
2
), 11.86
(eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
571; HRMS(ESI) calcd
for C
29
H
33
N
6
O
3
NaF
3
[M þNa]
þ
593.2464, found 593.2485.
4.2.17. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-((3-(trifluoromethyl)phenyl)
amino)pyridazin-1(6H)-yl) methyl)phenyl)piperidine-1,4-
dicarboxamide (15e)
Yield: 74%:
1
H NMR (400 MHz, CD
3
OD)
d
7.93 (s, 1H), 7.57 (d,
J¼9.2 Hz,1H), 7.46 (s, 1H), 7.32 (t, J¼8.0 Hz, 2H), 7.21 (t, J¼7.8 Hz,
1H), 7.12 (d, J¼7.7 Hz,1H), 7.09 (d, J¼7.6 Hz,1H), 7.00 (d, J¼9.7 Hz,
1H), 6.76(d, J¼9.7 Hz, 1H), 5.12 (s,2H), 4.18 (d, J¼13.2 Hz, 2H), 3.45e
3.34 (m, 2H), 3.30 (t, J¼7.1 Hz, 2H), 2.99e2.82 (m, 2H), 2.82e2.73 (m,
1H), 1.69 (dd, J¼19.6, 8.4 Hz, 4H), 1.17 (t, J¼7.1 Hz, 3H), 1.05 (t,
J¼7.1 Hz, 3H);
13
C NMR (125MHz, MeOD-d
4
)
d
174.63,158.28,156.16,
144.83, 141.02, 139.97, 137.01, 130.45, 129.02, 128.36, 127.95, 125.36,
123.20, 123.03, 121.08, 120.67, 120.38, 117.01, 113.70, 54.15 (eCH
2
e
Phe), 43.31 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.24 (eCON(CH
2
CH
3
)
2
), 38.26 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.40
(eNHCON(CH
2
CH
2
)
2
CHCOe), 13.80 (eCON(CH
2
CH
3
)
2
), 11.87 (e
CON(CH
2
CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
571; HRMS(ESI) calcd for
C
29
H
33
N
6
O
3
NaF
3
[M þNa]
þ
593.2464, found 593.2449.
4.2.18. N
4
,N
4
-Diethyl-N
1
-(3-((6-oxo-3-((4-(trifluoromethyl)phenyl)
amino)pyridazin-1(6H)-yl) methyl)phenyl)piperidine-1,4-
dicarboxamide (15f)
Yield: 58%:
1
H NMR (300 MHz, CD
3
OD-d
4
)
d
7.62 (d, J¼8.6 Hz,
2H), 7.54e7.45 (m, 3H), 7.35e7.29 (m, 1H), 7.26 (t, J¼7.7 Hz,1H), 7.18
(d, J¼9.7 Hz,1H), 7.09 (d, J¼7.2 Hz,1H), 6.94 (d, J¼9.7 Hz,1H), 4.25e
4.15 (m, 2H), 3.50e3.31 (m, 4H), 3.32e2.78 (m, 3H), 1.77e1.66 (m,
3H), 1.23 (t, J¼7.1 Hz, 3H),1.09 (t, J¼7.1 Hz, 3H);
13
C NMR (126 MHz,
DMSO-d
6
)
d
173.40, 157.48, 155.21, 155.14, 144.53, 143.87, 143.78,
141.31, 141.21, 137.65, 131.83, 128.75, 128.37, 128.30, 126.40,
126.14, 123.99, 122.11, 120.87, 120.61, 119.99, 119.89, 119.01, 118.91,
117.30, 117.24, 53.73 (eCH
2
ePhe), 43.71 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.48 (eCON(CH
2
CH
3
)
2
), 39.76 (eCON(CH
2
CH
3
)
2
), 37.86
(eNHCON(CH
2
CH
2
)
2
CHCOe), 28.93 (eNHCON(CH
2
CH
2
)
2
CHCOe),
15.34 (eCON(CH
2
CH
3
)
2
), 13.39 (eCON(CH
2
CH
3
)
2
); LCMS(ESI) m/z
[M þH]
þ
571; HRMS(ESI) calcd for C
29
H
33
N
6
O
3
NaF
3
[M þNa]
þ
593.2464, found 593.2482.
