Design, synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase

Article abstract of DOI:10.1016/j.bmc.2012.10.045

Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic and palliative, for the treatment of Alzheimer's disease (AD). Donepezil is one of most used AChEIs in AD therapy, acting as a dual binding site, reversi

Full text of DOI:10.1016/j.bmc.2012.10.045

Bioorganic & Medicinal Chemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of coumarin alkylamines
as potent and selective dual binding site inhibitors of acetylcholinesterase
⇑
Marco Catto , Leonardo Pisani, Francesco Leonetti, Orazio Nicolotti, Paolo Pesce, Angela Stefanachi,
⇑
Saverio Cellamare, Angelo Carotti
Dipartimento di Farmacia, Università degli Studi di Bari ‘‘Aldo Moro’’, via E. Orabona 4, Bari 70125, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic
and palliative, for the treatment of Alzheimer’s disease (AD). Donepezil is one of most used AChEIs in
AD therapy, acting as a dual binding site, reversible inhibitor of AChE with high selectivity over butyr-
ylcholinesterase (BChE). Through a combined target- and ligand-based approach, a series of coumarin
alkylamines matching the structural determinants of donepezil were designed and prepared. 6,7-Dimeth-
oxycoumarin derivatives carrying a protonatable benzylamino group, linked to position 3 by suitable
linkers, exhibited fairly good AChE inhibitory activity and a high selectivity over BChE. The inhibitory
potency was strongly influenced by the length and shape of the spacer and by the methoxy substituents
on the coumarin scaffold. The inhibition mechanism, assessed for the most active compound 13 (IC50
7.6 nM) resulted in a mixed-type, thus confirming its binding at both the catalytic and peripheral binding
sites of AChE.
Received 31 July 2012
Revised 25 October 2012
Accepted 29 October 2012
Available online xxxx
Keywords:
Alzheimer’s disease
Coumarin derivatives
Acetylcholinesterase
Dual binding site inhibitors
Selective cholinesterase inhibition
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
cholinergic transmission is involved in a variety of physiological
systems, AChE is a pursued pharmacological target for treatment
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder associated with cognitive, functional and behavioral
impairments.1 It affects brain regions that control thought, mem-
ory and language, and evolves to a devastating status for patients
and caregivers. Nearly 50 million people worldwide suffer from
AD. Social and economic burdens of this disease are massive due
to estimated direct and indirect annual costs of 100 billion dollars
in patient care per year only in US. Despite the huge efforts of both
industrial and academic scientists, the pathophysiological events
causing the onset and progression of AD remain obscure and, as
a consequence, the setup of appropriate pharmacological, dis-
ease-modifying therapies are still an unmet medical need.2
The current therapies for AD, yet symptomatic and palliative,
rely mainly on the restoration of acetylcholine levels3 and on the
partial antagonism of NMDA receptor4 (Chart 1). Marketed cholin-
ergic drugs are inhibitors of acetylcholinesterase (AChE) often used
in combination with diverse conventional drugs (e.g., antidepres-
sants, antioxidants, and neuroprotectants).
of several pathologies besides AD.5 AChE inhibitors (AChEIs) have
therapeutic application in surgical anesthesia in the treatment of
neuromuscular blockade,6 in myasthenia gravis7 and in glaucoma.8
Another cholinesterase, namely butyrylcholinesterase (BChE), is
involved in metabolic degradation of acetylcholine and differs from
AChE for tissue distribution and sensitivity to substrates and inhib-
itors (Chart 1).
The X-ray crystallographic structure of AChE solved at a high
resolution for a large number of enzyme–ligand complexes9 re-
veals two main binding sites: the catalytic binding site, comprising
the Ser-His-Glu catalytic triad, and the secondary/peripheral anio-
nic binding site (PAS), connected by a deep, hydrophobic gorge.
AChEIs can bind to the catalytic or the peripheral site, or, as in
the case of dual binding site (DBS) inhibitors, to both the sites
(e.g., donepezil, Chart 1). Ligand- and structure-based design has
facilitated the discovery of potent and often selective AChE inhib-
itors, prepared by assembling two identical or different moieties
through a linker of appropriate length and chemical nature.10 This
straightforward strategy has been successfully applied to the de-
sign of AChE inhibitors potentially addressing additional targets,11
according to multitargeted-ligand strategy, particularly promising
in AD12 as well as other multifactorial pathologies such as tumor
diseases.13
AChE (EC 3.1.1.7) is a serine hydrolase located either in periph-
eral (muscles) and/or central nervous (cholinergic) system. Since
⇑
Corresponding authors. Tel.: +39 080 5442803; fax: +39 080 5442230 (M.C.);
tel.: +39 080 5442782; fax: +39 080 5442230 (A.C.).
E-mail addresses: marco.catto@uniba.it (M. Catto), angelo.carotti@uniba.it
(A. Carotti).
