Quinolone-benzylpiperidine derivatives as novel acetylcholinesterase inhibitor and antioxidant hybrids for Alzheimer Disease

Article abstract of DOI:10.1016/j.bmc.2014.02.046

Design, synthesis and evaluation of new acetylcholinesterase inhibitors by combining quinolinecarboxamide to a benzylpiperidine moiety are described. Then, a series of hybrids have been developed by introducing radical scavengers. Molecular modeling was performed and structure activity relationships are discussed. Among the series, most potent compounds show effective AchE inhibitions, high selectivities over butyrylcholinesterase and high radical scavenging activities. On the basis of this work, the ability of quinolone derivatives to serve in the design of N-benzylpiperidine linked multipotent molecules for the treatment of Alzheimer Disease has been established.

Full text of DOI:10.1016/j.bmc.2014.02.046

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinolone–benzylpiperidine derivatives as novel
acetylcholinesterase inhibitor and antioxidant hybrids
for Alzheimer Disease
⇑
Marc Pudlo , Vincent Luzet, Lhassane Ismaïli, Isabelle Tomassoli , Anne Iutzeler, Bernard Refouvelet
Nanomedicine Lab, EA—4662, UFR SMP, 19 rue Ambroise Paré Université de Franche Comté, 25030 Besançon Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Design, synthesis and evaluation of new acetylcholinesterase inhibitors by combining quinolinecarboxa-
mide to a benzylpiperidine moiety are described. Then, a series of hybrids have been developed by intro-
ducing radical scavengers. Molecular modeling was performed and structure activity relationships are
discussed. Among the series, most potent compounds show effective AchE inhibitions, high selectivities
over butyrylcholinesterase and high radical scavenging activities. On the basis of this work, the ability of
quinolone derivatives to serve in the design of N-benzylpiperidine linked multipotent molecules for the
treatment of Alzheimer Disease has been established.
Received 16 December 2013
Revised 20 February 2014
Accepted 24 February 2014
Available online xxxx
Keywords:
Acetylcholinesterase
Antioxidant
Ó 2014 Elsevier Ltd. All rights reserved.
Hybrid
Quinolone
N-Benzylpiperidine
1. Introduction
lated during the last decade suggest a neuroprotective effect of
currently marketed anti-Alzheimer AChE inhibitors.7 In order to
Alzheimer’s Disease (AD) is the major cause of dementia affect-
ing approximately 10% of the population over the age of 65-year-
old and its incidence rises exponentially with age.1 This incurable
neurodegenerative disease is characterized by progressive failure
of thought, memory and language. The primary therapeutic ap-
proach to address cognitive loss associated with AD is based on
acetylcholinesterase (AChE).2 This symptomatic treatment led to
the suggestion that AD is associated with an impairment in cholin-
ergic transmission.3
The AD pathogenesis involves numerous pathway and several
theories. The widely accepted amyloid theory states that an accu-
mulation of b-amyloid peptides, a main component of the senile
plaques, initiates the pathogenic cascade.4 Then, takes place
inflammation, neuronal dysfunctions and imbalance of kinase
and phosphatase activities causing the characteristic neurofibril-
lary tangles. Another theory suggests that neurofibrillary tangles
occur prior to the senile plaques.5 In any case, oxidative stress is
a main feature of neuroinflammation and leads to neuronal dam-
ages.6 Beyond a symptomatic effect, clinical evidences accumu-
improve AChE inhibitors as a pharmacological instrument to retard
progressive neurodegeneration, scientists are engaged in the
development of multipotent compounds.8 As oxidative stress plays
a central role in AD pathogenesis, it is a key target to retard AD’s
progression9 and multitargeted drugs have been designed to com-
bine AChE inhibition and antioxidant properties.10
The multitarget approach is based on the unique structural
properties of AChE and the interaction of the enzyme with the
inhibitors.11 The structure of AChE reveals two main binding sites:
the catalytic binding site, comprising the Ser-His-Glu catalytic
triad, and the peripheral anionic binding site, connected by a deep
and hydrophobic gorge. Dual binding site inhibitors bind to both
the catalytic and the peripheral site. They are designed by assem-
bling a heterocyclic ring to an N-benzylpiperidine or to a tacrine
through a linker of appropriate length. Heterocyclic ring interacts
with the peripheral anionic site while the second moiety binds to
the gorge and the catalytic site.12 In hybrid design, the moiety that
scavenges Reactive Oxygen Species (ROS) generally interacts with
the peripheral anionic site13 which is implicated in other biological
processes.14
The quinolone structures include motifs exhibiting a wide
variety of biological activities15 One of which is ROS scavenging
ability.16 Quinolone scaffold has already been efficiently used for
⇑
Corresponding author. Tel.: +33 3 81 66 55 42; fax: +33 3 81 66 56 55.
E-mail address: marc.pudlo@univ-fcomte.fr (M. Pudlo).
Address: Department of Pharmaceutical Sciences, College of Pharmacy, University
of Hawai’i at Hilo, Hilo, HI96720, USA.
http://dx.doi.org/10.1016/j.bmc.2014.02.046
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

2
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
design of dual AChE inhibitor.17 However quinolones have never
been associated to a benzylpiperidine moiety for this purpose.
Moreover, such derivatives of coumarin, the oxygen analog of
quinolone, are efficient and widely studied AChE inhibitors.18 We
introduce here new acetylcholinesterase inhibitors, whose struc-
tures include a quinolone and a benzylpiperidine moiety. To assess
their potential in hybrids design a series of compounds displaying
simultaneously acetylcholinesterase inhibition and antioxidant
activity have been developed. Inhibitions of AChE and BuChE have
been determined using Ellman’s method and the antioxidant prop-
erties evaluated by radical scavenging activities using 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) and anion superoxide. In adition,
molecular modelling study was performed to determine the bind-
ing mode of the best inhibitor to the AChE.
amount of DMF in CH2Cl2. Acyl chlorides were directly engaged
in amidification with 4-amino-1-benzylpiperidine or 2-(1-benzyl-
4-piperidinyl)methylamine in presence of Et3N as base in CH2Cl2
to afford 5g–i. The difference can be explained by an intramolecu-
lar H-bond between hydroxyl substituent in position 4 (R2 = OH)
and the ester function in position 3 that presumably provides
transamidification. In addition 5d was chlorinated by POCl3, then
the 4-chloro substituted derivative was reacted with NH3 in etha-
nol to give 5j.
Compounds 8a–d and 9a–d were prepared according to the
route outlined in Scheme 2. Methoxylated derivatives of quinolone
(7) were prepared starting from 4,5-dimethoxyanthranilate (6a–c)
or N-substituted anthranilate with methoxybenzyl (6d). Then ben-
zylpiperidine moiety was coupled through amidification. The hy-
brid compounds were obtained by demethylation.
Methyl 2-aminobenzoate and methyl 2-amino-4,5-dim-
ethoxybenzoate were both used unchanged and modified by meth-
ylation or benzylation. Methylation was conducted by Chan–Lam
coupling21 with stoichiometric amount of copper(II), methylboron-
ic acid and pyridine under reflux in dioxane to give 6b. Benzylation
was mediated by reductive amination using sodium cyanoborohy-
dride as reducing agent and conducted to 6c and 6d respectively. In
this series, quinolones were prepared in 2 steps. Ethyl malonyl
chloride was reacting with 6a–d in presence of Et3N in CH2Cl2,
amide intermediates were succinctly purified and involved in
cyclization by NaOMe in MeOH to afford 7a–d with moderate to
good yield depending on steric hindrance. Then 7a–d were reacted
with 2-(1-benzyl-4-piperidinyl)methylamine in refluxing xylene to
give 8a–d. Lastly methyl groups were removed by BBr3 in CH2Cl2 to
give 9a–b.
2. Results and discussion
2.1. Synthetic pathways
The synthesis of compounds 5a–j has been approached as
shown in Scheme 1. The quinolone moiety (3) was easily prepared
by treatment of isatoic anhydride (1) or aminophenone (2) with
diethylmalonate and then the benzylpiperidine moiety was at-
tached trough amidification.
N-benzylisatoic anhydride 1c was obtained by N-alkylation of
isatoic anhydride 1a with benzyl bromide.19 Quinolones 3a–c were
prepared from isatoic anhydride and diethyl malonate using NaH
in DMF as previously described.20 Quinolones 3d and 3e were ob-
tained by reaction of diethyl malonate and respectively 20-amino-
acetophenone and 2-aminobenzophenone under reflux with DBU.
Amides 5a–f (R2 = OH) were obtained directly by the reaction of es-
ters 3a–c with 4-amino-1-benzylpiperidine (n = 0) or 2-(1-benzyl-
4-piperidinyl)methylamine (n = 1) in refluxing xylene. For amides
5g–h (R2 = Me) and 5i (R2 = Ph), we had to proceed through an acyl
chloride. First, 3d and 3e were saponified by NaOH in an ethanol–
water mixture under reflux. Carboxylic acids 4d and 4e were
converted into acyl chlorides with oxalyl chloride and a catalytic
2.2. In vitro analysis of compounds 5a–j, 8a–d and 9a–d
2.2.1. Cholinesterase assays
All newly synthesized compounds were tested for their
inhibitory activities toward AChE using Ellman’s method.22 Butyr-
ylcholinesterase inhibitory activities were also assessed to explore
R2
R2
O
c
b
CO2Et
O
O
O
N
NH2
N
O
R1
R1
2d : R2 = Me
2e : R2 = Ph
3a : R1 = H, R2 = OH
3b : R1 = Me, R2 = OH
3c : R1 = Bn, R2 = OH
3d : R1 = H, R2 = Me
3e : R1 = H, R2 = Ph
1a : R1 = H
1b : R1 = Me
1c : R1 = Bn
a
R2
O
R2
O
n
5a : R1 = H, R2 = OH, n = 0
5b : R1 = H, R2 = OH, n = 1
5c : R1 = Me, R2 = OH, n = 0
5d : R1 = Me, R2 = OH, n = 1
5e : R1 = Bn, R2 = OH, n = 0
5f : R1 = Bn, R2 = OH, n = 1
5g : R1 = H, R2 = Me, n = 0
5h : R1 = H, R2 = Me, n = 1
5i : R1 = H, R2 = Ph, n= 0
5j : R1 = Me, R2 = NH2, n= 1
d
N
H
O
N
3a, 3b, 3c
N
O
N
O
R1
R1
g
3d, 3e
R2
O
O
e
f
OH
N
n
R1
H2N
amine :
N
4d : R1 = H, R2 = Me
4e : R1 = H, R2 = Ph
Scheme 1. Reagents and conditions: (a) BnBr, DIPEA, DMA, 90 °C, 3 h, 95%; (b) diethyl malonate, NaH, DMF, reflux, 5 h, 64–71%; (c) diethyl malonate, DBU, reflux, overnight,
56–89%; (d) amine, xylene, reflux, 3 h, 56–99%; (e) NaOH, EtOH, H2O, reflux, overnight, 87–100%; (f) (i) oxalyl chloride, DMFcat, CH2Cl2, 0 °C to rt, 1 h; (ii) amine, Et3N, CH2Cl2,
0 °C to rt, 3 h, 57–72%; (g) POCl3, 1 h then NH3, EtOH, overnight, 55%.
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M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
O
R3
R3
O
O
O
R3
R3
b
R3
R3
a
O
O
NH
R1
NH
R1
NH2
6a : R1 = H, R3 = OMe
6b : R1 = Me, R3 = OMe
6c : R1 = Bn, R3 = OMe
6d : R1 = 4-OH-3,5-diOMe-Bn, R3 = H
H2N
OH
O
O
OH
O
O
O
O
N
R3
R3
R3
R3
c, d
R3
R3
N
H
OEt
N
N
N
NH
R1
e
R1
R1
8a : R1 = H, R3 = OMe
8b : R1 = Me, R3 = OMe
8c : R1 = Bn, R3 = OMe
8d : R1 = 4-OH-3,5-diOMe-Bn, R3 = H
6a : R1 = H, R3 = OMe
7a : R1 = H, R3 = OMe
7b : R1 = Me, R3 = OMe
7c : R1 = Bn, R3 = OMe
6b : R1 = Me, R3 = OMe
6c : R1 = Bn, R3 = OMe
f
6d : R1 = 4-OH-3,5-OMe-Bn, R3 = H
7d : R1 = 4-OH-3,5-OMe-Bn, R3 = H
9a : R1 = H, R3 = OH
9b : R1 = Me, R3 = OH
9c : R1 = Bn, R3 = OH
9d : R1 = 3,4,5-triOH-Bn, R3 = H
Scheme 2. Reagents and conditions: (a) MeB(OH)2, Cu(OAc)2, Py, dioxane, reflux, 24 h, 66%; (b)ArCHO, NaBH3CN, AcOHcat, MeOH, rt, overnight, 41–61%; (c) ethyl malonyl
chloride, Et3N, CH2Cl2, 0 °C to rt, 3 h; (d) NaOMe, MeOH, rt, overnight, 82–98% for 2 steps; (e) xylene, reflux, 3 h, 82–98%; (f) BBr3, CH2Cl2, ꢀ30 °C to rt, overnight, 55–87%.