4.2.19. N
1
-(3-((3-((2,6-Dimethylphenyl)amino)-6-oxopyridazin-
1(6H)-yl)methyl)phenyl)-N
4
,N
4
-diethylpiperidine-1,4-
dicarboxamide (16a)
Yield: 76%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.01 (s,1H), 7.51 (d,
J¼9.1 Hz, 1H), 7.31 (d, J¼7.0 Hz, 2H), 7.16e7.02 (m, 5H), 6.79 (d,
J¼9.7 Hz, 2H), 4.92 (s, 2H), 4.3e4.15 (m, 2H), 3.51e3.31 (m, 4H),
3.10e2.25 (m, 3H), 2.14 (s, 6H),1.77e1.63 (m, 4H), 1.19 (t, J¼7.0 Hz,
3H), 1.04 (t, J¼7.0 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.74,
158.58, 156.17, 147.34, 139.60, 136.96, 135.97, 135.60, 130.43, 128.02,
127.62, 126.56, 125.95, 122.47, 120.84, 120.01, 53.60 (eCH
2
ePhe),
43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.81 (eCON(CH
2
CH
3
)
2
), 40.25
(eCON(CH
2
CH
3
)
2
), 38.32 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.43 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 17.07, 13.72 (eCON(CH
2
CH
3
)
2
), 11.78 (e
CON(CH
2
CH
3
)
2
); HRMS(ESI) m/z[M þH]
þ
531; HRMS(ESI) calcd
for C
30
H
38
N
6
O
3
Na [M þNa]
þ
553.2903, found 553.2876.
4.2.20. N
4
,N
4
-Diethyl-N
1
-(3-((3-((2-ethylphenyl)amino)-6-
oxopyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-
dicarboxamide (16b)
Yield: 70%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.07 (s, 1H), 7.68 (d,
J¼8.0 Hz, 1H), 7.52 (d, J¼8.8 Hz, 2H), 7.36 (s, 1H), 7.26 (t, J¼6.6 Hz,
1H), 7.21e7.08 (m, 3H), 6.99 (m, 2H), 6.85e6.76 (m, 1H), 5.06 (s, 2H),
4.24 (m, 2H), 3.50e3.28 (m, 4H), 3.10e2.28 (m, 3H), 2.66 (q,
J¼7.5 Hz, 2H), 1.78e1.62 (m, 4H), 1.24e1.09 (m, 6H), 1.15e1.10 (m,
3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.73, 158.68, 156.21,
146.84, 139.75, 137.22, 137.06, 136.55, 130.19, 128.25, 128.13,
127.68, 125.83, 123.96, 123.11, 122.64, 120.75, 120.04, 53.86 (eCH
2
e
Phe), 43.28 (eNHCON(CH
2
CH
2
)
2
CHCOe), 41.80 (eCON(CH
2
CH
3
)
2
),
40.25 (eCON(CH
2
CH
3
)
2
), 38.31 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.41 (eNHCON(CH
2
CH
2
)
2
CHCOe), 23.76 (eNHePheCH
2
CH
3
),
13.71 (eCON(CH
2
CH
3
)
2
), 13.16 (eNHePheCH
2
CH
3
), 11.78 (e
CON(CH
2
CH
3
)
2
); HRMS(ESI) m/z[M þH]
þ
531; HRMS(ESI) calcd
for C
30
H
38
N
6
O
3
Na [M þNa]
þ
553.2903, found 553.2903.