In the course of our research aimed at the discovery of new
molecular leads with therapeutic potential in neurodegenerative
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.045
Please cite this article in press as: Catto, M.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.045

2
M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
OH
N
O
N
O
O
N
O
NH2
N
Rivastigmine
Galantamine
Tacrine
IC50 AChE: 100-200 nM
IC50 BChE: 20-40 nM
IC50 AChE: 0.7-1.5 μM
IC50 BChE: 0.3-1.0 μM
IC50 AChE: 0.3-0.7 μM
IC50 BChE: 7-20 μM
NH2
N
O
H3CO
H3CO
Donepezil
Memantine
(NMDA antagonist)
IC50 AChE: 2-10 nM
IC50 BChE: 2-10 μM
Chart 1. Marketed anti-AD drugs. For cholinergic drugs, inhibitory activity ranges on AChE and BChE are reported.
diseases (NDs), we have described several series of coumarin deriv-
atives endowed with high AChE,14 monoamine oxidase (MAO)15
and dual MAO–AChE16 inhibitory activities. Other studies on cou-
marin derivatives as cholinesterase inhibitors, have been recently
reviewed.17 Actually, coumarin scaffold represents a widely occur-
ring, nature friendly privileged structure, whose functionalization
is straightforward. With this skilled and easy chemistry in hand,
we designed and prepared new potent and selective coumarin
derivatives as cholinesterase inhibitors, in particular 6,7-dime-
thoxy-3-substituted coumarins, tethered to a benzylamine moiety
placed at an appropriate distance from the heterocyclic ring.
In order to explore wide structure–activity relationships three
readily available coumarin scaffolds were selected (Chart 2). The
classic Ellman spectrophotometric test18 was then performed to
assess the AChE and BChE inhibitory activities of the designed
compounds, whose structures and ChE inhibition data are reported
in Table 1.
Amides 9–12 were prepared from aminocoumarins II and com-
mercial -chloroacyl chlorides, and subsequent nucleophilic sub-
stitution with benzylmethylamine (Scheme 3).
-
Coumarin IIb was also used as starting material for the synthe-
sis of amide 13, through the coupling with commercial cis-N-Boc-
3-aminocyclohexanecarboxylic acid and final N-benzylation
(Scheme 4). Compound 14 was prepared through two simple con-
secutive alkylation and nucleophilic substitution reactions from
IIIa (Scheme 4).
Finally, derivatives 15–19 were prepared from 3-hydrox-
ycoumarins IIIa–d by Mitsunobu condensation reaction with
appropriate piperidinyl alcohols (Scheme 5).
2.2. Cholinesterase inhibition
Inhibitory activities on AChE (from bovine erythrocytes) and
BChE (from equine serum) were determined by the spectrophoto-
metric method of Ellman18 and are reported in Table 1 as IC50
(lM)
or as percentage of inhibition at 10 lM.
All compounds, with the exception of aminoethers 14, 15 and 17,
acted as selective inhibitors of AChE over BChE, with IC50s spanning
2. Results and discussion
2.1. Chemistry
from 66 lM of compound 10 to 7.6 nM of amide 13, the latter show-
6,7-Dimethoxycoumarin-3-carboxylic acid I,19 3-aminocouma-
rin IIa,20 3-amino-6,7-dimethoxycoumarin IIb,21 and 3-hydroxy-
coumarin derivatives IIIa,22 IIIb,23 IIIc19 and IIId21 were prepared
according to reported literature procedures. To achieve the final
amides 1–3, non-commercial amines Va–c were also prepared
(Scheme 1). Amides and esters 1–8 were synthesized by condensa-
tion of I with suitable amines or alcohols through the use of cou-
pling reagents (Scheme 2).
ing inhibitory potency toward AChE very close to that of donepezil
(4.2 nM). Amides 1–3 with a polymethylene bridge showed a signif-
icant increase of potency with the elongation of the linker. IC50 signif-
icantly decreased from compound 1 (IC50 = 9.0 lM, one of the less
active compounds of the whole data set) to 2 (IC50 = 86 nM), whilst
3 resulted among the most active inhibitors of the entire seriesexam-
ined (IC50 = 21 nM). The small difference in the inhibitoryactivitiesof
amide 2 and its isosteric ester 4 (86 vs111 nMIC50)contrastedindeed
with the four-fold drop of inhibitoryactivityof compound 12, an ana-
logue of2bearinganinvertedamide function. Incomparisonwiththe
corresponding open-chain analogues 1 and 2, piperazine and piperi-
dine amides 5 and 6, respectively, showed a slightly increased affin-
ity. Even in this case, the small oversizing of linker from 5 to 6 led to a
O
O
COOH
O
R1
R2
NH2
O
R1
R2
OH
O
gain of affinity as high as two orders of magnitude (from 4.0 lM to
O
O
O
28 nM). Transformation of amide group of 6 into ester 7 resulted in
an even higher potency (IC50 14 vs 28 nM), while a further elongation
of the spacer in aminoester 8 led to a less active, but yet potent
(IC50 = 73 nM) and selective AChE inhibitor. Compound 9, with a
short methylene linker and aminoether 14 carrying a longer triethyl-
ene linker, showed low inhibitory potency. In the latter case, the lack
of methoxy substituents in positions 6 and 7 of the coumarin ring
I
IIa,b
IIIa-d
a) R1 = R2 = H
b) R1 = H; R2 = OCH3
a) R1 = R2 = H
b) R1 = H; R2 = OCH3
c) R1 = OCH3; R2 = H
d) R1 = R2 = OCH3
Chart 2. Coumarin scaffolds I–III.