Table 1
required as phenyl group cancel out activity (5i). An amino group
has similar activity than a hydroxyl group (5j = , 5d). However,
Cholinesterase activities and selectivities of compounds 5a–j
R2
O
presence of a methyl group in position 4 retains activity but re-
verses the methylene linkage finding (5g > 5h).All compounds
showed selectivity toward AChE over BuChE with ratios from 3
to above 25. These results were within the range of the reference
compounds we tested.
n
N
H
N
N
O
R1
These data oriented the choice of the scaffold for the design of
AChE inhibitors—antioxidant hybrids. Since the best results were
obtained from compounds with a methylene linkage (n = 1) and a
hydoxyl substituent in position 4 (R2 = OH), we selected this
moiety as model for hybrid design. As N-quinoline substitution in-
creases the activity, we prepared N-methyl, N-benzyl and N-phe-
nolic derivatives.
On one hand, we prepared molecules bearing 6,7-dimethoxy
and 6,7-dihydroxy moieties on the quinolone ring (Table 2,
R3 = OMe, OH). On the other hand, we prepared some compounds
with phenolic and polyphenolic moieties to replace the N-benzyl
substituent (Table 2, R1 = Sy, SyOH). Compound 8a (R1 = H,
Compounds R1
R2
n
IC50
(
l
M) SEa
Selectivity
AChE
BuChE
5a
5b
H
H
OH
OH
0
1
0
1
0
1
0
1
0
1
2.27 0.65
1.41 0.17
1.52 0.13
0.37 0.05
0.39 0.10
0.49 0.11
2.46 0.60
0.47 0.10
>10
>10
6.02 1.74
>10
4.26 0.54
>10
>10
>10
>10
>10
1.14 0.17
> 4
4
5c
5d
Me OH
Me OH
>6
11
>25
>20
>4
>21
—
5e
5f
5g
Bn
Bn
H
OH
OH
Me
Me
Ph
5h
H
5i
H
5j
Me NH2
0.38 0.07
0.033 0.012 1.82 0.09
0.065 0.009 0.0033 0.0004 0.05
3
55
Donepezil
Tacrine
R3 = OMe) has a good AChE inhibition with an IC50 of 0.11
and a high selectivity for AChE as no BuChE inhibition was ob-
served at 60 M. Product 9a (R1 = H, R3 = OH) representing the O-
demethylated analog retains activity range. No BuChE inhibition
was observed at 10 M. Compound 8b (R1 = Me, R3 = OMe) and
lM
a
AChE from electric eel and BuChE from equine serum were used. IC50 values
represent the inhibitor concentrations required to decrease enzyme activity by 50%.
Results are presented as the average Standard Error of three independent assays.
l
l
9c (R1 = Bn, R3 = OH) could not be dissolved in the conditions of
cholinesterase assay, the hydrochlorides were prepared. Unfortu-
nately compound 8b (R1 = Me, R3 = OMe) and the catechol 9b
(R1 = Me, R3 = OH) have unsatisfactory activity on AChE. Compound
8c (R1 = Bn, R3 = OMe) presents moderate inhibition of AChE while
9c (R1 = Bn, R3 = OH) was ineffective in cholinesterase assays.
Compound 8d (R1 = Sy) shows moderate AChE inhibition but no
selectivity. Moreover O-demethylation into triphenolic 9d
(R1 = SyOH) clearly affects the inhibitory activity.
selectivity toward both enzymes. Tacrine and donepezil were
taken as reference compounds (Table 1).
For the first time N-benzylpiperidinyl-dihydroquinoline-3-car-
boxamides were evaluated as AChE inhibitors. Somes informations
could be gleaned from these results. AChE inhibition increases
when N-quinoline is substituted (R1, 5c, 5e > 5a). Modifying the
chain length between the quinolone and the benzylpiperidine moi-
ety affects the affinity. Thus compounds with a methylene linkage
(n = 1: 5b, 5d and 5h) between the N-amide and the piperidine ring
generaly present a higher activity than compounds without the
spacer (n = 0: respectively 5a, 5c and 5g). Results concerning
residues in position 4 (R2) were also instructive. A small group is
Hydroxy, or even better, methoxy substituents on the quinolone
ring (R3 = OH, OMe) appear to improve selectivity toward AChE
over BuChE. Compounds 8a and 9a did not show any inhibition
of butyrylcholinesterase activity under 60 lM and 10 lM
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4
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 2
Hydroxy and methoxy groups on the quinolone ring or the N-
benzyl substituent were introduced in order to target a radical
scavenging activity. We used catechol moieties for their well
known radical scavenging activities and a moiety derived from
syringaldehyde, a natural phenolic compound showing potent
anti-oxidant activity.24
Cholinesterase activities and selectivities of compounds 8a–d and 9a–d
OH
O
R3
R3
N
H
N
N
R1
O
The best activities were obtained when the dihydroxy moiety
was on the quinolone ring (Table 3, R3 = OH, 9a, 9b, 9c). Recently
it was reported16a,b that quinolone and iso-quinoline alkaloids with
only one hydroxyl showed lower antioxidant activity than 9a–c.
Thus the catechol moiety is crucial for improving radical scaveng-
ing activity. Hydroxyl substituents must be on the quinolone ring
to significantly affect the radical scavenging activity. Thus it is
likely that the 3,4-insaturation of the quinolone ring is important
for antioxidant activity.25 By comparison, syringaldehyde deriva-
tive 8d (R1 = Sy) and its polyhydroxylated analogue 9d (R1 = SyOH)
are less active. These derivatives have no hydroxyl substituent on
the quinolone ring and their antioxidant activity depends only on
the presence of the phenolic functional groups on the N-benzyl
substituent (R1). Moreover, since 9b (R1 = Me) and 9c (R1 = Bn)
have slightly lower antioxidant activity than 9a (R1 = H), hydrogen
atom on the N-quinolone appears to be involved in high scaveng-
ing activity.
Compounds R1
R3
IC50
(
l
M) SEa
BuChE
>60
Selectivity
AChE
8a
9a
H
OMe 0.11 0.03
>545
>20
hb: 0.23 0.03
0.48 0.14
H
OH
>10
hb: 0.98 0.35
8bꢂHCl
9b
Me
Me
OMe 2.93 0.63
OH 2.02 0.41
OMe 0.27 0.07
OH
H
>10
>10
>60
>10
0.33 0.26
>10
1.82 0.09
>3
5
>222
-
0.6
>2
55
8c
Bn
9cꢂHCl
Bn
>10
8d
Syc
0.56 0.15
3.89 0.50
0.033 0.012
0.065 0.009
9d
SyOHc
H
Donepezil
Tacrine
0.0033 0.0004 0.55
a
AChE from electric eel and BuChE from equine serum were used. IC50 values
represent the required inhibitor concentrations to decrease enzyme activity by 50%.
Results are presented as the average Standard Error of three independent assays.
b
The superoxide anion (OÅ2ꢀ) is the first product of the univalent
reduction of oxygen and is generated in many biological processes.
Hence, the superoxide anion scavenging activity of hybrid
compounds was explored using the non-enzymatic phenazine
methosulfate NADH-system.26 All phenolic derivatives were
found to have moderate activity. But 9a is again the most active
product. Unlike the DPPH test, in this assay the position of the
hydroxyl did not affect the anion superoxide scavenging activity
(9a ꢁ 9d).
AChE from human erythrocytes was used.
Sy = 4-OH-3,5-diOMe-Bn and SyOH = 3,4,5-triOH-Bn.
c
respectively. However, compound 8d (R1 = Sy, R3 = H) is slightly
selective for butyrylcholinesterase.
To sum up, 8a and 9a are the most potent compounds in terms
of inhibition and selectivity within the series. To further explore
their potenties, we tested their activity on human cholinesterase
and observed that inhibitory activities are close but lower with
an IC50 of 0.23 lM and 0.98 lM for 8a and 9a respectively. In add-
2.2.3. Structure activities relationships summary
titon, based on the low solubility of N-substituted derivatives, we
can say that the NH quinoline unsubstitution of 8a and 9a im-
proves solubility.
In term of AChE inhibition, compounds with methylene linkage
between the quinolone-caboxamide and the benzylpiperidine ring
generally present higher activity than equivalent compounds with-
out the spacer (5b > 5a, 5d > 5c, 5h > 5g). In this case, a hydroxyl in
position 4 seems to be required. Unlike the first series, in the hy-
brid design N-quinolone substituents clearly reduced solubility
and best inhibitions were obtained with N-unsubstituted quino-
lone derivatives.
In term of radical scavenging activity the best activities were
obtained when a catechol moiety was on the quinolone ring
(Table 3, R3 = OH, 9a, 9b, 9c) and were improved by an unsubstitut-
ed N-quinolone. EC50 (DPPH) of compound 9a was found to be
similar as quercetin and two fold higher than curcumin.
Among these series compounds 8a and 9a were the most potent
in term of AChE inhibition and radical scavenging activity. The
inhibitory activity was verified on human AChE. These two
compounds showed close but lower inhibitory activity. Struc-
ture–activities relationships for compounds 9a are summarized
in Figure 1.
2.2.2. Antioxidant assays
Each compound was tested for radical scavenging activity. The
effect of the synthesized compounds with the stable free radical
2,2-diphenyl-1-picrylhydrazyl (DPPH) was first investigated.23 In
the absence of phenolic substituents, no radical scavenging activity
was observed. However all phenolic derivatives interacted with
DPPH with EC50 values ranging from 12.2 to 107
lM (Table 3).
Table 3
Antioxidant activities of compounds 9a–d
Compounds
R1
R3
EC50
(
l
M) SEa
OÅ2ꢀ
DPPH
9a
9b
9c
8d
H
OH
OH
OH
H
12.2 0.4
21.7 0.6
20.8 0.7
90.5 6.3
107.0 7.3
10.3 0.3
26.6 0.9
138.8 6.6
40% 2%b
Insoluble
33% 3%b
140.4 0.7
49.9 7.9
ndd
Me
Bn
Syc
SyOHc
9d
H
Quercetine
Curcumine
increase AChE
small group
activity and selectivity
H
H bond donnor and acceptor
O
O
O
a
EC50 values represent the required compound concentrations to reduce radical
HO
HO
activity by 50%. Results are presented as the average Standard Error of three
independent assays.
N
H
N
b
N
H
Since this experiment was limited by the solubility of compounds in aqueous
solution, two results are expressed by the rate of radicals being scavenged at the
concentration of 100 lM. Data are expressed as the average of three independent
NH are essential for solubility
catechol moiety, insaturation and NH substitution
improve antioxidant activity
measurements (SE = Standard Error).
c
Sy = 4-OH-3,5-diOMe-Bn and SyOH = 3,4,5-triOH-Bn.
Not determined.
d
Figure 1. Structure–activities relationships of compound 9a.
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M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 2. Compound 9a (balls and sticks in yellow) in the binding site of hAChE (lines in grey) from 4ey7. Amino-acids and hydrogens have been removed to highlight the
interactions. The peripheral anionic site is represented by Trp 286 and Tyr 72, the gorge is represented by Phe338 and Tyr337 and the catalytic pocket is represented by Trp86.
p
interactions are in orange solid lines and Hydrogen Bonding interactions are in green dotted lines.
2.3. Molecular modeling studies of compounds 9a
hybrid 9a shows high radical scavenging activity without altering
the inhibition of AChE. The catechol moiety of 9a provides antiox-
idant activity and is also involved in an additional interaction with
AChE. Our work established the ability of quinolone derivatives to
serve in the design of multipotent hit molecules for the treatment
of AD. New generation of quinolones derivatives are currently
under investigation.
We performed molecular docking simulation for 9a to Torpedo
californica AChE27 (tAChE) and Human AChE28 (hAChE) using the
CDocker program29 implemented in Discovery studio 2.5 software.