4.2.21. N
4
,N
4
-Diethyl-N
1
-(3-((3-((2-isopropylphenyl)amino)-6-
oxopyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-
dicarboxamide (16c)
Yield: 89%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.10 (s, 1H), 7.49 (m,
4H), 7.27 (d, J¼7.4 Hz, 1H) , 7.2 2 (d, J¼9.8 Hz, 1H), 7.18e7.01 (m, 3H),
6.91 (d, J¼7.5 Hz, 1H), 6.79 (d, J¼9.8 Hz,1H), 5.03 (s, 2H), 4.30e4.15
(m,2H),3.50e3.25(m,5H),2.99e2.73 (m, 3H), 1.75e1.61 (m, 4H) ,
1.2 5e1.05 (m, 9H), 1.02 (t, J¼7.0 Hz , 3H);
13
C NMR (125 MHz, MeOD-
d
4
)
d
174.73, 158.71, 156.21, 147.50, 142.44, 139.71, 137.06, 136.38,
130.20, 128.11, 127.46, 125.67, 125.31, 124.83, 124.61, 122.46, 120.59,
120.00, 53.92 (eCH
2
ePhe), 43.27 (eNHCON(CH
2
CH
2
)
2
CHCOe),
41.80 (eCON(CH
2
CH
3
)
2
), 40.24 (eCON(CH
2
CH
3
)
2
), 38.30 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 28.40 (eNHCON(CH
2
CH
2
)
2
CHCOe), 27.21
(PheCH(CH
3
)
2
e), 22.22 (PheCH(CH
3
)
2
e), 13.71 (eCON(CH
2
CH
3
)
2
),
11.77 (eCON(CH
2
CH
3
)
2
); HRMS(ESI) m/z[M þH]
þ
545; HRMS(ESI)
calcd for C
31
H
40
N
6
O
3
Na [M þH]
þ
567.3060, found 567.3058.
4.2.22. 4-(Morpholine-4-carbonyl)-N-(3-((6-oxo-3-(o-tolylamino)
pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1-carboxamide (17a)
Yield: 89%:
1
H NMR (300 MHz, acetone-d
6
)
d
7.65 (s,1H), 7.37 (d,
J¼7.7 Hz, 1H), 7.20e7.15 (d, J¼9.2 Hz, 2H), 6.92 (d, J¼9.6 Hz, 2H),
6.85e6.70 (m, 3H), 6.64e6.51 (m, 2H), 6.46 (d, J¼9.5 Hz,1H), 3.90e
3.75 (m, 2H), 3.30e3.20 (s, 4H), 3.19e3.08 (m, 2H), 2.57e2.48 (m,
5H), 1.88 (s, 3H), 1.75e1.60 (m, 4H);
13
C NMR (126 MHz, MeOD-d
4
)
d
174.05, 158.78, 156.32, 146.49, 139.86, 138.07, 137.17, 130.29, 130.13,
130.03,128.28, 127.99, 125.95, 123.39,122.78, 121.95, 120.89, 120.16,
66.61 (CON(CH
2
CH
2
)
2
O), 66.41(CON(CH
2
CH
2
)
2
O), 53.99 (eCH
2
e
Phe), 45.83 (CON(CH
2
CH
2
)
2
O), 43.35 (eNHCON(CH
2
CH
2
)
2
CHCOe),
42.05 (CON(CH
2
CH
2
)
2
O), 37.75 (eNHCON(CH
2
CH
2
)
2
CHCOe),
28.21 (eNHCON(CH
2
CH
2
)
2
CHCOe); LCMS(ESI) m/z[M þH]
þ
531;
HRMS(ESI) calcd for C
29
H
34
N
6
O
4
Na [M þNa]
þ
553.2539, found
553.2514.