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M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3
Table 1
Chemical structures and cholinesterase inhibitory activity of compounds 1–19
Scaffold/structure
Entry
X
R1
R2
AChEa
BChEa,b
1
2
3
4
NH(CH2)2N(CH3)
NH(CH2)3N(CH3)
NH(CH2)4N(CH3)
O(CH2)3N(CH3)
9.0 0.7
0.086 0.004
0.021 0.006
0.111 0.003
(15 1)
(24 2)
(23 2)
(38 1)
O
O
O
X
O
O
N
5
4.0 1.1
(12 1)
N
HN
N
6
7
0.028 0.001
0.014 0.001
(19 1)
(12 2)
O
N
O
8
0.073 0.017
4.7 0.2
N
9
10
11
12
CH2
(CH2)2
(CH2)2
(CH2)3
OCH3
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
26
66
14
3
5
1
(11 1)
(0)
(0)
H
R1
R2
N
X
N
O
0.33 0.08
(43 1)
O
O
H
O
N
N
H
13
14
0.0076 0.0003
33
4
O
O
O
O
O
O
N
4.5 0.2
4.0 0.5
O
15
16
17
18
19
—
—
—
—
CH2
H
OCH3
H
OCH3
OCH3
H
H
OCH3
OCH3
OCH3
4.5 0.3
7.4 0.5
7.9 0.4
(34 2)
(46 1)
(36 3)
3.9 0.8
R1
O
N
12
1
X
1.5 0.1
0.021 0.003
R2
Donepezil
O
O
0.0042 0.0009
1.9 0.3
a
Inhibitory activities are expressed as IC50
Data in parentheses are % of inhibition at 10 M concentration.
(
l
M) and are the mean SEM of three independent measurements.
l
b
A
B
+
Cl
CN
n-1
NH
N
CN
n-1
N
NH2
n
Va-c
IVa-c
a) n = 2
b) n = 3
c) n = 4
Scheme 1. Synthesis of amines Va–c. Reaction conditions: (A) anhydrous K2CO3, acetone, reflux; (B) lithium aluminum hydride, anhydrous THF, 0 °C.
might be responsible for the considerable reduction of AChE affinity
and consequent complete loss of AChE/BChE selectivity. The same
consideration holds for the five-fold drop of inhibitory activity ob-
served going from compound 11 to 10, confirming to some extent
the previous findings reported for donepezil.24 To better understand
the role of methoxy substituents in similar analogues of 14, bearing
the typical AChE-binding motif, that is the N-benzylpiperidine, deriv-
atives15–18werepreparedalongwithcompound19asa homologue
of 18. As can be inferred from the data in Table 1, the unsubstituted
compound 15, closely related to 14, showed equal potency, while
monomethoxy-substituted derivatives 16 and 17 were even weaker,
although with a fair recovering of selectivity over BChE. 6,7-Dime-
thoxy derivative 18 showed only low micromolar inhibitory activity
while maintaining goodselectivity. Very interestingly, the elongation
of spacer of 18 with a methylene group led to compound 19, which
was endowed with a very high potency (IC50 = 21 nM) and a BChE/
AChE selectivity ratio of 186.
drug donepezil, but by far higher than its open-chain analogues 9,
11 and 12.
The mechanism of AChE inhibition by 13 was also assessed by
means of a kinetic study whose results are reported in a typical
Lineweaver–Burk plot in Figure 1. This study confirmed a revers-
ible, mixed-type model of inhibition, in accordance with a likely
dual binding site mode of interactions with AChE. The inhibition
constant Ki was equal to 8.6 1.5 nM.
3. Conclusions
The compelling need to find new drugs to combat AD and other,
less important but widespread cholinergic pathologies still calls for
the development of new, potent and safer AChE inhibitors. Starting
with reliable expertise in the synthesis and biopharmacological
evaluation of coumarin derivatives14–16 and taking into account
the main structural determinants for an effective, dual binding site
inhibition of AChE, we designed, prepared and tested as cholines-
terase inhibitors a pool of 3-substituted coumarins, mostly deco-
rated with methoxy groups at positions 6 and 7. The data
Greater attention was awarded to the outstanding potency of
amide 13, a cis-3-amino-cyclohexanecarboxylic acid derivative,
whose AChE inhibitory potency was comparable to the reference
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M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
O
O
O
O
O
O
N
H
N
O
N
n
O
O
O
O
1-3
4
N
H
Va-c
A
C
C
O
O
O
O
O
O
C
C
N
O
Br
N
I
N
HO
Br
NH
O
O
O
O
VI
5
N
YH
O
O
O
N
Y
O
O
6 (Y = NH)
7 (Y = O)
8 (Y = OCH2)
Scheme 2. Synthesis of compounds 1–8. Reaction conditions: (A) as in Scheme 1; (C) DIC, HOBt, dichloromethane, rt.
H
N
H
N
O
R1
R2
R1
R2
i
A
n Cl
n N
IIa,b
+
Cl
n
Cl
O
O
N
H
O
O
O
O
9-12
VIIa-d
a) n = 1, R =R =OCH
1
1
2
2
3
b) n = 2, R =R =H
c) n = 2, R =R =OCH
1
1
2
2
3
d) n = 3, R =R =OCH
3
Scheme 3. Synthesis of compounds 9–12. Reaction conditions: (A) as in Scheme 1; (i) triethylamine, anhydrous THF, 0 °C.