The position of compound 9a in the binding site of hAChE is
shown in Figure 2. Compound 9a binds in the same way to tAChE
and hAChE. Hereinafter, aminoacids and distances of the bindings
in AChE are indicated in brackets. The phenyl moiety displays a
4. Experimental section
4.1. Synthesis
weak
sented by Trp 86 (4.06–5.22 Å). Piperidinium is snaking along the
gorge, making cation-
interactions30 with aromatic rings in
Tyr337 (3.83 Å) and even less in Phe338 (6.21 Å). The quinolone
moiety is linked to the peripheral anionic site by -stacking inter-
p-stacking interaction in the central catalytic pocket repre-
p
Starting materials reagents and analytical grade solvents were
purchased from Sigma–Aldrich and Acros. All reaction were rou-
tinely checked by TLC using Merck Kieselgel 60 F254 aluminium
plates and visualized by UV light. IR spectra were performed on a
Perkin Elmer Spectrum65 with UATR and principal absorptions
are given in cmꢀ1. Weak absorptions, noted (w), are probably
due to intramolecular hydrogen bonds or to the propensity of
2(1H)-quinolinones to form dimers. Broad signal are noted (b).
1H and 13C NMR spectra were recorded in the specified deuterated
solvent at 300 MHz and 75 MHz on a Brucker AC 300 instrument.
Chemical shifts are expressed in parts per million (d) relative to
the solvent signal and the couplings constants J are given in Hertz
(Hz). The following abbreviations were used: s (singlet), d (dou-
blet), t (triplet), q (quadruplet), dd (doublet of doublet), m (multi-
plet), bs (broad signal). Elemental analysis were carried out at the
Service Central d’Analyses, ISA—UMR CNRS 5280—69 360 Solaize.
ESI-MS analyses were carried out at the Service Commun d’Ana-
lyse, ICMR—UMR CNRS 6229—51 100 Reims.
p
actions with Trp286 (4.06–5.71 Å). These data are in agreement
with the literature.27,28 Compounds 9a displays two other interac-
tions: an H-bond between the hydroxyl group in position 4
(R2 = OH) of the quinolone and a hydroxyl of the Tyr124 (2.32–
3.05 Å) and another one between the carbonyle in position 2 of
the quinolone and a NH of the Phe295 (1.97 Å). In addition, unlike
compound 8a (data not shown) the hydroxyl in position 6
(R3 = OH) of the quinolone displays an H-Bond donor interaction
in AChE with a hydroxyl of the Tyr72 (2.01–2.64 Å). Docking sim-
ulations clearly demonstrate that the radical scavenger moiety is
placed by the peripheral anionic site.
3. Conclusions
We have designed synthesized and evaluated two series of
quinolone-3-carboxamides linked to benzylpiperidine. All
compounds except one are selective toward AChE versus BuChE,
provide micromolar range inhibition and among them nine deriv-
atives were found to inhibit AChE enzyme in submicromolar range.
All phenolic compounds of the hybrid series have shown moderate
to high radical scavenging activities. Compounds 8a and 9a are the
most potent. Compound 8a is an effective inhibitor of AChE and
4.1.1. Typical procedure for preparation of compounds 3a–e
To a suspension of NaH (1 equiv) in DMF at 0 °C was slowly
added diethyl malonate (10 equiv) and a solution of the corre-
sponding isatoic anhydride (1 equiv) in DMF. Then the reaction
mixture was heated to reflux. After completion of the reaction
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

6
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(TLC monitoring) the crude mixture was quenched with water and
acidified with concentrated hydrochloric acid. The resulting solid
was filtered, washed with water and diethylether and then dried
in vacuo to give the desired compound.
of HCl 1 N, a satured solution of NaHCO3 and then brine and dried
over sodium sulfate. The organic layer was filtred and solvent was
evaporated under vacuum. The compound was obtained as a white
solid (662 mg, 89%). IR (ATR)
c
cmꢀ1: 3200–3050 (w), 2981, 2935,
2836, 1729, 1644, 1557. 1H NMR (DMSO D6), d ppm: 12.28 (s, 1H),
7.63–7.54 (m, 4H), 7.41 (d, J = 8.2 Hz, 1H), 7.36–7.28 (m, 2H), 7.20–
7.10 (m, 2H), 3.95 (q, J = 7.0 Hz, 2H), 0.85 (t, J = 7.0 Hz, 3H). 13C
NMR (DMSO D6), d ppm: 164.9, 158.4, 148.3, 138.5, 133.9, 131.4,
128.8, 128.4, 128.3, 127.1, 126.9, 122.4, 118.1, 115.6, 60.5, 13.4.
Anal. Calcd For C18H15NO3: C, 73.71; H, 5.15; N, 4.78. Found: C,
73.60; H, 5.21; N, 4.62.
4.1.1.1. Ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbox-
ylate 3a.
According to the general procedure, the reaction was
carried out with NaH (0.408 g, 17 mmol), diethyl malonate (27.2 g,
170 mmol) and isatoic anhydride (2.8 g, 17 mmol). The compound
was obtained as a white solid (2.81 g, 71%). IR (ATR)
c
cmꢀ1: 3406,
3193, 1658, 1604. 1H NMR (DMSO D6), d ppm: 11.47 (s, 1H), 7.94
(d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.27 (d, J = 8.1 Hz, 1H),
7.20 (t, J = 7.5 Hz, 1H), 4.34 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz,
3H). Anal. Calcd For C12H11NO4: C, 61.80; H, 4.75; N, 6.01. Found:
C, 61.72; H, 4.78; N, 6.06.
4.1.4. 4-Methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid
4d
To a solution of ethyl 4-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxylate 3d (1.5 g, 6.49 mmol) in ethanol was added a solution
of KOH 4 N (6.5 mL, 26 mmol) and the mixture was heated to re-
flux overnight. After cooling, the reaction mixture was diluted with
water and washed with ethyl acetate. The aqueous layer was acid-
ified and extracted with ethyl acetate. Crude mixture was dried
over magnesium sulfate and concentrated in vacuo to give the
4.1.1.2. Ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-
3-carboxylate 3b.
According to the general procedure, the
reaction was carried out with NaH (0.408 g, 17 mmol), diethyl mal-
onate (27.2 g, 170 mmol) and methylisatoic anhydride (3 g,
17 mmol). The compound was obtained as a white solid (2.98 g,
71%). IR (ATR)
c
cmꢀ1: 3200–3000 (w), 2996, 2977, 1657, 1624,
compound (1.15 g, 87%) as a white solid. IR (ATR) c
cmꢀ1: 3250–
1593, 1561. 1H NMR (DMSO D6), d ppm: 13.02 (bs, 1H), 8.01 (d,
J = 8.0 Hz, 1H), 7.72 (dd, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H),
7.28 (t, J = 8.0 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.52 (s, 3H), 1.30
(t, J = 7.1 Hz, 3H). 13C NMR (DMSO D6), d ppm: 169.7, 166.1,
158.9, 140.8, 134.1, 124.8, 122.1, 115.2, 114.7, 101.2, 61.7, 29.2,
14.3. Anal. Calcd for C13H13NO4: C, 63.15; H, 5.30; N, 5.67. Found:
C, 63.09; H, 5.24; N, 5.76.
2600 (l), 3136 (w), 3041, 2940, 2885, 1697, 1625, 1598, 1538. 1H
NMR (DMSO D6), d ppm: 13.69 (bs, 1H), 12.31 (bs, 1H), 7.88 (d,
J = 7.8 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.29
(t, J = 7.8 Hz, 1H), 2.56 (s, 3H). 13C NMR (DMSO D6), d ppm:
166.8, 159.8, 146.5, 137.6, 131.4, 125.6, 124.9, 122.4, 119.0,
115.6, 15.9. Anal. Calcd for C11H9NO3: C, 65.02; H, 4.46; N, 6.89.
Found: C, 64.82; H, 4.66; N, 6.99.
4.1.1.3. Ethyl 1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-
4.1.5. 2-Oxo-4-phenyl-1,2-dihydroquinoline-3-carboxylic acid
4e
3-carboxylate 3c.
According to the general procedure, the
reaction was carried out with NaH (0.24 g, 10 mmol), diethyl mal-
onate (16 g, 100 mmol) and methylisatoic anhydride (2.53 g,
10 mmol). The compound was obtained as a white solid (2.07 g,
To a solution of ethyl 2-oxo-4-phenyl-1,2-dihydroquinoline-3-
carboxylate 3e (1.172 g, 4 mmol) in ethanol was added a solution
of KOH 4 N (4 mL, 16 mmol) and the mixture was heated to reflux
overnight. After cooling, reaction mixture was diluted with water
and washed with ethyl acetate. Aqueous layer was acidified and
extracted with ethyl acetate. Crude mixture was dried over magne-
sium sulfate and concentrated in vacuo to give the compound
64%). IR (ATR)
c
cmꢀ1: 3250–3100 (w), 2986, 2937, 1669, 1622,
1560. 1H NMR (CDCl3), d ppm: 14.36 (s, 1H), 8.20 (d, J = 7.8 Hz,
1H), 7.53 (t, J = 7.4 Hz, 1H), 7.31–7.18 (m, 7H), 5.51 (s, 2H), 4.52
(q, J = 7.1 Hz, 2H), 1.49 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO D6), d
ppm: 171.9, 166.6, 158.2, 136.2, 134.4, 134.2, 128.6, 127.0, 126.3,
126.2, 121.9, 116.2, 115.0, 114.8, 62.3, 45.6, 14.1. Anal. Calcd for
(1.06 g, 100%) as a white solid. IR (ATR)
c
cmꢀ1: 3220 (w), 3200-
2200 (l), 3001, 2940, 2835, 1695, 1646, 1577, 1555. 1H NMR
(DMSO D6), d ppm: 13.06 (bs, 1H), 12.31 (bs, 1H), 7.62–7.47 (m,
4H), 7.42 (d, J = 8.1 Hz, 1H), 7.38–7.30 (m, 2H), 7.15 (t, J = 7.5 Hz,
1H), 7.07 (d, J = 8.1 Hz, 1H). 13C NMR (DMSO D6), d ppm: 166.1,
159.0, 147.5, 138.2, 134.3, 131.1, 128.5, 128.4, 128.2, 127.1,
126.9, 122.2, 118.6, 115.5. Anal. Calcd for C16H11NO3: C, 72.45; H,
4.18; N, 5.28. Found: C, 72.33; H, 4.29; N, 5.44.
C19H17NO4: C, 70.58; H, 5.30; N, 4.33. Found: C, 70.69; H, 5.41; N,
4.22.
4.1.2. Ethyl 4-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxylate 3d
A mixture of 1-(2-aminophenyl)ethanone (2 g, 16.9 mmol),
diethyl malonate (3.76 g, 23.5 mmol) and DBU (719 mg,
4.73 mmol) was heated to reflux overnight. After cooling, the resi-
due was recristallized in ethyl acetate. The compound was washed
by ethyl acetate and dichloromethane and was obtained as a white
4.1.6. Typical procedure for preparation of compounds 5a-i
Method A: Compounds 5a–f: Quinolinone ester 3a–c (1 equiv)
was added to a solution of the amine (1.1 equiv) in xylene and the
mixture was refluxed. After completion of the reaction (TLC moni-
toring), solvent was removed in vacuo and products were obtained
by precipitation, column chromatography and/or recrystallization.
Method B: Compounds 5g–i: To a solution of acid 4d–e (1 equiv)
at 0 °C in dry dichloromethane, was added oxalyl chloride
(1.2 equiv) and dry N,N-dimethylformamide (0.2 equiv). After
30 min, the reaction mixture was allowed to warm up at rt. After
completion of this step (TLC monitoring) the solvent was evapo-
rated. The residue was diluted with dichloromethane and added
to a solution of 4-Amino-1-benzylpiperidine (n = 0) or 2-(1-ben-
zyl-4-piperidinyl)methylamine (n = 1) (1 equiv) and triethylamine
(4 equiv) at 0 °C in dry dichloromethane. After 3 h at rt, reaction
mixture was washed with NaHCO3 5% and brine. The organic layer
was dried over sodium sulfate, filtered and concentred in vacuo.