4.2.23. N
4
,N
4
-Dimethyl-N
1
-(3-((6-oxo-3-(o-tolylamino)pyridazin-
1(6H)-yl)methyl)phenyl) piperidine-1,4-dicarboxamide(17b)
Yield: 93%:
1
H NMR (300 MHz, acetone-d
6
)
d
8.04 (s, 1H), 7.72 (d,
J¼8.7 Hz, 1H), 7.51 (m, 2H), 7.33 (s, 1H), 7.28 (d, J¼9.8 Hz, 1H),
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e103 101

7.21e7.09 (m, 3H), 7.00e6.70 (m, 2H), 6.82 (d, J¼9.8 Hz, 1H), 5.07
(s, 2H), 4.30e4.15 (m, 2H), 3.10 (s, 3H), 2.92e2.80 (m, 3H), 2.86 (s,
3H), 2.24 (s, 3H), 1.76e1.54 (m, 4H);
13
C NMR (125 MHz, MeOD-d
4
)
d
175.45, 158.78, 156.32, 146.49, 139.89, 138.07, 137.17, 130.29, 130.14,
130.04,128.27,128.00, 125.95,123.40, 122.75, 121.96,120.88,120.14,
53.98 (eCH
2
ePhe), 43.40 (eNHCON(CH
2
CH
2
)
2
CHCOe), 38.25 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 36.19 (eCON(CH
3
)
2
), 34.65 (e
CON(CH
3
)
2
), 28.01 (eNHCON(CH
2
CH
2
)
2
CHCOe), 16.90; LCMS(ESI)
m/z[M þH]
þ
489; HRMS(ESI) calcd for C
27
H
32
N
6
O
3
Na [M þNa]
þ
511.2434, found 511.2453.
4.2.24. N
4
-Ethyl-N
4
-methyl-N
1
-(3-((6-oxo-3-(o-tolylamino)
pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-
dicarboxamide(17c )
Yield: 75%:
1
H NMR (300 MHz, CD
3
OD)
d
7.48 (d, J¼8.0 Hz, 1H),
7.31 (m, 2H), 7.22 (t, J¼7.9 Hz, 2H), 7.18e7.05 (m, 2H), 6.96 (t,
J¼7.8 Hz, 2H), 6.91e6.82 (m, 1H), 5.09 (s, 2H), 4.25e4.10 (m, 2H),
3.52e3.27 (m, 2H), 3.08 (s, 1.5H), 3.03e2.78 (m, 4.5H), 2.18 (s, 3H),
1.80e1.58 (m, 4H),1.21 (t, J¼7.1 Hz,1.5H),1.08 (t, J¼7.1 H z, 1. 5H) ;
13
C
NMR (125 MHz, MeOD-d
4
)
d
175.27, 174.89, 158.78, 156.32, 146.48,
139.89, 138.08, 137.18, 130.29, 130.14, 130.01, 128.27, 127.98,
125.96, 123.39, 122.76, 121.94, 120.86, 120.15, 53.98 (eCH
2
ePhe),
44.14 (eCON(CH
3
)CH
2
CH
3
), 43.41 (eNHCON(CH
2
CH
2
)
2
CHCOe),
43.38 (eNHCON(CH
2
CH
2
)
2
CHCOe), 42.53 (eCON(CH
3
)CH
2
CH
3
),
38.45 (eNHCON(CH
2
CH
2
)
2
CHCOe), 38.20 (eNHCON(CH
2
CH
2
)
2
CHCOe), 33.79 (eCON(CH
3
)CH
2
CH
3
), 32.17 (eCON(CH
3
)CH
2
CH
3
),
28.54 (eNHCON(CH
2
CH
2
)
2
CHCOe), 27.95 (eNHCON(CH
2
CH
2
)
2
CHCOe), 16.89, 13.02 (eCON(CH
3
)CH
2
CH
3
), 11.04 (eCON(CH
3
)
CH
2
CH
3
); LCMS(ESI) m/z[M þH]
þ
503; HRMS(ESI) calcd for
C
28
H
34
N
6
O
3
Na [M þNa]
þ
525.2590, found 525.2600.