H
N
H
N
TFA
A
O
O
O
O
C
N
H
IIb
+
NHBOC
Br
NHBoc
O
O
HOOC
O
O
O
O
O
13
VIII
Cl
N
O
A
NaH
DMF, rt
IIIa
Br
Cl
+
N
H
O
O
O
O
IX
14
Scheme 4. Synthesis of compounds 13 and 14. Reaction conditions (A) and (C) as in Schemes 1 and 2, respectively.
CH2
N
O
O
OH
R1
R2
n
CH2
ADDP, Bu P
3
N
IIIa-d
n
+
THF, rt
O
15-18 (n = 0)
19 (n = 1)
Scheme 5. Synthesis of compounds 15–19.
reported in Table 1 revealed a number of very potent compounds
with AChE IC50s as low as 30 nM (i.e., compounds 3, 6, 7, 13 and
19) with compound 13 being the most active and selective AChE
inhibitor of the entire series of compounds examined
(IC50 = 7.6 nM) and acting as a dual binding site inhibitor. Most
inhibitors also showed high selectivity over BChE.
The outstanding inhibitory activity of compound 13, retained also
toward human AChE (IC50 = 43 nM), deserves further investigation,
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M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
5
0.0015
0.0010
0.0005
30 mmol of anhydrous potassium carbonate (4.14 g) in 30 mL of
anhydrous acetone for 3 h (reaction conditions A, Scheme 1). After
cooling to rt and filtration, the solution was evaporated to dryness
and the residue separated on SP1. A pale yellow oil was obtained
that was identified through ESI-MS and NMR and used without fur-
ther purification (IVa–c, yield 80–93%). A solution of 20 mmol of
nitrile IVa–c in 50 mL of anhydrous THF was cannulated into a sus-
pension of 1.25 g of lithium aluminum hydride (33 mmol) in 40 mL
of anhydrous THF, kept under vigorous magnetic stirring at 0 °C
(reaction conditions B). After 1 h the suspension was cautiously
added with water until effervescence disappeared, then filtered
and evaporated to dryness. Amines Va–c were obtained as pale yel-
low oils in a 61–84% yield, identified through ESI-MS and NMR and
used without further purification.
0
5E-9
10E-9
15E-9
4.1.2. Synthesis of compounds 1–8 (Scheme 2)
One twenty five milligram of 3-carboxycoumarin I19
(0.5 mmol) and 70 mg of diisopropylcarbodiimide (DIC;
0.55 mmol) were dissolved in 10 mL of anhydrous dichlorometh-
ane. After 3 min 74 mg of N-hydroxy-1,2,3-benzotriazole hydrate
(HOBt; 0.55 mmol) were added (reaction conditions C), followed
by 0.55 mmol of appropriate amine (Va for final compound 1;
Vb for 2; Vc for 3; N-benzylpiperazine for 5; 4-amino-N-benzylpi-
peridine for 6) or alcohol (3-bromopropanol for VI; N-benzyl-4-
hydroxypiperidine for 7; N-benzylpiperidin-4-ylmethanol for 8).
The reaction was stirred at rt until TLC monitoring showed the dis-
appearance of I (4–8 h). The mixture was then filtered and evapo-
rated to dryness, giving a residue that was separated on SP1 and
crystallized.
-10
10
20
30
1/[Substrate], mM-1
-0.0005
Figure 1. Lineweaver–Burk plot of inhibition kinetics of 13: reciprocals of enzyme
activity (bovine AChE) vs reciprocals of substrate (S-acetylthiocholine) concentra-
tion in the presence of different concentrations (0–15 nM) of inhibitor 13.
firstly, in the evaluation of the inhibitory activities of each of the re-
solved enantiomers aiming at the discovery of a very potent eutomer
and at the detection of the main enantioselective interactions at the
AChE-binding sites. As an additional goal, compound 13 will be eval-
uated in vitro in the test of AChE-promoted beta-amyloid aggrega-
tion25 and in in vivo models of AD. Data from these studies could
give the definitive proof-of-concept of the potential of this coumarin
derivative as a multipotent hit molecule for the treatment of AD.
4.1.2.1. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
[2-(N-benzyl-N-methylamino)ethyl]amide (1). Yellow crystals,
yield 63%, mp: 208–209 °C. 1H NMR (DMSO-d6) d: 2.31 (s, 3H), 2.69
(s, 2H), 3.63 (br, 4H), 3.95 (s, 3H), 3.99 (s, 3H), 6.90 (s, 1H), 7.00 (s,
1H), 7.24–7.44 (m, 5H), 8.80 (s, 1H), 9.14 (br, 1H, exch.). Anal. Calcd
for C22H24N2O5: C, 66.65; H, 6.10; N, 7.07. Found: C, 66.25; H, 6.43;
N, 7.16.
4. Experimental section
4.1. Chemistry
4.1.2.2. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
[3-(N-benzyl-N-methylamino)propyl]amide (2). Yellow crystals,
yield 93%, mp: 144–145 °C. 1H NMR (CDCl3) d: 1.83 (qn, J = 6.9 Hz,
2H), 2.22 (s, 3H), 2.48 (t, J = 6.9 Hz, 2H), 3.47–3.53 (m, 4H), 3.95 (s,
3H), 3.99 (s, 3H), 6.88 (s, 1H), 7.00 (s, 1H), 7.27–7.34 (m, 5H), 8.82
(s, 1H), 9.00 (br, 1H, exch.). Anal. Calcd for C23H26N2O5: C, 67.30; H,
6.38; N, 6.82. Found: C, 66.96; H, 6.05; N, 6.61.