Pure product was obtained by recrystallization.
solid (2.21 g, 56%). IR (ATR)
c
cmꢀ1: 3312 (w), 3001, 2981, 1728,
1647, 1608, 1561, 1505. 1H NMR (DMSO D6), d ppm: 11.99 (s,
1H), 7.80 (d, J = 7.7 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.33 (d,
J = 7.7 Hz, 1H), 7.25 (t, J = 7.7 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 2.39
(s, 3H), 1.29 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO D6), d ppm:
166.0, 158.4, 144.2, 137.9, 131.1, 126.4, 125.3, 122.1, 118.4,
115.4, 60.9, 15.6, 13.9. Anal. Calcd for C13H13NO3: C, 67.52; H,
5.67; N, 6.06. Found: C, 67.66; H, 5.64; N, 6.11.
4.1.3. Ethyl 2-oxo-4-phenyl-1,2-dihydroquinoline-3-
carboxylate 3e
A
mixture of (2-aminophenyl)(phenyl)methanone (500 mg,
2.54 mmol), diethyl malonate (568 mg, 3.55 mmol) and DBU
(54 mg, 0.354 mmol) was heated to reflux overnight. After cooling,
the residue was dissolved in ethyl acetate, washed with a solution
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
4.1.6.1. N-(1-Benzylpiperidin-4-yl)-4-hydroxy-2-oxo-1,2-dihy-
droquinoline-3-carboxamide 5a. The compound was pre-
pared according to method A from ethyl 4-hydroxy-2-oxo-1,
2-dihydroquinoline-3-carboxylate 3a (0.4 g, 1.7 mmol) and 1-ben-
zylpiperidin-4-amine (355 mg, 1.87 mmol) for 3 h to reflux. Com-
pound precipitated after cooling and was filtered and washed by
2.82 (m, 2H), 1.92 (m, 2H), 1.62 (m, 3H), 1.26 (m, 2H). 13C NMR
(DMSO D6), d ppm: 170.5, 164.3, 161.7, 139.5, 138.4, 134.1,
128.6, 128.0, 126.7, 124.4, 122.4, 115.4, 115.3, 95.7, 62.2, 52.7,
43.7, 35.4, 29.4, 29.0. Anal. Calcd for C24H27N3O3: C, 71.09; H,
6.71; N, 10.36. Found: C, 71.36; H, 6.88; N, 10.24.
diethyl ether. The product was obtained as
a
white solid
4.1.6.5. 1-Benzyl-N-(1-benzylpiperidin-4-yl)-4-hydroxy-2-oxo-
(359 mg, 56%). IR (ATR)
c
cmꢀ1: 3250, 3300–3000 (w), 3100–
1,2-dihydroquinoline-3-carboxamide 5e.
was prepared according to method A from ethyl 1-benzyl-4-hydro-
xy-2-oxo-1,2-dihydroquinoline-3-carboxylate 3c (161 mg,
Title compound
3000 (w), 2922, 2803, 2765, 1636, 1603, 1568. 1H NMR (DMSO
D6), d ppm: 11.28, (bs, 1H), 10.43 (s, 2H), 7.96 (d, J = 7.6 Hz, 1H),
7.66 (t, J = 7.6 Hz, 1H), 7.43–7.17 (m, 7H), 4.00–3.71 (m, 1H), 3.48
(s, 2H), 2.70 (m, 2H), 2.17 (t, J = 10.0 Hz, 2H), 2.00–1.80 (m, 2H),
1.54 (m, 2H). 13C NMR (DMSO D6), d ppm: 172.5, 169.8, 162.8,
138.6, 138.4, 133.6, 128.6, 128.1, 126.8, 124.0, 122.2, 115.9,
115.8, 95.4, 62.0, 51.2, 45.6, 31.2. Anal. Calcd for C22H23N3O3: C,
70.01; H, 6.14; N, 11.13. Found: C, 69.68; H, 6.20; N, 10.72.
0.5 mmol) and 1-benzylpiperidin-4-amine (104.5 mg, 0.55 mmol)
during 3 h of reflux. Solvent was removed in vacuo and compound
was obtained by column chromatography (dichloromethane–
methanol: 1–3% gradient) as a white solid (229 mg, 98%). IR
(ATR)
c
cmꢀ1: 3196, 3100–3000(w), 2937, 2910, 2790, 1627,
1560. 1H NMR (DMSO D6), d ppm: 17.53 (s, 1H), 10.30 (bs, 1H),
8.13 (dd, J = 7.8 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 8.6 Hz,
1H), 7.40–7.14 (m, 11H), 5.55 (s, 2H), 3.96–3.84 (m, 1H), 3.48 (s,
2H), 2.71 (m, 2H), 2.27–2.11 (m, 2H), 1.92 (d, J = 9.8 Hz, 2H), 1.58
(m, 2H). 13C NMR (DMSO D6), d ppm: 171.6, 169.7, 162.1, 138.9,
138.3, 136.4, 134.1, 128.7, 128.6, 128.0, 126.9, 126.7, 126.1,
124.6, 122.5, 115.7, 115.1, 95.6, 61.9, 51.1, 45.8, 44.5, 31.0. Anal.
Calcd for C29H29N3O3: C, 74.50; H, 6.25; N, 8.99. Found: C, 74.23;
H, 6.39; N, 8.78.
4.1.6.2. N-((1-Benzylpiperidin-4-yl)methyl)-4-hydroxy-2-oxo-
1,2-dihydroquinoline-3-carboxamide 5b.
The compound
was prepared according to method A from ethyl 4-hydroxy-2-
oxo-1,2-dihydroquinoline-3-carboxylate 3a (250 mg, 1.07 mmol)
and (1-benzylpiperidin-4-yl)methanamine (241 mg, 1.18 mmol)
for 3 h to reflux. Compound precipitated after cooling and was fil-
tered and washed by diethyl ether. The product was obtained as a
white solid (300 mg, 79%). IR (ATR) c
cmꢀ1: 3673, 3246, 3300–3000
(w), 3100–3000 (w), 2991, 2936, 2905, 2805, 2754, 1634, 1562. 1H
NMR (DMSO D6), d ppm: 11.85 (bs, 1H), 10.41 (s, 2H), 7.96 (d,
J = 7.9 Hz, 1H), 7.67 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 7.9 Hz, 1H),
7.29–7.43 (m, 6H) 3.43 (s, 2H), 3. 29 (m, 2H), 2.81 (m, 2H), 1.90
(t, J = 11.2 Hz, 2H), 1.63 (m, 3H), 1.24 (m, 2H). 13C NMR (DMSO
D6) d ppm: 171.1, 162.8, 162.2, 139.1, 139.2, 134.1, 129.2, 128.6,
127.2, 124.5, 122.8, 116.4, 116.3, 97.4, 62.8, 53.3, 44.2, 36.1, 30.0.
Anal. Calcd for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C,
70.34; H, 6.47; N, 10.66.
4.1.6.6.
1-Benzyl-N-((1-benzylpiperidin-4-yl)methyl)-4-
Title
hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamide 5f.
compound was prepared according to method A from ethyl
1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate 3c
(209 mg, 0.65 mmol) and (1-benzylpiperidin-4-yl)methanamine
(146 mg, 0.71 mmol) during 3 h of reflux. Solvent was removed
in vacuo and compound was obtained by column chromatography
(ethyl acetate–cyclohexane: 1–1) as a white solid (250 mg, 80%). IR
(ATR)
c
cmꢀ1: 3184, 3100–3000 (w), 2929, 2802, 2760, 1624, 1585,
1554. 1H NMR (DMSO D6) d ppm: 17.62 (s, 1H), 10.40 (bs, 1H), 8.12
(d, J = 7.9 Hz, 1H), 7.69 (t, J = 7.9 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H),
7.40–7.19 (m, 11H), 5.55 (s, 2H), 3.46 (s, 2H), 3.33 (d, 2H), 2.83
(d, J = 10.7 Hz, 2H), 1.95 (t, J = 11.7 Hz, 2H), 1.66 (m, 3H),
1.14–1.36 (m, 2H). 13C NMR (DMSO D6) d ppm: 172.3, 172.2,
171.1, 139.5, 138.8, 137.1, 134.7, 129.2, 129.1, 128.6, 127.5,
127.3, 126.8, 125.2, 123.2, 121.6, 116.2, 116.1, 62.7, 53.2, 45.1,
44.4, 35.9, 29.9. Anal. Calcd for C30H31N3O3: C, 74.82; H, 6.49;
N, 8.73. Found: C, 74.54; H, 6.56; N, 8.64.
4.1.6.3. N-(1-Benzylpiperidin-4-yl)-4-hydroxy-1-methyl-2-oxo-
1,2-dihydroquinoline-3-carboxamide 5c.
The compound
was prepared according to method A from ethyl 4-hydroxy-1-
methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate 3b (247 mg,
1 mmol) and 1-benzylpiperidin-4-amine (209 mg, 1.1 mmol) dur-
ing 3 h of reflux. Compound precipitated after cooling and was fil-
tered and washed by diethyl ether as a white solid (246 mg, 63%).
IR (ATR)
c
cmꢀ1: 3250–3000 (w), 3100–3000 (w), 3061, 2936,
2800, 2756, 1638, 1555. 1H NMR (DMSO D6), d ppm: 10.51 (s,
1H), 10.49 (s, 1H), 8.09 (d, J = 7.7 Hz, 1H), 7.80 (t, J = 7.7 Hz, 1H),
7.62 (d, J = 7.7 Hz, 1H), 7.47–7.12 (m, 6H), 3.95–3.79 (m, 1H),
3.63 (s, 3H), 3.49 (s, 2H), 2.70 (m, 2H), 2.20 (t, J = 9.8 Hz, 2H),
2.00–1.80 (m, 2H), 1.56 (m, 2H). 13C NMR (DMSO D6), d ppm:
169.7, 165.1, 156.2, 139.5, 138.4, 134.0, 128.6, 128.0, 126.7,
124.4, 122.4, 115.2, 113.8, 95.7, 62.0, 51.0, 45.6, 31.1, 28.9. Anal.
Calcd for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C,
70.21; H, 6.47; N, 10.61.
4.1.6.7.
N-(1-Benzylpiperidin-4-yl)-4-methyl-2-oxo-1,2-dihy-
According to method B
droquinoline-3-carboxamide 5g.
from 4-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid 4d
(142 mg, 0.7 mmol), oxalyl chloride (107 mg, 0.84 mmol), dimeth-
ylformamide (11 lL) in dry dichloromethane (5 mL), and then
1-benzylpiperidin-4-amine (133 mg, 0.7 mmol), triethylamine
(283 mg, 2.8 mmol) in dry dichloromethane (5 mL). Title com-
pound was obtained by recrystallization in diethyl ether as a white
solid (190 mg, 72%). IR (ATR)
c
cmꢀ1: 3306, 3100–3000 (w), 2950,
4.1.6.4.
N-((1-Benzylpiperidin-4-yl)methyl)-4-hydroxy-1-
Title
2850, 2811, 1663, 1630, 1562, 1525. 1H NMR (DMSO D6), d ppm:
11.78 (s, 1H), 8.21 (br d, J = 7.5 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H),
7.52 (t, J = 7.6 Hz, 1H), 7.39–7.15 (m, 7H), 3.82–3.62 (m, 1H), 3.46
(s, 2H), 2.78 (d, J = 11.5 Hz, 2H), 2.36 (s, 3H), 2.06 (t, J = 10.5 Hz,
2H), 1.90–1.73 (m, 2H), 1.56–1.37 (m, 2H). 13C NMR (DMSO D6),
d ppm: 164.6, 159.2, 142.8, 138.5, 137.8, 130.3, 129.9, 128.5,
128.0, 126.6, 125.0, 121.7, 119.0, 115.1, 62.0, 51.7, 46.0, 31.3,
15.4. Anal. Calcd for C23H25N3O2: C, 73.57; H, 6.71; N, 11.19. Found:
C, 72.79; H, 6.78; N, 11.07.
methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide 5d.
compound was prepared according to method A from ethyl
4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
3b (250 mg, 1.01 mmol) and (1-benzylpiperidin-4-yl)methan-
amine (226 mg, 1.11 mmol) during 3 h of reflux. Solvent was
removed in vacuo and compound was obtained by column chro-
matography (dichloromethane–methanol: 1–4% gradient) as a
white solid (258 mg, 63%). IR (ATR)
c
cmꢀ1: 3670, 3250–3000
(w), 3100–3000 (w), 2938, 2911, 2794, 2750, 1627, 1558, 1503.