4.2.25. N
4
-Isopropyl-N
4
-methyl-N
1
-(3-((6-oxo-3-(o-tolylamino)
pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-dicarboxamide
(17d )
Yield: 68%:
1
H NMR (300 MHz, CD
3
OD)
d
7.49 (d, J¼8.0 Hz, 1H),
7.33e7.27 (m, 2H), 7.27e7.19 (m, 2H), 7.19e7.06 (m, 2H), 6.97 (t,
J¼7.7 Hz, 2H), 6.88 (d, J¼9.7 Hz, 1H), 5.10 (s, 2H), 4.80e4.71 (m,
0.5H), 4.39e4.24 (m, 0.5H), 4.25e4.12 (m, 2H), 3.04e2.81 (m, 4.5H),
2.77 (s, 1.5H), 2.19 (s, 3H),1.82e1.59 (m, 4H), 1.24 (d, J¼6.6 Hz, 3H),
1.10 (d, J¼6.8 Hz, 3H);
13
C NMR (125 MHz, MeOD-d
4
)
d
174.92,
174.88, 158.78, 156.33, 146.49, 139.88, 138.07, 137.17, 130.28,
130.13, 130.04, 128.26, 127.97, 125.95, 123.40, 122.76, 121.97,
120.87, 120.16, 53.97 (eCH
2
ePhe), 44.38 (eCON(CH
3
)CH(CH
3
)
2
),
43.44 (eNHCON(CH
2
CH
2
)
2
CHCOe), 43.40 (eNHCON (CH
2
CH
2
)
2
CH
COe), 39.00 (eNHCON(CH
2
CH
2
)
2
CHCOe), 38.43 (eNHCON(CH
2
CH
2
)
2
CHCOe), 28.65 (eNHCON(CH
2
CH
2
)
2
CHCOe), 27.99 (eNHCON
(CH
2
CH
2
)
2
CHCOe), 27.24 (eCO N(CH
3
)CH(CH
3
)
2
), 25.43 (eCON
(CH
3
)CH(CH
3
)
2
), 19.49 (eCON(CH
3
)CH(CH
3
)
2
), 18.10 (eCON(CH
3
)
CH(CH
3
)
2
), 16.88 (eNHePheCH
3
); LCMS(ESI) m/z[M þH]
þ
517;
HRMS(ESI) calcd for C
29
H
36
N
6
O
3
Na [M þNa]
þ
539.2747, found
539.2733.
4.2.26. N
4
-Ethyl-N
4
-isopropyl-N
1
-(3-((6-oxo-3-(o-tolylamino)
pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-
dicarboxamide(17e )
Yield: 55%:
1
H NMR (300 MHz, CD
3
OD)
d
7.49 (d, J¼8.0 Hz, 1H),
7.33 (d, J¼7.8 Hz, 2H), 7.29e7.19 (m, 2H), 7.19e7.05 (m, 2H), 6.98 (d,
J¼7.6 Hz, 2H), 6.88 (d, J¼9.7 Hz, 1H), 5.10 (s, 2H), 4.65e4.50 (m,
0.5H), 4.30e4.12 (m, 2.5H), 3.43e3.20 (m, 2H), 3.10e2.75 (m, 3H),
2.19 (s, 3H), 1.90e1.75 (m, 4H),1.28e1.08 (m, 9H);
13
C NMR (125MHz,
MeOD-d
4
)
d
175.20, 174.34, 158.63, 156.15, 146.35, 139.79, 137.97,
137.03, 130.16, 130.06, 129.80, 128.19, 127.93, 125.87, 123.24, 122.62,
121.74, 120.81, 120.03, 53.98 (eCH
2
ePhe), 45.73 (eCON(CH
3
)
CH(CH
3
)
2
), 43.32 (eNHCON(CH
2
CH
2
)
2
CHCOe), 42.02 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 39.25 (eNHCON(CH
2
CH
2
)
2
CHCOe),
38.43 (eNHCON(CH
2
CH
2
)
2
CHCOe), 37.02 (eCON(CH
2
CH
3
)
CH(CH
3
)
2
), 35.27 (eCON(CH
2
CH
3
)CH(CH
3
)
2
), 28.52 (eNHCO
N(CH
2
CH
2
)
2
CHCOe), 28.50 (eNHCON(CH
2
CH
2
)
2
CHCOe), 20.27 (e
CON(CH
2
CH
3
)CH(CH
3
)
2
), 19.13 (eCON(CH
2
CH
3
)CH(CH
3
)
2
), 16.87 (e
NHePheCH
3
), 16.17 (eCON(CH
2
CH
3
)CH(CH
3
)
2
), 13.71 (e
CON(CH
2
CH
3
)CH(CH
3
)
2
); LCMS(ESI) m/z[M þH]
þ
531; HRMS(ESI)
calcd for C
30
H
38
N
6
O
3
Na [M þNa]
þ
553.2903, found 553.2926.