Starting materials, reagents and analytical grade solvents were
purchased from Sigma–Aldrich Europe (Steinheim, Germany). All
reactions were routinely checked by TLC using Merck Kieselgel
60 F254 aluminum plates and visualized by UV light or iodine. 1H
NMR spectra were recorded in the specified deuterated solvent at
300 MHz on a Varian Mercury 300 instrument. Chemical shifts
are expressed in parts per million (d) relative to the solvent signal
and the coupling constants J are given in Hertz (Hz). The following
abbreviations were used: s (singlet), d (doublet), t (triplet), qn
(quintuplet), dd (double doublet), m (multiplet), br (broad signal);
signals due to NH protons were located by deuterium exchange
with D2O. ESI-MS analyses were performed on an Agilent 1100
LC–MSD trap system VL. Chromatographic separations were per-
formed on Biotage SP1 purification system using flash cartridges
4.1.2.3. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
[4-(N-benzyl-N-methylamino)butyl]amide (3). Yellow crystals,
yield 64%, mp: 142–143 °C. 1H NMR (CDCl3) d: 1.63–1.71 (m, 4H),
2.45–2.55 (m, 5H), 3.45–3.47 (m, 4H), 3.95 (s, 3H), 3.99 (s, 3H),
6.89 (s, 1H), 7.00 (s, 1H), 7.30–7.40 (m, 5H), 8.82 (s, 1H), 8.86 (br,
1H, exch.). Anal. Calcd for C24H28N2O5: C, 67.91; H, 6.65; N, 6.60.
Found: C, 67.95; H, 6.73; N, 6.82.
4.1.2.4. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
(4-benzylpiperazin-1-yl)amide (5). Yellow crystals, yield 69%,
mp: 203–204 °C. 1H NMR (CDCl3) d: 2.48–2.54 (m, 4H), 3.40 (t,
J = 4.2 Hz, 2H), 3.54 (s, 2H), 3.78 (t, J = 4.2 Hz, 2H), 3.92 (s, 3H),
3.96 (s, 3H), 6.84 (s, 1H), 6.86 (s, 1H), 7.29–7.32 (m, 5H), 7.85 (s,
1H). Anal. Calcd for C23H24N2O5: C, 67.63; H, 5.92; N, 6.86. Found:
C, 67.38; H, 5.98; N, 6.90.
prepacked with KP-Sil™ 32–63 lm, 60 Å silica; elution gradient
was automatically set with ‘‘TLC plate measurement’’ mode, with
dichloromethane/methanol (90:10 v/v) as TLC eluent. All the final
compounds were recrystallized from ethyl alcohol. Melting points
(mp) were taken on a Gallenkamp MFB 595010M apparatus (open
capillary method) and are uncorrected. Elemental analyses were
performed on a EuroEA 3000 analyser for C, H, N; experimental re-
sults agreed to within 0.40% of theoretical values.
4.1.2.5. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
N-(1-benzylpiperidin-4-yl)amide (6). Yellow crystals, yield 53%,
mp: 189–190 °C. 1H NMR (CDCl3) d: 2.08 (br, 4H), 2.39 (br, 2H),
2.91 (br, 2H), 3.70 (s, 2H), 3.94 (s, 3H), 3.99 (s, 3H), 4.05 (br, 1H),
4.1.1. Synthesis of intermediate amines Va–c (Scheme 1)
Twenty five millimole of N-methylbenzylamine (3.03 g,
3.22 mL) and 30 mmol of
--chloroalkanonitrile were refluxed with
Please cite this article in press as: Catto, M.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.045

6
M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
6.89 (s, 1H), 6.99 (s, 1H), 7.36–7.38 (m, 5H), 8.79 (s, 1H), 8.87 (br,
1H, exch.). Anal. Calcd for C24H26N2O5: C, 68.23; H, 6.20; N, 6.63.
Found: C, 67.86; H, 6.18; N, 6.98.
4.1.3.3. 3-(N-Benzyl-N-methylamino)-N-(6,7-dimethoxy-2-oxo-
2H-chromen-3-yl)propionamide (11). White crystals, yield 63%,
mp: 178–179 °C. 1H NMR (CDCl3) d: 2.36 (s, 3H), 2.57 (t, J = 6.0 Hz,
2H), 2.74 (t, J = 6.0 Hz, 2H), 3.68 (s, 2H), 3.92 (s, 3H), 3.94 (s, 3H),
6.85 (s, 1H), 6.89 (s, 1H), 7.26–7.34 (m, 3H), 7.40–7.43 (m, 2H),
8.64 (s, 1H), 11.07 (br, 1H, exch.). Anal. Calcd for C22H24N2O5: C,
66.65; H, 6.10; N, 7.07. Found: C, 66.40; H, 6.03; N, 7.07.
4.1.2.6. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
(1-benzylpiperidin-4-yl)ester (7). Yellow crystals, yield 58%,
mp: 147–148 °C. 1H NMR (CDCl3) d: 1.66 (br, 2H), 1.93 (br, 2H),
2.49 (br, 2H), 2.85 (br, 2H), 3.64 (s, 2H), 3.94 (s, 3H), 3.98 (s, 3H),
5.12 (br, 1H), 6.88 (s, 1H), 6.94 (s, 1H), 7.28–7.39 (m, 5H), 8.46 (s,
1H). Anal. Calcd for C24H25NO6: C, 68.07; H, 5.95; N, 3.31. Found:
C, 67.71; H, 6.07; N, 3.37.