1H NMR (DMSO D6), d ppm: 10.45 (s, 2H), 8.08 (d, J = 7.7 Hz, 1H),
7.80 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.37 (t, J = 7.7 Hz,
1H), 7.31–7.15 (m, 5H), 3.62 (s, 3H), 3.44 (s, 2H), 3.31 (m, 2H),
4.1.6.8. N-((1-Benzylpiperidin-4-yl)methyl)-4-methyl-2-oxo-1,
2-dihydroquinoline-3-carboxamide 5h. According to method
B
from 4-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

8
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4d (142 mg, 0.7 mmol), oxalyl chloride (107 mg, 0.84 mmol),
dimethylformamide (11 L) in dry dichloromethane (5 mL), and
mixture was filtered through CeliteÒ and solvent was evaporated
under vacuum. The compound was obtained by column chroma-
tography (cyclohexane–dichloromethane: 7–1) and appeared as a
l
then (1-benzylpiperidin-4-yl)methanamine (143 mg, 0.7 mmol),
triethylamine (283 mg, 2.8 mmol) in dry dichloromethane (5 mL).
Title compound was obtained by recrystallization in diethyl ether
white solid (720 mg, 66%). IR (ATR)
c
cmꢀ1: 3374, 2996, 2960,
2935, 2917, 2850, 1661, 1579, 1519. 1H NMR (DMSO D6), d ppm:
7.58 (bs, 1H), 7.37 (s, 1H), 6.13 (s, 1H), 3.92 (s, 3H), 3.82 (s, 6H),
2.91 (s, 3H). 13C NMR (DMSO D6), d ppm: 168.0, 156.0, 149.5,
139.0, 114.1, 100.0, 94.8, 56.6, 55.9, 51.6, 30.0. Anal. Calcd for
as a white solid (158 mg, 58%). IR (ATR)
c
cmꢀ1: 3334, 3100–
3000 (w), 2939, 2915, 2801, 2765, 1641, 1535, 1504. 1H NMR
(DMSO D6), d ppm: 11.76 (s, 1H), 8.26 (bt, J = 4.9 Hz, 1H), 7.77 (d,
J = 7.7 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.37–7.16 (m, 7H), 3.46 (s,
2H), 3.11 (t, J = 6.0 Hz, 2H), 2.81 (d, J = 11.35 Hz, 2H), 2.36 (s, 3H),
2.02–1.89 (m, 2H), 1.84–1.62 (m, 2H), 1.60–1.42 (m, 1H), 1.31–
1.10 (m, 2H). 13C NMR (DMSO D6), d ppm: 165.4, 159.3, 142.9,
138.4, 137.7, 130.3, 130.0, 128.6, 127.9, 126.6, 125.0, 121.8,
119.0, 115.1, 62.2, 52.7, 44.1, 35.5, 29.5, 15.6. Anal. Calcd for
C11H15NO4: C, 58.66; H, 6.71; N, 6.22. Found: C, 58.76; H, 6.69;
N, 6.24.
4.1.9. Methyl 2-(benzylamino)-4,5-dimethoxybenzoate 6c
To
a solution of methyl 2-amino-4,5-dimethoxybenzoate
(469 mg, 2.2 mmol) in a dichloromethane–methanol (1–1), benzal-
dehyde (279 mg, 2.2 mmol) and acetic acid (0.5 mL) were added.
The solution was stirred at room temperature for 1 h and NaBH3CN
(330 mg, 5.24 mmol) was added. After overnight reaction, water
was added and the crude product was extracted with ethyl acetate
and washed with brine. Organic layers were dried over magnesium
sulfate and concentrated in vacuo. The compound was obtained by
recrystallization in cyclohexane to give the title compound
C24H27N3O2: C, 74.01; H, 6.99; N, 10.79. Found: C, 73.63; H, 7.16;
N, 10.77.
4.1.6.9. N-(1-Benzylpiperidin-4-yl)-2-oxo-4-phenyl-1,2-dihydro-
quinoline-3-carboxamide 5i.
oxo-4-phenyl-1,2-dihydroquinoline-3-carboxylic
(185.5 mg, 0.7 mmol), oxalyl chloride (107 mg, 0.84 mmol),
dimethylformamide (11 L) in dry dichloromethane (5 mL), and
According to method B from 2-
acid 4e
l
(404 mg, 61%) as a white solid. IR (ATR) c
cmꢀ1: 3343, 3013,
then 1-benzylpiperidin-4-amine (133 mg, 0.7 mmol), triethyl-
amine (283 mg, 2.8 mmol) in dry dichloromethane (5 mL). Title
compound was obtained by recrystallization in a dichlorometh-
ane–diethyl ether mixture as a white solid (176 mg, 57%). IR
2961, 2835, 1673, 1620, 1582, 1518. 1H NMR (DMSO D6) d ppm:
8.01 (bs, 1H), 7.44-7.22 (m, 6H), 6.28 (s, 1H), 4.47 (s, 2H), 3.76 (s,
3H), 3.70 (s, 3H), 3.64 (s, 3H). 13C NMR (DMSO D6) d ppm: 168.6,
155.1, 148.2, 139.3, 139.0, 128.7, 127.2, 127.1, 113.5, 101.1, 95.1,
56.5, 55.6, 51.3, 47.5. Anal. Calcd for C17H19NO4: C, 67.76; H,
6.36; N, 4.65. Found: C, 67.86; H, 6.49; N, 4.67.
(ATR)
c
cmꢀ1: 3277, 3200–3000 (w), 3058, 2940, 2800, 2755,
1665, 1634, 1541. 1H NMR (DMSO D6), d ppm: 12.02 (s, 1H), 7.95
(d, J = 7.4 Hz, 1H), 7.59–7.15 (m, 12H), 7.10 (t, J = 7.5 Hz, 1H),
7.03 (d, J = 7.5 Hz, 1H), 3.46–3.35 (m, 1H), 3.30 (s, 2H), 2.57 (d,
J = 11.5 Hz, 2H), 1.88 (t, J = 10.5 Hz, 2H), 1.36 (d, J = 10.5 Hz, 2H),
1.20–1.06 (m, 2H). 13C NMR (DMSO D6), d ppm: 163.4, 159.2,
146.7, 138.5, 138.2, 134.4, 130.4, 128.7, 128.5, 128.1, 128.0,
127.9, 127.8, 127.7, 126.6, 121.8, 118.8, 115.2, 61.9, 51.4, 45.5,
30.8. Anal. Calcd for C28H27N3O2: C, 76.86; H, 6.22; N, 9.60. Found:
C, 76.38; H, 6.28; N, 9.59.
4.1.10. Methyl 2-(4-hydroxy-3,5-dimethoxybenzylamino)
benzoate 6d
To a solution of methyl anthranilate (1.02 g, 6.62 mmol) in
dichloromethane–methanol
(1–1),
syringaldehyde
(1.00 g,
6.62 mmol) and acetic acid (2 mL) were added. The solution was
stirred at room temperature for 1 h and NaBH3CN (105 mg,
26.5 mmol) was added. After overnight reaction, water was added
and the crude product was extracted with dichloromethane and
washed with brine. Organic layer was dried over magnesium sul-
fate and concentrated in vacuo. The compound was obtained by
column chromatography (hexane–dichloromethane: 2–1) to give
4.1.7. 4-Amino-N-((1-benzylpiperidin-4-yl)methyl)-1-methyl-2-
oxo-1,2-dihydroquinoline-3-carboxamide 5j
A solution of N-((1-benzylpiperidin-4-yl)methyl)-4-hydroxy-1-
methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide 5d (150 mg,
0.37 mmol) in phosphorous oxychloride (2 mL) was stirred at room
temperature for 1 h. The mixture was poured into saturated
NaHCO3 and the resulting precipitate was collected and engaged
in reaction without further purification. The precipitates and
ammonia were heated overnight in ethanol to 100 °C in a sealed
tube. The solvent was evaporated and the residue was purified
by column chromatography (dichloromethane–methanol: 9–1)
the title compound (610 mg, 41%) as a white solid. IR (ATR)
c
cmꢀ1: 3361, 2930, 2845, 1680, 1610, 1578, 1511. 1H NMR (DMSO
D6) d ppm: 8.08 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H), 7.31 (t, J = 7.8 Hz,
1H), 6.77–6.60 (m, 2H), 6.59 (s, 2H), 5.48 (s, 1H), 4.35 (s, 2H),
3.86 (s, 9H). 13C NMR (DMSO D6) d ppm: 169.1, 151.0, 147.2,
134.6, 133.7, 131.6, 130.0, 115.0, 111.8, 110.2, 103.8, 56.3, 51.5,
47.4. Anal. Calcd for C17H19NO5: C, 64.34; H, 6.03; N, 4.41. Found:
C, 64.42; H, 6.09; N, 4.44.
and appeared as a white solid (81 mg, 55%). IR (ATR)
c :
cmꢀ1
3324, 3400–2900, 2918, 2851, 1727, 1633, 1620, 1568, 1547. 1H
NMR (DMSO D6), d ppm: 8.45 (t, J = 7.8 Hz, 1H), 7.88 (dd, J = 7.8,
1.4 Hz, 1H), 7.63 (dt, J = 7.8, 1.4 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H),
7.32–7.20 (m, 6H), 3.47 (s, 3H), 3.43 (s, 2H), 3.33 (s, 2H), 2.79 (m,
2H), 1.89 (t, J = 10.8 Hz, 2H), 1.63 (m, 3H), 1.26–1.18 (m, 2H),
1.07 (s, 2H). 13C NMR (DMSO D6), d ppm: 168.9, 166.9, 156.8,
141.1, 134.2, 132.4, 129.1, 128.6, 127.2, 124.1, 122.6, 116.5,
111.7, 85.7, 62.8, 53.2, 48.3, 36.3, 29.9, 28.3. Anal. Calcd for
4.1.11. Typical procedure for preparation of compounds 7a–d
A solution of ethyl malonyl chloride (1.1 equiv) in dry dichloro-
methane (distilled on P2O5) was slowly added to a solution of ami-
nobenzoate 6a–d (1 equiv) and triethylamine (3 equiv) at 0 °C
under nitrogen atmosphere in dry dichloromethane. After 30 min
the reaction mixture was allowed to warm up at rt. After comple-
tion of the reaction, (1–3 h, TLC monitoring: a drop of the reaction
mixture is diluted in methanol to form an ester which is compared
to ethyl malonic acid), the mixture was washed with NaHCO3 5%
and brine. The organic layer is dried over sodium sulfate, filtered
and concentrated in vacuo.
C24H28N4O2: C, 71.26; H, 6.98; N, 13.85. Found: C, 70.96; H, 6.99;
N, 13.95.
4.1.8. Methyl 4,5-dimethoxy-2-(methylamino)benzoate 6b
This residue was engaged without further purification into the
next step.
To
a solution of methyl 2-amino-4,5-dimethoxybenzoate
(1.02 g, 4.84 mmol) in dioxane was added copper-(II) acetate
(2.2 g, 12.1 mmol) and pyridine (956 mg, 12.1 mmol). The mixture
was stired 15 min., methyl boronic acid (688 mg, 12.1 mmol) was
added and the reaction was refluxed 24 h. After cooling, the
The residue was diluted in dry methanol (distilled on Na0),
freshly prepared NaOMe (2.2 equiv) was added and the reaction
was stirred at rt overnight. The mixture was diluted in water and
acidified with a solution of hydrochloric acid below pH 2. The
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
product precipitated and/or was extracted with dichloromethane.
The organic layer was washed with brine, dried over magnesium
sulfate and concentrated in vacuo. The compound was obtained
by carefull column chromatography.
62.3, 56.4, 45.8, 14.2. Anal. Calcd for C21H21NO7: C, 63.15; H,
5.30; N, 3.51. Found: C, 62.92; H, 5.24; N, 3.54.
4.1.12. Preparation of compounds 8a–d
Conpounds were prepared according to method A of amidifica-
tion (4.1.6).
4.1.11.1. Methyl 4-hydroxy-6,7-dimethoxy-2-oxo-1,2-dihydro-
quinoline-3-carboxylate 7a. According to the general procedure
(4.1.11.) from ethyl malonyl chloride (433 mg, 2.11 mmol); methyl
4,5-dimethoxy-2-aminobenzoate (593 mg, 1.92 mmol); triethyl-
amine (582 mg, 5.76 mmol) for 3 h at rt. Then NaOMe (228 mg,
4.22 mmol) in methanol overnight. The title compound was ob-
tained by column chromatography (dichloromethane–methanol:
4.1.12.1. N-((1-Benzylpiperidin-4-yl)methyl)-4-hydroxy-6,7-dime-
thoxy-2-oxo-1,2-dihydroquinoline-3-carboxamide 8a.