4.2.27. N
4
,N
4
-Diisopropyl-N
1
-(3-((6-oxo-3-(o-tolylamino)
pyridazin-1(6H)-yl)methyl) phenyl)piperidine-1,4-dicarboxamide
(17f )
Yield: 62%:
1
H NMR (300 MHz, acetone-d
6
)
d
7.97 (s,1H), 7.77e7.66
(m,1H), 7.51 (m, 2H), 7.34e7.21(m,2H),7.21e7.10 (m, 3H), 6.99e6.70
(m, 2H), 6.81 (d, J¼9.8 Hz, 1H), 5.06 (s, 2H),4.25e4.11(m,4H),3.55(s,
1H), 3.01e2.74 (m, 3H), 2.23 (s, 3H), 1.74e1.62 (m, 4H), 1. 36e1.19 ( m,
12H) ;
13
C NMR (125 MHz, MeOD-d
4
)
d
158.80, 156.36, 146.51,
139.88, 138.07, 137.17, 130.29, 130.12, 130.08, 128.25, 127.97, 125.94,
123.41, 122.76, 122.00, 120.85, 120.16, 53.97 (eCH
2
ePhe), 45.77 (e
CON(CH(CH
3
)
2
)
2
), 43.43 (eNHCON(CH
2
CH
2
)
2
CHCOe), 39.84 (e
NHCON(CH
2
CH
2
)
2
CHCOe), 28.56 (eNHCON(CH
2
CH
2
)
2
CHCOe),
19.52 (eCON(CH(CH
3
)
2
)
2
), 16.86; LCMS(ESI) m/z[M þH]
þ
545;
HRMS(ESI) calcd for C
31
H
40
N
6
O
3
Na [M þNa]
þ
567.3060, found
567.3073.
5. Materials and methods
5.1. Biological assays
Cholinesterase (ChE) activity was evaluated using modified
Ellman’s spectrophotometric method [36]. Individual compounds
were dissolved in dimethyl sulfoxide (DMSO) to a concentration of
10 mM and diluted to appropriate concentrations in double-
distilled water. For in vitro tests of inhibition, 4 mL reaction mix-
tures consisting 0.1 mL of test compound, selective substrate
[0.6 mL acetylthiocholine iodide for AChE (2 mM) or 0.8 mL S-
butyrylthiocholine iodide for BuChE (2 mM)], 1 mL of phosphate
buffered solution (0.1 mM), and 0.1 mL of enzyme (homogenate of
rat cerebral cortex for AChE, ratserum for BuChE) were incubated at
37

C for 8 min, and the reaction was terminated with 1 mL of 3%
(w/v) sodium dodecylsulfate (SDS). Finally,1 mL of 0.2% (w/v) 5,5
0
-
Dithiobis (2-nitrobenzoic acid) (DTNB) was added, and the yellow
anion, 5-thio-2-nitrobenzate, was measured at 440 nm. All samples
were assayed in duplicate.
5.2. Docking
The crystal structure of donepezileAChE complex (code ID:
1EVE) was downloaded from the Protein Data Bank. Further
preparation of the protein included deleting the waters, addition of
hydrogen atoms and applying CHARMm forcefield. The 3D Struc-
tures of 4,12b and 15a were built and performed geometry opti-
mization by molecular mechanics.
Docking studies were performed using the CDOCKER protocol of
Discovery Studio 2.1 program. The binding sphere was generated at
the center of the donepezil with a radius of 11.5 Å. Then the ligand
donepezil was deleted and the docking results were obtained using
the default settings except the value of random conformations was
set to 300 and the value of top hits was set to 20. The docking
results were analyzed using Discovery Studio 3.5 Visualizer.
Acknowledgments
This work was supported by grant from National Science &
Technology Major Project “Key New Drug Creation and Manu-
facturing Program”(2012ZX09301001-001).
W. Xing et al. / European Journal of Medicinal Chemistry 63 (2013) 95e103102

Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.056.
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