4.1.3.4. 4-(N-Benzyl-N-methylamino)-N-(6,7-dimethoxy-2-oxo-
2H-chromen-3-yl)butyramide (12). Yellow crystals, yield 53%,
mp: 104–105 °C. 1H NMR (CDCl3) d: 2.16 (br, 2H), 2.42–2.62 (m,
5H), 2.84 (br, 2H), 3.81–3.96 (m, 2H), 3.93 (s, 3H), 3.94 (s, 3H),
6.84 (s, 1H), 6.86 (s, 1H), 7.30–7.44 (m, 3H), 7.48–7.58 (m, 2H),
8.50 (br, 1H, exch.), 8.57 (s, 1H). Anal. Calcd for C23H26N2O5: C,
67.30; H, 6.38; N, 6.82. Found: C, 67.28; H, 6.57; N, 7.20.
4.1.2.7. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
[(1-benzylpiperidin-4-yl)methyl]ester (8). Brown crystals, yield
57%, mp: 171–172 °C. 1H NMR (CDCl3) d: 1.90–2.17 (m, 5H),
2.60–2.67 (m, 4H), 3.97 (s, 3H), 3.99 (s, 2H), 4.00 (s, 3H), 4.15 (d,
J = 3.3 Hz, 1H), 4.25 (d, J = 3.3 Hz, 1H), 6.79 (s, 1H), 7.45–7.47 (m,
3H), 7.61–7.63 (m, 2H), 7.67 (s, 1H), 9.16 (s, 1H). Anal. Calcd for
4.1.4. Synthesis of compound 13 (Scheme 4)
3-Aminocoumarin IIb (2.7 mmol, 600 mg) and cis-N-Boc-3-ami-
nocyclohexanecarboxylic acid (660 mg) were reacted in reaction
conditions C to yield the desired coumarin intermediate VIII as a
brown solid (57% yield; NMR and ESI-MS characterization).
446 mg of VIII (1.0 mmol) were dissolved in 20 mL of dichloro-
methane. The solution was cooled to 0 °C using an external ice
bath, and trifluoroacetic acid (1.7 mL, 22 mmol) was added drop-
wise. The reaction solution was slowly allowed to warm to room
temperature and stirred overnight. The mixture was then cooled
to 0 °C and a 20% sodium carbonate aqueous solution (45 mL)
was slowly added. The organic layer was evaporated to dryness
and the resulting yellow solid was reacted with one equivalent of
benzyl bromide (reaction conditions A) to produce, after purifica-
tion, the final compound 13.
C25H27NO6: C, 68.64; H, 6.22; N, 3.20. Found: C, 68.30; H, 6.23;
N, 3.32.
3-Carboxycoumarin I was reacted in the same reaction condi-
tions C with 3-bromo-1-propanol. After SP1 separation, compound
VI was obtained as a yellow solid and used without further purifi-
cation (identification: ESI-MS and NMR; yield 64%). Reaction of VI
with N-methylbenzylamine under the above reported conditions A
yielded compound 4, purified as above.
4.1.2.8. 6,7-Dimethoxy-2-oxo-2H-chromene-3-carboxylic acid
[3-(N-benzyl-N-methylamino)propyl]ester (4). Brown crystals,
yield 64%, mp: 97–98 °C. 1H NMR (CDCl3) d: 2.45–2.55 (m, 2H),
2.72 (s, 3H), 3.45–3.47 (m, 4H), 3.95 (s, 3H), 3.99 (s, 3H), 4.41 (t,
J = 6.9 Hz, 2H), 6.85 (s, 1H), 7.03 (s, 1H), 7.43–7.45 (m, 3H), 7.62–
7.64 (m, 2H), 8.63 (s, 1H). Anal. Calcd for C23H25NO6: C, 67.14; H,
6.12; N, 3.40. Found: C, 66.74; H, 5.99; N, 3.17.
4.1.4.1. cis-3-(Benzylamino)cyclohexanecarboxylic acid (6,7-
dimethoxy-2-oxo-2H-chromen-3-yl)-amide (13). Yellow crys-
tals, yield 51%, mp: 81–82 °C. 1H NMR (DMSO-d6) d: 1.18–1.36
(m, 2H), 1.72–1.90 (m, 3H), 2.30–2.08 (m, 3H), 2.66–2.82 (m,
1H), 2.94–3.10 (m, 2H), 3.78 (s, 3H), 3.82 (s, 3H), 4.10–4.20 (m,
1H), 7.06 (s, 1H), 7.26 (s, 1H), 7.38–7.44 (m, 3H), 7.44–7.60 (m,
2H), 8.57 (s, 1H), 9.05 (br, 1H, exch.), 9.62 (s, 1H, exch.). Anal. Calcd
for C25H28N2O5: C, 68.79; H, 6.47; N, 6.42. Found: C, 68.48; H, 6.57;
N, 6.20.