Title
compound was prepared from 7a (338 mg, 1.15 mmol) and (1-ben-
zylpiperidin-4-yl)methanamine (259 mg, 1.27 mmol) for 3 h to reflux
in xylene. After cooling to room temperature the product precipitated,
and was washed by diethyl ether to give a white solid (447 mg, 86%). IR
9–1) as a white solid (350 mg, 65%). IR (ATR)
c
cmꢀ1: 3200–3000
(w), 2957, 2902, 2830, 1668, 1632, 1610, 1513. 1H NMR (DMSO
D6), d ppm: 13.68 (s, 1H), 11.36 (s, 1H), 7.26 (s, 1H), 6.78 (s, 1H),
3.86 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H). 13C NMR (DMSO D6), d
ppm: 172.4, 170.3, 159.5, 155.5, 145.4, 137.3, 105.7, 104.3, 97.7,
97.2, 56.3, 56.1, 52.8. Anal. Calcd for C13H13NO6: C, 55.91; H,
4.69; N, 5.02. Found: 55.41; H, 4.86; N, 5.09.
(ATR) c
cmꢀ1: 3237, 3161, 3200–3000 (w), 2915, 2799, 2935, 1639,
1511. 1H NMR (CDCl3), d ppm: 17.14 (s, 1H), 10.14 (t, J = 5.7 Hz, 1H),
9.80 (s, 1H), 7.43 (s, 1H), 7.35–7.20 (m, 5H), 6.55 (s, 1H), 3.96 (s, 3H),
3.95 (s, 3H), 3.49 (s, 2H), 3.42–3.25 (m, 2H), 2.90 (d, J = 11.3 Hz, 2H),
1.96 (t, J = 11.0 Hz, 2H), 1.76 (d, J = 12.5 Hz, 2H), 1.53–1.63 (m, 1H),
1.43–1.28 (m, 2H). 13C NMR (CDCl3), d ppm: 172.9, 171.1, 163.7,
155.0, 146.2, 138.5, 134.3, 129.0, 128.1, 126.9, 108.5, 104.7, 97.3, 95.2,
63.3, 56.2, 56.1, 53.3, 44.6, 36.3, 30.2. Anal. Calcd for C25H29N3O5: C,
66.50; H, 6.47; N, 9.31. Found: C, 65.91; H, 6.44; N, 9.51.
4.1.11.2. Ethyl 4-hydroxy-6,7-dimethoxy-1-methyl-2-oxo-1,2-
dihydroquinoline-3-carboxylate 7b.
According to the gen-
eral procedure (4.1.11.) from ethyl malonyl chloride (433 mg,
2.89 mmol); 6b (593 mg, 2.63 mmol); triethylamine (799 mg,
7.91 mmol) for 3 h at rt. Then NaOMe (312 mg, 5.78 mmol) in
methanol overnight. The title compound was obtained by column
chromatography (dichloromethane–methanol: 9–1) as a white so-
4.1.12.2.
dimethoxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxam-
ide 8b. Title compound was prepared from 7b (152 mg,
N-((1-Benzylpiperidin-4-yl)methyl)-4-hydroxy-6,7-
0.519 mmol) and (1-benzylpiperidin-4-yl)methanamine (127 mg,
0.622 mmol) for 3 h to reflux in xylene. Solvent was removed in va-
cuo and title compound was obtained as a white solid (198 mg,
82%) by column chromatography (dichloromethane–methanol:
lid (577 mg, 65%). IR (ATR)
c
cmꢀ1: 3200–3000 (w), 3002, 2965,
2935, 2830, 1643, 1595, 1567, 1516. 1H NMR (DMSO D6), d ppm:
13.38 (bs, 1H), 7.39 (s, 1H), 6.92 (s, 1H), 4.33 (q, J = 7.1 Hz, 2H),
3.84 (s, 3H), 3.83 (s, 3H), 3.55 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). 13C
NMR (DMSO D6), d ppm: 171.3, 167.7, 158.9, 155.4, 145.3, 138.0,
106.9, 105.5, 98.5, 98.8, 61.6, 56.6, 56.3, 29.6, 14.5. Anal. Calcd
for C15H17NO6: C, 58.63; H, 5.58; N, 4.56. Found: C, 58.43; H,
5.66; N, 4.59.
1–4% gradient). IR (ATR)
c
cmꢀ1: 3191 (w), 3200–3000 (w), 3000,
2922, 2795, 2753, 1637, 1574, 1525. 1H NMR (CDCl3), d ppm:
17.16 (s, 1H), 10.43 (s, 1H), 7.56 (s, 1H), 7.37–7.17 (m, 5H), 6.74
(s, 1H), 4.03 (s, 3H), 3.98 (s, 3H), 3.68 (s, 3H), 3.50 (s, 2H), 3.42–
3.30 (m, 2H), 2.91 (d, J = 11.1 Hz, 2H), 1.98 (t, J = 11.2 Hz, 2H),
1.77 (d, J = 11.9 Hz, 2H), 1.64–1.53 (m, 1H), 1.50–1.29 (m, 2H).
13C NMR (CDCl3), d ppm: 171.3, 171.1, 162.6, 154.6, 145.5, 138.5,
136.1, 129.1, 128.1, 126.8, 109.1, 105.3, 96.7, 95.5, 63.4, 56.2
(2C), 53.3, 44.5, 36.0, 30.1, 29.3. Anal. Calcd for C26H31N3O5: C,
67.08; H, 6.71; N, 9.03. Found: C, 67.29; H, 6.77; N, 8.95.
4.1.11.3. Ethyl 1-benzyl-4-hydroxy-6,7-dimethoxy-2-oxo-1,2-
dihydroquinoline-3-carboxylate 7c.
According to the gen-
eral procedure (4.1.11.) from ethyl malonyl chloride (157 mg,
1.05 mmol); 6c (301 mg, 0.95 mmol); triethylamine (288 mg,
2.85 mmol) for 3 h at rt. Then NaOMe (103 mg, 1.9 mmol) in meth-
anol overnight. The compound was obtained by column chroma-
tography (dichloromethane–ethyl acetate: 9–1) as a white solid
4.1.12.3.
hydroxy-6,7-dimethoxy-2-oxo-1,2-dihydroquinoline-3-carbox-
amide 8c. According to method A of amidification (4.1.4.)
1-Benzyl-N-((1-benzylpiperidin-4-yl)methyl)-4-
(240 mg, 66%). IR (ATR)
c
cmꢀ1: 3100–3000 (w), 2946, 2838,
1623, 1598, 1567. 1H NMR (CDCl3), d ppm: 14.37 (s, 1H), 7.50 (s,
1H), 7.34–7.20 (m, 5H), 6.61 (s, 1H), 5.51 (s, 2H), 4.52 (q,
J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.75 (s, 3H), 1.49 (t, J = 7.1 Hz, 3H).
13C NMR (CDCl3), d ppm: 173.1, 171.3, 160.0, 155.0, 145.1, 137.4,
136.8, 128.8, 127.3, 126.6, 107.7, 105.3, 97.9, 96.2, 62.2, 56.2,
56.0, 46.0, 14.3. Anal. Calcd for C21H21NO6: C, 65.79; H, 5.52; N,
3.65. Found: C, 65.27; H, 5.62; N, 3.62.
from 7c (158 mg, 0.4 mmol) and (1-benzylpiperidin-4-yl)methan-
amine (93 mg, 0.44 mmol) for 4 h to reflux in xylene. The solvent
was removed in vacuo and the compound was obtained as a white
solid (196 mg, 91%) by crystallization from pentane. IR (ATR)
c
cmꢀ1: 3186, 3200–3000 (w), 3002, 2924, 2791, 2746, 1628, 1569,
1517. 1H NMR (DMSO D6), d ppm: 17.39 (bs, 1H), 10.45 (bs, 1H),
7.39 (s, 1H); 7.28 (m, 10H), 6.89 (s, 1H), 5.57 (s, 2H), 3.81 (s, 3H),
3.74 (s, 3H), 3.42 (s, 2H), 3.29 (m, 2H), 2.80 (d, J = 10.0 Hz, 2H),
1.90 (t, J = 10.0 Hz, 2H), 1.65 (d, J = 12.0 Hz, 3H), 1.26 (m, 2H). 13C
NMR (DMSO D6), d ppm: 171.3, 172.2, 162.4, 154.9, 145.7, 139.1,
137.4, 135.7, 129.3, 129.2, 128.6, 128.5, 127.6, 127.2, 108.5,
104.9, 99.2, 94.8, 62.9, 56.4, 56.1, 53.3, 45.2, 44.4, 36.1, 30.1. Anal.
Calcd for C32H35N3O5: C, 70.96; H, 6.51; N, 7.76. Found: C, 70.53; H,
6.60; N, 7.67.
4.1.11.4. Ethyl 4-hydroxy-1-(4-hydroxy-3,5-dimethoxybenzyl)-
2-oxo-1,2-dihydroquinoline-3-carboxylate 7d.
the general procedure (4.1.11.) from ethyl malonyl chloride (1.07 g,
7.11 mmol); methyl 2-(4-hydroxy-3,5-dimethoxybenzylami-
According to
no)benzoate 6d (600 mg, 5.93 mmol); triethylamine (1.80 g,
17.79 mmol) for 4 h at rt. Then NaOMe (312 mg, 5.78 mmol) in
methanol overnight. The compound was obtained by column chro-
matography (dichloromethane) as a white solid (925 mg, 46%). IR
4.1.12.4.
hydroxy-3,5-dimethoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-
carboxamide 8d. According to method A of amidification
N-((1-Benzylpiperidin-4-yl)methyl)-4-hydroxy-1-(4-
(ATR)
c
cmꢀ1: 3361, 3100–3000 (w), 2981, 2939, 1615, 1592,
1560, 1520. 1H NMR (CDCl3), d ppm: 14.35 (s, 1H), 8.20 (d,
J = 7.0 Hz, 1H), 7.55 (t, J = 7.0 Hz, 1H), 7.39–7.03 (m, 3H), 6.42 (s,
2H), 5.42 (s, 2H), 4.53 (q, J = 6.8 Hz, 2H), 3.80 (s, 6H), 1.49 (t,
J = 6.8 Hz, 3H). 13C NMR (CDCl3), d ppm: 172.7, 171.9, 159.6,
147.4, 141.0, 134.2, 127.7, 125.7, 122.0, 115.1, 115.0, 103.7, 97.9,
(4.1.4.) from 7d (117 mg, 0.293 mmol) and (1-benzylpiperidin-4-
yl)methanamine (90 mg, 0.44 mmol) for 4 h of reflux in xylene.
The solvent was removed in vacuo and the title compound was ob-
tained as a white solid (157 mg, 96%) by column chromatography
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

10
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(dichloromethane–methanol: 8%). IR (ATR)
c
cmꢀ1: 3221, 3400–
135.1, 131.9, 131.8, 130.0, 129.3, 129.1, 126.8, 108.8, 107.9, 102.3,
3000, 2924, 2803, 1628, 1561, 1517. 1H NMR (DMSO D6), d ppm:
17.57 (bs, 1H), 10.47 (bs, 1H), 8.33 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H),
7.71 (t, J = 7.8 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.40–7.18 (m, 6H),
6.53 (s, 2H), 5.41 (s, 2H), 3.65 (s, 6H), 3.43 (s, 2H), 3.36–3.28 (m,
2H), 2.80 (d, J = 10.2 Hz, 2H), 1.90 (t, J = 11.1 Hz, 2H), 1.70–1.50
(m, 3H), 1.35–1.14 (m, 2H). 13C NMR (DMSO D6), d ppm: 172.1,
171.2, 162.7, 148.6, 139.6, 139.0, 135.3, 134.6, 129.2, 128.6,
127.3, 127.1, 125.1, 123.1, 116.4, 107.9, 104.9, 96.2, 62.8, 56.5,
53.2, 45.3, 44.4, 35.9, 30.0. Anal. Calcd for C32H35N3O6: C, 68.92;
H, 6.33; N, 7.54. Found: C, 68.56; H, 6.29; N, 7.56.
94.0, 59.7, 51.8, 51.7, 45.3, 33.9, 27.1. HRMS (ESI, (M+H)+) calc
514.2342, found 514.2328.
4.1.13.4. N-((1-benzylpiperidin-4-yl)methyl)-4-hydroxy-2-oxo-
ide 9d.
According to the typical procedure 8d (42 mg,
0.075 mmol) and BBr3 1 M in dichloromethane (0.45 mL,
0.45 mmol). The compound was obtained obtained as a white solid
(18 mg, 45%) by column chromatography (dichloromethane–meth-
anol: 20%). IR (ATR) c
cmꢀ1: 3500–2800 (l), 3220, 2955, 2919, 2851,
1727, 1626, 1562. 1H NMR (DMSO D6), d ppm: 17.56 (s, 1H), 10.44
(bs, 1H), 8.78 (s, 2H), 8.11 (d, J = 8.1 Hz, 1H), 8.03 (s, 1H), 7.71 (t,
J = 8.1 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.39–7.26 (m, 6H), 6.09 (s,
2H), 5.30 (bs, 2H), 3.46 (s, 2H), 3.36–3.28 (m, 2H), 2.98–2.78 (m,
2H), 1.94–1.82 (m, 2H), 1.78–1.58 (m, 3H), 1.38–1.20 (m, 2H). 13C
NMR (DMSO D6), d ppm: 172.2, 171.2, 167.2, 146.8, 139.7, 134.8,
134.6, 128.8 128.7, 128.6, 127.8, 127.0, 125.1, 125.0, 116.5, 116.0,
105.6, 96.2, 67.7, 53.2, 45.4, 45.0, 31.0, 29.4. HRMS (ESI, (M+H)+)
calc 530.2271, found 530.2267.
4.1.13. Typical procedure for the preparation of 9a–d
To a solution of the appropriate methoxy derivative 8a–d
(1 equiv) in dry dichloromethane at ꢀ30 °C was slowly added bor-
on tribromide (1 M in dichloromethane, 3 equiv per methoxy func-
tion) under nitrogen atmosphere. The mixture was allowed slowly
to warm up to room temperature and stirred overnight. The reac-
tion was quenched by careful addition of brine and product was
extracted by ethyl acetate. Organic layers were evaporated under
reduce pressure and the product was obtained by column chroma-
tography and/or crystallization.
4.2. In vitro assays
4.2.1. Inhibition of AChE and BuChE
4.1.13.1. N-((1-Benzylpiperidin-4-yl)methyl)-4,6,7-trihydroxy-
The method of Ellman et al. was followed.22 Human erythro-
cyte AChE, Torpedo califonica AChE and equine BChE were
obtained from Sigma–Aldrich. Stock solutions were prepared by
dissolving lyophilized enzymes in phosphate buffer solution
(pH = 8.0). Solutions of tested compounds were prepared starting
from 10 mM stock solutions in DMSO diluted with aqueous
assay medium to a final content of organic solvent always under
1%. Five concentrations of each compound were used in order to
obtain inhibition of AChE and BuChE comprised between 20 and
80%.
2-oxo-1,2-dihydroquinoline-3-carboxamide 9a.
According
to the typical procedure from 8a (91 mg, 0.202 mmol) and and
BBr3 1 M in dichloromethane (1.22 mL, 1.22 mmol). The compound
was obtained by column chromatography (dichloromethane–
methanol: 1–5%) and recrystallization from acetone as a white so-
lid (58 mg, 68%). IR (ATR)
c
cmꢀ1: 3227, 3600–2800 (l), 2972, 2689,
1659, 1615, 1549. 1H NMR (DMSO D6), d ppm: 16.66 (s, 1H), 11.48
(s, 2H), 11.15–11.00 (m, 1H), 10.56–10.42 (m, 1H), 7.49–7.35 (m,
5H), 6.91 (s, 1H), 6.59 (s, 1H), 4.29–4.15 (m, 2H), 3.31–3.20 (m,
2H), 3.14–3.02 (m, 1H), 2.99–2.79 (m, 2H), 1.86–1.40 (m, 6H). 13C
NMR (DMSO D6), d ppm: 171.8, 171.7, 162.8, 153.6, 143.1, 136.0,
134.8, 131.8, 130.0, 129.3, 107.7, 106.8, 101.3, 94.2, 59.8, 51.8,
43.0, 34.0, 27.0. Anal. Calcd for C23H25N3O5: C, 65.24; H, 5.95; N,
9.92. Found: C, 64.94; H, 5.91; N, 9.86%. HRMS (ESI, (M+H)+) calcd
424, 1794, found 424, 1798.
The assay solution in a total volume of 3 mL consisted of 0.1 M
phosphate buffer solution pH = 8.0 and contained 5,50-dithio-bis(2-
nitrobenzoic acid), DTNB (2625
sample (3 L, 0.01–10 final concentration), AChE (29
0.035 u.i./mL final concentration) or BuChE (60 L, 0.05 u.i./mL fi-
nal concentration) and substrate (acetylthiocholine iodide, ATCh
or butyrylthiocholine iodide, BTCh respectively, 105 L, 0.35 mM
lL, 0.35 mM final concentration),
l
l
M
lL,
l
l
4.1.13.2. N-((1-Benzylpiperidin-4-yl)methyl)-4,6,7-trihydroxy-1-
final concentration). Increasing concentration of tested compounds
were added to the assay solution and pre-incubated for 10 min at
room temperature with the enzyme followed by the addition of
substrate and absorbance was measured after 15 min. Assay were
done with a blank containing all components except the enzyme in
order to account for non enzymatic reactions and one sample
where only inhibitor was replaced by the buffer solution was al-
ways present to yield the 100% of ChE activity (control). Absor-
bance values were recorded at 412 nm in quadruplicate and the
values were averaged. The pourcentage inhibition was calculated
as [(Acontrol ꢀ Ablank) ꢀ (Asample ꢀ Ablank)/(Acontrol ꢀ Ablank)] ꢃ 100.
Bovine Serum Albumin (BSA) is usually added up to 0.5% of the
total reaction volume to reduce the coating of target enzyme dur-
ing the incubation. In our assay, no obvious effect from BSA on the
activity of the compounds was found. This indicates that the new
compounds selectively inhibit the target enzymes but do not inter-
act with BSA.
methyl-2-oxo-1,2-dihydroquinoline-3-carboxamide 9b.
Accord-
ing to the typical procedure from 8b (52.3 mg, 0.11 mmol) and BBr3
1 M in dichloromethane (0.67 mL, 0.67 mmol). The compound was
obtained as a white solid (42 mg, 87%) by column chromatography
(dichloromethane–methanol: 15%). IR (ATR)
c
cmꢀ1: 3327, 3203,
3500–3000 (l), 2954, 2732, 1646, 1564, 1529. 1H NMR (DMSO D6),
d ppm: 16.97 (s, 1H), 10.52 (t, J = 5.9 Hz, 1H), 10.43 (s, 1H), 9.68
(s, 1H), 7.59–7.38 (m, 5H), 7.36 (s, 1H), 6.90 (s, 1H), 4.26 (s, 2H),
3.52 (s, 3H), 3.43–3.33 (m, 2H), 3.07–2.80 (m, 2H), 1.92–1.69
(m, 5H), 1.58–1.33 (m, 2H). 13C NMR (DMSO D6), d ppm: 171.0,
170.0, 161.3, 153.1, 142.2, 135.8, 135.2, 131.2, 129.4, 128.7, 108.1,
107.0, 101.1, 93.6, 55.4, 54.8, 51.3, 42.6, 33.4, 28.8. HRMS
(ESI, (M+H)+) calc 438.2029, found 438.2030.
4.1.13.3. 1-benzyl-N-((1-benzylpiperidin-4-yl)methyl)-4,6,7-trihy-
droxy-2-oxo-1,2-dihydroquinoline-3-carboxamide 9c.
Accord-
ing to the typical procedure from 8c (52.3 mg, 0.11 mmol) and
BBr3 1 M in dichloromethane (0.67 mL, 0.67 mmol). The compound
was obtained as a white solid (42 mg, 87%) by column chroma-
The concentration of compound which determined 50% inhibi-
tion of the AChE activity (IC50) was calculated using a sigmoidal hill
slope model.
tography (dichloromethane–methanol 15%). IR (ATR)
c :
cmꢀ1
3600–2800 (l), 3390, 3064, 2937, 1626, 1555. 1H NMR (DMSO D6),
d ppm: 11.49 (bs, 1H), 11.30 (bs, 1H), 10.53 (bs, 2H), 7.70–7.10 (m,
11H), 6.76 (s, 1H), 5.41 (s, 2H), 4.29 (s, 2H), 3.92–3.60 (m, 4H),
3.54–3.22 (m, 2H), 1.98–1.74 (m, 3H), 1.58–1.32 (m, 2H). 13C NMR
(DMSO D6), d ppm: 171.7, 171.1, 162.2, 153.6, 143.0, 137.2, 136.4,
4.2.2. DPPH free radical scavenging activity
Assay for the scavenging of stable free radical 1,1-diphenyl-2-
picrylhydrazyl (DPPH) was done as previously reported.23 A freshly
prepared solution of DPPH (0.15 mM, 2.7 mL) in methanol was
added to a methanol solution of tested compounds or reference
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
compounds (quercetin and curcumin) at different concentrations.
The concentrations of the tested compounds were used in order
to obtain activities comprised between 20% and 80%. The mixture
was stirred at room temperature in the dark for 2 h and the
absorbance was measured in quadruplicate at 517 nm. Radical
Scavenging Activity (RSA%) was calculated as [(Acontrol ꢀ Asample)/
Acontrol] ꢃ 100 where Acontrol represents absorbance of control with-
out test sample and Asample represents absorbance in the presence
of the test sample. The scavenging activity was expressed as the
EC50, the concentration of compounds required for scavenging
50% of the DPPH radicals in the solution. EC50 values were calcu-
lated from linear fits.
agreed with the crystallographic results with 1.34 and 1.59 Å
all-atom root-mean-square deviations for hAChE and tAChE
respectively. The docking (CDOCKER)energy including the intra-
molecular energy for the ligand and the ligand–protein interac-
tions energy of compd 9a and donepezil in hAChE were
52.86 kcal molꢀ1 and 33.95 kcal molꢀ1 respectively.
Acknowledgments
This work was supported by a grant from the CONSEIL REGIO-
NAL DE FRANCHE COMTE grant N° 2011-07304. Authors thank
Dr. F. Chérioux for fruitfull discussions.
4.2.3. Superoxide anion scavenging assay
References and notes
The technic of Robak and Gryglewski.26 based on the reactions
of NADH, phenazine methosulfate (PMS), molecular oxygen and
nitroblue tetrazolium (NBT) was used to evaluate the superoxide
anion scavenging. The NADH/PMS/O2/NBT system involves the
intermediate formation of the superoxide anion radical from the
interaction of reduced PMS with O2; the superoxide anion radical
then reduces NBT to the highly coloured formazan. The reaction
was followed by measuring the absorbance of the formazan at
578 nm. The incubation mixture contained in 2 mL of 16 mM
1. Seshadri, S.; Wolf, P. A.; Beiser, A.; Au, R.; McNulty, K.; White, R.; D’Agostino, R.
B. Neurology 1997, 49, 1498.
2. (a) Singh, M.; Kaur, M.; Kukreja, H.; Chugh, R.; Silakari, O.; Singh, D. Eur. J. Med.
Chem. 2013, 70, 165; (b) Marlatt, M. W.; Webber, K. M.; Moreira, P. I.; Lee, H.-G.;
Casadesus, G.; Honda, K.; Zhu, X.; Perry, G.; Smith, M. A. Curr. Med. Chem. 2005,
12, 1137.
3. (a) Francis, P. T.; Palmer, A. M.; Snape, M.; Wilcock, G. K. J. Neurol. Neurosurg.
Psychiatry 1999, 66, 137; (b) Dunnett, S. B.; Fibiger, H. C. Prog. Brain Res. 1993,
98, 413.
4. Jakob-Roetne, R.; Jacobsen, H. Angew. Chem., Int. Ed. 2009, 48, 3030.
5. Ballatore, C.; Lee, V. M.-Y.; Trojanowski, J. Q. Nat. Rev. Neurosci. 2007, 8, 663.
6. Smith, M. A.; Perry, G.; Pryor, W. A. Free Radical Biol. Med. 2002, 32, 32.
7. (a) Munoz-Torrero, D. Curr. Med. Chem. 2008, 15, 2433; (b) Soreq, H.; Seidman,
S. Nat. Rev. Neurosci. 2001, 2, 294; (c) Hashimoto, M.; Kazui, H.; Matsumoto, K.;
Nakano, Y.; Yasuda, M.; Mori, E. Am. J. Psychiatry 2005, 162, 676.