4.1.3. Synthesis of compounds 9–12 (Scheme 3)
Two twenty one milligram of 3-aminocoumarin IIa20 or 3-ami-
no-6,7-dimethoxycoumarin II21 (1.0 mmol) and 607 mg of triethyl-
amine (0.84 mL; 6.0 mmol) were dissolved in 8 mL of anhydrous
THF. A solution of 1.0 mmol of the appropriate
--chloroacyl chlo-
ride (n = 1–3; see Scheme 3) in 2 mL of anhydrous THF was
dropped through a dropping funnel under vigorous magnetic stir-
ring in the reaction flask kept in ice-cold water. After 1 h the sus-
pension was filtered and the solution evaporated to dryness,
4.1.5. Synthesis of compound 14 (Scheme 4)
One sixty two milligram of 3-hydroxycoumarin IIIa22
(1.0 mmol) and 24 mg of sodium hydride (1.0 mmol) were dis-
solved in 5 mL of anhydrous DMF, then 0.79 g of 1-bromo-3-chlo-
ropropane (0.49 mL, 5.0 mmol) were added. The mixture was
reacted at 120 °C for 1 h and poured into crushed ice. Extraction
with diethyl ether (3 ꢁ 10 mL) and evaporation of solvent to dry-
ness gave IX as a brown solid, used as such for the subsequent
reaction (yield 72%). 170 mg of IX (0.7 mmol) reacted with N-
methylbenzylamine in the above reported conditions A to give,
after chromatographic separation, pure compound 14, which was
finally crystallized as hydrochloride.
yielding
--chloroamides VIIa–d in quantitative yield. Crude
VIIa–d were reacted with N-methylbenzylamine in the above re-
ported conditions A to give final compounds 9–12. Compound 10
was crystallized as hydrochloride.
4.1.3.1. 2-(N-Benzyl-N-methylamino)-N-(6,7-dimethoxy-2-oxo-
2H-chromen-3-yl)acetamide (9). Yellow crystals, yield 62%, mp:
177–178 °C. 1H NMR (CDCl3) d: 2.48 (s, 3H), 3.27 (s, 2H), 3.71 (s,
2H), 3.92 (s, 3H), 3.94 (s, 3H), 6.85 (s, 1H), 6.88 (s, 1H), 7.32–7.39
(m, 3H), 7.46–7.48 (m, 2H), 8.60 (s, 1H), 9.82 (br, 1H, exch.). Anal.
Calcd for C21H22N2O5: C, 65.96; H, 5.80; N, 7.33. Found: C, 66.28; H,
5.57; N, 7.26.
4.1.5.1. 3-[3-(N-Benzyl-N-methylamino)propyloxy]-6,7-Dime-
thoxy-2-oxo-2H-chromene (14). White crystals, yield 59%, mp
(hydrochloride): 202–204 °C (dec.). 1H NMR (CDCl3) d: 2.08 (qn,
J = 6.6 Hz, 2H), 2.25 (s, 3H), 2.59 (t, J = 6.6 Hz, 2H), 3.53 (s, 2H),
4.08 (t, J = 6.6 Hz, 2H), 6.82 (s, 1H), 7.18–7.43 (m, 9H). Anal. Calcd
for C20H21NO3ꢀHCl: C, 66.75; H, 6.16; N, 3.89. Found: C, 66.60; H,
6.01; N, 4.22.
4.1.3.2. 3-(N-Benzyl-N-methylamino)-N-(2-oxo-2H-chromen-3-
yl)propionamide (10). White crystals, yield 62%, mp: 216–218 °C
(dec.). 1H NMR (DMSO-d6) d: 2.63 (s, 3H), 3.11 (t, J = 6.0 Hz, 2H),
3.24 (t, J = 6.0 Hz, 2H), 4.31 (s, 2H), 7.26–7.34 (m, 3H), 7.29–7.70
(m, 9H), 8.56 (s, 1H), 10.05 (s, 1H, exch.), 10.91 (br, 1H, exch.). Anal.
Calcd for C20H20N2O3 ꢀ HCl: C, 64.42; H, 5.68; N, 7.51. Found: C,
64.80; H, 5.37; N, 7.16.
4.1.6. Synthesis of compound 15–19 (Scheme 5)
In a flame-dried flask flushed with Ar stream, 1.0 mmol of the
appropriate 3-hydroxycoumarin IIIa–d19,21–23 were dissolved
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M. Catto et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
7
together with 0.76 g of N,N0-azobis(dicarbonyldipiperidine)
(ADDP; 3.0 mmol), 0.61 g of tributylphosphine (0.75 mL, 3.0 mmol)
and the suitable alcohol (4-hydroxy-N-benzylpiperidine for com-
pounds 15–18; N-benzyl-4-piperidinemethanol for 19) in 15 mL
of anhydrous THF. After 24 h the reaction was filtered, evaporated
to dryness and separated on SP1, affording title compounds that
were finally crystallized.
0.1 M phosphate buffer, pH 8.0), 100
DTNB in 0.1 M phosphate buffer (pH 7.0) containing 6 mM NaH-
l
L of a 3.3 mM solution of
CO3, 100 lL of a solution of the inhibitor (six to seven concentra-
tions ranging from 1 ꢁ 10ꢂ10 to 1 ꢁ 10ꢂ4 M), and 500
lL of
phosphate buffer, pH 8.0. After incubation for 20 min at 25 °C, acet-
ylthiocholine iodide (100 lL of 5 mM aqueous solution) was added,
and AChE-catalyzed hydrolysis was followed by measuring the in-
crease of absorbance at 412 nm for 3.0 min at 25 °C. The concentra-
tion of compound which determined 50% inhibition of the AChE
activity (IC50) was calculated by non-linear regression of the re-
sponse–log(concentration) curve, using GraphPad PrismÒ (v. 5).
BChE inhibitory activity was assessed similarly using butyrylthi-
ocholine iodide as the substrate. Kinetic studies were performed
under the same incubation conditions, using six concentrations
of substrate (from 0.033 to 0.2 mM) and four concentrations of
inhibitor 13 (0–15 nM). Apparent inhibition constants and kinetic
parameters were calculated within the ‘Enzyme kinetics’ module
of Prism.
4.1.6.1.
3-(1-Benzylpiperidin-4-yloxy)-2-oxo-2H-chromene
(15). White crystals, yield 59%, mp: 141–142 °C. 1H NMR (CDCl3)
d: 1.88–2.04 (m, 4H), 2.35 (br, 2H), 2.79 (br, 2H), 3.56 (s, 2H),
4.42 (br, 1H), 6.87 (s, 1H), 7.21–7.40 (m, 9H). Anal. Calcd for
C21H21NO3: C, 75.20; H, 6.31; N, 4.18. Found: C, 74.89; H, 6.28;
N, 4.36.
4.1.6.2.
3-(1-Benzylpiperidin-4-yloxy)-6-methoxy-2-oxo-2H-
chromene (16). White crystals, yield 49%, mp: 151–152 °C. 1H
NMR (CDCl3) d: 1.75–1.86 (m, 2H), 2.35 (br, 2H), 2.74–2.76 (m,
4H), 3.52 (s, 2H), 3.82 (s, 3H), 4.51 (br, 1H), 7.00 (dd, J = 9.1 Hz,
3.0 Hz, 1H), 7.08 (d, J = 3.0 Hz, 1H), 7.20–7.37 (m, 7H). Anal. Calcd
for C22H23NO4: C, 72.31; H, 6.34; N, 3.83. Found: C, 71.94; H,
6.37; N, 3.94.
Acknowledgments
This work was supported by a grant from MIUR (Rome, Italy;
PRIN project 20085HR5JK_005).
4.1.6.3.
3-(1-Benzylpiperidin-4-yloxy)-7-methoxy-2-oxo-2H-
chromene (17). White crystals, yield 51%, mp: 127–128 °C. 1H
NMR (CDCl3) d: 1.84–1.95 (m, 2H), 2.03 (br, 2H), 2.33 (br, 2H),
2.78 (d, J = 6.0 Hz, 2H), 3.55 (s, 2H), 3.84 (s, 3H), 4.36 (br, 1H),
6.82 (s, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.89 (s, 1H), 7.25–7.34 (m,
6H). Anal. Calcd for C22H23NO4: C, 72.31; H, 6.34; N, 3.83. Found:
C, 71.93; H, 6.32; N, 4.12.
References and notes
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4.1.6.4. 3-(1-Benzylpiperidin-4-yloxy)-6,7-dimethoxy-2-oxo-2H-
chromene (18). White crystals, yield 44%, mp: 62–63 °C. 1H NMR
(CDCl3) d: 1.86–2.10 (m, 2H), 2.12 (br, 2H), 2.43 (br, 2H), 2.84 (br,
2H), 3.62 (s, 2H), 3.90 (s, 3H), 3.91 (s, 3H), 4.35 (br, 1H), 6.78 (s, 1H),
6.82 (s, 1H), 6.90 (s, 1H), 7.27–7.41 (m, 2H), 7.42–7.70 (m, 3H).
Anal. Calcd for C23H25NO5: C, 69.86; H, 6.37; N, 3.54. Found: C,
69.93; H, 6.42; N, 3.32.
4.1.6.5. 3-[(1-Benzylpiperidin-4-yl)methoxy]-6,7-dimethoxy-2-
oxo-2H-chromene (19). White crystals, yield 58%, mp: 60–
61 °C. 1H NMR (CDCl3) d: 1.82–2.15 (m, 2H), 2.24 (br, 2H), 2.45
(br, 2H), 2.93 (br, 2H), 3.62 (br, 2H), 3.91 (s, 3H), 3.92 (s, 3H),
3.96–4.06 (m, 2H), 4.20 (br, 1H), 6.81 (s, 1H), 6.82 (s, 1H), 6.83 (s,
1H), 7.41–7.45 (m, 3H), 7.62–7.66 (m, 2H). Anal. Calcd for
C24H27NO5: C, 70.40; H, 6.65; N, 3.42. Found: C, 70.63; H, 6.52; N,
3.33.
4.2. Biological tests
All materials were purchased from Sigma–Aldrich Europe. The
inhibition assays of AChE from bovine erythrocytes (0.36 U/mg),
and BChE from equine serum (13 U/mg), were run in phosphate
buffer 0.1 M, at pH 8.0. Acetylthiocholine iodide was used as sub-
strate and 5,50-dithiobis(2-nitrobenzoic acid) (DTNB) as the chro-
mophoric reagent.18 Inhibition assays were carried out on an
Agilent 8453E UV–vis spectrophotometer equipped with a cell
changer. Solutions of tested compounds were prepared starting
from 10 mM stock solutions in DMSO that were diluted with aque-
ous assay medium to a final content of organic solvent always less
than 1%. AChE inhibitory activity was determined in a reaction
mixture containing 200 lL of a solution of AChE (0.415 U/mL in
Please cite this article in press as: Catto, M.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.10.045