8. (a) León, R.; Garcia, A. G.; Marco-Contelles, J. Med. Res. Rev. 2013, 33, 139; (b)
Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347; (c) Mehta, M.; Abdu Adem, A.;
Sabbagh, M. Int. J. Alzheimers Dis. 2012, 8. article ID 728983.
9. (a) Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. J. Med. Chem.
Article ASAP.; (b) Liu, Q.; Xie, F.; Rolston, R.; Moreira, P. I.; Nunomura, A.; Zhu,
X.; Smith, M. A.; Perry, G. Mini-Rev. Med. Chem. 2007, 7, 171.
10. (a) Rosini, M.; Simoni, E.; Bartolini, M.; Tarozzi, A.; Matera, R.; Milelli, A.;
Hrelia, P.; Andrisano, V.; Bolognesi, M. L.; Melchiorre, C. Eur. J. Med. Chem.
2011, 46, 5435; (b) Bolognesi, M. L.; Cavalli, A.; Bergamini, C.; Fato, R.;
Lenaz, G.; Rosini, M.; Bartolini, M.; Andrisano, V.; Melchiorre, C. J. Med.
Chem. 2009, 52, 7883.
Tris–HCl buffer at pH = 8, NADH (400
tion), nitroblue tetrazolium (400 L, 50
PMS (400 L, 10 M final concentration) and compounds to be
tested at various concentrations (from 12.5 to 200 M). Controls
lL, 78
lM final concentra-
l
lM final concentration),
l
l
l
were realized by replacing PMS by the buffer and quercetine was
used as reference for the assay. Radical Scavenging Activity
(RSA%) was calculated as [(Acontrol ꢀ Asample)/Acontrole] ꢃ 100 where
Acontrol represents absorbance of control and Asample represents
absorbance in the presence of PMS. The scavenging activity was ex-
pressed as the EC50, the concentration of compounds required for
scavenging 50% of the superoxide anion radicals in the solution.
EC50 values were calculated from linear fits.
11. Sussman, J. L.; Harel, M.; Silman, I. Chem. Biol. Interact. 1993, 87, 187.
12. (a) Silva, D.; Chioua, M.; Samadi, A.; Agostinho, P.; Garção, P.; Lajarín-Cuesta,
R.; de los Ríos, C.; Iriepa, I.; Moraleda, I.; Gonzalez-Lafuente, L.; Mendes, E.;
Pérez, C.; Rodríguez-Franco, M. I.; Marco-Contelles, J.; Carmo Carreiras, M. ACS
Chem. Neurosci. 2013, 4, 547; (b) Belluti, F.; Bartolini, M.; Bottegoni, G.; Bisi, A.;
Cavalli, A.; Andrisano, V.; Rampa, A. Eur. J. Med. Chem. 2011, 46, 1682; (c)
Leonetti, F.; Catto, M.; Nicolotti, O.; Pisani, L.; Cappa, A.; Stefanachi, A.; Carotti,
A. Bioorg. Med. Chem. 2008, 16, 7450; (d) Gemma, S.; Gabellieri, E.; Huleatt, P.;
Fattorusso, C.; Borriello, M.; Catalanotti, B.; Butini, S.; De, A. M.; Novellino, E.;
Nacci, V.; Belinskaya, T.; Saxena, A.; Campiani, G. J. Med. Chem. 2006, 49, 3421;
(e) Carlier, P. R.; Han, Y. F.; Chow, E. S. H.; Li, C. P. L.; Wang, H.; Lieu, T. X.; Wong,
H. S.; Pang, Y. P. Bioorg. Med. Chem. 1999, 7, 351; (f) Pang, Y. P.; Quiram, P.;
Jelacic, T.; Hong, F.; Brimijoin, S. J. Biol. Chem. 1996, 271, 23646.
4.3. Docking study
The hAChE and tAChE three-dimensional structures were ob-
tained from Protein Data Bank (PDB), accession code 4ey728 and
1eve27 respectively. The CDocker program29 implement in Discov-
ery studio 2.5 software was used. Inhibitors and unnecessary water
molecules were removed, hydrogens were added and CHARMm
forcefield was applyed. The crystal structure was defined as the
receptor and the active site was defined as a sphere within 8 Å
reach of the inhibitor. The docking was performed using the de-
fault parameters. Ligands were ionized by a pH-based method:
ionization state are predicted for a pH range of 5.5–9.5 using esti-
mated pKa values included in drugbank database. For each ligand a
generation of 3D coordinates was realized by calculation using
CATALYST algorithm. Random ligand conformations were gener-
ated using high-temperature molecular dynamics. Ten conforma-
tions were generated from equilibrium and minimization of the
starting ligand structure. 1000 steps of molecular dynamics and a
targeted temperature of 1000 K was used to generate random
starting conformations. Then ligand conformers were translated
into the binding site sphere. Candidates poses were then created
using random rigid body rotation (with a maximum of 800
rotations and a 300 kcal molꢀ1 energy threshold) followed by sim-
ulated annealing (with 2000 heating phase steps, a target temper-
ature of 700 K, 5000 steps of cooling phase and a cooling targeted
temperature of 300 K). CHARMm forcefield was used to determine
the CDocker score used to sort the poses of each input ligand. The
ten best poses are saved and the result can be considered as con-
vergent when all poses are about in the same position. Donepezil
(E2020) was used to validate our docking model, the prediction
13. (a) Chen, X.; Zenger, K.; Lupp, A.; Kling, B.; Heilmann, J.; Fleck, C.; Kraus, B.;
Decker, M. J. Med. Chem. 2012, 55, 5231; (b) Fernández-Bachiller, M. I.; Pérez,
C.; Monjas, L.; Rademann, J.; Rodríguez-Franco, M. I. J. Med. Chem. 2012, 55,
1303; (c) Ferna´ndez-Bachiller, M. I.; Pe´rez, C.; Gonza´lez-Mun~oz, G. C.; Conde,
´
´
´
S.; Lopez, M. G.; Villarroya, M.; Garcia, A. G.; Rodriguez-Franco, M. I. J. Med.
Chem. 2010, 53, 4927; (d) Decker, M.; Kraus, B.; Heilmann, J. Bioorg. Med. Chem.
2008, 16, 4252; (e) Rodríguez-Franco, M. I.; Fernández-Bachiller, M. I.; Pérez,
C.; Hernández-Ledesma, B.; Bartolomé, B. J. Med. Chem. 2006, 49, 459; (f) Rosini,
M.; Andrisano, V.; Bartolini, M.; Bolognesi, M. L.; Hrelia, P.; Minarini, A.; Andrea
Tarozzi, A.; Melchiorre, C. J. Med. Chem. 2005, 48, 360.
14. Jonhson, G.; Moore, S. W. Curr. Pharm. Des. 2006, 12, 217.
15. (a) Larsson, E. L.; Jansson, A.; Ng, F. M.; Then, S. W.; Panicker, R.; Liu, B.;
Sangthongpitag, K.; Pendharkar, V.; Tai, S.; Hill, J.; Dan, C.; Ho, S. Y.; Cheong, W.
W.; Poulsen, A.; Blanchard, S.; Lin, G. R.; Alam, J.; Keller, T. H.; Nordlund, P. J.
Med. Chem. 2013, 56, 4497; (b) Jönsson, S.; Andersson, G.; Fex, T.; Fristedt, T.;
Hedlund, G.; Jansson, K.; Abramo, L.; Fritzson, I.; Pekarski, O.; Runström, A.;
Sandin, H.; Thuvesson, I.; Björk, A. J. Med. Chem. 2004, 47, 2075; (c) Tsuji, K.;
Spears, G. W.; Nakamura, K.; Tojo, T.; Seki, N.; Sugiyama, A.; Matsuo, M. Bioorg.
Med. Chem. Lett. 2002, 12, 85.
16. (a) Detsi, A.; Bouloumbasi, D.; Prousis, K. C.; Koufaki, M.; Athanasellis, G.;
Melagraki, G.; Afantitis, A.; Igglessi-Markopoulou, O.; Kontogiorgis, C.;
Hadjipavlou-Litina, D. J. J. Med. Chem. 2007, 50, 245; (b) Teichert, A.;
Schmidt, J.; Porzel, A.; Arnold, N.; Wessjohann, L. J. Nat. Prod. 2008, 71, 1092;
(c) Chung, H. S.; Woo, W. S. J. Nat. Prod. 2001, 64, 1579; (d) Naito, Y.;
Yoshikawa, T.; Tanigawa, T.; Sakurai, K.; Yamasaki, K.; Uchida, M.; Kondo, M.
Free Radical Biol. Med. 1995, 18, 117.
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046

12
M. Pudlo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
17. (a) Carlier, P. R.; Du, D.-M.; Han, Y.-F.; Liu, J.; Perola, E.; Williams, I. D.; Pang, Y.-
P. Angew. Chem., Int. Ed. 2000, 39, 1775; (b) Carlier, P. R.; Du, D.-M.; Han, Y.-F.;
Liu, J.; Pang, Y.-P. Bioorg. Med. Chem. Lett. 1999, 9, 2335.
23. (a) Foti, M. C.; Daquino, C.; Geraci, C. J. Org. Chem. 2004, 69, 2309; (b) Eklund, P.
C.; Långvik, O. K.; Wärnå, J. P.; Salmi, T. O.; Willför, S. M.; Sjöholm, R. E. Org.
Biomol. Chem. 2005, 3, 3336.
18. (a) Asadipour, A.; Alipour, M.; Jafari, M.; Khoobi, M.; Emami, S.; Nadri, H.;
Sakhteman, H.; Moradi, A.; Sheibania, V.; Moghadam, F. H.; Shafiee, A.;
Foroumadi, A. Eur. J. Med. Chem. 2013, 70, 623; (b) Catto, M.; Pisani, L.; Leonetti,
F.; Nicolotti, O.; Pesce, P.; Stefanachi, A.; Cellamare, S.; Carotti, A. Bioorg. Med.
Chem. 2013, 21, 146; (c) Bag, S.; Ghosh, S.; Tulsan, R.; Sood, A.; Zhou, W.;
Schifone, C.; Foster, M.; LeVine, H., III; Török, B.; Török, M. Bioorg. Med. Chem.
Lett. 2013, 23, 2614; and for a recent review see: (d) Anand, P.; Singh, B.; Singh,
N. Bioorg. Med. Chem. 2012, 20, 1175.
19. Beutner, G. L.; Kuethe, J. T.; Yasuda, N. J. Org. Chem. 2007, 72, 7058.
20. Tomassoli, I.; Ismaili, L.; Pudlo, M.; de los Ríos, C.; Soriano, E.; Colmena, I.;
Gandía, L.; Rivas, L.; Samadi, A.; Marco-Contelles, J.; Refouvelet, B. Eur. J. Med.
Chem. 2011, 46, 1.
24. (a) Colaric, M.; Veberic, R.; Solar, A.; Hudina, M.; Stampar, F. J. Agric. Food Chem.
2005, 53, 6390; (b) Belkheiri, N.; Bouguerne, B.; Bedos-Belval, F.; Duran, H.;
Bernis, C.; Salvayre, R.; Nègre-Salvayre, A.; Baltas, M. Eur. J. Med. Chem. 2010,
45, 3019; (c) Li, L.; Seeram, N. P. J. Agric. Food Chem. 2010, 58, 11673.
25. Weber, W. M.; Hunsaker, L. A.; Abcouwer, S. F.; Decka, L. M.; Vander Jagtb, D. L.
Bioorg. Med. Chem. 2005, 13, 3811–3820.
26. Robak, J.; Gryglewski, R. J. Biochem. Pharmacol. 1988, 37, 837.
27. pdb code 1eve, Kryger, G.; Silman, I.; Sussman, J. L. Struct. Fold Des. 1999, 7, 297.
28. pdb code 4ey7, Cheung, J.; Rudolph, M. J.; Burshteyn, F.; Cassidy, M. S.;
Gary, E. N.; Love, J.; Franklin, M. C.; Height, J. J. Med. Chem. 2012, 55,
10282.
29. Wu, G.; Robertson, D. H.; Brooks, C. L., III; Vieth, M. J. Comput. Chem. 2003, 24,
1549.
30. Dougherty, D. A. Acc. Chem. Res. 2013, 46, 885–893.
21. González, I.; Mosquera, J.; Guerrero, C.; Rodríguez, R.; Cruces, J. Org. Lett. 2009,
11, 1677.
22. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
Please cite this article in press as: Pudlo, M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.046